Structural, biochemical characterization and molecular mechanism of Cerastokunin: A new Kunitz-type peptide with potential inhibition of thrombin, factor Xa and platelets

Structural, biochemical characterization and molecular mechanism of Cerastokunin: A new Kunitz-type peptide with potential inhibition of thrombin, factor Xa and platelets | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Structural, biochemical characterization and molecular mechanism of Cerastokunin: A new Kunitz-type peptide with potential inhibition of thrombin, factor Xa and platelets Noussaiba Saghour, Fatah Chérifi, Samah Saoud, Younes Zebbiche, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4354127/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Aug, 2024 Read the published version in The Protein Journal → Version 1 posted 10 You are reading this latest preprint version Abstract The current investigation focused on separating Cerastes cerastes venom to produce the first Kunitz-type peptide. Three stages of chromatography were used to purify a 7.75 kDa peptide called Cerastokunin with pI 8.48 till homogeneity based on antitrypsin activity. Cerastokunin was found to include 67 amino acid residues that were obtained by de novo sequencing using LC-MALDI-MSMS. Upon alignment with kunitz-type peptides, there was a high degree of similarity. Cerastokunin's 3D structure had 12% α-helices and 21% β-strands. Cerastokunin showed a strong anticoagulant potential by completely eliminating the protease activity of thrombin and trypsin as well as blocking the intrinsic and extrinsic coagulation pathways. In both PT and aPPT, Cerastokunin increased the blood clotting time in a dose-dependent way. Using Lys48 and Gln192 for direct binding, Cerastokunin inhibited thrombin, Factor Xa and trypsin as shown by molecular docking. Cerastokunin exhibited a dose-response blockade of PARs-dependent pathway platelet once stimulated by thrombin. In vivo study showed a substantial reduction in tail thrombus of mice-carrageenan model; in contrast to antithrombotic medications, this antithrombosis was boosted by a greater dose of Cerastokunin. Throughout the trial course, no in vivo toxicity was observed in challenged mice at any of Cerastokunin doses up to 6 mg/kg. Cerastokunin Kunitz-Type peptide Antithrombotic Anti-platelet FXa inhibitor Thrombin inhibitor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 1. Introduction Thrombo-inflammatory disorders are a common characteristic complication of diverse diseases including coagulopathies, infectious diseases, and various cancers [ 1 – 3 ]. Apart from monoclonal antibodies for certain types of cancers, synthetic molecules are mainly used for therapeutic purposes for all these concern conditions. However, chemical medicines are showing more and more side-effects, especially when they target critical diseases like cardiovascular disorders. In this regard, researchers are interested in discovering and identifying new biomolecules with biological activities purposely as an alternative biotherapy that consists of potential innovative biotherapeutics that might replace traditional chemical drugs. Venomous animals including Viperidae snake’s venoms present a rich source of various peptides and proteins doted of a myriad of biological activities that have been previously purified and characterized [ 4 ]. Some of these biomolecules can be used as therapeutics in coagulopathies related to blood clotting factors’ deficiencies or to their genetic anomalies in terms of expression or function. They either act as substitutes to coagulation factors by mimicking their functions or by interacting with them. Other snake venom components interfere as whether activators or inhibitors or even both with various steps of platelet functions [ 5 , 6 ]. Cerastes cerastes venom is a heterogeneous cocktail of bioactive molecules that interact with the hemostatic system by activating or inhibiting coagulation pathways, with a persistent bleeding resulting from a synergistic action of different components [ 7 , 8 ]. Actually, a variety of biomolecules from Cerastes cerastes venom were isolated and their mechanisms underlying the pharmacological action on hemostasis were well elucidated. The structural characterization reveals some as enzymatic proteins like metalloproteinases (CCSV-MPase), serine proteases (Afâacytin, RP-34 and Cc3-SPase), a 5’-nucleotidase (Cc-5’NTase), phospholipases A2 (Cc1-PLA2 and Cc2-PLA2), a phosphodiesterase (Cc-PDE) and others are peptides with non catalytic activity including a C-type lectin (Cc-Lec) and two desintegrins (Cerastategrin and Ophidiastatin) [ 9 – 14 ]. However, there are still numerous bioactive components to explore from Cerastes cerastes venom. In our knowledge, until today none kunitz type peptide has been yet isolated from this venom. Snake venom Kunitz type peptides commonly known as BPTI (Bovin Pancreatic Trypsin Inhibitors), compose an abundant family of serine proteases inhibitors that are mostly present in Elapidae and Viperidae venoms, according to many proteome analyses. In fact, BPTI are hugely found in Daboia russelii venom (28%) whilst around only 16% in Dendroaspis angusticeps venom, this may explain their contribution in the hemostasis disruption of prey since BPTI interact efficiently with blood coagulation cascade and are able to abolish more than serine proteases actively involved in coagulation [ 15 ]. Aside this versatile abundance, functionally, snakes’ venom kunitz-type peptides were subdivided into two groups: (i) non neurotoxic snake venom kunitz type inhibitors that display an inhibitory activity of trypsin and/or chymotrypsin, and, (ii) neurotoxic kunitz-type peptides that inhibit potassium and calcium channels, which do not display serine proteases’ inhibitory activity [ 16 ]. Apart from these two previous classes, a third class was introduced to this classification based on bifunctional kunitz type peptides generated from a structural evolution of snakes’ venom kunitz-type peptides [ 17 ]. Interestingly, several researches on kunitz type peptides of snake venoms have shown that they provide a source of promising pharmaceutical applications. Actually, these peptides contribute in different biological processes, especially, on the blood clotting, fibrinolysis and inflammation pathways, and present a potent anti-tumoral effect due to their multifunctional features [ 18 ]. Furthermore, kunitz type inhibitors appear to present a selectivity towards their targets which makes them excellent candidates for drug development. For instance, Textilinin-1, an antihemorrhagic component that shows fewer side effects, a better efficiency and higher selectivity than Trasylol, commonly known as Aprotinin, a BPTI of reference [ 19 ]. The main objective of the current study was to purify the first antithrombotic kunitz-type peptide from Cerastes cerastes venom, its in vivo , in vitro and in silico characterization to be performed and the mechanism of action as anti-thrombin, anti-FXa and antiplatelet peptide to be elucidated as well. In the future, the new peptide may be developed as a new diagnostic and/therapeutic biodrug for blood coagulopathies. 2. Materials and Methods 2.1. Biological materials and reagents 2.1.1. Animals NMRI mice (20–25 g) were obtained and maintained at the animal breeding of Faculty of Biological Sciences of USTHB (Algiers, Algeria) and experimental protocols were conducted according to the Ethics Committee of European Community in Experimentation for Animals’ Welfare ( https://ec.europa.eu/food/animals/welfare/strategy_en ), and approved after expertise performed by the Ethical Committee of Animal Experimentation (ECAE). The ECAE is a fully constituted Committee in accordance with the National Statement on Ethical conduct in research involving laboratory Animal-Law N°.88 − 08 of 26th January, 19988 related to veterinary medicine and the protection of animal health. 2.1.2. Venom Cerastes cerastes venom was provided by the Faculty of Biological Sciences, USTHB (Algiers, Algeria) in freeze- lyophilized form, and conserved at − 20°C in a protected zone from light until use. The blood hemostasis kits were procured from Biolabo. 2.1.3. Medicines Apixaban (Biopexa 5mg lot 015 n°19/12A200/263) and Rivaroxaban (Trombix 20 mg lot 005822 n°16/12F1688447). All Chemical Reagents are of analytical grade and were purchased from Sigma Aldrich- Merck group. 2.2 Purification of anti-trypsin activity peptide from Cerastes crude venom Three successive chromatographies were carried out in order to purify Cerastokunin from the crude venom of Cerastes cerastes; gel filtration chromatography followed by an affinity chromatography. Finally, a Reverse High Performance Liquid Chromatography (RP-HPLC) was performed to check the purity of the purified Cerastokunin. First, lyophilized crude venom (460 mg) was dissolved in 5 mL of saline buffer 0.9% and submitted to G-75 Sephadex size exclusion chromatography on column (60 × 2.5 cm) (Pharmacia) equilibrated with 100 mM ammonium acetate buffer (pH 8.5). Fractions’ elution occurred at flow rate of 18 mL/h with the same buffer of equilibration and 3 mL of the fractionated protein and peptide peaks were collected and monitored at 280 nm. The obtained fractions were pooled, desalted by a direct elution on a column (10 x 1.5 cm) of G-25 Sephadex gel (GE 17-0032-02 Healthcare, Sweden) and concentrated. Afterwards, hemolytic activity or phospholipase A2 [ 20 ], casein protease [ 21 ], modified by [ 22 ] and anti-trypsin [ 23 ] activities were assessed. The third Gel-filtration (GF3) fraction of low molecular weight molecules was pooled (tubes 210–427). GF3 doesn’t exhibit any hemolytic nor casein protease activities while it does show a potent inhibition on trypsin-activity against a natural substrate (casein). Based on this activity, GF3 was re-subjected to a certain repeated runs of affinity chromatography on Trypsin-Sepharose CL4B column (1.4 × 8.5 cm). Before performing the affinity chromatography, immobilization of trypsin as ligand to Sepharose CL4B was conducted and performed as described by Thernyck and Avrameas in 1991. Briefly, an amount of BrCN activated Sepharose CL4B gel (2.5 g) was extensively washed with HCl solution (1 mM) by successive centrifugations at 3000x g during 2 min each, trypsin solution [10 mg trypsin dissolved in 10 mL sodium carbonate/bicarbonate buffer (0.1 M, pH 8.3) containing NaCl (1 M)] was added to the gel, and the mixed solution was incubated on shaker at + 4°C all over the night then centrifuged for 2 min at 3000x g . The blocking buffer (glycine (0.1 M, pH 8.2) was added and the mixture was incubated at + 4°C for 16 hours, the excess of trypsin non-linked to the gel was eliminated by three successive centrifugations at 3000x g within 2 minutes each. The Trypsin-Sepharose CL4B gel was submitted on a chromatography column (5 x 1 cm) and equilibrated with PBS buffer (0.1 M, pH 7.4), then concentrated GF3 (1.5 mL, 7.381 mg) was subjected to the affinity chromatography and left at least 1 hour on the column before elution run to enable sufficient interaction between fraction containing anti-trypsin peptides and immobilized trypsin onto column. The affinity chromatography began by first removing unbound proteins and peptides by simple elution with PBS buffer (0.1 M, pH 7.4). Then, the same buffer with highest molarity (1 M, pH7.4) was used to elute trypsin-high affinity peptides. The flow rate was maintained at 18 mL/h and 3 mL of the fractionated proteins and peptide peaks were collected and monitored at 280 nm. Finally, the purity of the obtained Cerastokunin, was checked by injecting 10 µL (14.5 µg) of the concentrated fraction of trypsin-high affinity, on a C18 RP-HPLC column (250 mm × 4.6 mm, 5 µm) previously equilibrated with 0.1% (v/v) trifluoroacetic acid (TFA) (Solvent A), then the purified peptide was eluted with a linear gradient over 70 min from 5 to 100% (v/v) acetonitrile 80% (ACN) containing 0.1% (v/v) TFA (Solvent B) under a flow rate of 1 ml/min as described by [ 23 , 24 ]. The elution of peptides was monitored at 280 nm and 10 µL of blank solution containing 95% solvent A (v/v) and 5% of solvent B (v/v), was subjected to the same HPLC protocol in order to detect the additional peaks relatives to solvent absorbance on the Ultraviolet spectrum. 2.3. Mass spectrometry analysis on MSMS-MALDI device The analysis using mass spectrometry was carried out in accordance with [ 25 ]. Cerastokunin was first precipitated in six volumes of cold acetone (overnight, -20°C), after which the peptide was reduced with five milligrams of Tris (2-CarboxyEthyl) Phosphine hydrochloride and alkylated with ten milligrams of 2-iodoacetamide to create the analytical sample. The sample was then cleaved by incubating it for six hours at 37°C with 1 µg/µL of Lys-C endopeptidase and 2 mM CaCl2. The treated peptide was then centrifuged at 5,000 xg for 10 minutes after being incubated with trypsin for 5 hours. The resultant peptide fragments were extracted once the enzymatic digestion was complete, diluted in solvent A (5% ACN/0.1% TFA), and put into an Ultimate 3000 HPLC system fitted with a Probot fractionation device (15 cm x 75 µm x 3 µm, 100Å) with a separate C18 PepMap column (Dionex LC-Packings, Sunnyvale, CA, USA). The peptide separation was then initiated after a 5-minute wash of the utilized column. Following the column wash, a linear gradient of solvent B containing 80% CAN with 0.1% TFA (0 to 50%) was applied to start the separation process. This was done for 45 minutes, and then a final wash phase was carried out for 5 minutes using solvent B at 95%. The separation of peptide fragments was carried out at a consistent temperature of roughly 40°C. Lastly, the collected fractions were immediately spotted onto a MALDI plate with a matrix of succinic acid (CHCA) (2 mg/mL). Using CHCA containing 10 fmoles of Glu-1-fibrino peptide B ([M + H] + = 1570.670), the mass spectra were calibrated and for the MS/MS spectra, the 2 kV positive CID ON technique was chosen. In positive reflector ionization mode (m/z range: 800 to 4,000), 4,800 MALDI TOF/TOF (Applied Biosystems/MDS SCIEX, Framingham, MA, USA) analyzed MALDI MS data with 3000 laser shots/spot with a satisfactory S/N (signal/noise) quality for precursor selection. A MASCOT search engine 2.1 (Matrix Science Ltd., UK) integrated into GPS-Explorer Software 3.5 on the NCBInr database was used to analyze MS/MS queries. The research in MASCOT was calibrated with 0.3 Da for trypsin cleavage and 50 ppm mass accuracy for the precursor. The collected values were verified by considering only protein scores of 53 and protein indexes of 95%. Additionally, two Scaffold software reanalyses of the discovered proteins were conducted utilizing and a workflow incorporating both Peptide- and Protein-Prophet was chosen for the MS/MS independent search engines (MASCOT and X! Tandem). In this instance, only proteins with peptide confidence indexes greater than 95 and protein confidence indexes greater than 95% were taken into account. 2.4 Antiplatelet activity of Cerastokunin on thrombin-induced platelet aggregation 2.4.1. Platelet-rich plasma and platelet-poor plasma preparation Healthy volunteers who had not taken any medications that would affect their hemostasis for at least three weeks prior to the blood collection were used to donate human blood. 3.8% of the boots used for blood i.v. extraction were citrated. Platelet-rich plasma (PRP) was obtained by centrifuging the collected blood for seven minutes at 150 xg , whereas platelet-poor plasma (PPP) was obtained by centrifuging the blood for twenty minutes at 2,000 xg [ 12 ]. 2.4.2 Evaluation of the anti-platelet activity by aggregometry Purified peptide's antiplatelet activity was measured using the Chronolog aggregometer (Chrono-Log, ServiBIO 540 VS USA) for five minutes after 200 µL of PRP was added. PPP was used as a blank control. Thrombin, used at 0.45 U/mL, was pre-incubated for 30 min with 10 and 20 µg (1.31 and 2.6 nmole) of Cerastokunin at 37°C. Cerastokunin's suppression of platelet aggregation was evaluated in relation to thrombin-induced platelet aggregation, which was observed when thrombin (0.45 U/mL) was added to PRP (200 µL). The maximal platelet aggregation activity was identified as the platelet aggregation attained with thrombin. 2.4.3 Dose-response inhibitory activity of platelet aggregation In order to assess its dose-response effect, various increasing Cerastokunin doses ranging from 1 to 20 µg/mL (1, 2, 3, 5, 10, 15, and 20 µg/ml) or (0.13, 0.258, 0.387, 0.65, 1.29, 1.94, 2.58 µM) were pre-incubated with 0.45 U/mL thrombin at 37°C for 30 min in a 96-well microplate prior to adding 90 µL of PRP in each well. The absorbance was recorded at 540 nm by a microtiter reader Bio-Tek Instrument. Thrombin and heparin were used as positive and negative aggregation activity controls, respectively, and the platelet aggregation percent was deduced by comparison to thrombin-induced aggregation considered as maximal aggregation activity. Percent aggregation = [(A 0 - Af)/ (A 0 -A)] ×100%, where Ao is the absorbance of PRP at 540 nm prior to addition of tested molecules; Af is the absorbance of PRP at 540 nm post addition of tested molecules ; and A is PPP absorbance at 540 nm [ 26 ]. 2.5 In vitro studies of anti-blood coagulation of Cerastokunin 2.5.1 Measurement of Prothrombin Time (PT) In this experiment, the inhibitory action of Cerastokunin on the extrinsic coagulation pathway was assessed. Graded concentrations of peptide (0–20 µg/mL equivalent to 0-2.58 µM respectivly) were incubated with 100 µL of PRP for 3 min at 37°C then, 200 µL of Prothrombin Time reagent (Biolabo, France) was added following the manufacturer’s instruction and the clotting time was optically measured in seconds. 2.5.2 Measurement of Activated Partial Thromboplastin Time (aPTT) To evaluate the probable involvement of Cerastokunin on the intrinsic pathway of blood coagulation, a mixture of 100 µL of PPP and different concentrations of Cerastokunin (0–20 µg/mL equivalent to 0-2.58 µM respectively) was incubated with 100 µL of aPTT reagent (Biolabo, France) at 37°C for 3min, as recommended by the manufacturer, and clotting time was recorded in seconds after 0.025 M CaCl 2 (100 µL) addition. 2.6 In vivo antithrombotic effect of Cerastokunin and toxicity assessment 2.6.1 Antithrombosis assay in Carrageenan-induced mouse tail thrombosis model The mouse model was performed as described by [ 27 , 28 ] with male NMRI mice (20-25g), with four individuals in each group. Cerastokunin dissolved in sterile Phosphore buffer (1×M, pH 7.4) was administered intravenously at doses of 2, 4 and 6 mg/kg body weight. Blank control animals received an equivalent volume of sterile 0.9% saline by intravenous injection. Apixaban and Rivaroxaban, two antithrombotic drugs, were orally administered, at a therapeutic dose of 0.2 mg/kg body weight as positive controls of antithrombotic activity. In order to induce tail-thrombus formation, each mouse received 100 µL (1%, intraperitoneal route) containing 60 mg/kg body weight of κ-Carrageenan (type I, Sigma) dissolved in 0.9% NaCL. The κ-Carrageenan was injected 30 min post-treatment in the case of saline and tested peptide whereas, in the case of drugs, κ-Carrageenan was administrated 60 min post-treatment since the drugs were injected by non-systemic route. Six hours after κ-Carrageenan injection in all experiments, the tested doses of Cerastokunin were re-injected into mice through the caudal vein. All mice were placed in a room at 20°C for 48 hours and the length of thrombus in mouse tail was measured and recorded at 24 and 48 hours post-treatment, respectively. Experimental protocol was conducted according to the Ethics Committee of European Community in Experimentation for Animals’ Welfare ( https://ec.europa.eu/food/animals/welfare/strategy_en ). 2.6.2 Evaluation of toxicity and in vivo anticoagulant activity The toxicity was examined throughout the two days of the antithrombosis experiment previously given in section 2.6.1 . Mice were observed at regular intervals of time for death or any physical or behavioral changes. 48 hours post-treatment, mice were sacrificed, thoracic blood was collected in plastic petri dish and the blood clotting time from Cerastokunin-treated mice or control group of mice was recorded to evaluate the in vivo anticoagulant role of Cerastokunin, if any. 2.7 Bioinformatics analysis 2.7.1. Structure and sequence homology analysis Homology study of primary sequence of Cerastokunin with other kunitz-type peptides’ sequences from other species was performed using the BLAST program, ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ) [ 29 ] through the National Center for Biotechnology Information databases (NCBI, http://www.ncbi.nlm.nih.gov/ ). The generated Cerastokunin homologous sequences were used to perform a sequence alignment which was analyzed through Clustal W program [ 30 ] ( https://www.ebi.ac.uk/jdispatcher/msa/clustalo ) and a phylogenetic tree was then constructed using sequences that demonstrated the best homology with Cerastokunin sequence. The physicochemical parameters were also analyzed in silico by submitting the amino-acid sequence of Cerastokunin on Protein Calculator v3.4 software ( https://protcalc.sourceforge.net/ ). Finally, two dimensional structure was elaborated through Phyre2 online server ( http://www.sbg.bio.ic.ac.uk/~phyre2 ) [ 31 ]. Afterwards, inhibitor-3 sequence (Model ID : A0A1S5QJJ1 (A0A1S5QJJ1_VIPAA); from Vipera ammodytes ammodytes Kunitz/BPTI venom), that presents 70.15% of identity with Cerastokunin primary sequence, was used as template and the three tridimensional structure was modeled using Swiss-Model server ( https://swissmodel.expasy.org/ ) [ 32 ]. 2.7.2. Molecular docking The protein-ligand complexes of : FXa-Apixaban, FXa-Rivaroxaban, Thrombin-Apixaban, Thrombin-Rivaroxaban, Trypsin-Apixaban and Trypsin-Rivaroxaban, were all modeled through SwissDock online-server ( http://www.swissdock.ch/docking ), using the following protein databank structures : Rivaroxaban [2w26.pdb], Apixaban [2p16.pdb], Trypsin 3d65.pdb]), Thrombin [3u69.pdb], FXa [1hcg.pdb]. Complexes with the lowest energy (ΔG) were selected and the results were visualized using UCSF Chimera. ClusPro database, version 2.0 ( https://cluspro.bu.edu/publications.php ) was used for protein-protein docking of Cerastokunin-Serine proteases (Trypsin, thrombin and FXa), the modeled Cerastokunin 3D structure was used, the complex at position 0 was selected and the results were visualized using PyMol. 2.8. Statistical analysis Statistical study of obtained results was done by Normalization and one-way ANOVA analysis significant difference with multiple comparison test to compare between data. Results were expressed as the mean ± SEM and considered statistically significant at p-values < 0.05 *, very significant p-values < 0.01 ** and highly significant p-values < 0.001 ***. 3. Results 3.1 Purification of Cerastokunin from Cerastes cerastes venom Cerastes cerastes venom was fractionated on G-75 Sephadex gel-filtration column in three peaks yielded on molecular size exclusion (GF1-3). The third peak (GF3) contained low molecular weight peptides was considered for extracting a kunitz-type peptide as this type of peptides are characterized by a low molecular weight around 7 kDa [17]. So, GF3 (tubes 210-427) (Fig1.A) was pooled. GF3 displayed an inhibitory activity on trypsin-casein interaction since it didn’t reveal any casein protease or hemolysis activities (Fig1.B) . However, both peaks of GF1 and GF2 exhibited highest activity of trypsin as well as PLA2 activity, therefore, GF3 fraction was desalted and concentrated and then, applied to a trypsin-affinity chromatography (Fig1.C) and resolved in two peaks, the homogeneity of the second peak compounds with high affinity towards trypsin was analyzed through RP-HPLC column at 280 nm. The purity of this peak consisting of Cerastokunin peptide was approved since, only one amino acid peak was generated with a retention time of 24.197 min (Fig1.D) excluding the mobile phase relative two peaks (with a retention time of 17 and 61.933 min) due to acetonitrile which is an organic solvent that absorbs in the ultraviolet spectrum (Fig1.E). Purification process performed for the isolation of Cerastokunin and the retrieved data are summed up according to antiplatelet activity (Table 1) . The peptide was purified to an extend of about thirty-fold (purification factor equals to 29.85) and accounted only for 1.02% (protein by weight) in Cerastes cerastes venom. Cerastokunin retained about 20.895% of the total antiplatelet activity of the whole venom. 3.2. Mass measurement of Cerastokunin on MALDI-MSMS To further check the homogeneity of Cerastokunin, purity and calculation of its molecular weight, we proceeded with MALDI-TOF-MS analysis (Table 2) . As demonstrated in [Fig.1. (F1)], the spectra obtained by Tandem mass spectrometry evidenced the presence of different isoforms with slight molecular mass variation probably due to the variability in the length of the final sequences which are obtained from the proteolytic cleavage of their precursors known as spectrum estimation as described in Fig.1.(F2). Taken together, all these data provided the main mass corresponding to 7,746.89 Da for the new purified peptide termed Cerastokunin. 3.3 Functional study of Cerastokunin 3.3.1 Dose-dependent inhibition of thrombin-induced platelet aggregation of Cerastokunin The antiplatelet role of Cerastokunin on the aggregation of platelets activated by thrombin was studied following two independent assays. Obtained results derived from the first experiment showed that Cerastokunin exhibited in vitro antithrombotic activity considering that it inhibited 83% of the thrombin-dependent platelets activity at 20 µg/mL (2.58 µM), as the platelet aggregation decreased from 60 % to 10 % in the presence of Cerastokunin (Fig.2 A and Fig.2B). The dose of 2.58 µM was taken in this assay because it was the minimal effective dose against thrombin-induced platelet aggregation, as for a lower dose (1.29 µM) was ineffective when assessed (data not shown). In the second experiment performed on a 96-well microplate, the incubation of thrombin with various increasing doses of Cerastokunin revealed that platelet-rich plasma was prevented to aggregate due to the inhibition of thrombin because of its close binding to Cerastokunin. In fact, the peptide deactivated the platelets with a dose-dependent manner (Fig.3) , the highest inhibition was recorded with 1.29, 1.94 and 2.58 µM of Cerastokunin which corresponded to high significant difference of platelet absorbance compared to control (Fig.3.A) . In addition, it seems that the platelet inhibition was significantly higher with Cerastokunin (2.58 in µM) compared to heparin as illustrated in Fig.3.B 3.2.2 Anticoagulant function of Cerastokunin In addition to the antiplatelet activity of Cerastokunin, the inhibition of blood clotting was evaluated for both extrinsic and intrinsic blood coagulation pathways. The involvement of thrombin and other plasma factors in both pathways was studied in presence of Cerastokunin and experimentally checked by monitoring Prothrombin time (PT) and activated Partial Thromboplastin Time (aPTT). Both PT and aPTT were dose-dependently prolonged in blood samples pretreated with various doses of Cerastokunin. This indicated that Cerastokunin significantly inhibited both intrinsic and extrinsic blood coagulation pathways. In fact, it appeared that Cerastokunin with only lower doses was able to significantly delay both extrinsic and intrinsic pathways of coagulation. Moreover, the peptide potentially targeted the intrinsic pathways as it high significantly prolonged PT compared to control time (20 ± 2 s vs. 8 ± 0.5 s, n=3, p<0.001) and at only the dose of 0.258 µM (2 µg/ mL). On counterpart, Cerastokunin prolonged more efficiently PT (96 ± 5.5 s, n = 3, p-values < 0.001) but with higher doses (2.58 µM) (Fig.4.A) . However, obtained results illustrated in Fig.4.B , showed that aPTT was not prolonged in a highly significant manner until the dose of 0.65 µM (39 ± 2.5 s vs. 13 ± 1 s, n = 3, p-values < 0.001) and at 2.58 µM the activated partial thromboplastin time was extended to 84 ± 5 s (n=3, p-values < 0.001) (Fig.4.B). Taken together, all these recorded time values lead to emphasize that Cerastokunin inhibited both intrinsic and extrinsic pathway coagulation factors, with a more significant effect on the extrinsic pathway, resulting in a potent anticoagulant role. 3.3 In vivo validation of Cerastokunin anti-thrombosis and safety Cerastokunin is a safe peptide since it was non-toxic up to the highest intravenously administered dose of 6 mg/kg into mice. Cerastokunin-treated mice did not present any clinical manifestations or abnormal behavioral changes within 48 hours post-injection. In addition, in carrageenan-induced mouse tail thrombosis model, a reduction on thrombus length was observed 24 hours after treatment in Cerastokunin-pretreated mouse tail, particularly at doses of 2 and 6 mg/kg ( Fig.5A ). For both doses, the antithrombosis was pronounced and highest than observed with Apixaban and Rivaroxaban, two antithrombotic drugs of reference [33–35]. At 48 hours post-treatment, the measurements of the thrombosis length along mice tail showed a dose-dependent antithrombotic effect. Interestingly, the obtained value of Cerastokunin (6 mg/kg) revealed an inhibition of about 75 ± 2% (p-values < 0.001) of thrombus formation against 51.79 ± 3 % (p-values < 0.001) and 17.92 ± 0.1% (p-values < 0.001) recorded with Apixaban and Rivaroxaban respectively ( Fig.5B and Fig.5C ). 3.4 In vivo anticoagulant effect In order to deepen the investigation of the Cerastokunin anticoagulant effect, supplementary in vivo experiment was carried out, completing the results previously obtained from in vitro assays. Hence, blood samples were collected from mice previously challenged with the same doses of Cerastokunin as the toxicity protocol, and clotting time was recorded after 48 hours post-injection. As illustrated in Fig.5D , Cerastokunin extended the clotting time proportionally to administered doses (2 mg/kg to 6 mg/kg). Blood clotting time was extended from 16 ± 1 s (p-values < 0.001) to 31 ± 2.5 s (p-values < 0.001) and to 201 ± 5 s (p-values < 0.001) with 2 mg/kg and 6 mg/kg respectively whereas less values were measured with Rivaroxaban (120 ± 5 s, p-values < 0.001) and Apixaban (186 ± 6 s, p-values < 0.001). 3.5 Bioinformatics studies 3.5.1. Structural features of Cerastokunin Multiple-alignment of the primary sequence of Cerastokunin by BLAST server confirmed that the peptide belongs to kunitz family (inhibitors of serine proteases) ( Fig.6 ). This sequence also showed homology with other Viperidae venom peptides belonging to the same family (Kunitz-type peptides) including those extracted from the venoms of Macrovipera lebetina transmediterranea ( Kunitz-type serine protease inhibitor PIVL), Vipera berus nikolskii ( Kunitz-type serine protease inhibitor ki-VN), Vipera ammodytes ( Kunitz/BPTI inhibitor-2, Kunitz/BPTI inhibitor-3, Kunitz/BPTI inhibitor-5 and Kunitz/BPTI inhibitor-7), Pseudocerastes persicus (Chain A, Trypsin Inhibitor , Kunitz-type serine protease inhibitor PPTI ) and Eristicophis macmahoni (Kunitz-type serine protease inhibitor ), all of which share more than 65% of identity with Cerastokunin ( Table 3 ). Furthermore, the phylogenetic tree was created with the aim of elucidating the relationship between various proteases’ inhibitors studied from snake venoms. Analysis of the constructed phylogenetic tree shows that Cerastokunin belongs to the same group as Chain A, Trypsin Inhibitor, Kunitz-type serine protease inhibitor PPTI purified from the venom of Pseudocerastes persicus and Venom trypsin inhibitor from the venom of Eristicophis macmahoni , which belong to the Viperidae family (Fig.7). The physicochemical characterization of Cerastokunin was carried out by Protein calculator V3.4 server ( Table 4 ), and generated results indicate that it has a low molecular weight of 7,746.89 Da and consists of amino acid residues equal to 67. Its primary sequence contains 6 cysteine residues which contribute to its structural stability. The kunitz peptide is classified as basic and hydrophilic peptide. Finally, the predicted two and three dimensional Cerastokunin structures, illustrated in the Fig.8A and Fig.8B , were generated through two bioinformatics tools Phyre-2 and Swiss-Model respectively. The build modeled molecule of Cerastokunin includes β-strands (21%), α-helixes (12%) and disordered domains (16%). 3.5.2. Interaction studies with molecular docking Molecular docking of protein-protein selected complexes on position 0 show, interactions between Cerastokunin and two essential coagulation molecules in this study; plasma coagulation factor FXa and thrombin (FIIa). These interactions are established with eight (8) bonds between FXa and Cerastokunin and involved the amino acid residues « Glu97, Thr98, Tyr99 (double bonds), Glu39, Arg143, Gln192, Gln192 and Gln61 » of FXa with their pairs identified in Cerastokunin sequence « Asn17, Asn17, Tyr19 (double bonds), Tyr23, Thr34, Phe35, Tyr19 and Lys48 ». As for Cerastokunin-thrombin binding, nine (9) bonds linked both through the following thrombin residues « Lys372, Pro369, Trp412, Arg413, Glu466, Glu522, Glu522, Glu549 (double bonds) and Arg553 (double bonds) ». These residues interacted with Cerastokunin through « Asn17, Tyr13, Tyr19, Thr34, Gly41, Asn17 (two bonds), Ala18, Lys48 (two bonds), Tyr37 (two bonds) » (Table 5) . Obtained results pointed out that the ligand–protein complexes with the lowest energy (DG) resulted from the in silico simulation of FXa and thrombin interactions with Apixaban and Rivaroxaban, showed that Apixaban interacted with FXa with only one hydrogen bond involving the amino acid Gln192 of FXa with a bond length of 2.84Å and DG = -8.35 Kcal/mol. Similarly, Rivaroxaban blocked the coagulation by linking FXa with only one hydrogen bond through Gly193 with a bond length= 2.5Å (DG = -7.69 Kcal/mol) ( Fig.9 ). The selected complex with the lowest energy (DG= -7.86 Kcal/mol)) between thrombin and Apixaban showed two (2) hydrogen bonds, H21 and nitrogen N6 which established bonds with the amino acid residues of thrombin Arg310 (bond length = 2.3 Å) and Asn537 (bond length = 2.75 Å). However, lowest energy of complex Thrombin-Rivaroxaban was formed by only one bond established between the amino acid Trp370 of thrombin and the oxygen (O13) of Rivaroxaban with bond length of 2.37 Å and DG= -7.43 Kcal/mol ( Fig.10, Table 5 ). Trypsin-Cerastokunin complex on position 0 was strongly established through the formation of seven (7) bonds involving « Tyr39, Tyr151, Gly216, Gln192, Gly219, Ser190 (2 involved bonds) and Asp189 » of trypsin and « Pro21, Tyr19, Asn17, Cys16, Pro15, Asn17 (double bonds) and Asn17 » residues from Cerastokunin (Table 5) . However, the lowest energy of the complex Trypsin–Rivaroxaban involved only one (1) hydrogen bond with the amino acid Gln175 of trypsin (bond length = 2.95 Å, DG= -8.18 Kcal/mol), whilst the lowest energy (DG= -8.96 Kcal/mol) for the complex Trypsin- Apixaban involved two (2) hydrogen bonds where Ser96 and Gln192 contributed with bond length= 2.75 Å and 2.28 Å respectively ( Fig.11 ). 4. Discussion Thromboembolic disorders known as the main cause of mortality in between cardiovascular diseases are generally treated with anticoagulant agents which present in association to their efficient anti-thrombotic effect, a bleeding risk or hemorrhagic complications [ 36 ]. In order to prevent these side-effects, new therapeutic approaches have been more and more investigated, these could equilibrate the altered hemostasis in patients without causing any bleeding risk [ 37 ]. In this regard, snake venoms are worldwide considered as a rich source of pharmacologically active biomolecules. Basic on their ability to interfere with the blood-coagulation factors, platelet-aggregation and fibrinolysis as well, once isolated, many of which are considered as a potential anti-thrombotic agents [ 6 ] and some are already used as marketed clinical medicines such as Captopril® from Bothrops jararaca venom which is an inhibitor of angiotensin-converting enzyme. This biotherapeutic was the first successful snake’s venom component designed drug currently indicated for hypertension therapy. Afterwards, other venom-derived drugs have been commercialized such as Aggrastat® (Tirofiban), initially identified from Echis carinatus venom and consists of a disintegrin-based drug that inhibits Glycoprotein IIb/IIIa platelets, it is currently prescribed as antiplatelet medicine [ 38 ]. In this current study, Cerastokunin with dual antithrombotic and antiplatelet activity was characterized and its mechanism of action was fully elucidated. Multiple alignment of the primary sequence validated the presence of putative serine protease binding sites and indicated a high similarity of Cerastokunin sequence with a variety of Viperidae venoms’ kunitz type peptides. Based on these structural data, the purified peptide was named Cerasto ( Cerastes cerastes ) –Kunin (kunitz type serine proteases’ inhibitor). The structural characterization of Cerastokunin demonstrated that it is a new member of kunitz type peptides’ family, known as serine proteases’ inhibitors for trypsin, chymotrypsin and other plasma coagulation factors including thrombin and Factor Xa [ 38 , 39 ] Cerastokunin is a single-chain, basic (pI 8.48) and hydrophilic peptide composed of 67 amino acid residues including six residues of cysteine that contribute to its stability by forming a three disulfide bridges. With a molecular weight of 7,746.89 Da Cerastokunin is similar to typical bovine pancreatic trypsin inhibitor (BPTI), a family characterized by small molecular masses ranging between 6,000 and 7,000 Da [ 17 ]. Some of known extracted Kunitz type peptides are Dendroaspis polylepis ‘ venom kunitz-type peptide of 7,128.4 Da that inhibits elastase-1 and cathepsin L [ 24 ], Vipera russelii russelii’s Vipegrin is a 6,800 Da serine proteases inhibitor that inhibits trypsin and could inhibit collagen and ADP-induced platelet aggregation through its disintegrin-like activity [ 39 ]. Recently, different groups of trypsin inhibitors were extracted from Buckwheat, a pseudocereal, each group with a range of molecular masses, including BTI I, IIa, IIb, and IIIa that are formed of permanent inhibitors with 6,000 to 7,000 Da [ 40 ] . The three dimensional structure of Cerastokunin includes two antiparallel beta strands separated by a β-hairpin loop and a stable alpha helix at the C-terminal end with a disordered helix at the N-terminal extremity. The disorder state should be explained by the presence of a conserved proline in this sequence domain. All these structural features further approve that the purified Cerastokunin is a kunitz type serine protease inhibitor [ 4 , 18 , 23 , 41 ]. As example, the BPTI is the first discovered and well known kunitz peptide that inhibits a spectrum of serine proteinases including trypsin, chymotrypsin, plasmin and plasma kallikrein [ 42 , 43 ] [ 44 ]. BPTI has also a conserved three dimensional structure containing three disulfide bridges and a two regions of helix α, a short helix3 10:H1 in the N-terminal extremity while the second helix α: H2 is longer than the first and localized in the C-terminal extremity. Between the two helices, the central region consists of double β strands linked by a one- tower loop that forms a β-hairpin [ 45 ]. In terms of functions, snake venom Kunitz-type/BPTIs are doted of a panel of pharmacological activities which have been figured out by the identification of several isoforms of these peptides. A previous analytical study reported that the sequences of kunitz type peptides undergo through a gene duplication followed by subsequent diversification resulting in sequence evolution [ 46 , 47 ]. In vivo anticoagulant activity of Cerastokunin was examined on Carrageenan-induced mouse tail thrombosis model, a non-complicated and non-invasive animal modelization that is known to be used on the evaluation of new antithrombotic and anticoagulant substances like aspirin and heparin [ 48 ]. Interestingly, we found that the anticoagulant effect of Cerastokunin is more important than that of Rivaroxaban and Apixaban, direct factor Xa inhibitors [ 34 , 35 ]. Our results correlate with those of Joannsin, a kunitz type peptide from the venom of Prospirobolus joannsi , composed of 72 amino acid residues with three intramolecular disulfide bridges, that has shown a similar anticoagulant effect in addition of an anti-Factor Xa and anti-trypsin activity [ 28 ]. Furthermore, when this anticoagulant effect was assessed in vitro using activated partial thromboplastin time (aPTT) and prothrombin time (PT) reagents, the blood coagulation was inhibited on both extrinsic and intrinsic coagulation pathways; this data may be of a great interest to explain the potent in vivo antithrombotic effect of Cerastokunin. In fact, Cerastokunin significantly lengthened the clotting time of plasma by exhibiting an inhibitory effect on factors involved in the initial steps of extrinsic pathway of the blood coagulation and behaved as a blocker of extrinsic coagulation pathway. Additionally, our study showed that Cerastokunin also acted by inhibiting the intrinsic pathway since it delayed blood clotting through prothrombin or/and FX blockade (Fig. 12 ). It is well-known that, thrombin interacts at least with two types of human platelet-membrane receptors, which consist on the protease activated receptors (PAR1/PAR4) and GPIb (alpha). PARs, being a seven-transmembrane G-protein coupled receptors can be activated by proteolysis of their N-terminal part by thrombin resulting on a transmembrane signaling cascade contributing to thrombus formation [ 49 ]. These clots are formed by building a cross-linked platelet-fibrinogen thrombus resulting from the interaction of activated glycoprotein (GP) IIb/IIIa with dimeric fibrinogen molecules. On the other hand, thrombin binding to GP Ib(alpha) platelet-receptor activates the internal calcium mobilization signaling pathway inducing the platelet release reaction. This second thrombin-induced platelet activation pathway, is associated to a fibrin-integrin platelets cross-linking [ 50 ] and leads to an enhancement of PAR1 signaling pathway and an activation of glycoprotein V (GPV) on a synergistic interacting way [ 49 ]. Similar to other snake kunitz peptides, Cerastokunin blocked the aggregation of platelets by preventing thrombin to bind to its PARs and/or GPIb(alpha) platelet receptors through a direct thrombin inhibition. The same mechanism has been reported and elucidated for Rusvikunin, a Kunitz-type protease inhibitor peptide purified from Daboia russelii venom inhibited amidolytic activity of trypsin, plasmin and fibrinogen clotting as well as plasma clotting activity of thrombin [ 23 ]. Rusvikunin exhibited also an antiplatelet aggregation activity and an in vitro prolongation of blood clotting time [ 26 ]. Aside the fact that BPTI, pancreatic Kunitz inhibitor, was initially used like a crucial tool for studying protein structural conformations and protein-protein interactions. Many reports documented that BPTI, possesses a highest affinity to serine proteases that have a primary specificity to substrates with « Lys /Arg » residues, such as Thrombin and FXa. This affinity depends on structural factors outside the catalytic site, especially the residue Glu192 or Gln192 in the mutant variant which is involved in this type of BPTI- Serine protease(s) interaction that leads to inhibitory effect [ 51 ]. This data might explain the implication of Lys48 of Cerastokunin in the interaction with both FXa and thrombin, as well as, the involvement of the residue Gln192 of FXa on both FXa-Cerastokunin and FXa-Apixaban interactions. In addition, the study of the interaction between FXa and Tissue Factor Pathway Inhibitor (TFPI), has demonstrated that the residue Arg143 of FXa is involved in this inhibitory interaction similar to FXa-Cerastokunin docking results. Furthermore, the two residues Tyr17 and Lys34 of TFPI were also implicated by similarity, this may explain the involvement of Tyr 19, Tyr 23 and Lys48 residues of Cerastokunin [ 52 ]. AH-884 is a variant of kunitz-like trypsin inhibitor peptide from the skin secretion of Amolops hainanensis , named Kunitzin-AH presenting trypsin inhibitory activity. The docking simulation of Trypsin with AH-884 has demonstrated that four (4) amino acid residues Ser789, Asp792, Lys702, and Tyr 681 trypsin bond to Arg1, Asn5 and Cys7 residues of the AH-884 [ 53 ]. These reported results matched up with the docking data of the current study that illustrated the involvement of Cerastokunin Cys16 and Asn17 in the interaction with trypsin. Furthermore, in another reported study of pTTI (Purified trypsin inhibitor from tamarind seeds), the docking result of interaction between pTTI and trypsin has shown that Pro57 is one of the peptide residue that has the greatest interaction with trypsin [ 54 ]. Hence again, this provides a supplementary argument and fully correlates with our findings that the conserved Pro15 and Pro21 residues of Cerastokunin are importantly involved in the interaction with trypsin. Obtained results of molecular docking are valuable and showed that the affinity of Cerastokunin towards FXa, thrombin and trypsin is higher than that of Apixaban and Rivaroxaban, since Cerastokunin required between 7 to 9 bonds to interact with FXa, thrombin and trypsin whilst each one of the two drugs bonds to each protease by only 1 to 2 hydrogen bonds. This set, can be considered as additional support for an explanation of its significant antiplatelet effect, as well as, its potent anticoagulant effect exhibited on both extrinsic and intrinsic coagulation pathways as elaborated in in vivo and in vitro experiments. The low molecular weight of Cerastokunin would guarantee a fast biodistribution and bioavailability. Its structure stability established by three intramolecular disulfide bridges and its potent dual function as antithrombotic and platelet blocker with a non-enzymatic trypsin inhibitory activity would be features of interest as a potential therapeutic and/or diagnostic biodrug for coagulopathies. Conclusion The first kunitz-type peptide from Cerastes cerastes venom was purified in this study, and in vitro, in vivo, and in silico investigations were used to characterize its structure and function. The acquired data demonstrated that Cerastokunin is a 67 amino acid basic peptide that is two folded and has three disulfide bridges. In addition to its strong anticoagulant and antiplatelet action, Cerastokunin also exhibits non-enzymatic anti-trypsin, anti-thrombin, and anti-FXa actions. The comprehension of the mechanism underlying the thrombosis inhibition of venom Kunitz-type peptides could be enhanced by all of these studies. When considered collectively, the data point to Cerastokunin's potential as a novel therapeutic agent for coagulopathies due to its important antiplatelet, antithrombotic, and anticoagulant properties, all of which occur without causing any in vivo harm. Abbreviations ΔG : lowest energy A :Ala : Alanine ACN: Acetonitrile aPPT: Activated Partial Thromboplastin Time BPTI: Bovin pancreatic trypsin inhibitors or Aprotinin BrCN: Cyanogen bromide C : Cys : Cysteine CaCl2: Calcium chloride Cc: Cerastes cerastes CHCA: α-Cyano-4-hydroxycinnamic acid or Sinapinic Acid Cm: Centimeter D : Asp : Aspartate E : Glu : Glutamate F : Phe : Phenylalanine FXa: Blood-clotting Factor ten active G : Gly : Glycine GF: Gel-filtration fraction H : His : Histidine HCL : Hydrochloric acid i.v: Intravenous I: Ile : Isoleucine K : Lys : Lysine L : Leu : Leucine LC-MALDI-MSMS: Liquid chromatography-matrix-assisted laser desorption/ionization tandem mass spectrometry M : Met : Methionine N : Asn : Asparagine NaCl : Sodium chloride NMRI mice: The Naval Medical Research Institute mice P : Pro : Proline PARs: Protease Activated Receptors PBS buffer: Phosphate-buffered saline pI: Isoelectric point PLA2: Phospholipase A2 PPP: Platelet-poor plasma PRP: Platelet-rich plasma PT: Prothrombin Time Q : Gln : Glutamine R : Arg : Arginine RP-HPLC: Revers high performance liquid chromatography S : Ser : Serine S: Seconds Sepharose CL4B: 4% cross-linked agarose chromatography base matrix SV: Snake- venom T : Thr : Threonine TFA: Trifluoroacetic acid TFPI : Tissue Factor Pathway Inhibitor U : SeC : Selenocysteine V : Val : Valine W : Trp : Tryptophan Y : Tyr : Tyrosine Declarations Acknowledgements The authors express their gratitude to Dr. Khouloud BENALI, Dr. Nour Elyakine KERAGHEL and Dr. Abderrahmane KORI YAHIA (Faculty of pharmacy, University of Algiers 1) for their technical assistance. We would like to express our gratitude for Prof. Dr. CHARALLAH Salima (Chair of Ethic Committee) and Dr. BENMILOUD Abdelouafi and all the members of the Ethical Committee of Animal Experimentation (CEEA-USTHB) for their invaluable assistance and their thoroughness of expertise before delivering an approved ethical certificate for the use of animals. The CEEA is a fully constituted Committee in accordance with the National Statement on Ethical conduct in research involving laboratory Animal-Law N°.88-08 of 26th January, 19988 related to veterinary medicine and the protection of animal health. We also extend our sincere gratitude to the Hospital-University Center of Mustapha Bacha, Algiers, Algeria's Ethical Committee of Human Sample' Use, for granting permission to use human samples in accordance with their approved ethical guidelines. Their knowledge was crucial in validating the experimental design. Authors’ Contributions Noussaiba Saghour performed all the experiments, collected the data and wrote the article, Fatah Chérifi designed the study, supervised the experimental part, reviewed and edited the manuscript and contributed to revising the manuscript for clarity and coherence, Saoud Samah performed the computational experiments of docking studies of this work, validated and verified the accuracy and reliability of results related to molecules’ interactions, Younes Zebbiche contributed in the purification procedure and provided the technical assistance, Amel Meribai assisted with providing the required materials for animal model, Nadjia Bekkari provided the necessary reagents for the main part of this work and provided the documents for some techniques, Taright-Mahi Samya validated the experimental design of human samples and assisted in providing the ethical approval of use of blood samples, Fatima Laraba-Djebari assisted in the conceptualization stage of the study, provided data, logistics and reagents throughout this work and secured funding acquisition and financial support for the research. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Conflict of interest The authors declare that they have no conflict of interest. References Chérifi, F., & Laraba-Djebari, F. (2021). Bioactive Molecules Derived from Snake Venoms with Therapeutic Potential for the Treatment of Thrombo-Cardiovascular Disorders Associated with COVID-19. The Protein Journal , 40 (6), 799–841. https://doi.org/10.1007/s10930-021-10019-4 Karbach, S., Lagrange, J., & Wenzel, P. (2019). Thromboinflammation and Vascular Dysfunction. Hämostaseologie , 39 (02), 180–187. https://doi.org/10.1055/s-0038-1676130 Bhuria, V., Baldauf, C. K., Schraven, B., & Fischer, T. (2022). Thromboinflammation in Myeloproliferative Neoplasms (MPN)—A Puzzle Still to Be Solved. 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Retrieved from https://www.tandfonline.com/doi/abs/10.1080/14756366.2021.1876686 Tables Table 2: Peptide spectra and their respective masses derived from Mass Spectrometry scans of Cerastokunin (only fragment or peptides with a mass bigger than 500 Dalton were displaying and reported in the table) Peptides were digested by Lys C, Maximum number of missed cleavages (MC): 0All cysteines have been treated with Iodoacetamide to form carbamidomethyl-cysteine (Cys_CAM), with acrylamide adducts (Cys_PAM).Methionines have been oxidized to form methionine sulfoxide (MSO).Using monoisotopic masses of the occurring amino acid residues and giving peptide masses as [M+H]+). Fragment Number Start Residue End Residue Modification (Cystein and Methionin) Weight with modifications (Da) MeasuredMass (Da) Peptide Sequence 1 2 30 Cys_CAM: 7, 16 3560.6475 3560.6475 DRPDFCLLPLDYGPCNAYMP RYFYNIFEK Cys_PAM: 7, 16 3702.7218 MSO: 20 3576.6425 2 32 48 Cys_CAM: 32, 40 1893.8006 1779.7577 CQTFVYGGCGGNANNFK Cys_PAM: 32, 40 1921.8320 3 49 62 Cys_CAM: 53, 57 1746.8374 1632.7944 TIEECRHTCVASRK Cys_PAM: 53, 57 1774.8687 4 63 67 ---------- 570.3358 570.3358 GIQPR Mw (average mass): 7746.89 / Mw (monoisotopic mass): 7741.66 Table 3 : Alignment and sequence homology of Cerastokunitz with related proteins; using Blast in NCBI (only Kunitz-type peptides sharing more than 65% of identity to Cerastokunitz and with best hits are listed). Protein Species Max score Total score Query Cover E value Identity (%) Accession Number Kunitz/BPTI inhibitor-3 Vipera ammodytes ammodytes 102 102 100% 4e-26 70.15% AMH40735.1 Kunitz/BPTI inhibitor-7 Vipera ammodytes ammodytes 102 102 100% 4e-26 70.15% AMH40739.1 Kunitz-type serine protease inhibitor PIVL Macrovipera lebetina transmediterranea 102 102 100% 2e-26 68.66% I2G9B4.1 Inhibitor,chymotrypsin Vipera ammodytes 95.1 95.1 100% 1e-23 68.66% 0909196A Chain A, Trypsin Inhibitor Pseudocerastes persicus 91.7 91.7 92% 3e-22 67.74% 6A5I_A Kunitz/BPTI inhibitor-5 Vipera ammodytes ammodytes 94.7 94.7 100% 3e-23 67.16% AMH40737.1 Kunitz/BPTI inhibitor-2 Vipera ammodytes ammodytes 93.6 93.6 100% 8e-23 67.16% AMH40734.1 Kunitz-type serine protease inhibitor PPTI Pseudocerastes persicus 92.0 92.0 94% 2e-22 66.67% C0HLB2.1 Kunitz-type serine protease inhibitor ki-VN Vipera berus nikolskii 98.6 98.6 100% 1e-24 65.67% E5AJX3.1 Kunitz-type serine protease inhibitor 3 Vipera ammodytes ammodytes 93.6 93.6 100% 9e-23 65.67% P00992.2 Kunitz-type serine protease inhibitor Eristicophis macmahoni 82.4 82.4 86% 1e-18 65.52% P24541.1 Table 4 : Physicochemical characterization of Cerastokunin sequence Table 5 : Summarized data retrieved from the different molecular dockings performed in order to determine the interactions between Cerastokunin and coagulation factors and trypsin compared to Apixaban and Rivaroxaban. Protein- Protein docking bonds on position 0 Cerastokunin-FXa interaction FXa involved residues Cerastokunin involved residues Glu97 Asn17 Thr98 Asn17 Tyr99 (double bonds) Tyr19 (double bonds) Glu39 Tyr23 Arg143 Thr34 Gln192 Phe35 Gln192 Tyr19 Gln61 Lys48 Cerastokunin-Thrombin interaction Thrombin involved residues Cerastokunin involved residues Lys372 Asn17 Pro369 Tyr13 Trp412 Tyr19 Arg413 Thr34 Glu466 Gly41 Glu522 Asn17 (2 bonds) Glu522 Ala18 Glu 549(double bonds) Lys48 (2 bonds) Arg553 (double bonds) Tyr37 (2 bonds) Cerastokunin-Trypsin interaction Trypsin involved residues Cerastokunin involved residues Tyr39 Pro21 Tyr151 Tyr19 Gly216 Asn17 Gln192 Cys16 Gly219 Pro15 Ser190 (2 involved bonds) Asn17 (double bonds) Asp189 Asn17 Protein-Ligand docking Protein-Ligand docking of FX-Apixaban bond FX involved residue Bond length D G Gln192 84Å -8.35 Kcal/mol Protein- ligand docking of FX-Rivaroxaban bond FX involved residue Bond length D G Gly 193 2.5Å -7.69 Kcal/mol Protein- ligand docking of Thrombin-Apixaban bond Thrombin involved residues Bonds length D G Arg310 (with H21 of Apixaban) 2.3 Å -7.86 Kcal/mol Asn537 (with the N6 of Apixaban) 2.75 Å Protein- ligand docking of Thrombin-Rivaroxaban bond Thrombin involved residue Bond length D G TRP 370 (with the O13 of Rivaroxaban) 2.37 Å -7.43 Kcal/mol Protein- ligand docking of Thrombin-Apixaban bond Trypsin involved residues Bonds length D G Ser96 2.75 Å -8,96 Kcal/mol Gln192 2.28 Å Protein- ligand docking of Trypsin-Rivaroxaban bond Trypsin involved residue Bond length D G Gln175 2.95 Å -8,18 Kcal/mol Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 02 Aug, 2024 Read the published version in The Protein Journal → Version 1 posted Editorial decision: Revision requested 05 Jun, 2024 Reviews received at journal 22 May, 2024 Reviews received at journal 20 May, 2024 Reviewers agreed at journal 08 May, 2024 Reviewers agreed at journal 06 May, 2024 Reviewers agreed at journal 06 May, 2024 Reviewers invited by journal 05 May, 2024 Editor assigned by journal 02 May, 2024 Submission checks completed at journal 02 May, 2024 First submitted to journal 01 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4354127","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":299877732,"identity":"c07560c9-8cf2-4ad7-b708-e395fa05b326","order_by":0,"name":"Noussaiba Saghour","email":"","orcid":"","institution":"USTHB, University of Sciences and Technology Houari Boumediene, Faculty of Biological Sciences","correspondingAuthor":false,"prefix":"","firstName":"Noussaiba","middleName":"","lastName":"Saghour","suffix":""},{"id":299877734,"identity":"433c8d4a-5e2e-4d10-b70f-267a78ec95b2","order_by":1,"name":"Fatah Chérifi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYBAC/hlAIsFGgh9IsTF8ALJBoh/waZG4AdKSJiHZwMbAxjgDogVE4wYGESAyjQGshZmHKC3Szcc+PEiwkOCf3/zssW2bXR4/ewNjcwU+LTLHkmckJEhISBxjMzfObUsuluw5wNh4Bp8WiRxjhsQfEnUMxxjMpHPbmBM33Ehgf9iAV0v+ZwaQLfLH2L9JW7bVg7QwNuLVEpHDDNZicIzHTJqx7TBhLRI30ozBWgyP5ZRJ9pw7njiz52AjXi38M5IfM/5IqJOQO3x8m8SPsurEfvbmg3i1oAJGNjBJvAYg+EOK4lEwCkbBKBgpAAAZ2036YWoDGwAAAABJRU5ErkJggg==","orcid":"","institution":"USTHB, University of Sciences and Technology Houari Boumediene, Faculty of Biological Sciences","correspondingAuthor":true,"prefix":"","firstName":"Fatah","middleName":"","lastName":"Chérifi","suffix":""},{"id":299877736,"identity":"14bd6979-c739-4820-916d-442dae933cc4","order_by":2,"name":"Samah Saoud","email":"","orcid":"","institution":"University of Algiers Benyoucef Benkhedda","correspondingAuthor":false,"prefix":"","firstName":"Samah","middleName":"","lastName":"Saoud","suffix":""},{"id":299877738,"identity":"e706779d-a78d-49c4-97d5-3fcaa0e83430","order_by":3,"name":"Younes Zebbiche","email":"","orcid":"","institution":"University of Algiers Benyoucef Benkhedda","correspondingAuthor":false,"prefix":"","firstName":"Younes","middleName":"","lastName":"Zebbiche","suffix":""},{"id":299877740,"identity":"59564872-d9b7-4fd1-913a-d48625255a26","order_by":4,"name":"Amel Meribai","email":"","orcid":"","institution":"National Agronomic High School","correspondingAuthor":false,"prefix":"","firstName":"Amel","middleName":"","lastName":"Meribai","suffix":""},{"id":299877742,"identity":"0c37d4a4-b598-48df-b7ea-15fa8640a724","order_by":5,"name":"Nadjia Bekkari","email":"","orcid":"","institution":"USTHB, University of Sciences and Technology Houari Boumediene, Faculty of Biological Sciences","correspondingAuthor":false,"prefix":"","firstName":"Nadjia","middleName":"","lastName":"Bekkari","suffix":""},{"id":299877744,"identity":"3699959c-efe6-49a4-8c9a-58b96b8ff99e","order_by":6,"name":"Samya Taright-Mahi","email":"","orcid":"","institution":"University of Algiers Benyoucef Benkhedda","correspondingAuthor":false,"prefix":"","firstName":"Samya","middleName":"","lastName":"Taright-Mahi","suffix":""},{"id":299877746,"identity":"bb9a33b4-8d97-4823-ad2f-7b2a87af7da1","order_by":7,"name":"Fatima Laraba-Djebari","email":"","orcid":"","institution":"USTHB, University of Sciences and Technology Houari Boumediene, Faculty of Biological Sciences","correspondingAuthor":false,"prefix":"","firstName":"Fatima","middleName":"","lastName":"Laraba-Djebari","suffix":""}],"badges":[],"createdAt":"2024-05-01 11:56:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4354127/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4354127/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10930-024-10226-9","type":"published","date":"2024-08-02T15:56:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56062322,"identity":"e3e6162e-76e2-41f0-aad6-9057661c55c9","added_by":"auto","created_at":"2024-05-08 05:16:58","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":420188,"visible":true,"origin":"","legend":"\u003cp\u003eCerastokunin purification.\u003cstrong\u003e \u003c/strong\u003eFractionation of crude \u003cem\u003eCerastes cerastes\u003c/em\u003e venom on size-exclusion Gel sephadex G-75 column (60 × 2.5 cm). GF1, GF2 and GF3 indicate elution peaks of the three pooled proteins fractions (\u003cstrong\u003eA\u003c/strong\u003e). Activities assays of obtained fractions (\u003cstrong\u003eB\u003c/strong\u003e). Affinity chromatography of GF3 peak in (A) using a trypsin-Sepharose CL4B column (\u003cstrong\u003eC\u003c/strong\u003e). Validation of purity of trypsin high-affinity eluted peptide (Cerastokunin) on a C18 RP-HPLC column. Cerastokunin was eluted at 24.019 min, the two other absorbance appeared peaks are relative to the blank solution, as it is known that acetonitrile does absorb in the ultraviolet spectrum (\u003cstrong\u003eD\u003c/strong\u003e). The HPLC chromatography of the blank solution containing 95% solvent A (v/v) and 5% of solvent B (v/v) (\u003cstrong\u003eE\u003c/strong\u003e). Molecular mass determination of Cerastokunin using MALDI TOF-MSMS (\u003cstrong\u003eF\u003c/strong\u003e). Tandem MS-MS Mass Spectrometry data of Cerastokunin \u003cstrong\u003e(F.1)\u003c/strong\u003e. Spectrum model errors of Cerastokunin \u003cstrong\u003e(F.2)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"FIGURE1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4354127/v1/4711d6603e18c7eaa0ea66ca.jpg"},{"id":56062301,"identity":"1e109b5b-f50b-4583-b2bd-e7d9d9ba3b8f","added_by":"auto","created_at":"2024-05-08 05:16:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":175245,"visible":true,"origin":"","legend":"\u003cp\u003eAntiplatelet activity of Cerastokunin on platelet aggregation induced by thrombin.\u003c/p\u003e","description":"","filename":"FIGURE2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4354127/v1/2fc7d8f1dcbb451aee77d488.jpg"},{"id":56062661,"identity":"35d80e99-d897-4f6f-b2b5-a7130fa79ff6","added_by":"auto","created_at":"2024-05-08 05:24:36","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":182947,"visible":true,"origin":"","legend":"\u003cp\u003eConcentration-dependent effect of Cerastokunin platelet-aggregation modulation. (\u003cstrong\u003eA\u003c/strong\u003e) Absorbance at 540 nm depending on Cerastokunin concentration. (\u003cstrong\u003eB\u003c/strong\u003e) Percent aggregation depending on Cerastokunin ranging concentrations. Values are mean ± SE. * P\u0026lt; 0.05, \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;** P\u0026lt; 0.01 and *** P\u0026lt; 0.001 compared with control (Thrombin + PRP). Analyzed by ANOVA one-way test for three or more samples comparison. Percent aggregation = [(A\u003csub\u003e0\u003c/sub\u003e- Af)/ (A\u003csub\u003e0\u003c/sub\u003e-A)] ×100% , where Ao is the absorbance of PRP at 540 nm prior to addition of tested molecules; Af is the absorbance of PRP at 540 nm post addition of tested molecules ; and A is PPP absorbance at 540 nm .\u003c/p\u003e","description":"","filename":"FIGURE3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4354127/v1/0b623092a5ef72f666106452.jpg"},{"id":56062278,"identity":"7ee18b2c-5bba-40f8-8e72-fce96ae5f9b1","added_by":"auto","created_at":"2024-05-08 05:16:36","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":181324,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro \u003c/em\u003eanticoagulant effect of Cerastokunin on activated Partial Thromboplastin Time (aPTT) (\u003cstrong\u003eA\u003c/strong\u003e) and on prothrombin time (PT) (\u003cstrong\u003eB\u003c/strong\u003e). Values are mean ± SE (n = 3). * P\u0026lt; 0.05, \u0026nbsp;** P\u0026lt; 0.01 and *** P\u0026lt; 0.001 compared with blank control.\u003c/p\u003e","description":"","filename":"FIGURE4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4354127/v1/65eb7831b3630e71d71163c4.jpg"},{"id":56062283,"identity":"b9fcb36d-7e51-45b8-805f-e86c87e329a3","added_by":"auto","created_at":"2024-05-08 05:16:36","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":294305,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo \u003c/em\u003eantithrombotic effect of Cerastokunin 24 hours post treatment (\u003cstrong\u003eA\u003c/strong\u003e) and 48 hours post-treatment (\u003cstrong\u003eB\u003c/strong\u003e), representative images of the thrombus length of each group at 48 hours after treatment: \u003cstrong\u003e(a)\u003c/strong\u003eRivaroxaban (0.2 mg/kg), \u003cstrong\u003e(b)\u003c/strong\u003e Apixaban (0.2mg/kg), \u003cstrong\u003e(c)\u003c/strong\u003e 6 mg/kg Cerastokunin \u003cstrong\u003e(d)\u003c/strong\u003e 4 mg/kg Cerastokunin \u003cstrong\u003e(e)\u003c/strong\u003e 2 mg/kg Cerastokunin \u003cstrong\u003e(f)\u003c/strong\u003eblank control (NaCl 0.9%) (\u003cstrong\u003eC\u003c/strong\u003e) and \u003cem\u003eIn vivo\u003c/em\u003e anticoagulant effect of Cerastokunin compared with those of Apixaban and Rivaroxaban (\u003cstrong\u003eD\u003c/strong\u003e), Values are mean ± SE. * P\u0026lt; 0.05, ** P\u0026lt; 0.01 and *** P\u0026lt; 0.001 compared with blank control.\u003c/p\u003e","description":"","filename":"FIGURE5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4354127/v1/facf580a343c430fa13ee772.jpg"},{"id":56062663,"identity":"31aeccf9-a072-4b1a-b93f-df7498082753","added_by":"auto","created_at":"2024-05-08 05:24:36","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":409387,"visible":true,"origin":"","legend":"\u003cp\u003eAlignment and sequence homology of Cerastokunin with related proteins; using BLAST in NCBI (\u003cstrong\u003eA). \u003c/strong\u003eCerastokunin sequence aligned with homologous Inhibitors sequences from snake venom using CLUSTAL O(1.2.4) (\u003cstrong\u003eB\u003c/strong\u003e). *Indicates identical residues in all sequences; \u0026nbsp;(:) = highly conserved; (.) = moderately conserved. The GenBank accession number of aligned sequences were: Cerastokunin (present study), Chain A, Trypsin Inhibitor (6A5I_A), Kunitz-type serine protease inhibitor PPTI (C0HLB2.1), Kunitz-type serine protease inhibitor (P24541.1), Kunitz-type serine protease inhibitor 3 (P00992.2), Kunitz/BPTI inhibitor-7 (AMH40739.1), Kunitz/BPTI inhibitor-3 (AMH40735.1), Inhibitor,chymotrypsin (0909196A), Kunitz/BPTI inhibitor-5 (AMH40737.1), Kunitz/BPTI inhibitor-2 (AMH40734.1), Kunitz-type serine protease inhibitor PIVL(I2G9B4.1), Kunitz-type serine protease inhibitor ki-VN (E5AJX3.1).\u003c/p\u003e","description":"","filename":"FIGURE6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4354127/v1/6e90f43a9ee5cb1959fba24d.jpg"},{"id":56062290,"identity":"89543fec-cef0-4fc4-9abd-73f2c1251e2d","added_by":"auto","created_at":"2024-05-08 05:16:37","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":109310,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenic tree and evolution relationships of Cerastokunin with Kunitz-type peptides sharing more than 65% of identity to Cerastokunin.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4354127/v1/02d6adee8b49263333b49388.jpg"},{"id":56062298,"identity":"1a83bcf2-a64c-4389-9fa6-2d1d46c9ad41","added_by":"auto","created_at":"2024-05-08 05:16:38","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":340168,"visible":true,"origin":"","legend":"\u003cp\u003eModeled structure of Cerastokunin. Prediction of two (\u003cstrong\u003eA\u003c/strong\u003e) and three (\u003cstrong\u003eB\u003c/strong\u003e) dimensional structures of Cerastokunin using Phyre-2 server and Swiss Model database respectively, construction was done based on the template (A0A1S5QJJ1_VIPAA) for 3D modeling.\u003c/p\u003e","description":"","filename":"FIGURE8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4354127/v1/8903e30a5ad3e9bd0e47b821.jpg"},{"id":56062297,"identity":"5eddf539-9805-45c3-b8c5-2936c7dfb34c","added_by":"auto","created_at":"2024-05-08 05:16:37","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":574698,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction of Cerastokunin with FXa comparatively to Apixaban and Rivaroxaban interactions. 3D Diagram illustrations of \u0026nbsp;Cerastokunin (in blue) complex with FXa (1hcg.pdb) (in green\u003cstrong\u003e) (A)\u003c/strong\u003e, \u0026nbsp;Apixaban (2p16.pdb)-FXa complex \u003cstrong\u003e(B)\u003c/strong\u003e, Rivaroxaban (2w26.pdb)-FXa complex \u003cstrong\u003e(C)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"FIGURE9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4354127/v1/85380152711f084dc50c52cb.jpg"},{"id":56062293,"identity":"4990451f-2886-40ff-999b-cca8da74ca15","added_by":"auto","created_at":"2024-05-08 05:16:37","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":568242,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction of Cerastokunin thrombin comparatively to Apixaban and Rivaroxaban interactions. 3D Diagram illustrations of \u0026nbsp;Cerastokunin (in blue) complex with Thrombin (3u69.pdb) (in green) \u003cstrong\u003e(A)\u003c/strong\u003e, Apixaban (2p16.pdb)-Thrombin \u003cstrong\u003e(B)\u003c/strong\u003e, Rivaroxaban (2w26.pdb)-Thrombin \u003cstrong\u003e(C).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"FIGURE10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4354127/v1/7acc233eb0b10ee4968049e6.jpg"},{"id":56062662,"identity":"d0ecc44e-9ade-4f3f-a9fc-b49844a014de","added_by":"auto","created_at":"2024-05-08 05:24:36","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":582972,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction of Cerastokunin-Trypsin comparatively to Apixaban and Rivaroxaban interactions. 3D Diagram illustrations of \u0026nbsp;Cerastokunin (in blue) complex with Trypsin (3d65.pdb) (in green) \u003cstrong\u003e(A)\u003c/strong\u003e, Apixaban (2p16.pdb)-Trypsin \u003cstrong\u003e(B)\u003c/strong\u003e, Rivaroxaban (2w26.pdb)-Trypsin \u003cstrong\u003e(C).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"FIGURE11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4354127/v1/c9b2efeefcee41326cfa8e7f.jpg"},{"id":56062288,"identity":"c96fd2aa-f385-46eb-93f1-13ad97385ac3","added_by":"auto","created_at":"2024-05-08 05:16:37","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":624412,"visible":true,"origin":"","legend":"\u003cp\u003eProposed mechanism of action of Cerastokunin on blood coagulation and platelet aggregation. P115 RhoGEF: p115 Rho guanine nucleotide exchange factor, RhoA: Ras homolog family member A, PKA: cAMP-dependent protein kinase, SFK: Src family of protein tyrosine kinases, Rac1:Ras family relates small GTPase 1. XII(a): Coafilation factor XII (activated), III (a): Coagulation factor III (activated) or tissue factor, VII: Coagulation factor VII, V: Coagulation factor V, VIII: Coagulation factor VIII, Xa: activated coagulation factor X, IX: coagulation factor IX, XIII (a): Coagulation factor XIII (activated) or fibrin stabilizing factor, G: G protein, PL: phospholipids, PLC : phospholipase C , IP3: inositol triphosphate, DAG: diacylglycerol, PKC: protein kinase C, PI3K: Phosphoinositide 3-kinase, alpha granule, : dense granule, FvW: von Willebrand factor, PF4: platelet factor 4, -TG: beta-thromboglobulin, PDGF: Platelet-derived growth factor.\u003c/p\u003e","description":"","filename":"FIGURE12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4354127/v1/d17cdd7ec8c77b92316848c8.jpg"},{"id":61793298,"identity":"5ca46258-1666-4da9-83e7-01252c757f7c","added_by":"auto","created_at":"2024-08-05 16:09:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6015987,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4354127/v1/33202ed1-c648-4c90-bb4b-f8e4f6f9a480.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Structural, biochemical characterization and molecular mechanism of Cerastokunin: A new Kunitz-type peptide with potential inhibition of thrombin, factor Xa and platelets","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThrombo-inflammatory disorders are a common characteristic complication of diverse diseases including coagulopathies, infectious diseases, and various cancers [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Apart from monoclonal antibodies for certain types of cancers, synthetic molecules are mainly used for therapeutic purposes for all these concern conditions. However, chemical medicines are showing more and more side-effects, especially when they target critical diseases like cardiovascular disorders. In this regard, researchers are interested in discovering and identifying new biomolecules with biological activities purposely as an alternative biotherapy that consists of potential innovative biotherapeutics that might replace traditional chemical drugs.\u003c/p\u003e \u003cp\u003eVenomous animals including \u003cem\u003eViperidae\u003c/em\u003e snake\u0026rsquo;s venoms present a rich source of various peptides and proteins doted of a myriad of biological activities that have been previously purified and characterized [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Some of these biomolecules can be used as therapeutics in coagulopathies related to blood clotting factors\u0026rsquo; deficiencies or to their genetic anomalies in terms of expression or function. They either act as substitutes to coagulation factors by mimicking their functions or by interacting with them. Other snake venom components interfere as whether activators or inhibitors or even both with various steps of platelet functions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. \u003cem\u003eCerastes cerastes\u003c/em\u003e venom is a heterogeneous cocktail of bioactive molecules that interact with the hemostatic system by activating or inhibiting coagulation pathways, with a persistent bleeding resulting from a synergistic action of different components [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Actually, a variety of biomolecules from Cerastes cerastes venom were isolated and their mechanisms underlying the pharmacological action on hemostasis were well elucidated. The structural characterization reveals some as enzymatic proteins like metalloproteinases (CCSV-MPase), serine proteases (Af\u0026acirc;acytin, RP-34 and Cc3-SPase), a 5\u0026rsquo;-nucleotidase (Cc-5\u0026rsquo;NTase), phospholipases A2 (Cc1-PLA2 and Cc2-PLA2), a phosphodiesterase (Cc-PDE) and others are peptides with non catalytic activity including a C-type lectin (Cc-Lec) and two desintegrins (Cerastategrin and Ophidiastatin) [\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, there are still numerous bioactive components to explore from \u003cem\u003eCerastes cerastes\u003c/em\u003e venom. In our knowledge, until today none kunitz type peptide has been yet isolated from this venom.\u003c/p\u003e \u003cp\u003eSnake venom Kunitz type peptides commonly known as BPTI (Bovin Pancreatic Trypsin Inhibitors), compose an abundant family of serine proteases inhibitors that are mostly present in \u003cem\u003eElapidae\u003c/em\u003e and \u003cem\u003eViperidae\u003c/em\u003e venoms, according to many proteome analyses. In fact, BPTI are hugely found in \u003cem\u003eDaboia russelii\u003c/em\u003e venom (28%) whilst around only 16% in \u003cem\u003eDendroaspis angusticeps\u003c/em\u003e venom, this may explain their contribution in the hemostasis disruption of prey since BPTI interact efficiently with blood coagulation cascade and are able to abolish more than serine proteases actively involved in coagulation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Aside this versatile abundance, functionally, snakes\u0026rsquo; venom kunitz-type peptides were subdivided into two groups: (i) non neurotoxic snake venom kunitz type inhibitors that display an inhibitory activity of trypsin and/or chymotrypsin, and, (ii) neurotoxic kunitz-type peptides that inhibit potassium and calcium channels, which do not display serine proteases\u0026rsquo; inhibitory activity [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Apart from these two previous classes, a third class was introduced to this classification based on bifunctional kunitz type peptides generated from a structural evolution of snakes\u0026rsquo; venom kunitz-type peptides [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Interestingly, several researches on kunitz type peptides of snake venoms have shown that they provide a source of promising pharmaceutical applications. Actually, these peptides contribute in different biological processes, especially, on the blood clotting, fibrinolysis and inflammation pathways, and present a potent anti-tumoral effect due to their multifunctional features [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Furthermore, kunitz type inhibitors appear to present a selectivity towards their targets which makes them excellent candidates for drug development. For instance, Textilinin-1, an antihemorrhagic component that shows fewer side effects, a better efficiency and higher selectivity than Trasylol, commonly known as Aprotinin, a BPTI of reference [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe main objective of the current study was to purify the first antithrombotic kunitz-type peptide from \u003cem\u003eCerastes cerastes\u003c/em\u003e venom, its \u003cem\u003ein vivo\u003c/em\u003e, \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein silico\u003c/em\u003e characterization to be performed and the mechanism of action as anti-thrombin, anti-FXa and antiplatelet peptide to be elucidated as well. In the future, the new peptide may be developed as a new diagnostic and/therapeutic biodrug for blood coagulopathies.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Biological materials and reagents\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1. Animals\u003c/h2\u003e \u003cp\u003eNMRI mice (20\u0026ndash;25 g) were obtained and maintained at the animal breeding of Faculty of Biological Sciences of USTHB (Algiers, Algeria) and experimental protocols were conducted according to the Ethics Committee of European Community in Experimentation for Animals\u0026rsquo; Welfare (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ec.europa.eu/food/animals/welfare/strategy_en\u003c/span\u003e\u003cspan address=\"https://ec.europa.eu/food/animals/welfare/strategy_en\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and approved after expertise performed by the Ethical Committee of Animal Experimentation (ECAE). The ECAE is a fully constituted Committee in accordance with the National Statement on Ethical conduct in research involving laboratory Animal-Law N\u0026deg;.88\u0026thinsp;\u0026minus;\u0026thinsp;08 of 26th January, 19988 related to veterinary medicine and the protection of animal health.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2. Venom\u003c/h2\u003e \u003cp\u003e \u003cem\u003eCerastes cerastes\u003c/em\u003e venom was provided by the Faculty of Biological Sciences, USTHB (Algiers, Algeria) in freeze- lyophilized form, and conserved at \u0026minus;\u0026thinsp;20\u0026deg;C in a protected zone from light until use. The blood hemostasis kits were procured from Biolabo.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.1.3. Medicines\u003c/h2\u003e \u003cp\u003eApixaban (Biopexa 5mg lot 015 n\u0026deg;19/12A200/263) and Rivaroxaban (Trombix 20 mg lot 005822 n\u0026deg;16/12F1688447). All Chemical Reagents are of analytical grade and were purchased from Sigma Aldrich- Merck group.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Purification of anti-trypsin activity peptide from \u003cem\u003eCerastes\u003c/em\u003e crude venom\u003c/h2\u003e \u003cp\u003eThree successive chromatographies were carried out in order to purify Cerastokunin from the crude venom of \u003cem\u003eCerastes cerastes;\u003c/em\u003e gel filtration chromatography followed by an affinity chromatography. Finally, a Reverse High Performance Liquid Chromatography (RP-HPLC) was performed to check the purity of the purified Cerastokunin. First, lyophilized crude venom (460 mg) was dissolved in 5 mL of saline buffer 0.9% and submitted to G-75 Sephadex size exclusion chromatography on column (60 \u0026times; 2.5 cm) (Pharmacia) equilibrated with 100 mM ammonium acetate buffer (pH 8.5). Fractions\u0026rsquo; elution occurred at flow rate of 18 mL/h with the same buffer of equilibration and 3 mL of the fractionated protein and peptide peaks were collected and monitored at 280 nm. The obtained fractions were pooled, desalted by a direct elution on a column (10 x 1.5 cm) of G-25 Sephadex gel (GE 17-0032-02 Healthcare, Sweden) and concentrated. Afterwards, hemolytic activity or phospholipase A2 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], casein protease [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], modified by [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and anti-trypsin [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] activities were assessed. The third Gel-filtration (GF3) fraction of low molecular weight molecules was pooled (tubes 210\u0026ndash;427). GF3 doesn\u0026rsquo;t exhibit any hemolytic nor casein protease activities while it does show a potent inhibition on trypsin-activity against a natural substrate (casein). Based on this activity, GF3 was re-subjected to a certain repeated runs of affinity chromatography on Trypsin-Sepharose CL4B column (1.4 \u0026times; 8.5 cm). Before performing the affinity chromatography, immobilization of trypsin as ligand to Sepharose CL4B was conducted and performed as described by Thernyck and Avrameas in 1991. Briefly, an amount of BrCN activated Sepharose CL4B gel (2.5 g) was extensively washed with HCl solution (1 mM) by successive centrifugations at 3000x\u003cem\u003eg\u003c/em\u003e during 2 min each, trypsin solution [10 mg trypsin dissolved in 10 mL sodium carbonate/bicarbonate buffer (0.1 M, pH 8.3) containing NaCl (1 M)] was added to the gel, and the mixed solution was incubated on shaker at +\u0026thinsp;4\u0026deg;C all over the night then centrifuged for 2 min at 3000x\u003cem\u003eg\u003c/em\u003e. The blocking buffer (glycine (0.1 M, pH 8.2) was added and the mixture was incubated at +\u0026thinsp;4\u0026deg;C for 16 hours, the excess of trypsin non-linked to the gel was eliminated by three successive centrifugations at 3000x\u003cem\u003eg\u003c/em\u003e within 2 minutes each. The Trypsin-Sepharose CL4B gel was submitted on a chromatography column (5 x 1 cm) and equilibrated with PBS buffer (0.1 M, pH 7.4), then concentrated GF3 (1.5 mL, 7.381 mg) was subjected to the affinity chromatography and left at least 1 hour on the column before elution run to enable sufficient interaction between fraction containing anti-trypsin peptides and immobilized trypsin onto column. The affinity chromatography began by first removing unbound proteins and peptides by simple elution with PBS buffer (0.1 M, pH 7.4). Then, the same buffer with highest molarity (1 M, pH7.4) was used to elute trypsin-high affinity peptides. The flow rate was maintained at 18 mL/h and 3 mL of the fractionated proteins and peptide peaks were collected and monitored at 280 nm. Finally, the purity of the obtained Cerastokunin, was checked by injecting 10 \u0026micro;L (14.5 \u0026micro;g) of the concentrated fraction of trypsin-high affinity, on a C18 RP-HPLC column (250 mm \u0026times; 4.6 mm, 5 \u0026micro;m) previously equilibrated with 0.1% (v/v) trifluoroacetic acid (TFA) (Solvent A), then the purified peptide was eluted with a linear gradient over 70 min from 5 to 100% (v/v) acetonitrile 80% (ACN) containing 0.1% (v/v) TFA (Solvent B) under a flow rate of 1 ml/min as described by [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The elution of peptides was monitored at 280 nm and 10 \u0026micro;L of blank solution containing 95% solvent A (v/v) and 5% of solvent B (v/v), was subjected to the same HPLC protocol in order to detect the additional peaks relatives to solvent absorbance on the Ultraviolet spectrum.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Mass spectrometry analysis on MSMS-MALDI device\u003c/h2\u003e \u003cp\u003eThe analysis using mass spectrometry was carried out in accordance with [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Cerastokunin was first precipitated in six volumes of cold acetone (overnight, -20\u0026deg;C), after which the peptide was reduced with five milligrams of Tris (2-CarboxyEthyl) Phosphine hydrochloride and alkylated with ten milligrams of 2-iodoacetamide to create the analytical sample. The sample was then cleaved by incubating it for six hours at 37\u0026deg;C with 1 \u0026micro;g/\u0026micro;L of Lys-C endopeptidase and 2 mM CaCl2. The treated peptide was then centrifuged at 5,000\u003cem\u003exg\u003c/em\u003e for 10 minutes after being incubated with trypsin for 5 hours. The resultant peptide fragments were extracted once the enzymatic digestion was complete, diluted in solvent A (5% ACN/0.1% TFA), and put into an Ultimate 3000 HPLC system fitted with a Probot fractionation device (15 cm x 75 \u0026micro;m x 3 \u0026micro;m, 100\u0026Aring;) with a separate C18 PepMap column (Dionex LC-Packings, Sunnyvale, CA, USA). The peptide separation was then initiated after a 5-minute wash of the utilized column. Following the column wash, a linear gradient of solvent B containing 80% CAN with 0.1% TFA (0 to 50%) was applied to start the separation process. This was done for 45 minutes, and then a final wash phase was carried out for 5 minutes using solvent B at 95%. The separation of peptide fragments was carried out at a consistent temperature of roughly 40\u0026deg;C. Lastly, the collected fractions were immediately spotted onto a MALDI plate with a matrix of succinic acid (CHCA) (2 mg/mL). Using CHCA containing 10 fmoles of Glu-1-fibrino peptide B ([M\u0026thinsp;+\u0026thinsp;H] \u003csup\u003e+\u003c/sup\u003e = 1570.670), the mass spectra were calibrated and for the MS/MS spectra, the 2 kV positive CID ON technique was chosen. In positive reflector ionization mode (m/z range: 800 to 4,000), 4,800 MALDI TOF/TOF (Applied Biosystems/MDS SCIEX, Framingham, MA, USA) analyzed MALDI MS data with 3000 laser shots/spot with a satisfactory S/N (signal/noise) quality for precursor selection. A MASCOT search engine 2.1 (Matrix Science Ltd., UK) integrated into GPS-Explorer Software 3.5 on the NCBInr database was used to analyze MS/MS queries. The research in MASCOT was calibrated with 0.3 Da for trypsin cleavage and 50 ppm mass accuracy for the precursor. The collected values were verified by considering only protein scores of 53 and protein indexes of 95%. Additionally, two Scaffold software reanalyses of the discovered proteins were conducted utilizing and a workflow incorporating both Peptide- and Protein-Prophet was chosen for the MS/MS independent search engines (MASCOT and X! Tandem). In this instance, only proteins with peptide confidence indexes greater than 95 and protein confidence indexes greater than 95% were taken into account.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Antiplatelet activity of Cerastokunin on thrombin-induced platelet aggregation\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Platelet-rich plasma and platelet-poor plasma preparation\u003c/h2\u003e \u003cp\u003eHealthy volunteers who had not taken any medications that would affect their hemostasis for at least three weeks prior to the blood collection were used to donate human blood. 3.8% of the boots used for blood i.v. extraction were citrated. Platelet-rich plasma (PRP) was obtained by centrifuging the collected blood for seven minutes at 150\u003cem\u003exg\u003c/em\u003e, whereas platelet-poor plasma (PPP) was obtained by centrifuging the blood for twenty minutes at 2,000\u003cem\u003exg\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Evaluation of the anti-platelet activity by aggregometry\u003c/h2\u003e \u003cp\u003ePurified peptide's antiplatelet activity was measured using the Chronolog aggregometer (Chrono-Log, ServiBIO 540 VS USA) for five minutes after 200 \u0026micro;L of PRP was added. PPP was used as a blank control. Thrombin, used at 0.45 U/mL, was pre-incubated for 30 min with 10 and 20 \u0026micro;g (1.31 and 2.6 nmole) of Cerastokunin at 37\u0026deg;C. Cerastokunin's suppression of platelet aggregation was evaluated in relation to thrombin-induced platelet aggregation, which was observed when thrombin (0.45 U/mL) was added to PRP (200 \u0026micro;L). The maximal platelet aggregation activity was identified as the platelet aggregation attained with thrombin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 Dose-response inhibitory activity of platelet aggregation\u003c/h2\u003e \u003cp\u003eIn order to assess its dose-response effect, various increasing Cerastokunin doses ranging from 1 to 20 \u0026micro;g/mL (1, 2, 3, 5, 10, 15, and 20 \u0026micro;g/ml) or (0.13, 0.258, 0.387, 0.65, 1.29, 1.94, 2.58 \u0026micro;M) were pre-incubated with 0.45 U/mL thrombin at 37\u0026deg;C for 30 min in a 96-well microplate prior to adding 90 \u0026micro;L of PRP in each well. The absorbance was recorded at 540 nm by a microtiter reader Bio-Tek Instrument. Thrombin and heparin were used as positive and negative aggregation activity controls, respectively, and the platelet aggregation percent was deduced by comparison to thrombin-induced aggregation considered as maximal aggregation activity. Percent aggregation = [(A\u003csub\u003e0\u003c/sub\u003e- Af)/ (A\u003csub\u003e0\u003c/sub\u003e-A)] \u0026times;100%, where Ao is the absorbance of PRP at 540 nm prior to addition of tested molecules; Af is the absorbance of PRP at 540 nm post addition of tested molecules ; and A is PPP absorbance at 540 nm [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.5 \u003cem\u003eIn vitro\u003c/em\u003e studies of anti-blood coagulation of Cerastokunin\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Measurement of Prothrombin Time (PT)\u003c/h2\u003e \u003cp\u003eIn this experiment, the inhibitory action of Cerastokunin on the extrinsic coagulation pathway was assessed. Graded concentrations of peptide (0\u0026ndash;20 \u0026micro;g/mL equivalent to 0-2.58 \u0026micro;M respectivly) were incubated with 100 \u0026micro;L of PRP for 3 min at 37\u0026deg;C then, 200 \u0026micro;L of Prothrombin Time reagent (Biolabo, France) was added following the manufacturer\u0026rsquo;s instruction and the clotting time was optically measured in seconds.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 Measurement of Activated Partial Thromboplastin Time (aPTT)\u003c/h2\u003e \u003cp\u003eTo evaluate the probable involvement of Cerastokunin on the intrinsic pathway of blood coagulation, a mixture of 100 \u0026micro;L of PPP and different concentrations of Cerastokunin (0\u0026ndash;20 \u0026micro;g/mL equivalent to 0-2.58 \u0026micro;M respectively) was incubated with 100 \u0026micro;L of aPTT reagent (Biolabo, France) at 37\u0026deg;C for 3min, as recommended by the manufacturer, and clotting time was recorded in seconds after 0.025 M CaCl\u003csub\u003e2\u003c/sub\u003e (100 \u0026micro;L) addition.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.6 \u003cem\u003eIn vivo\u003c/em\u003e antithrombotic effect of Cerastokunin and toxicity assessment\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1 Antithrombosis assay in Carrageenan-induced mouse tail thrombosis model\u003c/h2\u003e \u003cp\u003eThe mouse model was performed as described by [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] with male NMRI mice (20-25g), with four individuals in each group. Cerastokunin dissolved in sterile Phosphore buffer (1\u0026times;M, pH 7.4) was administered intravenously at doses of 2, 4 and 6 mg/kg body weight. Blank control animals received an equivalent volume of sterile 0.9% saline by intravenous injection. Apixaban and Rivaroxaban, two antithrombotic drugs, were orally administered, at a therapeutic dose of 0.2 mg/kg body weight as positive controls of antithrombotic activity. In order to induce tail-thrombus formation, each mouse received 100 \u0026micro;L (1%, intraperitoneal route) containing 60 mg/kg body weight of κ-Carrageenan (type I, Sigma) dissolved in 0.9% NaCL. The κ-Carrageenan was injected 30 min post-treatment in the case of saline and tested peptide whereas, in the case of drugs, κ-Carrageenan was administrated 60 min post-treatment since the drugs were injected by non-systemic route. Six hours after κ-Carrageenan injection in all experiments, the tested doses of Cerastokunin were re-injected into mice through the caudal vein. All mice were placed in a room at 20\u0026deg;C for 48 hours and the length of thrombus in mouse tail was measured and recorded at 24 and 48 hours post-treatment, respectively. Experimental protocol was conducted according to the Ethics Committee of European Community in Experimentation for Animals\u0026rsquo; Welfare (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ec.europa.eu/food/animals/welfare/strategy_en\u003c/span\u003e\u003cspan address=\"https://ec.europa.eu/food/animals/welfare/strategy_en\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2 Evaluation of toxicity and \u003cem\u003ein vivo\u003c/em\u003e anticoagulant activity\u003c/h2\u003e \u003cp\u003eThe toxicity was examined throughout the two days of the antithrombosis experiment previously given in section \u003cspan refid=\"Sec17\" class=\"InternalRef\"\u003e2.6.1\u003c/span\u003e. Mice were observed at regular intervals of time for death or any physical or behavioral changes. 48 hours post-treatment, mice were sacrificed, thoracic blood was collected in plastic petri dish and the blood clotting time from Cerastokunin-treated mice or control group of mice was recorded to evaluate the \u003cem\u003ein vivo\u003c/em\u003e anticoagulant role of Cerastokunin, if any.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Bioinformatics analysis\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1. Structure and sequence homology analysis\u003c/h2\u003e \u003cp\u003eHomology study of primary sequence of Cerastokunin with other kunitz-type peptides\u0026rsquo; sequences from other species was performed using the BLAST program, (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"http://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] through the National Center for Biotechnology Information databases (NCBI, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The generated Cerastokunin homologous sequences were used to perform a sequence alignment which was analyzed through Clustal W program [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/jdispatcher/msa/clustalo\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/jdispatcher/msa/clustalo\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and a phylogenetic tree was then constructed using sequences that demonstrated the best homology with Cerastokunin sequence. The physicochemical parameters were also analyzed \u003cem\u003ein silico\u003c/em\u003e by submitting the amino-acid sequence of Cerastokunin on Protein Calculator v3.4 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://protcalc.sourceforge.net/\u003c/span\u003e\u003cspan address=\"https://protcalc.sourceforge.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Finally, two dimensional structure was elaborated through Phyre2 online server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.sbg.bio.ic.ac.uk/~phyre2\u003c/span\u003e\u003cspan address=\"http://www.sbg.bio.ic.ac.uk/~phyre2\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Afterwards, inhibitor-3 sequence (Model ID : A0A1S5QJJ1 (A0A1S5QJJ1_VIPAA); from \u003cem\u003eVipera ammodytes ammodytes\u003c/em\u003e Kunitz/BPTI venom), that presents 70.15% of identity with Cerastokunin primary sequence, was used as template and the three tridimensional structure was modeled using Swiss-Model server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2. Molecular docking\u003c/h2\u003e \u003cp\u003eThe protein-ligand complexes of : FXa-Apixaban, FXa-Rivaroxaban, Thrombin-Apixaban, Thrombin-Rivaroxaban, Trypsin-Apixaban and Trypsin-Rivaroxaban, were all modeled through SwissDock online-server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swissdock.ch/docking\u003c/span\u003e\u003cspan address=\"http://www.swissdock.ch/docking\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), using the following protein databank structures : Rivaroxaban [2w26.pdb], Apixaban [2p16.pdb], Trypsin 3d65.pdb]), Thrombin [3u69.pdb], FXa [1hcg.pdb]. Complexes with the lowest energy (ΔG) were selected and the results were visualized using UCSF Chimera.\u003c/p\u003e \u003cp\u003eClusPro database, version 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cluspro.bu.edu/publications.php\u003c/span\u003e\u003cspan address=\"https://cluspro.bu.edu/publications.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used for protein-protein docking of Cerastokunin-Serine proteases (Trypsin, thrombin and FXa), the modeled Cerastokunin 3D structure was used, the complex at position 0 was selected and the results were visualized using PyMol.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical study of obtained results was done by Normalization and one-way ANOVA analysis significant difference with multiple comparison test to compare between data. Results were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and considered statistically significant at p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 *, very significant p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.01 ** and highly significant p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.001 ***.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Purification of Cerastokunin from \u003cem\u003eCerastes cerastes\u003c/em\u003e venom\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCerastes cerastes\u003c/em\u003e venom was fractionated on G-75 Sephadex gel-filtration column in three peaks yielded on molecular size exclusion (GF1-3). The third peak (GF3) \u0026nbsp;contained \u0026nbsp;low molecular weight peptides was considered for extracting a kunitz-type peptide as this type of peptides are characterized by a low molecular weight around 7 kDa\u0026nbsp;[17]. So, GF3 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(tubes 210-427) \u003cstrong\u003e(Fig1.A)\u0026nbsp;\u003c/strong\u003ewas pooled. GF3 displayed an inhibitory activity on trypsin-casein interaction since it didn\u0026rsquo;t reveal any casein protease or hemolysis activities \u003cstrong\u003e(Fig1.B)\u003c/strong\u003e. However, both peaks of GF1 and GF2 exhibited highest activity of trypsin as well as PLA2 activity, therefore, GF3 fraction was desalted and concentrated and then, applied to a trypsin-affinity chromatography \u003cstrong\u003e(Fig1.C)\u003c/strong\u003e and resolved in two peaks, the homogeneity of the second peak compounds with high affinity towards trypsin was analyzed through RP-HPLC column at \u0026nbsp; 280 nm. The purity of this peak consisting of Cerastokunin peptide \u0026nbsp;was approved since, only one amino acid peak was generated with a retention time of 24.197 min \u003cstrong\u003e(Fig1.D)\u003c/strong\u003e excluding the mobile phase relative two peaks (with a retention time of 17 and 61.933 min) \u0026nbsp;due to acetonitrile which is an organic solvent that absorbs in the ultraviolet spectrum \u003cstrong\u003e(Fig1.E).\u0026nbsp;\u003c/strong\u003ePurification process performed for the isolation of Cerastokunin and the retrieved data are summed up according to antiplatelet activity \u003cstrong\u003e(Table 1)\u003c/strong\u003e. The peptide was purified to an extend of about thirty-fold (purification factor equals to 29.85) and accounted only for 1.02% (protein by weight) in \u003cem\u003eCerastes cerastes\u003c/em\u003e venom. Cerastokunin retained about 20.895% of the total antiplatelet activity of the whole venom.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Mass measurement of Cerastokunin on MALDI-MSMS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further check the homogeneity of Cerastokunin, purity and calculation of its molecular weight, we proceeded with MALDI-TOF-MS analysis\u003cstrong\u003e\u0026nbsp;(Table 2)\u003c/strong\u003e. As demonstrated in \u003cstrong\u003e[Fig.1. (F1)],\u003c/strong\u003e the spectra obtained by Tandem mass spectrometry evidenced the presence of different isoforms with slight molecular mass variation probably due to the variability in the length of the final sequences which are obtained from the proteolytic cleavage of their precursors known as spectrum estimation as described in \u003cstrong\u003eFig.1.(F2).\u0026nbsp;\u003c/strong\u003eTaken together, all these data provided the main mass corresponding to 7,746.89 Da for the new purified peptide termed Cerastokunin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Functional study of Cerastokunin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.1 Dose-dependent inhibition of thrombin-induced platelet aggregation of Cerastokunin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antiplatelet role of Cerastokunin on the aggregation of platelets activated by thrombin was studied following two independent assays. Obtained results derived from the first experiment showed that Cerastokunin exhibited \u003cem\u003ein vitro\u003c/em\u003e antithrombotic activity considering that it inhibited 83% of the thrombin-dependent platelets activity at 20 \u0026micro;g/mL (2.58 \u0026micro;M), as the platelet aggregation decreased from 60 % to 10 % in the presence of\u0026nbsp;Cerastokunin\u0026nbsp;\u003cstrong\u003e(Fig.2 A and Fig.2B).\u003c/strong\u003e The dose of 2.58 \u0026micro;M was taken in this assay because it was the minimal effective dose against thrombin-induced platelet aggregation, as for a lower dose (1.29 \u0026micro;M) was ineffective when assessed (data not shown). \u0026nbsp;In the second experiment performed on a\u0026nbsp;96-well microplate, the incubation of thrombin with various increasing doses of Cerastokunin\u0026nbsp;revealed that\u0026nbsp;platelet-rich plasma\u0026nbsp;was prevented to aggregate due to the inhibition of thrombin because of its close binding to Cerastokunin. In fact, the peptide deactivated the platelets with a dose-dependent manner \u003cstrong\u003e(Fig.3)\u003c/strong\u003e, the highest inhibition was recorded with 1.29, 1.94 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; and 2.58 \u0026micro;M of Cerastokunin which corresponded to high significant difference of platelet absorbance compared to control \u003cstrong\u003e(Fig.3.A)\u003c/strong\u003e. In addition, it seems that the platelet inhibition was significantly higher with Cerastokunin (2.58 in \u0026micro;M) compared to heparin as illustrated in \u003cstrong\u003eFig.3.B\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e3.2.2 Anticoagulant function of Cerastokunin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In addition to the antiplatelet activity of Cerastokunin, the inhibition of blood clotting was evaluated for both extrinsic and intrinsic blood coagulation pathways. \u0026nbsp;The involvement of thrombin and other plasma factors in both pathways was studied in presence of Cerastokunin and experimentally checked by monitoring Prothrombin time (PT) and activated Partial Thromboplastin Time (aPTT). Both PT and aPTT were dose-dependently prolonged in blood samples pretreated with various doses of Cerastokunin. This indicated that Cerastokunin significantly inhibited both intrinsic and extrinsic blood coagulation pathways. In fact, it appeared that Cerastokunin with only lower doses was able to significantly delay both extrinsic and intrinsic pathways of coagulation. Moreover, the peptide potentially targeted the intrinsic pathways as it high significantly prolonged PT compared to control time (20 \u0026plusmn; 2 s vs. 8 \u0026plusmn; 0.5 s, n=3, p\u0026lt;0.001) and at only the dose of 0.258 \u0026micro;M (2 \u0026micro;g/ mL). On counterpart, Cerastokunin prolonged more efficiently PT (96 \u0026plusmn; 5.5 s, n = 3, p-values \u0026lt; 0.001) but with higher doses (2.58 \u0026micro;M) \u003cstrong\u003e(Fig.4.A)\u003c/strong\u003e. However, obtained results illustrated in \u003cstrong\u003eFig.4.B\u003c/strong\u003e, showed that aPTT was not prolonged in a highly significant manner until the dose of 0.65 \u0026micro;M \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(39 \u0026plusmn; 2.5 s vs. 13 \u0026plusmn; 1 s, n = 3, p-values \u0026lt; 0.001) and at 2.58 \u0026micro;M the activated partial thromboplastin time was extended to 84 \u0026plusmn; 5 s\u0026nbsp;(n=3, p-values \u0026lt; 0.001) \u003cstrong\u003e(Fig.4.B).\u003c/strong\u003e Taken together, all these recorded time values lead to emphasize that Cerastokunin inhibited both intrinsic and extrinsic pathway coagulation factors, with a more significant effect on the extrinsic pathway, resulting in a potent anticoagulant role.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 \u003cem\u003eIn vivo\u003c/em\u003e validation of Cerastokunin anti-thrombosis and safety\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCerastokunin is a safe peptide since it was non-toxic up to the highest intravenously administered dose of 6 mg/kg into mice. Cerastokunin-treated mice did not present any clinical manifestations or abnormal behavioral changes within 48 hours post-injection. In addition, in carrageenan-induced mouse tail thrombosis model, a reduction on thrombus length was observed 24 hours after treatment in Cerastokunin-pretreated mouse tail, particularly at doses of 2 and 6 mg/kg (\u003cstrong\u003eFig.5A\u003c/strong\u003e). For both doses, the antithrombosis was pronounced and highest than observed with Apixaban and Rivaroxaban, two antithrombotic drugs of reference\u0026nbsp;[33\u0026ndash;35].\u0026nbsp;At 48 hours post-treatment, the measurements of the thrombosis length along mice tail showed a dose-dependent antithrombotic effect. Interestingly, the obtained value of Cerastokunin (6 mg/kg) revealed an inhibition of about 75 \u0026plusmn; 2% \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; (p-values \u0026lt; 0.001) of thrombus formation \u0026nbsp;against 51.79 \u0026nbsp;\u0026plusmn; 3 % (p-values \u0026lt; 0.001) \u0026nbsp;and \u0026nbsp; \u0026nbsp; \u0026nbsp;17.92 \u0026nbsp;\u0026plusmn; 0.1% (p-values \u0026lt; 0.001) \u0026nbsp;recorded with Apixaban and Rivaroxaban respectively (\u003cstrong\u003eFig.5B\u003c/strong\u003e and \u003cstrong\u003eFig.5C\u003c/strong\u003e). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 \u003cem\u003eIn vivo\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eanticoagulant effect\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to deepen the investigation of the Cerastokunin anticoagulant effect, supplementary \u003cem\u003ein vivo\u003c/em\u003e experiment was carried out, completing the results previously obtained from \u003cem\u003ein vitro\u003c/em\u003e assays. Hence, blood samples were collected from mice previously challenged with the same doses of Cerastokunin as the toxicity protocol, and clotting time was recorded after 48 hours post-injection. As illustrated in \u003cstrong\u003eFig.5D\u003c/strong\u003e, Cerastokunin extended the clotting time proportionally to administered doses (2 mg/kg to 6 mg/kg). Blood clotting time was extended from 16 \u0026plusmn; 1 s (p-values \u0026lt; 0.001) \u0026nbsp;to 31 \u0026nbsp;\u0026plusmn; 2.5 s (p-values \u0026lt; 0.001) and to 201 \u0026nbsp;\u0026plusmn; 5 s (p-values \u0026lt; 0.001) with 2 mg/kg \u0026nbsp;and 6 mg/kg respectively whereas less values were measured with Rivaroxaban (120 \u0026plusmn; 5 s, p-values \u0026lt; 0.001) \u0026nbsp;and Apixaban (186 \u0026plusmn; 6 s, p-values \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Bioinformatics studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5.1. Structural features of Cerastokunin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMultiple-alignment of the primary sequence of Cerastokunin by BLAST server confirmed that the peptide belongs to kunitz family (inhibitors of serine proteases) (\u003cstrong\u003eFig.6\u003c/strong\u003e). This sequence also showed homology with other\u003cem\u003e\u0026nbsp;Viperidae\u003c/em\u003e venom peptides belonging to the same family (Kunitz-type peptides) including those extracted from the venoms of \u003cem\u003eMacrovipera lebetina transmediterranea (\u003c/em\u003eKunitz-type serine protease inhibitor PIVL), \u003cem\u003eVipera berus nikolskii (\u003c/em\u003eKunitz-type serine protease inhibitor ki-VN), \u003cem\u003eVipera ammodytes (\u003c/em\u003eKunitz/BPTI inhibitor-2, Kunitz/BPTI inhibitor-3, Kunitz/BPTI inhibitor-5 and Kunitz/BPTI inhibitor-7), \u003cem\u003ePseudocerastes persicus\u003c/em\u003e (Chain A, Trypsin Inhibitor\u0026nbsp;,\u0026nbsp;Kunitz-type serine protease inhibitor PPTI ) and\u0026nbsp;\u003cem\u003eEristicophis macmahoni\u003c/em\u003e (Kunitz-type serine protease inhibitor ), all of which share more than 65% of identity with Cerastokunin (\u003cstrong\u003eTable 3\u003c/strong\u003e). Furthermore, the phylogenetic tree was created with the aim of elucidating the relationship between various proteases\u0026rsquo; inhibitors studied from snake venoms. Analysis of the constructed phylogenetic tree shows that Cerastokunin belongs to the same group as Chain A, Trypsin Inhibitor, Kunitz-type serine protease inhibitor PPTI purified from the venom of \u003cem\u003ePseudocerastes persicus\u003c/em\u003e and Venom trypsin inhibitor from the venom of \u003cem\u003eEristicophis macmahoni\u003c/em\u003e, which belong to the\u003cem\u003e\u0026nbsp;Viperidae\u003c/em\u003e family \u003cstrong\u003e(Fig.7).\u003c/strong\u003e The physicochemical characterization of Cerastokunin was carried out by Protein calculator V3.4 server (\u003cstrong\u003eTable 4\u003c/strong\u003e), and generated results indicate that it has a low molecular weight of 7,746.89 Da and consists of amino acid residues equal to 67. Its primary sequence contains 6 cysteine residues which contribute to its structural stability. The kunitz peptide is classified as basic and hydrophilic peptide. Finally, the predicted two and three dimensional Cerastokunin structures, illustrated in the \u003cstrong\u003eFig.8A and Fig.8B\u003c/strong\u003e, were generated through two bioinformatics tools Phyre-2 and Swiss-Model respectively. The build modeled molecule of Cerastokunin includes \u0026beta;-strands (21%), \u0026alpha;-helixes (12%) and disordered domains (16%).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5.2. Interaction studies with molecular docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular docking of protein-protein selected complexes on position 0 show, interactions between Cerastokunin and two essential coagulation molecules in this study; plasma coagulation factor FXa and thrombin (FIIa). These interactions are established with eight (8) bonds between FXa and Cerastokunin and involved the amino acid residues \u0026laquo;\u0026nbsp;Glu97, Thr98, Tyr99 (double bonds), Glu39, Arg143, Gln192, Gln192 and Gln61 \u0026raquo; of FXa with their pairs identified in Cerastokunin sequence \u0026laquo; Asn17, Asn17, Tyr19 (double bonds), Tyr23, Thr34, Phe35, Tyr19 and Lys48 \u0026raquo;. As for Cerastokunin-thrombin binding, nine (9) bonds linked both through the following thrombin residues \u0026laquo; Lys372, Pro369, Trp412, Arg413, Glu466, Glu522, Glu522, Glu549 (double bonds) and Arg553 (double bonds) \u0026raquo;. These residues interacted with Cerastokunin through \u0026laquo; Asn17, Tyr13, Tyr19, Thr34, Gly41, Asn17 (two bonds), Ala18, Lys48 (two bonds), Tyr37 (two bonds) \u0026raquo; \u003cstrong\u003e(Table 5)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eObtained results pointed out that the ligand\u0026ndash;protein complexes with the lowest energy (DG) resulted from the\u0026nbsp;\u003cem\u003ein silico\u003c/em\u003e simulation of FXa and thrombin interactions with Apixaban and Rivaroxaban, showed that Apixaban interacted with FXa with only one hydrogen bond involving the amino acid Gln192 of FXa with a bond length of 2.84\u0026Aring; and \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;DG = -8.35 Kcal/mol. Similarly, Rivaroxaban blocked the coagulation by linking FXa with only one hydrogen bond through Gly193 with a bond length= 2.5\u0026Aring; (DG = -7.69 Kcal/mol) (\u003cstrong\u003eFig.9\u003c/strong\u003e). The selected complex with the lowest energy (DG= -7.86 Kcal/mol))\u0026nbsp;between thrombin and Apixaban showed two (2) hydrogen bonds, H21 and nitrogen N6 which established bonds with the amino acid residues of thrombin Arg310 \u0026nbsp;(bond length = 2.3 \u0026Aring;)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAsn537 \u0026nbsp;(bond length\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e= 2.75 \u0026Aring;). However, lowest energy of complex Thrombin-Rivaroxaban was formed by only one bond established between the amino acid Trp370 of thrombin and the oxygen (O13) of Rivaroxaban with bond length of 2.37 \u0026Aring; and\u0026nbsp;DG= -7.43 Kcal/mol (\u003cstrong\u003eFig.10, Table 5\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTrypsin-Cerastokunin complex on position 0 was strongly established through the formation of seven (7) bonds involving \u0026laquo; Tyr39, Tyr151, Gly216, Gln192, Gly219, Ser190 (2 involved bonds) and Asp189 \u0026raquo; of trypsin and \u0026laquo; Pro21, Tyr19, Asn17, Cys16, Pro15, Asn17 (double bonds) and Asn17 \u0026raquo; residues from Cerastokunin \u003cstrong\u003e(Table 5)\u003c/strong\u003e. However, the lowest energy of the complex Trypsin\u0026ndash;Rivaroxaban involved only one (1) hydrogen bond with the amino acid Gln175 of trypsin\u0026nbsp;(bond length = 2.95 \u0026Aring;,\u0026nbsp;DG= -8.18 Kcal/mol), whilst the lowest energy (DG= -8.96 Kcal/mol) for the complex Trypsin- Apixaban\u0026nbsp;involved two (2)\u0026nbsp;hydrogen bonds where Ser96 and Gln192 contributed with bond length= 2.75 \u0026Aring; and 2.28 \u0026Aring; respectively (\u003cstrong\u003eFig.11\u003c/strong\u003e).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThromboembolic disorders known as the main cause of mortality in between cardiovascular diseases are generally treated with anticoagulant agents which present in association to their efficient anti-thrombotic effect, a bleeding risk or hemorrhagic complications [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In order to prevent these side-effects, new therapeutic approaches have been more and more investigated, these could equilibrate the altered hemostasis in patients without causing any bleeding risk [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this regard, snake venoms are worldwide considered as a rich source of pharmacologically active biomolecules. Basic on their ability to interfere with the blood-coagulation factors, platelet-aggregation and fibrinolysis as well, once isolated, many of which are considered as a potential anti-thrombotic agents [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and some are already used as marketed clinical medicines such as Captopril® from \u003cem\u003eBothrops jararaca\u003c/em\u003e venom which is an inhibitor of angiotensin-converting enzyme. This biotherapeutic was the first successful snake’s venom component designed drug currently indicated for hypertension therapy. Afterwards, other venom-derived drugs have been commercialized such as Aggrastat® (Tirofiban), initially identified from \u003cem\u003eEchis carinatus\u003c/em\u003e venom and consists of a disintegrin-based drug that inhibits Glycoprotein IIb/IIIa platelets, it is currently prescribed as antiplatelet medicine [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this current study, Cerastokunin with dual antithrombotic and antiplatelet activity was characterized and its mechanism of action was fully elucidated. Multiple alignment of the primary sequence validated the presence of putative serine protease binding sites and indicated a high similarity of Cerastokunin sequence with a variety of \u003cem\u003eViperidae\u003c/em\u003e venoms’ kunitz type peptides. Based on these structural data, the purified peptide was named Cerasto (\u003cem\u003eCerastes cerastes\u003c/em\u003e) –Kunin (kunitz type serine proteases’ inhibitor).\u003c/p\u003e \u003cp\u003eThe structural characterization of Cerastokunin demonstrated that it is a new member of kunitz type peptides’ family, known as serine proteases’ inhibitors for trypsin, chymotrypsin and other plasma coagulation factors including thrombin and Factor Xa [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eCerastokunin is a single-chain, basic (pI 8.48) and hydrophilic peptide composed of 67 amino acid residues including six residues of cysteine that contribute to its stability by forming a three disulfide bridges. With a molecular weight of 7,746.89 Da Cerastokunin is similar to typical bovine pancreatic trypsin inhibitor (BPTI), a family characterized by small molecular masses ranging between 6,000 and 7,000 Da [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Some of known extracted Kunitz type peptides are \u003cem\u003eDendroaspis polylepis\u003c/em\u003e ‘ venom kunitz-type peptide of 7,128.4 Da that inhibits elastase-1 and cathepsin L [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], \u003cem\u003eVipera russelii russelii’s\u003c/em\u003e Vipegrin is a 6,800 Da serine proteases inhibitor that inhibits trypsin and could inhibit collagen and ADP-induced platelet aggregation through its disintegrin-like activity [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Recently, different groups of trypsin inhibitors were extracted from Buckwheat, a pseudocereal, each group with a range of molecular masses, including BTI I, IIa, IIb, and IIIa that are formed of permanent inhibitors with 6,000 to 7,000 Da [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] .\u003c/p\u003e \u003cp\u003eThe three dimensional structure of Cerastokunin includes two antiparallel beta strands separated by a β-hairpin loop and a stable alpha helix at the C-terminal end with a disordered helix at the N-terminal extremity. The disorder state should be explained by the presence of a conserved proline in this sequence domain. All these structural features further approve that the purified Cerastokunin is a kunitz type serine protease inhibitor [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. As example, the BPTI is the first discovered and well known kunitz peptide that inhibits a spectrum of serine proteinases including trypsin, chymotrypsin, plasmin and plasma kallikrein [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. BPTI has also a conserved three dimensional structure containing three disulfide bridges and a two regions of helix α, a short helix3 10:H1 in the N-terminal extremity while the second helix α: H2 is longer than the first and localized in the C-terminal extremity. Between the two helices, the central region consists of double β strands linked by a one- tower loop that forms a β-hairpin [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn terms of functions, snake venom Kunitz-type/BPTIs are doted of a panel of pharmacological activities which have been figured out by the identification of several isoforms of these peptides. A previous analytical study reported that the sequences of kunitz type peptides undergo through a gene duplication followed by subsequent diversification resulting in sequence evolution [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. \u003cem\u003eIn vivo\u003c/em\u003e anticoagulant activity of Cerastokunin was examined on Carrageenan-induced mouse tail thrombosis model, a non-complicated and non-invasive animal modelization that is known to be used on the evaluation of new antithrombotic and anticoagulant substances like aspirin and heparin [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Interestingly, we found that the anticoagulant effect of Cerastokunin is more important than that of Rivaroxaban and Apixaban, direct factor Xa inhibitors [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Our results correlate with those of Joannsin, a kunitz type peptide from the venom of \u003cem\u003eProspirobolus joannsi\u003c/em\u003e, composed of 72 amino acid residues with three intramolecular disulfide bridges, that has shown a similar anticoagulant effect in addition of an anti-Factor Xa and anti-trypsin activity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Furthermore, when this anticoagulant effect was assessed \u003cem\u003ein vitro\u003c/em\u003e using activated partial thromboplastin time (aPTT) and prothrombin time (PT) reagents, the blood coagulation was inhibited on both extrinsic and intrinsic coagulation pathways; this data may be of a great interest to explain the potent \u003cem\u003ein vivo\u003c/em\u003e antithrombotic effect of Cerastokunin. In fact, Cerastokunin significantly lengthened the clotting time of plasma by exhibiting an inhibitory effect on factors involved in the initial steps of extrinsic pathway of the blood coagulation and behaved as a blocker of extrinsic coagulation pathway. Additionally, our study showed that Cerastokunin also acted by inhibiting the intrinsic pathway since it delayed blood clotting through prothrombin or/and FX blockade (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt is well-known that, thrombin interacts at least with two types of human platelet-membrane receptors, which consist on the protease activated receptors (PAR1/PAR4) and GPIb (alpha). PARs, being a seven-transmembrane G-protein coupled receptors can be activated by proteolysis of their N-terminal part by thrombin resulting on a transmembrane signaling cascade contributing to thrombus formation [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. These clots are formed by building a cross-linked platelet-fibrinogen thrombus resulting from the interaction of activated glycoprotein (GP) IIb/IIIa with dimeric fibrinogen molecules. On the other hand, thrombin binding to GP Ib(alpha) platelet-receptor activates the internal calcium mobilization signaling pathway inducing the platelet release reaction. This second thrombin-induced platelet activation pathway, is associated to a fibrin-integrin platelets cross-linking [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] and leads to an enhancement of PAR1 signaling pathway and an activation of glycoprotein V (GPV) on a synergistic interacting way [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSimilar to other snake kunitz peptides, Cerastokunin blocked the aggregation of platelets by preventing thrombin to bind to its PARs and/or GPIb(alpha) platelet receptors through a direct thrombin inhibition. The same mechanism has been reported and elucidated for Rusvikunin, a Kunitz-type protease inhibitor peptide purified from \u003cem\u003eDaboia russelii\u003c/em\u003e venom inhibited amidolytic activity of trypsin, plasmin and fibrinogen clotting as well as plasma clotting activity of thrombin [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Rusvikunin exhibited also an antiplatelet aggregation activity and an \u003cem\u003ein vitro\u003c/em\u003e prolongation of blood clotting time [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAside the fact that BPTI, pancreatic Kunitz inhibitor, was initially used like a crucial tool for studying protein structural conformations and protein-protein interactions. Many reports documented that BPTI, possesses a highest affinity to serine proteases that have a primary specificity to substrates with « Lys /Arg » residues, such as Thrombin and FXa. This affinity depends on structural factors outside the catalytic site, especially the residue Glu192 or Gln192 in the mutant variant which is involved in this type of BPTI- Serine protease(s) interaction that leads to inhibitory effect [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. This data might explain the implication of Lys48 of Cerastokunin in the interaction with both FXa and thrombin, as well as, the involvement of the residue Gln192 of FXa on both FXa-Cerastokunin and FXa-Apixaban interactions. In addition, the study of the interaction between FXa and Tissue Factor Pathway Inhibitor (TFPI), has demonstrated that the residue Arg143 of FXa is involved in this inhibitory interaction similar to FXa-Cerastokunin docking results. Furthermore, the two residues Tyr17 and Lys34 of TFPI were also implicated by similarity, this may explain the involvement of Tyr 19, Tyr 23 and Lys48 residues of Cerastokunin [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAH-884 is a variant of kunitz-like trypsin inhibitor peptide from the skin secretion of \u003cem\u003eAmolops hainanensis\u003c/em\u003e, named Kunitzin-AH presenting trypsin inhibitory activity. The docking simulation of Trypsin with AH-884 has demonstrated that four (4) amino acid residues Ser789, Asp792, Lys702, and Tyr 681 trypsin bond to Arg1, Asn5 and Cys7 residues of the AH-884 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. These reported results matched up with the docking data of the current study that illustrated the involvement of Cerastokunin Cys16 and Asn17 in the interaction with trypsin. Furthermore, in another reported study of pTTI (Purified trypsin inhibitor from tamarind seeds), the docking result of interaction between pTTI and trypsin has shown that Pro57 is one of the peptide residue that has the greatest interaction with trypsin [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Hence again, this provides a supplementary argument and fully correlates with our findings that the conserved Pro15 and Pro21 residues of Cerastokunin are importantly involved in the interaction with trypsin.\u003c/p\u003e \u003cp\u003eObtained results of molecular docking are valuable and showed that the affinity of Cerastokunin towards FXa, thrombin and trypsin is higher than that of Apixaban and Rivaroxaban, since Cerastokunin required between 7 to 9 bonds to interact with FXa, thrombin and trypsin whilst each one of the two drugs bonds to each protease by only 1 to 2 hydrogen bonds. This set, can be considered as additional support for an explanation of its significant antiplatelet effect, as well as, its potent anticoagulant effect exhibited on both extrinsic and intrinsic coagulation pathways as elaborated in \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments.\u003c/p\u003e \u003cp\u003eThe low molecular weight of Cerastokunin would guarantee a fast biodistribution and bioavailability. Its structure stability established by three intramolecular disulfide bridges and its potent dual function as antithrombotic and platelet blocker with a non-enzymatic trypsin inhibitory activity would be features of interest as a potential therapeutic and/or diagnostic biodrug for coagulopathies.\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eThe first kunitz-type peptide from Cerastes cerastes venom was purified in this study, and in vitro, in vivo, and in silico investigations were used to characterize its structure and function. The acquired data demonstrated that Cerastokunin is a 67 amino acid basic peptide that is two folded and has three disulfide bridges. In addition to its strong anticoagulant and antiplatelet action, Cerastokunin also exhibits non-enzymatic anti-trypsin, anti-thrombin, and anti-FXa actions. The comprehension of the mechanism underlying the thrombosis inhibition of venom Kunitz-type peptides could be enhanced by all of these studies. When considered collectively, the data point to Cerastokunin's potential as a novel therapeutic agent for coagulopathies due to its important antiplatelet, antithrombotic, and anticoagulant properties, all of which occur without causing any \u003cem\u003ein vivo\u003c/em\u003e harm.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u0026Delta;G : lowest energy\u003c/p\u003e\n\u003cp\u003eA\u0026nbsp;:Ala\u0026nbsp;: Alanine\u003c/p\u003e\n\u003cp\u003eACN: Acetonitrile\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eaPPT: Activated Partial Thromboplastin Time\u003c/p\u003e\n\u003cp\u003eBPTI: Bovin pancreatic trypsin inhibitors or Aprotinin\u003c/p\u003e\n\u003cp\u003eBrCN: \u003cem\u003eCyanogen bromide\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eC\u0026nbsp;: Cys\u0026nbsp;: Cysteine\u003c/p\u003e\n\u003cp\u003eCaCl2:\u0026nbsp;Calcium chloride\u003c/p\u003e\n\u003cp\u003eCc: Cerastes cerastes\u003c/p\u003e\n\u003cp\u003eCHCA: \u0026alpha;-Cyano-4-hydroxycinnamic \u003cem\u003eacid\u003c/em\u003e or Sinapinic Acid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCm: Centimeter\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD\u0026nbsp;: Asp\u0026nbsp;: Aspartate\u003c/p\u003e\n\u003cp\u003eE\u0026nbsp;: Glu\u0026nbsp;: Glutamate\u003c/p\u003e\n\u003cp\u003eF : Phe : Phenylalanine\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFXa:\u0026nbsp;Blood-clotting Factor ten active\u003c/p\u003e\n\u003cp\u003eG\u0026nbsp;: Gly\u0026nbsp;: Glycine\u003c/p\u003e\n\u003cp\u003eGF: Gel-filtration fraction\u003c/p\u003e\n\u003cp\u003eH\u0026nbsp;: His\u0026nbsp;: Histidine\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHCL :\u0026nbsp;\u003c/em\u003eHydrochloric acid\u003c/p\u003e\n\u003cp\u003ei.v:\u0026nbsp;Intravenous\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eI: Ile\u0026nbsp;: Isoleucine\u003c/p\u003e\n\u003cp\u003eK\u0026nbsp;: Lys\u0026nbsp;: Lysine\u003c/p\u003e\n\u003cp\u003eL\u0026nbsp;: Leu\u0026nbsp;: Leucine\u003c/p\u003e\n\u003cp\u003eLC-MALDI-MSMS:\u0026nbsp;Liquid chromatography-matrix-assisted laser desorption/ionization tandem\u0026nbsp;mass spectrometry\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eM\u0026nbsp;: Met\u0026nbsp;: Methionine\u003c/p\u003e\n\u003cp\u003eN\u0026nbsp;: Asn\u0026nbsp;: Asparagine\u003c/p\u003e\n\u003cp\u003eNaCl :\u0026nbsp;Sodium chloride\u003c/p\u003e\n\u003cp\u003eNMRI mice:\u0026nbsp;The Naval Medical Research Institute mice\u003c/p\u003e\n\u003cp\u003eP\u0026nbsp;: Pro\u0026nbsp;: Proline\u003c/p\u003e\n\u003cp\u003ePARs: Protease Activated Receptors\u003c/p\u003e\n\u003cp\u003ePBS buffer:\u0026nbsp;Phosphate-buffered saline\u003c/p\u003e\n\u003cp\u003epI: \u0026nbsp;Isoelectric point\u003c/p\u003e\n\u003cp\u003ePLA2: Phospholipase A2\u003c/p\u003e\n\u003cp\u003ePPP: Platelet-poor plasma\u003c/p\u003e\n\u003cp\u003ePRP: Platelet-rich plasma\u003c/p\u003e\n\u003cp\u003ePT: Prothrombin Time\u003c/p\u003e\n\u003cp\u003eQ\u0026nbsp;: Gln\u0026nbsp;: Glutamine\u003c/p\u003e\n\u003cp\u003eR\u0026nbsp;: Arg\u0026nbsp;: Arginine\u003c/p\u003e\n\u003cp\u003eRP-HPLC: Revers high performance liquid chromatography\u003c/p\u003e\n\u003cp\u003eS\u0026nbsp;: Ser\u0026nbsp;: Serine\u003c/p\u003e\n\u003cp\u003eS: Seconds\u003c/p\u003e\n\u003cp\u003eSepharose CL4B:\u0026nbsp;4% cross-linked agarose\u0026nbsp;chromatography base matrix\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSV: Snake- venom\u003c/p\u003e\n\u003cp\u003eT\u0026nbsp;: Thr\u0026nbsp;: Threonine\u003c/p\u003e\n\u003cp\u003eTFA: Trifluoroacetic acid\u003c/p\u003e\n\u003cp\u003eTFPI : Tissue Factor Pathway Inhibitor\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eU\u0026nbsp;: SeC\u0026nbsp;: Selenocysteine\u003c/p\u003e\n\u003cp\u003eV\u0026nbsp;: Val\u0026nbsp;: Valine\u003c/p\u003e\n\u003cp\u003eW\u0026nbsp;: Trp\u0026nbsp;: Tryptophan\u003c/p\u003e\n\u003cp\u003eY : Tyr : Tyrosine\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their gratitude to Dr. Khouloud BENALI, Dr. Nour Elyakine KERAGHEL and Dr. Abderrahmane KORI YAHIA (Faculty of pharmacy, University of Algiers 1) for their technical assistance.\u003c/p\u003e\n\u003cp\u003eWe would like to express our gratitude for Prof. Dr. CHARALLAH Salima (Chair of Ethic Committee) and Dr. BENMILOUD Abdelouafi and all the members of the Ethical Committee of Animal Experimentation (CEEA-USTHB) for their invaluable assistance and their thoroughness of expertise before delivering an approved ethical certificate for the use of animals. The CEEA is a fully constituted Committee in accordance with the National Statement on Ethical conduct in research involving laboratory Animal-Law N°.88-08 of 26th January, 19988 related to veterinary medicine and the protection of animal health.\u003c/p\u003e\n\u003cp\u003eWe also extend our sincere gratitude to the Hospital-University Center of Mustapha Bacha, Algiers, Algeria's Ethical Committee of Human Sample' Use, for granting permission to use human samples in accordance with their approved ethical guidelines. Their knowledge was crucial in validating the experimental design.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNoussaiba Saghour performed all the experiments, collected the data and wrote the article, Fatah Chérifi designed the study, supervised the experimental part, reviewed and edited the manuscript and contributed to revising the manuscript for clarity and coherence, Saoud Samah performed the computational experiments of docking studies of this work, validated and verified the accuracy and reliability of results related to molecules’ interactions, Younes Zebbiche contributed in the purification procedure and provided the technical assistance, Amel Meribai assisted with providing the required materials for animal model, Nadjia Bekkari provided the necessary reagents for the main part of this work and provided the documents for some techniques, Taright-Mahi Samya validated the experimental design of human samples and assisted in providing the ethical approval of use of blood samples, \u0026nbsp;Fatima Laraba-Djebari assisted in the conceptualization stage of the study, provided data, logistics and reagents throughout this work and secured funding acquisition and financial support for the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCh\u0026eacute;rifi, F., \u0026amp; Laraba-Djebari, F. 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Retrieved from https://www.tandfonline.com/doi/abs/10.1080/14756366.2021.1876686\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cimg 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\" width=\"750\" height=\"420\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2:\u003c/strong\u003e Peptide spectra and their respective masses derived from Mass Spectrometry scans of Cerastokunin (only fragment or peptides with a mass bigger than 500 Dalton were displaying and reported in the table) Peptides were digested by Lys C, Maximum number of missed cleavages (MC): 0All cysteines have been treated with Iodoacetamide to form carbamidomethyl-cysteine (Cys_CAM), with acrylamide adducts (Cys_PAM).Methionines have been oxidized to form methionine sulfoxide (MSO).Using monoisotopic masses of the occurring amino acid residues and giving peptide masses as [M+H]+).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"697\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.169054441260744%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFragment Number\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.166189111747851%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eStart Residue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.166189111747851%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eEnd Residue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.911174785100286%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eModification (Cystein and Methionin)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.61031518624642%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeight with modifications\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Da)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.455587392550143%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMeasuredMass (Da)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.5214899713467%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePeptide Sequence\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.169054441260744%\" rowspan=\"3\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.166189111747851%\" rowspan=\"3\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.166189111747851%\" rowspan=\"3\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.911174785100286%\"\u003e\n \u003cp\u003eCys_CAM: 7, 16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.61031518624642%\"\u003e\n \u003cp\u003e3560.6475\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.455587392550143%\" rowspan=\"3\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3560.6475\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.5214899713467%\" rowspan=\"3\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eDRPDFCLLPLDYGPCNAYMP RYFYNIFEK\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"58.14977973568282%\"\u003e\n \u003cp\u003eCys_PAM: 7, 16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"41.85022026431718%\"\u003e\n \u003cp\u003e3702.7218\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"58.14977973568282%\"\u003e\n \u003cp\u003eMSO: 20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"41.85022026431718%\"\u003e\n \u003cp\u003e3576.6425\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.169054441260744%\" rowspan=\"2\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.166189111747851%\" rowspan=\"2\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.166189111747851%\" rowspan=\"2\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.911174785100286%\"\u003e\n \u003cp\u003eCys_CAM: 32, 40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.61031518624642%\"\u003e\n \u003cp\u003e1893.8006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.455587392550143%\" rowspan=\"2\"\u003e\n \u003cp\u003e1779.7577\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.5214899713467%\" rowspan=\"2\"\u003e\n \u003cp\u003eCQTFVYGGCGGNANNFK\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"58.14977973568282%\" valign=\"top\"\u003e\n \u003cp\u003eCys_PAM: 32, 40\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"41.85022026431718%\" valign=\"top\"\u003e\n \u003cp\u003e1921.8320\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.169054441260744%\" rowspan=\"2\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.166189111747851%\" rowspan=\"2\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.166189111747851%\" rowspan=\"2\"\u003e\n \u003cp\u003e62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.911174785100286%\" valign=\"top\"\u003e\n \u003cp\u003eCys_CAM: 53, 57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.61031518624642%\" valign=\"top\"\u003e\n \u003cp\u003e1746.8374\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.455587392550143%\" rowspan=\"2\"\u003e\n \u003cp\u003e1632.7944\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.5214899713467%\" rowspan=\"2\"\u003e\n \u003cp\u003eTIEECRHTCVASRK\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"58.14977973568282%\" valign=\"top\"\u003e\n \u003cp\u003eCys_PAM: 53, 57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"41.85022026431718%\" valign=\"top\"\u003e\n \u003cp\u003e1774.8687\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.169054441260744%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.166189111747851%\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.166189111747851%\"\u003e\n \u003cp\u003e67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.911174785100286%\"\u003e\n \u003cp\u003e----------\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.61031518624642%\"\u003e\n \u003cp\u003e570.3358\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.455587392550143%\"\u003e\n \u003cp\u003e570.3358\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.5214899713467%\"\u003e\n \u003cp\u003eGIQPR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"7\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMw (average mass): 7746.89 / Mw (monoisotopic mass): 7741.66\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e: Alignment and sequence homology of Cerastokunitz with related proteins; using Blast in NCBI (only Kunitz-type peptides sharing more than 65% of identity to Cerastokunitz and with best hits are listed).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"662\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.809667673716014%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45015105740181%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecies\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.099697885196375%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMax score\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal score\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eQuery Cover\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eE value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.969788519637461%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eIdentity (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.83987915407855%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAccession Number\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.809667673716014%\" valign=\"top\"\u003e\n \u003cp\u003eKunitz/BPTI inhibitor-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45015105740181%\" valign=\"top\"\u003e\n \u003cp\u003eVipera ammodytes ammodytes\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.099697885196375%\" valign=\"top\"\u003e\n \u003cp\u003e102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e4e-26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.969788519637461%\" valign=\"top\"\u003e\n \u003cp\u003e70.15%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.83987915407855%\" valign=\"top\"\u003e\n \u003cp\u003eAMH40735.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.809667673716014%\" valign=\"top\"\u003e\n \u003cp\u003eKunitz/BPTI inhibitor-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45015105740181%\" valign=\"top\"\u003e\n \u003cp\u003eVipera ammodytes ammodytes\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.099697885196375%\" valign=\"top\"\u003e\n \u003cp\u003e102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e4e-26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.969788519637461%\" valign=\"top\"\u003e\n \u003cp\u003e70.15%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.83987915407855%\" valign=\"top\"\u003e\n \u003cp\u003eAMH40739.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.809667673716014%\" valign=\"top\"\u003e\n \u003cp\u003eKunitz-type serine protease inhibitor PIVL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45015105740181%\" valign=\"top\"\u003e\n \u003cp\u003eMacrovipera lebetina transmediterranea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.099697885196375%\" valign=\"top\"\u003e\n \u003cp\u003e102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e2e-26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.969788519637461%\" valign=\"top\"\u003e\n \u003cp\u003e68.66%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.83987915407855%\" valign=\"top\"\u003e\n \u003cp\u003eI2G9B4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.809667673716014%\" valign=\"top\"\u003eInhibitor,chymotrypsin\u003c/td\u003e\n \u003ctd width=\"21.45015105740181%\" valign=\"top\"\u003e\n \u003cp\u003eVipera ammodytes\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.099697885196375%\" valign=\"top\"\u003e\n \u003cp\u003e95.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e95.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e1e-23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.969788519637461%\" valign=\"top\"\u003e\n \u003cp\u003e68.66%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.83987915407855%\" valign=\"top\"\u003e\n \u003cp\u003e0909196A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.809667673716014%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eChain A, Trypsin Inhibitor\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45015105740181%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003ePseudocerastes persicus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.099697885196375%\" valign=\"top\"\u003e\n \u003cp\u003e91.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e91.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e92%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e3e-22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.969788519637461%\" valign=\"top\"\u003e\n \u003cp\u003e67.74%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.83987915407855%\" valign=\"top\"\u003e\n \u003cp\u003e6A5I_A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.809667673716014%\" valign=\"top\"\u003eKunitz/BPTI inhibitor-5\u003c/td\u003e\n \u003ctd width=\"21.45015105740181%\" valign=\"top\"\u003eVipera ammodytes ammodytes\u003c/td\u003e\n \u003ctd width=\"7.099697885196375%\" valign=\"top\"\u003e\n \u003cp\u003e94.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e94.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e3e-23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.969788519637461%\" valign=\"top\"\u003e\n \u003cp\u003e67.16%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.83987915407855%\" valign=\"top\"\u003e\n \u003cp\u003eAMH40737.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.809667673716014%\" valign=\"top\"\u003eKunitz/BPTI inhibitor-2\u003c/td\u003e\n \u003ctd width=\"21.45015105740181%\" valign=\"top\"\u003eVipera ammodytes ammodytes\u003c/td\u003e\n \u003ctd width=\"7.099697885196375%\" valign=\"top\"\u003e\n \u003cp\u003e93.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e93.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e8e-23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.969788519637461%\" valign=\"top\"\u003e\n \u003cp\u003e67.16%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.83987915407855%\" valign=\"top\"\u003e\n \u003cp\u003eAMH40734.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.809667673716014%\" valign=\"top\"\u003eKunitz-type serine protease inhibitor PPTI\u003c/td\u003e\n \u003ctd width=\"21.45015105740181%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003ePseudocerastes persicus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.099697885196375%\" valign=\"top\"\u003e\n \u003cp\u003e92.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e92.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e94%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e2e-22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.969788519637461%\" valign=\"top\"\u003e\n \u003cp\u003e66.67%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.83987915407855%\" valign=\"top\"\u003e\n \u003cp\u003eC0HLB2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.809667673716014%\" valign=\"top\"\u003eKunitz-type serine protease inhibitor ki-VN\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"21.45015105740181%\" valign=\"top\"\u003eVipera berus nikolskii\u003c/td\u003e\n \u003ctd width=\"7.099697885196375%\" valign=\"top\"\u003e\n \u003cp\u003e98.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e98.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e1e-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.969788519637461%\" valign=\"top\"\u003e\n \u003cp\u003e65.67%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.83987915407855%\" valign=\"top\"\u003e\n \u003cp\u003eE5AJX3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.809667673716014%\" valign=\"top\"\u003e\n \u003cp\u003eKunitz-type serine protease inhibitor 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45015105740181%\" valign=\"top\"\u003e\n \u003cp\u003eVipera ammodytes ammodytes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.099697885196375%\" valign=\"top\"\u003e\n \u003cp\u003e93.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e93.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e9e-23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.969788519637461%\" valign=\"top\"\u003e\n \u003cp\u003e65.67%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.83987915407855%\" valign=\"top\"\u003e\n \u003cp\u003eP00992.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.809667673716014%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eKunitz-type serine protease inhibitor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45015105740181%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eEristicophis macmahoni\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.099697885196375%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e82.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e82.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e86%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.610271903323262%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1e-18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.969788519637461%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e65.52%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.83987915407855%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eP24541.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u003c/strong\u003e: Physicochemical characterization of Cerastokunin sequence\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAiYAAAK1CAYAAAD8PCWrAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAAFiUAABYlAUlSJPAAAABhaVRYdFNuaXBNZXRhZGF0YQAAAAAAeyJjbGlwUG9pbnRzIjpbeyJ4IjowLCJ5IjowfSx7IngiOjU1MCwieSI6MH0seyJ4Ijo1NTAsInkiOjY5M30seyJ4IjowLCJ5Ijo2OTN9XX2/Hr7cAABwkElEQVR4Xu3dP6/U2J7v/31+8Y1vgBBCw3kMBOg0AQH7hDcgaDgTELW0RzoZapKW5lxpJ/uIZJJGIiIYsUdXPAC6AwIYEfAYYIQQ4mn0r96mPtVru5fr364/Lvv9kqyyXS6Xq2qV/fVa9vr+6beJI0mSpB74/6aPkiRJe2dgIkmSesPARJIk9YaBiSRJ6g0DE0mS1BsGJpIkqTcMTCRJUm/8oR+TP/3pT9MxSZKk7Wp3p1YNTNoLSZIkbVot5rApR5Ik9YaBibQF//Zv/9acCbSHP//5z9Mllvff//3fzWt5LLEu5v/1r3+dzllP1t/evqyfYROyvn/+85/TOZfD5872tYdtKb+r//qv/5rOlbRJBibSFvz8889HZ2dnzfjbt2+bqkoeP3782AQtm/Dhw4fp2OX85S9/abYL5fax/pOTk4017T5//nw6thmvXr1qtg9sN9vJcHx83MzbBr6r8/Pz6ZSkbTAwkXaEgxr+53/+p3lcFq/jgJvXb0sOuE+fPv1D7cwhIWCRdLgMTKQdycH+4cOHzSPS5JMmFIKWNBVkXqbz+rymXfOS5WhiKMdRrndeEwS1Ovjuu++ax6D5hdeyTeU40qTC9qS5hm3N/Pb7PXv2rJnPUAZpmZemnrye6ax7Ht6nfK+y2SXNXTyfeeU4ys+R+bXte/PmzXSOpK2YnIldUJklaQ1nZ2fN/ynD8fHx9Jlv8jx4jmmGk5OT2TxkuUnQ0AyMn5+fN0O53kyXz/EIxllPXt/28ePH2bK8f9abbcGNGzeaoT3Oa1meoRznvVgm25f3zjrL5xhnfl7PY8Z5rvyuStnWDPkMYJrXtcd5T6bb47XPkdfkfZDXlO8laT35X5WsMZG2bHJAPpocXI9++eWXC2f0r1+/bh45C+c5pu/du9c0pXD2XmuSePfuXfN49erVo++//74ZXyQ1LY8fP57VhGReDdfHZHt//fXX6dzFJgfvo3/5l39pxicH71nTU/tamOvXrzeP1IDwHLUSk0Cg+dy8L96/f9884u7du0c//vgje6/pnD/i9ZNAYTr1++e7du1a88h6830vwudo43vItpU1XpI2z8BE2oEc4O/fv988ljjgMhCIcGDPAZiAZZMmZ/+z91p0vcoqAcmmEBBk+8qgK4HMIrxm2WBtWZ8+fZqOSdoVAxNpBwg4ckaf6x3u3LnTPKYWhWsbuJ6CaYIUztDbNRu3bt1qHr98+XLh+oe2z58/T8d+v+iWazvAe8x7LdheApku1FBcFjUy1IbwXnxWakzA559Xo7OMfOZ8D2xvvu/Ssnc2sZ35zOV3K2kLJmcnF1RmSVpR17UPuT4h/7PJAfnCNNc0ZBkekedZFll3+Vqu30C5bB7B++e5XDcRufaj9hzvFTyX5bJ+1lt+pnKZcpzleB/mtT8fym3Ie5brnQQFzbxS+TxDvoMo11m+V/ld5HOU28VQ/n5570y3v1tJ6+O/1GaX9JIkaS9qMYdNOZIkqTcMTCRJUm8YmEiSpN4wMJEkSb1hYCJJknrDwESSJPWGgYkkSeoNAxNJktQbBiaSJKk3DEwkSVJvGJhIkqTeMDCRJEm9YWAiSZJ6w8BEkiT1hoGJJEnqjT/9NjEdb/zpT3+ajkmSJG1XKwypBybthSRYNtTFsqEulg3NUysfNuVIkqTeWDsw+ec//3n05z//eTol/a6rbDDPMjNu2ygb//Vf/3X0b//2b9MpHRrKBGfNDP/zP/8znasxWzsw+fTpU7Mj+e///u/pHOmbrrLx66+/Tsc0VtsoG99///3Rzz//PJ3SIUkgQlX+2dnZ0ZMnT5ppjdtagQmF6fr160d37tw5evfu3XSuZNlQN8uGan788cfm8datW035kNYKTN6/f98Uonv37h09e/ZsOldarWxw5mwV7nhcpmzQVMM4NS3l/EzTHECTTsb/+te/NuPMi7zOZp/++Jd/+Zfp2NHRixcvZkGKxm2twOTz589Hf/nLX5pCZXOOSsuWDQ4Yd+/enVXhvnz5cvqMhuoyZYOmmhs3bhx99913zfyTk5Mm0GF9LAOadJj/+PHjo59++uno/Pz86M2bN81zBCofP35sXkuzkfus/iDAJGB8+vRpE1RKawUm/PFz9vHLL79YLauZZctGrgvggMFrNHybKBtv376djn0LdGoIVAhYkJo43o/AhvcmQHGf1R8EqglCrYEXVg5MONNg50BBYuBPbmESVikbLMtBgjPbnPFquPpQNvLeDDYZ9I+/iWLlwIR2wJyNwOYcxSplgzPW8sxWw7bvsnF8fDxrJqAWxSaD/qEs0IQncfZwQWXWzI0bN5rnT05OpnN+++38/LyZxzDZmUznaoguWzYyPjlI/DY5e55N57Usr8PE79dlE2WjHMrl//GPf8zG//73v8/Gy2UY//jx42yaQbsz7/tu//Yan1r5sEt6Lc2yoS6WDXWxbGieWvlY6+JXSZKkbTC7sCRJ2pt2jYlNOVqaZUNdLBvqYtnQPLXyYVOOJEnqjZUCE27vI7opB2+7UxtlgrIitW2ibLDfWYTO2ezC4HCUxxZppcDkw4cPTX8A6SiJR3pmdAegkpmn1WUTZWOZZoFXr17ZR86BIAUBaQL4XW3yES7VlMMf/8aNG0dfvnyZztHY0XmVGWRVY9lQDT38chzxREZxqcCESJeupW/evDmdo7HryiDLTodq2mSJTYbXVN9mKLPBalg2UTaobUlTEPMYZz7jNN8g6yEQYh7jeQ+GyOvyvPaD2hKOIyRo9P+vxm8tlVkX0DsfyzDQK6PGY1HZAD05RnrxjPTyGPQEmuV5LF+rw7KrskEPrtnvpIfY9Chbvp5lWBbMz2vyvjzHOJh2X7Y9y5QNlL+txqNWPtaqMZn8kZvBvAZqW5RBdnIQmY4dHd2+fXs6dnR07dq15voDDdc2ysbkQNZkIo5kE27jrDxoeqb2hm1gWzhT54y967XajeRPktZuysmFZVa9KagOJ2CdBLzNwM5+XuZp0tvzPAeH+/fvXzjAaFj6WDYIhLI9DBwYtV/+BsKlrjFhZ8FOQ8Kqmae5dfT58+ezA4OGq29lg+vinj59Oqsl4f2sMdkvTnLLmjKN2ORPf0Fl1gztfzzPkEywZZZQDdsyZSPt/SjLBvMzXis7GXSY5v12mywbGS+vdeP1eQ8ey+cyzvzyfbieIde1MJTbps3i++3SLgcan1r5sEt6LW3TZYMzJKrsI2fP9j9xeCwb6uIxRfPUyoeBiZa26bLRXh+3eXqdyWGybKiLxxTNUysfl7rGRLqMs7OzplBmsH1ZYdmQxqtaYyJJkrQL7RoTm3K0NMuGulg21MWyoXlq5cOmHEmS1BtLBybc409kw1B2qsbV8pmfHBcat02kttcw9bVssE32Y7I/fP85jkhLByZ0iER1y8nJSdOpWv7E3L5HL45crOZV88ImUttrmPpaNj58+GCvo3vCiS4pAzi+2OQjrNWUQxBinhzVELCa2l41lg3V/PTTT03OI09kFGsFJj/++GNz1kO1rFTqSm0PzozSFEizX9LTl82BVqcP1zplg31Mu2zkuSyf15avybzMj7/+9a+z+Xku88rnyzIZ5To9iG4OtSXUupNMke9YoursgsqsC9JtMF06sywpw9O9s4ZtUdlAWQ6SYj6SZp4ylHKUrsTBvHRJrsOyrbLRns++hvng9eXzmQ/GWRbZNspWli9fi3L7Gc+6WCb7uPK15XtpvmXKBsrfVuNRKx9r35VDe+zkj95EuVLMS21PmWH+o0ePLlyPNNnRT8eOjj5//jwd09CsUzao5gfzObNmOa4HoVakve/54YcfpmPfrhn5+vVrs84gcV9cuXKlWaYL7xVfvnxpanvYZtbH+04OotbubRi/LTXx0tqBCchlMTlTuVBVqvGiepsgYxLwNgM777LKngMO87k+yTIzLpssGwk2yoC2jSaZ09PTZp3BgS8B0OTMvMlevAr2ddl+BtalzfI7FS4VmIAdyrwzD43HotT2HCw4y6S8lGekGr51ywa1FXj58mVTI8Lyx8fHzXVu81C78erVq+nUN7yWi28TWKySEJDalqdPn85qSaixscZks7i+xNQDakz+oBdUZs3QxsrzDLQHBm2uXmMyfPPKBm3DPE9bf5QpzSkfuQ6gnC6fzzjlSYeF363LOmUDTGcej5HX5blySNkp91VZD/usclkGli/fI69jvCyfvLYso+Vn0Xx8X13KcuB3Ok618mGX9FqaZUNdtlE2qFGhuWWVmo15qDHh2pKyuYCaj0W1L7oc9xuap1Y+Lt2UI0mHgGtOuCA2aDrg9mVJ/WJ2YUmStDftGhObcrQ0y4a6WDbUxbKheWrlw6YcSZLUGysFJlyMRnTTHnLLnwQuKLSjJNVYNtRGmcixxFuwhZUCE/oYoA8BOjai6oXh5OTEHAe6wOzC6rKrssEBjgPePDzvgXC/8v1zLDk7Ozt68uRJM61x20gHawQn6Tpa48aOxgyyqtll2VjmAEcX+dq/3K7NHVKUD2kj15iQx4Iupj370KYyyGp4dpVdmIFeWgk86FEWmc+ANCfduHGjeT3yPOvRbpR9ytA7sH3KCBu9+LXsI0DjRBI+OsRqdzkOOsuiyvbNmzfNNLVtHBhev349axa0Kne4NlE2CE7osp55NCnzuuTsyvwsT9MA3dIT3DCe+QQiSaPBCRWvJ4BhnGVYj82Qu8NvSkBIMLmo+U0jMfkjXlCZdQFdNre7DE9XzzxquBaVDbBMOaR7cUwOCs28spzQ9XfKE/OZ1uHhd12kLBcMlykbvJbl2R+B17fXV06D1/MaukFH+X6Ml0P7tVof3+cy+M79/49PrXxspMaEM5lJgbpQLafx4SyzvDB6stM3u7AamywbaY5hfctIUw81IdSYdMm2MdiksHt+54pLBybsRKiCWzWFuIZnUxlkNTy7zi5conno/Px87okT60wzAtthk8Lu8dsSmEqcHVxQmTWTqtD2UFa/arj4rbukbFCFHpfJIKvDwu/XZdNlI6/Lc+WQpp+sn6ae8r3yGpbjOcZ5nn1YlmHQ5sz7Pvmt852nWU7jUisfdkmvpW2jbHDWvMkMstoPy4a6eEzRPLXysdG7ciRJki7D7MKSJGlv2jUmNuVoaZYNdbFsqItlQ/PUyodNOZIkqTfWCkzKbqIZuL1OQlk2uAWUWwBzSygXMzJonPpUNvL+2j9+i5QLCSsHJvRbQsdIVL0w0FHSjRs3/JOrOfCQnyRlg8SOZJ5OXxRl/xQal02VjU11zEdX9d7ts3+UC8oCZYLO7whSpJUCE2pG6Eyt3InQadHZ2dnRw4cPp3M0RpQNDjxlb5zs+JftnVPDtamywcmPwe2w0FFeAkSSwf7yyy/NuMZtpcCE3hepHWn3oEi2UGpObNIZr5SN9lko0yRJa6PanqpbDjY82sQzXOuWDVBDwjjdylPDwn6GafY1eS7Pp+t5zsLTPMByGWd+uc7yuZRDhsj68ry2i+MK5UTa6MWvZhdWqX3gKOXM9/T0tKnGhYHteCxTNpAsw1evXm1qWBhPeWE5xplfZhmmdobmAWpyWZ5xliFTMbJO0KQDanxZF13TE4RQFlln1m+N8PbxvZuSQthoYHLlypXpmMaIs9kSBwAODgy1M2PkwAAD2+Fap2zMw9k1eXWoBaEmpcQ6qY25du1aE6ikpoblu5TBENe9vH//vmlWIHBKTY2B83aRT8lEfsJKgcmtW7eqf1D+xJyBzEuSpWGjbKB99ittq2wQNGDVa1WWRVBDjUkG92/bQ4D54MGD6ZTGbqXAhDMPqjrLDJAEKffv32+q5DVelA125JQF2+NVWrVslAEA+5d2bQtYD/uibZ1h37x5s7nQPydhHDitMdmOBKyUE77jXAukEZucCVxQmfUHk51Ms1wGsnNq+JYpG2UmV4YblaywZBFlfsYZ8pwO0zK/3bJlAykTLJOygiw3CVZm43m+HM6KrLXl/orxsryV71Mux/rb69B6+P66tI8lDHz3Go9a+bhUl/SpSl12eR22VcqGxsWyoS6WDc1TKx+XuviVlVHNyooZrMKXJEmXYRI/Lc2yoS6WDXWxbGieWvmoBiaSJEm7sFRg0l5IgmVDXSwb6mLZ0Dy18rHRDtYkSZIuY6XAJDks2oOdainogyDlgjwkXBCdi6IpP+bEGS/LhmrKcmFfMcJKgQldOh8fHzc9LVL1wrCtXhd1eNjBbCK1vYZnU2WDHDvbwjZ6YNytfN+UCVIJPHnypJnWuF26KYfe+tbJdaFhYQezidT2Gp5NlQ1qV7YZ3LKN2r303kvqguvXrzfjGrdLBSbsKGzGETaR2j7V+hqWTZQN9jNJppcq/3aGYgbGqfmgqSjLZZz5kdcxIM1IbGf2aXl+m7U0Y1emHzCJn2KtwIQdBH9YHqUu7QNHqTzzLdPQaxxWLRtXr15talgYp9ofLMc4858/f94EOScnJ03NB01FNA2wPOMs8/r16+Z1BCg8x2tZnvdP5mECH9ZDMMM4y/A+Bs3bQ/BIOSA3URk8arzWCkz4k2eHIAU78tJlU9trODZdNjjTJpjgQNY+QWKd1MZcu3atCTxSU5Pgg7NyBmpJOBjW/PLLL01QwwGTbX/37t30GW0avyXHE363Z8+eTedqzC7VlNNVFavx2VZqex2+bZUNggaseoLEdvBaakIIXLpwsMxgE8P2+R0rLn3xK7zNTwSp7OQ3mdpew7CNssF6uENwnYPZmzdvjs7Pzy+8TxvrTrMC22ATw/bxm969e3c6pVGbnA1cUJk1c6OSXpzh7OxsuoSGjN96kU2kttfhWea320TZyHKTYGU2nufLgX1SxidB0YXxcjvy2rdv387ek+fL9TNoffO+v/J3ym+vcamVD7uk19IsG+pi2VAXy4bmqZWPjTTlSJIkbYLZhSVJ0t60a0xsytHSLBvqYtlQF8uG5qmVD5tyJElSb6wVmHDrHFEOQztLqMZtXtngtnJvLR+vbZUN+iWx2/jDl9QB9oUkqlAuqMy6ILd3BbfZMc0tdhq2y5YNbsEsbxHVcFg21GVR2QDLcCu3xqdWPlaqMaGjITPIqsayoS6WDc1DTckkcG3SFEhYKTBZNUuoxmPVspEq/fSoaTPPcG2ybJTJ/9IUlGXT1TzjtWaBvM5mn/4gaCWHEQkW/W0Ul774dV6WUI3bvLLxW1ODd3R079695vH09HSWZE3Dt07ZyHI8zxk2afIJbhgHQQ5d3ye78Pn5edP9PAhU6Nqe15otuD/ev3/fBJ38Xvw2JFX0t9HKgQl/7pIZZBWrlg3mczaNz58/N48apk2UDYKMBw8eNOPkyOmq+ue1qZ3hjBxmC+4nfts7d+7Mfi8CS38brRSYmEFWXdYpG5wRk+acM6S8XsOzybLx5cuX6djqOCPPYCbbfrh27drRp0+fplNHR9evX2/madxWCkyIaolol80SqvFYp2yQ3ZVq3FTJa5g2VTZu37599Pz582YcuQZlGcdmC+4lastovknNFsHozZs3m3GN2OTs4YLKrD/gFj+Wy+BtfuNw2bKReWUWUZafHLSmUzpU/K6LbKJsJAMwA8+X6/z73/8+Gz8rstYy/tFswXuz6Pt+O711nIHfU+NSKx92Sa+lbaNs5AzaGpPDZtlQF48pmqdWPi59V450GVzo5oFHNZYNaZzMLixJkvamXWNiU46WZtlQF8uGulg2NE+tfNiUI0mSemOlwITb94huGNInQTnPW/DGa9mywbjGZRtlI9mJS6y7fA8e7eL8MPA7+Vtp5reWyqwLuF2P27tKzPM2r+GzbKjLLsrGMreVs4zlrV8WlQ1+M5ax24BxqpUPm3Ik9Z61scOV9ARSGJhoZ6i+Zwiq3amStwpX88oGTTIk5qOHUJZhHs+nt1AemS5fTzMP8who0sTDuFmHpf5bKzD57rvvZn9mBhJkSZhXNsjqGhwYzs/Pm6yiHHA0fOuWDbot54z65OSkyTLMGTYJ+eLu3btNYj7WkbJE/yc5C+f1vNasw9JhWCswefv2bfNHznB8fDx9RmO3bNm4evVqkzuFTrRYTsO3jbJBIEFNCbl1GAhAuhCopMM2sw5L/WVTjvaCA0QOOhwUpNh12UigxGDWYWn/DEy0F1TX0+bPgYCzXKvQFauUjStXrjS1HlmmbBJaBrU2ubDWrMNST0zOEi6ozJohGyjPM+SWvHIeWTw1XJctGxnPbaI8Ms1yOmz8jl02UTa41TjTeY4BLJ/pPPcf//Efs3lmHd6vRd9r+ZtMAtHpXI1FrXzYJb2WZtlQF8uGulg2NE+tfNiUI0mSesPARJIk9Ua1KUeSJGkX2k05XmOipVk21MWyoS6WDc1TKx825UiSpN5YKTBJngkG80qo1C4b9AfBuP2T6DL7jXYOncuir5JNrk+Xl30FQ3rk1bit3JTDjuX69ev2kDhCq5YNDkjkJkk34Bqube03OFCRC4ccOTpM88oGv+/Lly+bckGA8unTpyYXksajVj5sypEk7U2C1Vu3bjXBq2Rgop2gSYfImKFMOa9xohxQo1Y27ZRNPl1V+iyfZcppyhflivF0K1+WubwP47wP6894mhJq22Kzz3aReDFevHhhTbwaBibaiYcPHzbVdWRwJXOsxosA4vz8vGnme/r0aTOPoCBlhOdu3LjRzC/xOs6oWebs7Kx5DdX+Wfb777+/kF34u+++m5W5vA8ZjsEBkfchzw5YJnl2CFSyLTQjJdDRdiRI5DfyuxYMTLR1nLmy42fnk4OIF8WO19WrV5vg9N27d83BHwQFN2/ebMYJMCgn7TLy5s2bo8ePHzfliMd5Cft4bYIUgpC8T9vx8fHsLJ0yitevXzfbl4Ml09qe/D4Em8+ePZvO1ZgZmGgnOACw88ngBbHjxW+fQIGDf01XEwq1HClDiy6I7WoOWgY1K3mfV69eTedqm2zGURiYaOs4EJWp6amSt8ZkvPj9qbLnQEStBmWBJpPyGg+Cjnbwevv27ab5J7J8GcSkFiVlLsEJ77lsoHLnzp2j09PT6dTv76PtSjmQOCO4oDJrpkw3bnrq8VmlbDBk+uPHj39ITa9h4Xft0i4blIXMu3HjxnSp35rxLEeZQaZTZsp1sR6UafPz/Nu3b/9Q5lhnpv/2t7/NxtuvR7ktrEvr4zvsUvvuNS618mGX9FqaZUNdLBvqYtnQPLXyYVOOJEnqDbMLS5KkvWnXmNiUo6VZNtTFsqEulg3NUysfNuVIkqTeWCkw4bY8ohsGbr+TwrKhLrsoG3Qh7y3ohykpARgu0/eMhmOlwIS+BY6Pj5vOh+idUQrLhrrsomzQCZqd9h2eBCJU5dPz65MnT5ppjZtNOZKkvTG7sNoMTLQTVLOX1fmZTq+ajHd1Q65hW7VslE1DYDnGOfvOeLnOsvfXzON57Z/ZhVVjYKKdaGcXptqdqtugml/jtErZIPCgu3iWp3mIYKPMMJzxZBamy/v37983wcnz58+beayP91Q/8NsQLJpdWGFgoq3j7JSDDjufHEA8YxVWLRvkwsl1KlxX0nXNShnMfP78uQlOyJ3D+xC08J6pSdF+mV1YbQYm2gnObtn5ZPBCRcWqZWPdgILak/J9ymYE7Z/NOIpLBybsJNIWLJVSNjjQ1LILX7t2rZnGu3fvpmMag3XLBtlnX758OZ36dqvpMm7evNk0FSSo4XXrBjjaDn53swurMTlzuKAya6bMuFkOb82+OQr81l0WlY1aduGPRbZX5vFI9lkdHn67LpsuG5nPwHJ5PstkvMxcy3uV05az3eH77lL+JvntNS618mGX9FqaZUNdLBvqYtnQPLXy4TUmkiSpN8wuLEmS9qZdY2JTjpZm2VAXy4a6WDY0T6182JQjSZJ6Y6XApJ0ltOzi2VuGx20XGWR1mDZZNswiPEztNAMat5Wbctgx/PTTT7NOkJi+c+eOneOMwKplQ+Nh2VCXRWWDYJV+Zuzwbpxq5eNSTTlEuexsDEokSevgGEI6AmvCFGsFJvTESJRD3grPgLQIZ8uUFx5Bsx/T9LyZ5zhrcsc0PquUjfK5jDOfx8xHOc9mxf7jOELuInIY+XsJawUmjx8/bh6/fv3aPErzkGzt+Ph4ltH1wYMHzY4ISeBFVlmNzyplY5kswmhnK1a/8Tsz8HtReyKtFZiQufP8/LzZKXiWq2WwwyHtPKhxy86I/CWcGXMgsfZtnC5TNtgXBVmE2R9xgKO2JEGM+6jDwG/O5QHS2teYkG48wYnJsLQIB5YPHz78oaxw0OHAxIHEA8g4bbpsUAPDazMY8B4OghPpUhe/EpxQheqFS6rJtQDxww8/NNP37t1rpnmeM2IOHAS5Zhgej22VDZavZStW//Fb3b59ezqlUZucUVxQmTUzCUCa5xnICErGzkwzkClSw8Vv3KUsG+WQDLL4+PFjs1wwXWaGZVqHid+vy6bKRjk9L4sw+6ZM8xrtF79Dl/K3MuPzONXKh13Sa2mbKBv//Oc/vb18gCwb6uIxRfPUyselmnKkVVBVm6p6qWTZkBQGJto6DjpExW/evPHiNl1g2ZDUVm3KkSRJ2oV2U47XmGhplg11sWyoi2VD89TKh005kiSpN1YKTJKamnbhmuS2KPsn0DhYNtRlk2WDZVi21jcJ61+3fNFvir2O7g+/m/sGzfzWUpl1Af0C1PoGoJ8BXmtfJsO17bJhPwaHa5dlg/5Oyj5Q1G+Lykb6pPH/P0618rFyU86dO3eax/YZy8uXL5teYDVelykbvIYsoxomy4a6kJBxEphOp6Q1rzEhe+eLFy+mU9Lvli0bqd6nGp9qdHIuTc6em3lMa3g2XTaYzjyCF8bppC2yHgbk1mSWSfNRmpcYz/tlnOUYL5sY8jqbfaTtWSswIUdOeQbDTuHWrVvTKY3ZMmWDHT3ZZH9ravGOmnT1b9++bXIuMc/+LIZpk2WDYIVpaltYhhw55Vk3AUTWw3wCieT2evz4cZMckBw89J8C3gOsn/nk2wEBUbaZQIXginXevXv3QhAkaXPWviuHpFv5Y3IWZAZPxaKywY6eAwtnnhwAcnDQ8G2qbCSQwOfPn6djv+O1WTfd3BNgpLaFQCXPlTUwpePj41n3+LwWr1+/Prp//36zbU+fPm2mJW3e2oEJ3Uc/e/as+WNfv359Oldarmyws+fMk4E2Zo3DvsoGNS6bQECUbXv16tV0rqRNWjswocqT6kwGc1yotKhsMP/JkyfTqW/V9xqHXZUNajzyWoIgmnLSDLQuLuA9PT2dTllupa2ZRP4XVGbNcJsez/OIydnD7BZAHnmO4cTbvgZpk2Uj0wyTM+QL8zKtw8Hv1mWTZeNvf/vbbJxbjDP+j3/8YzZOKv3yNQwoU+yXr338+PFsvGv92d58FgY+hxbju5qn/J49doxPrXzYJb2WZtlQF8uGulg2NE+tfKzdlCNJkrRpZheWJEl7064xsSlHS7NsqItlQ10sG5qnVj5sypEkSb2xUmBSdvFcDvTgqHGrlQ26+05X4d5aOV77Khusl/V3daIGnqeX2HXQUZy9v25GWUaklQKTDx8+NP0DlJ0M0X2z1C4blAt6yQQ9bWq89lU26JztxoKO1dimddEzbHqH1foIUunxN8cU6dJNOeSfsDt6tVEuOCi8e/duOkf6xrKhEnmLKA/WvCsuFZhYPa8u7GToWrxM0paq9bL6O1W461an6/CsWzYYZ16yBDOfZhrGGdqZgtuYl2VrB8FyXbwP8po0OzEg28Aj25zxbHPWX67TZp86aksoD+RIym+okfutpTLrgrKnxkXLalhWLRv06Ij07EjPm/SWyXKgl0emwTye12HaRdlgmmXLHmJB77AZR1mO6KmV58t183zW0X7PvJZ5eX/WzXpQzm8vz3K8F58pny+vQ54fm/K3mYfvpvy+NA618rFWjcnkj8mamhTiUillg6Fsf6esUIUPrjmAWYbHZRNlgyr/MrEfNRLkwGEdOduuZRumuZmke9TM5PqWNtbLduQ9S2xPfPnyZTp20SRImeXj+fTpU7Ntk4PtrMYE79+/bx71R3x3qanSuF2qKcessLosdtw5WFmeVFqlbDx48ODo+fPnTXBSSw6YJpXbt293XrCfJhne7/j4eDr3cgik8hkYEoCp7rKJFjUMl774VVqXWYbVZdWyQY0ItS3UltQObtRUlDUzNa9fv77UXTpt2Y7U5HDdiddQdOO7IXCUiOIvqMyaof2P5xnStqrxWKds0B6f+bmegGFykGiezzTDGNvfh4Lfr8umykb5fNbJY5TXdiDXfbRfn9eWWYVZZ7kN5frzyDJ5vv3ajJfr4LOWn7Hc1jHhs3fhO8r3k99d41IrH3ZJr6VZNtSlD2WDM+6bN2/aHNAz7jc0T6182JQjaRC6mnEkHRazC0uSpL1p15jYlKOlWTbUxbKhLpYNzVMrHzblSJKk3lgpMEl3y+Xg7W+CZUNdNlE2WEfZ+RZdxde6lddhKsuItFJg0pUl1B2ELBvqsomyUfa8CnpxNXnoMBCk8vtSNtpV+hqnSzXl0FnRDbOEqsKyoS6WDZXMLqy2SwUmFCS6jS6zhEpI2fhf/+t/NdWzyR6bHjzLTK90F67xuGzZyPI8l3HW2X5NOc9mxf6itoTyYHZhzfzWUpl1QdmbIkPZ06KGbd2ykd4wgx4e6fER6flRh23Rb3jZskHPrWXPqYwzL+NZrnx9lme58j20W8t+9+3fWONQKx9r1ZikrZihzBIqdZWNyQFjOvbtDIkeOpFqfc5uNWzbLBusO+hoLbUy1JawDljG+o3O8coLnDVel2rKkTbBnZG6XKZscMFtAiEGL5btP3vuFQxMtHNkjs31BOCuDQ8awqbKBq/55ZdfZrUkXLtgjUm/8RuZXViNyZnEBZVZM7T/8TxD2nE1HuuUjbdFdtVyfrl8rhXQ4eJ37LKJspFprlUpr1fhmpKM53oVBtbNOjPNa7QffP9dyt+I31LjUysfdkmvpVk21MWyoS6WDc1TKx825UiSpN4wMJEkSb1RbcqRJEnahXZTjteYaGmWDXWxbKiLZUPz1MqHTTmSJKk3Vg5M/vnPfzYRDgM5KZiWYNlQl8uWDfLn2A/JcNF3Tdl/jcZtpcCEHcrjx4+baheGG9OuniXLhrpsomy8evXKTvgGioDk6dOn0ylpxcDk69evF3Yq5KKQYNlQF8uG5vn555+Pzs7OplPSioFJzliojgV5DUziJywqG+Q84Tmq5MtpzpbyGg3TZctGHql5yTjNOjxmPsp5ps+XDtfK15iQu4KzH/782ZFI6CobHEyeP3/eVOODgwZZZEFujMzXcF2mbHBGnRqXjH/33XfNc2Qmfv/+ffPcw4cPm3nUyNy/f7+ZJ+nwrByYgJ0MOwCSZLFjkaJWNjjQcCDhoMT8N2/eNPNBanuNwybLxtu3b6djR0efP39uaksISFhPghgvlpUO00qBCX/0soqUHUHObjRui8oG0xyUGDjr1XjsqmwcHx/P1sPgxbLSYVq5xqSsIqUKlfZgCV1lg1T2T548acZhLdv4bLtsEIRQ45JaEgIha0ykAzU5s7igMmumnUqcYXK2M31WQ3eZstGef2Oa2t509MPAb9nlsmWDxzxPavyMn52dzcbb72G56g9+j3nK35HfV+NSKx92Sa+lWTbUxbKhLpYNzVMrH2td/CpJkrQNZheWJEl7064xsSlHS7NsqItlQ10sG5qnVj5sypEkSb2xUmDCrXxEN+3BW4Y1r2zkOW/fHKc+lQ26r3d/1S/tzNPSyjUmH6edIdG7Ysb9owtdZaPsUlzj1JeyQZ4eeqBVPyQQoTyQyK/s00bjtVJgwk6EP3YbKck1bpYNdbFsaJ4kdLx169bR9evXm3GNm9eYaKfKKttU49vUIywqGwxZpuzinpqXzAfPMU4TAQkDy+UZZx7vkfE0JfBekddZG7xdZcD64sULs9WrYWCinWlnhC2r8ZlvbpPxWqZs1LIHE0AkOzFNAQQSJP9jPY8fPz766aefjs7Pz2fJAZP8jwMi8+nGHqwz+XsIVPJedJnPtLYnQeLTp0/9rtUwMNHO5KAAMsLGgwcPpmMaq0Vlg9oSggcOYAlYmEdgkYCWs22WyXULBCp5LvPaSPyXs3Rei9evXzeBTw6WTGt7CBITWD579mw6V2NmYCLpIBBEcADLUKth29SFtARKeR+vhdkNm3EUBiaSeo8gpJY9mGAl14ZQK0JTTu1C21XcuXPn6PT0dDplNuxd4fek6UzijOCCyqw/YJkMZPXUOKxbNroywv6f//N/ZuM6bMv8hvmtGdYpG13ZgzMvy5bLlet8/PjxbPxvf/vbbLxcJuu9Mc1wzOA+7nL4DrvUvnuNS6182CW9lmbZUBfLhrpYNjRPrXzYlCNJknrD7MKSJGlv2jUmNuVoaZYNdbFsqItlQ/PUyodNOZIkqTdWCkzSdTMDPS4ivfYxeFvdeKXr8PZgl97aR9lg/5Rbi9V/lIWUC2mlwIQOcKhyoROjdDpEnwFnZ2dNr4l0I63xogykfGTcwETYddlg/1TrgE39Q580pAOgTDBIazXl0PlQ2akRLtupkQ6bGWTVxbKhechnRMBqDZdircCEJFlEuAQlL1++tCthzZVMrWn+S9X+v/7rvzaPmXbHND6rlI2y2TjLI+tIb7B5DcvnOebntZEsxHle+8GxhFo0EjkmC7TGbe2LX6k1ofvga9euTedIdZwZ03U4GVtBYjZ2RP/5n/85y21CFa5V7+OzStlguqzuJwAh+KA2hnnJOFxmJk6tTLIF814JcpKVmLw4eX/tHr8fA787tSfS2oEJtSYUJB6lRdjhcCDAu3fvLlTtm1143JYpGwQTBBXBazjTZlkyAFMzQpDRFdyybHz58uXo/fv3Te4daks4U2dfRrCi/eG39Jo0Ye3ARFoFB4wPHz6489cfrFM2rly5Mh37VqNCoLJqk8zJyUnz2gxlQKT98DcQDEy0Mz/88ENT/X7v3r3pHOmbRWWD4IUajlyDQI0HryGYobaE58/Pz5sal2XcvHmzqWlJMERXCAbN+8Vve/v27emURm1ypnBBZdYffPz4sVkuA9MavmXKRlku2llZKSc3btyYTl3MLKvDtsxvmN+aYZ2ywWsyL5loeR3jmd+ezjjrLtfJcmVmW57TdvD9dikzQfsbjFOtfNglvZa2ibLBmal3cQ2PZUNdPKZonlr5sClHO0NVrc04qrFsSAoDE20dBx2i4jdv3nhxmy6wbEhqqzblSJIk7UK7KcdrTLQ0y4a6WDbUxbKheWrlw6YcSZLUGysFJslBwUDbcGQeV9VrnNYtG3mdfUgM16bKBq9lnoYl+YwYpMZvLZVZF9AvAPf/l9J3gIZtW2WDPiZYTofLsqEu88oGZSJ92tCPiceR8amVj5Wbcuj6+fXr19Opb75+/drM17hZNtTFsqEa+q1JfqNHjx41vftKKwcmtbwWL168mBUujdeislGmnqf6VuNx2bKR52n2yTjNOmnuKZuDsh6bfQ4Lt4vfmGaF1ritdfErOSpevnw5nZJ+N69snJ6eNinmf/vtNxP6jdBlygYBzNnZ2YVxct2QW4XXpTaGoObjx4/NesgovEpSP+0XvxVlRForMKGHxmfPnjXjFCbT1ivmlY1Xr141WWE5m+XgoXHZdNk4OTk5+v7775txghnQFMBZd9azbFI/7R81aKYkENYKTKhy+/Of/9zsXGzGUWle2aBq/e7du83ZrFW247OrssE6MnigOww0xZWBqsZtrcAEDx8+bHYuUltX2aBqnUHjte2ycXx8PLvehOagrluR1R+5hZxA1d9MjclZxQWVWZ1YlrTVGofLlo0y7Ty3gfLI7YGZp8O1yu/HsquWjTI9/j/+8Y/ZOLebZpx1cGtxphm0f/N+h/J3z8BvqPGolQ+7pNfSLBvqYtlQF8uG5qmVj7WbciRJkjbN7MKSJGlv2jUmNuVoaZYNdbFsqItlQ/PUyodNOZIkqTdWCkzKLJDc1pVx+iaIzCuziGr40jV4eyjLhsapr2WDfRTb4e2p+1UeS9o9/mqcVgpM6J2Rzo/oAprOknhE2f8AvS3SXXR6ZNR48NtTJUcZybiBibDNskGAQadtq2IfRe+x2i861qNMMDAurdyUQ8H58uVLM06HOOxoyBIajNP1tMbl559/boLVNoJZjdu2y4YZig8XASUBKuWDgePLOkGmhmXlwIRug9+8eTOd+paYq8xHwXhtJ6TxSlV+HtkRpQrdKtxxa5eNDMkwnPn/+q//2jy2y0yS9n333XezJpmULwaUTTaZ325qTjN15uc1DGxDmhtYLuthvi6H48WdO3emU0dH169fN7+RVg9MqCUhq2eQ1TOJuXDt2rXpmPQNZ8zUrIEqfBKuMU3ZYZrq9CdPnjTPa1zaZYOBbuXpuh6cCBF4/Od//me1zJTNy+TFIXB4/vx5swxNygQpabJ5/PhxM//8/PxCLQvzmWZ+TrqYTpMT+ztOtnieJIHgOdMrSNuxcmACdhxUt3EWkR0I48y7evVqMy21tZN05eDw6NEjd/IjV5YNygXBBdo1sIvKDIFDkgMSqBBApDaOQAW5/q2cn9dkHsEzzdLUjJTY9yUxIOuWtHlrBSZUvbHDePny5dHNmzebdkHGmZc/uCStg30IgUGChMtIbcyqqHk5PT1taky0Pbdu3WpqwYJx5mnc1gpMuLg1zTeczXC2UxYuaRm5iJqglmuVpKA8cA1H+0L6RWWGGo1c+0Fgkwsr8enTp+axvOCyCzUvXri9fWUQysC4J7fijOCCyqyqyZlIk9kzeF07Y6iGZZmywTIZ3r5928wrM4gG5YeBeTzqsJW/bZeUAYZ5ZQMfP378Q7noKjNZR/ZHWV+5TpbJaxlYf5mxmNeW42Vm4/J1eT7jLKf5+J7moSzk++R30bjUyodd0mtpmywbnLFyHYFnR8Owjf0Gd7/keg5cpsxQi8IdH+X6tBseUzRPrXys1ZQjSdvE7br2hySNk9mFJUnS3rRrTGzK0dIsG+pi2VAXy4bmqZUPm3IkSVJvrBWYlF07M86wiT4HdPgsG+qyq7LB9Sl2F3942mkBNF4rByb84enDhKqXVL/QpXOZyE/jZNlQl02WjUVBBz270tW9DgcBCf3KUDbMTK+VrjHh7ObGjW9py8vOiYhw6YreWz+HzbKhLrsqG9Sy0FGagcfhmFc2QE0JvYl7K/c41crHSjUm9LbIDqbdYyIRrgeecVtUNih8DBxYQJ8UDGA+Oyer34dpU2WDQIZaFpLq5Xnm5/WgV9esi+UzXmsmyOssd/tD0Epvr/Qc7m+hWLkpJzsEqW1e2UhEnL4pyEPCDokDRbK9llmrNSybKBsEMmdnZ02m4DxPp2m8nvkc1Ah0GAfLs2wtezCBCjU4vJZkgAQ02r337983ZYPfh9+C39nfQt6Vo53hgMHZMz5//tw8UpV///79JgFkDlAan3XKBkEGQQdn2jx2Zahm3anRzcW25MKhFofXEqDwHto9fmuacfL7EEj6W2ilwISsj/yh8+cOItxUw2qclikbSf7IvGQQZYeUgw4HCQ3PNssGtSAsw0AtyiryOgavb9iPa9euzZIrghow5mncVgpM2FEQ0d69e3c655uHDx/6xx65ZcoG1xhQbfvixYvZGRLV8RycWIbXc2DSsGyrbNy+fbtpAohVrk8gC3GCIgKmjGu3aG6j+SZBK8HpzZs3m3GN2ORs4YLKrD+Y7CSa5TJoHDZRNsjoyjLBdDK53igyxuqw7KpsJBNtpssswCzPkOm///3vs/GzVvbgjx8/zqYZtD2Lvt8yuzC/n8alVj7skl5L20TZyFlvzoo1DJYNdfGYonlq5cOLX7VTXNjmgUc1lg1JMDDRTqS/CS9sU5tlQ1Kp2pQjSZK0C+2mHK8x0dIsG+pi2VAXy4bmqZUPm3IkSVJvrBSY0M8A0Q0DfQyAK+kzzzwH40ZfECkLy/YNUb6GcYZ2R1w6fKuUjSx7aH3asP/LflHLozyUZUOiCuWCyqwL6DeA+85L9AlA3wAatnllI/1CBOOLygR9VpR9l6SviXb5Uv9tumzU9jP7xLbYx8Z65pUNsA+gjDDYl9H41MqHTTnaiK9fvza5R2Kyk5mO1XFmRI+PZX4Tevic7PynUxqKVctGH5FYUJtHrRg18fT8y0DvwIdWU6bNMzDRRqT/CapjwU4m3Y0jzYBkdcWiVPgajlXLRlvZXExTSaZpOsmtxmXTUNnkXMo8ni/XyXiaE1hfnmOdeQ9y/ZBQkHGwrVkXyuanjLOt2Rbmo2y2KLd5rOi7hiR+Qa4ck/jJwEQbQxI1gg12uuVBhp358+fPqa9rptMOn526hm/VslEipw7PU9NCcECgc3Z21tS4kS/n7du3R69fv26WZd1ZH8ukjDGf5fI+YJptYn0ES9TW/fzzz02+ntTqEEgw7/j4ePY8AUW2iTN8pgm0WAYZZ1upEWQ7crBleV7HQEZkr6mQ/mhjgYmdIwkcgNjpcoaZs0t2zt99911zUGI+6eo1PuuUDWoaCBJ4Ps1BqX04OTlpatiQzMKsIzU0BAi8loM/z2d+xhlSe1IGCJy1815kOX716tV07u8Iggg62CaCowRFbQQyqREkgy7vkc/CgPfv3zePkn63cmDCH+3Lly/TqW9oQ9a4sXMvz3bZAZfXjzCdM0XOOpdJha9hWLVstFH7kOcZEmAsI8EM71FDzQc1JDQtJsghoOF9uK6krN0ppfaFoRa8dGF78jqGvOdYsR8oAzvGmadxWzkwoeqUs4USZzxj/4Pp6EK54Eww1ehUXz958qQZB2fLHFw4452XCl/DsUrZKFFOCGBTS0KAk/Eagpisg6CX9+FkivkJjpifcfZb1HqUNb68nvcg4EhNTIlrIsqLYdvb3CW1J3nvdsA2Rvy+fMf8JgxlzZZGbBK1X1CZ9Qe5rTMDt9Jp+OaVDcoAt1OW5WJyljp99ttra/MXpcLXYZj3261aNsoywXT52kmA0awv0+W+iNe11xespzYfvI73CKZ5H5bLOvM+mb5x48ZsXWwP8zP9+PHj2Xi5fbxHue2sYwz4rPOU30lZLjQOtfJhl/RammVDXQ65bOTiVW2H+w3NUysf3pUjSZJ6w+zCkiRpb6wxkSRJveU1JpIkaS+8xkSSJPWagYk0UHQOtmwfG5tEfxScBWWgvw7ufGG+JC1iYCINEEEAHZPReVjNtnrXpcMwejc9OTlpqmcZ6IyMvDDbYk/B0rAYmEgDRBfr9HaKdk+pBA/Pnj2bTm0WPbwSmJRdy9ODKoHKNlAjRB4aScNhYCINFN37g1wwQZBC8PDx47dkciBQSbNLmn54ZJrmIAbGWS7za7UU6V69nWYABCrpkp1u4vN+ad7JdLktjLe3jce8N4/UCDF05bSRdHgMTKSBIfggEVryV7Wbc6jRYKCZhcCAQOX8/LwJVliWYCA1HjQH/fTTT83yLEeuLGpiajUunz9/bh7JztslgQ/vTS1KghjeP8pxPkNqftrvnfw2rGeVRHqS+s3ARBoYakhIrEntQqQ2o50gLWn3r169OqvRePPmTfMIAoG8huAkwQ5BTFsChXlNK2QVTgI/AhjWs+xFse1EoTdv3pyOSRoSAxNpYKhZyIWnb9++beY9f/68edymBAoEH23t61wuoxYUSRoOAxNpQLjugtqPoLaDmg6aZGrBQYKJL1++zGouCGzWQY3L2dlZEziU16DwvrnOhaYbUtuDmhW2LTU1kSYhSSM1Oau6oDJL0gEgrT7/XwZSyeN4mr4/wyRomKXonwQFzTLl63gOWYahTN3fHq8p18fANpR43zzH9kTm5Xke29uW8bx3ls12Szos/H/b7JJekiTtRS3msClHkiT1RrXGRJIkaRfaNSY25WivDIS1Te7LpH6rxRwGJtqrsZU3/1+743ct9V/tf+o1JpIkqTcMTNRbyY1SG9KT6ZAkJ035+cpcMbX8NKsq17cJ9FGS9bWHTWxvl+TbMUeONDwGJuo1OutKNR/dozNe5lIZkjILL3lp6PCMbtjpvZXP/OOPPzbPXUaZe2YT6MAtPbGy7fw+DOlxdlvSSZuk4TEwUW+VGWlLHFzbeVOGgvwx9J6KJLw7RAQsmwikJI2PgYkOVpoMkhSOR4Y0AaW6n6Fs+kmTSV+bATigU6tBN/Lt5pDy85Tj7SYfHvkeMr/2WfP9lAFQXsuAvJ5l02yzKO9N+73yfZevzXvzfuV42TSU37Hcvto8ScNiYKKDlSaEpM4/PT1tEsglZT9oVuAgT9MIOKDRDMB8Hjnw9lE+w+PHj4/evXvXjKNsxirH2000NKs8ffq0yRRMDQxBTjuLL98V62A5AgK+C96P75XX812V6+X7ZZy8OjWsh6ChbGYh2OC9+b7ZDrIeo0wqWI5T05LmLGqP8jnANjLONq+bz0dS/xmY6GDRzJMDVw66ZdMPZ+K4c+dO88gyHIw58HIA5bFM8d8nfI4EHgQLqyibUB49ejQdOzr6+vXrdOwb3iMJ/wg28l3cuHGj+U7LLMF8V1wDw9DVjMZvQQCS7x2vX79u1odr1641j4tqXOLevXvTsW8SoLHNQ23Kk2RgogOXA++TJ08uZNWdhwMlB1CGsnalbzj4pvZglwhCUqMUCS6WQfCyae3aHknDZWCig8ZZP80LnN3TDFDDWTtYlmYfDryctXOwo6mhz+YFTp8/f56OrS+1EDdv3pw1j7x8+bJ53MR1HNRW8X0j29v+nbqahtpu3brVPLK8gYo0YJMzowsqs6StWVTeJge1ZpkMpLlvOz8//0MK/vI1DCwTmVdb17bxvl0mAdZs20pv377t3P488nzm813Uxlk/eGSdmc94lNvAd99eb1u5Hoa8R6lcZ/le5faXz2f85OTkwjgyr/2aGp6T1G+1/6ld0muvNlHeqP24cuXKhetLWO/kgLiVZoXL8P+1O37XUv/V/qc25ehg0dTAQHNEGZRIkg6XNSbaK8qbtC3uy6R+q8UcBibaq7GVN/9fu+N3LfVf7X9qU44kSeoNAxP1XtlNOuMMy94uyjUovG7ZTr20e+myvuyYrcTz5e+XLuxr3exLOnwGJuo1Aotnz541VX2p7qMn1HYvpl3oB+TGCp2DabfoBv+nn35qflsCjnaKAAISurQPglL6rGF5OoAz4JSGx8BEvUWtSLtrdLpbPy9yxOiw0bFbOlyjM7Z2770vXry4kAOIbupzBxad5XGbuKRhMTBRb9EDKbUd7VuB6ao9B7NU6zMQyKTppt2Ewy3FWS5YPvPSLFC+Ls/Zy+j25LfNd1z2CkvtSJnrB1me34eear1NXBoeAxP1Wtd1B6Dan7NmqvXJXEsgUzbdMD8HOrqlZ5rcMwQfYDnmMYD5eT1ZcLP8+/fvm+e1HfyOfOc00SWQTKBSCzwIWPh9yBht0CgNj4GJDhY1JwQS1Ha0M/A+ePBgOvYN1zGAM3CahjgAlk0EPF82Gb19+3Y6tpmcNOrG70gQyO9B0w0IMsssySXmszzBTPL6SBoOAxP1FknbuPCxfVZMUMFZc5pbCCqoMbkMr1XYvwSPINBMUxplgBqS9oWxp6en0zFJQ2Jgot6iGYamFJprSg8fPmzOmrluhICkvC5hkSdPnhz98MMPzWs44OVgR3MN87U//J7JcJwmttSkUINFzUrpzZs3s4zDkgZk8se/oDJL2pplylsyymaIMhPtjSLbbIYoMxSzrihfPzn4NfPK9yoz83ZlsF0V69Lv+N7L77uGZfL9l79zmXG5xu9a6r/a/9Qu6bVXYytv/r92x+9a6r/a/9SmHEmS1BsGJpIkqTdsytFeUd6kbXFfJvVbLeYwMNFeja28+f/aHb9rqf9q/1ObciRJUm8YmKiX6FSNSJqh7FgrnaoxpGt5xjU8yVtU+33L8iFpWAxM1EvkSKF6jw7WypwodIz28ePHpmM1uqNHWQ2YYEWHLcEovy3ZpJNkEcmtQ6drNtVIw2Ngot4jCGn3/lpD8PL06dPplA4Zvbom3xE9vn748KH5fRkIVAlOV+nxV9LhMDBR79H9PFmGyY/TxvxkIOYsGlTvM4/HNAfQBFRW/7fPwDM/Q3s+r88028HrGc+ZvTaL3+rLly/TqW+/89evX5ukfdSi8Vvnd5E0LAYmOgg025Rp8aPMCMxZNKje5ww7gQrTnF0zzXiq/9PskzNw5rMM4xwYnz9/3syjyYD8PJy5c1BkO0g4RxMDZ/bavDt37jTff/B7kmjx9evXzTS/C98/v4ukYTEw0UHgmhMORGSZXUWaAwhoSAYXBBYJasr5OTMnqR9J/jgr5z0TrKBMHJh52ixqyQhG+P4ZQBlg3qNHj5ppAsXyd5E0DAYmOhipsdjEBa6cfQdBSpoGOPgl6OC9UsPCwHPaHYIQvncCwWR+TuAY/G7+LtKwGJjooNCkwwFrVQQb1IDkmhBqRHKwOz09nQUfudPn5s2bzYW0ORvnuhLPzHeP753mG2pQQNMNvxeoBcv1RZIGZLIzvqAyS9qaeeWtTIn/8ePH6dzfmhT4SZGf51kWkzPoZjqPDMHr2sujfB+Gk5OTZj7vUc4jzX6my+eyLctgeS3G99313ea5Rd+l37XUf7X/qV3Sa6/6UN6oRaGZKDhLzxn6pvn/2h2/a6n/av9Tm3I0agQl7TtrPn36NB2TJO2aNSbaK8qbtC3uy6R+q8UcBibaq7GVN/9fu+N3LfVf7X9qU44kSeoNAxP1HhejElVnsBvycTC7sDROBibqNQ5Oz549a6r6GOjpk55YDU6GLf3N8JubXVgaF68x0V7NK28EH+kOvuzdkxoUgpV1OlrbN/9fyyEgJZ1AeuGlI7WkECAoaZeJGr9rqf9q/1NrTNRb7969q3Y5fuvWrebA9P/+3/9rCjUDZ9EZL5t+qPInwMl85LkMOTvPdLq8Z5wDYtZdnrVru/jdzC4sjZOBiQ5O8tzwSHU+6EI+Z9J0jkb1P8n5CGo462aa+QQdZ2dnTYTOIwOdqxF08Frmc2bOAY91M49+TphPl/baDbMLS+NlYKKDxYEqQQdBCQey1K4QbHAwS43J1atXm/m3b99uHnHt2rVZZ2oEHTkLJxihtgbMS/4cmC9nN8wuLI2XgYl6K0027QMP1fkEDAlCPn/+3EwnuVuQpI9lCTJyrQIHM65P4WB3//79C0EHZ+EZttUlvZZHEMJvQa2W2YWl8TAwUW8RTHA9wd27d6dzvl0Q+/jx41k1f64P4SCGXB+Ce/fuNUEINSPBdSa8NgFI0OyTa1AIhDKu/eJ3MLuwNDKTnfMFlVnS1ixT3sgwy3IZPk4zDSdbMNmBmZfnyT4bPMdycV5kCM6A8vWZl3HWNzkzb8Z5vIysW/MlgzC/fVueW/Rd+l1L/Vf7n3q7sPZq2+WNM+6yWYYaljKTcO7qSFPPtvn/2h2/a6n/av9Tm3I0KlxXUnrx4sXOghJJ0mLWmGivKG/Strgvk/qtFnMYmGivxlbe/H/tjt+11H+1/6lNOZIkqTcMTNRrXLxKRM2wzG283D7avoWUXl15vV3KH74y9YC/qTRMBibqLQIR+iyhmo+BzrQWSaK3IJChe3NeT++u9hJ6+FIeGCQNj4GJeosePstghF5gV0WX8+lgjQOZvYQetvat3ubKkYbHwES9ldt4qbIHQQV9knBAYl47C3Ab1fxPnz5tbhHm+XK5NO9Qg5L5zKutR/1EqgGSN0oaFgMT9RpdzVNrQvCQ6wkIWMifEskw3Pbq1aumS3uS/LGecjmei2QR/umnn2Zd26v/6KreGjBpeAxM1HtJ5sY1ImUunE0i+LGjtcNBTRfXDkkaHgMT9RZNNknSB2o12he3apzev3/fZJ+WNDwGJuq1sgt5Dka5BqTMGMy1Bsu4cuXKdOz3HDk6TGSItoZLGiYDE/Ua14dwfQkDQcrPP//czOeiRy5sZT7XGlCbQjMPTTKMcz1KefErz+V6BF6T1Pl37949+u6772av12Hw2hJpuOySXns1tvLm/2t3/K6l/qv9T60xkSRJvWFgIkmSesOmHO0V5U3aFvdlUr/VYg4DE+3V2Mqb/6/d8buW+q/2P7UpR5Ik9YaBiXor+W0Y0tFaOY/Mwctg2XRnr/5JvqLa78TvXXayB5bJ8pKGx8BEvUVX9MfHx00um2SVzTz6NyGh3zK6cumoH16+fNlU5TKUOYwISuhfppQghWXJl2TfM9LwGJhI2htqSx4/flyt/UgQWvr8+fMsRw5d0vN6ScNiYKKDxdlzqvQ5QNHNPONp4kmVf7qsz/OcZecRZfNQlM0LGdKNfaZ5fZbjvXjfzNfyqP1ID7+LAg2CEQIZluN3JSO0pGExMFHv0WV8ggEGsgyD5h0OaJxV00U5uVPSxEOQwJk1Bz26rAfPU/1PN/XMp3t7AgryrqRpgCAFBBesi/knJyfNOK9neZoXmE9Cwa9fvzbPZZt4zkSDy0vX8vktnzx50kx3yW9I6oFMSxoWAxP1HteIEAhkKKv3OaBR5Z8ak6tXrzbznz17dnTv3r1mvH1WTaARBBQ5uBHQEFiwrjKl/vXr15smBLA8B0UCJJZNbQzblGte2tdFaDn8lotqTPDp06cmiKHmxGSM0vAYmOjg/fDDD80FlAQJlz2Dzpk4QQbJ/whACHLKC23LIKmcr8tblJyPmrDbt283QQwB68OHD6fPSBoKAxMdPGpGCB6uXbs2nfPtupE0C6RWo4aajlwTwtk6r+PgyLw02VAjEyyfa1hYPuO6PL7zR48eTafqqC158+bNdErSIE12vBdUZklbM6+83bhxo3me4fz8/A/zzs7OmnmYBAy/Tc6gp1PfZDme4/Hk5GQ2L+tD5jEE6y7n876YBCsX5pfT5Wt4zxqe0++6fhPkd2s/V5aB9m9e4nlJ/Vb7n9olvfZqU+WNmotNNqtwxw/NBcH0zZs3FzY1LOL/a3f8rqX+q/1PbcqRWmiiaV8wS/PBZYMSSdJi1phoryhv0ra4L5P6rRZzVAMTSZKkXVgqMGkvJEmStGm1mMNrTCRJUm8YmGglZcp5hn30vMn7rtp/SPLkMCD5cdop9ZeRdS3TS+k+lbmE2r8TfYbkudp3wPfb9dpof6ebkPJ1mf5hys/NUG4nQ/qtyXfQVQb4fec9j6yD7d6U9vbX1l3+Pgz5TCXmt+W7SOqFtvwvyufLssJQ/jZZX1vXfGkZBiZaCWnp06U7HZCt09PqZQ4662I76RwtTk9Pp2Ore/HiRfNIb7N9xu3O5JWpIU9QniNXUBsd1mHeb9z+TjdhE0n5ys/NI9tZVhXz2fNI1/blbeGryrq6ENzMC2y6sL0ZauhMMM/zG7XV/mPMI+8Uy5edBgbP3717t1knj1kHPe3mvehtNwg+WF9b13xpWQYm2il2WuQ4uQx2kPvsCj41JUkOeOjI/1PW/vAbHbrkSUqAhQTUCRR4pG+aLtweTlm7TODCAX5V5fvxW9S63S+Xef/+/dGDBw+mU7+Xz1L+d3yertve6VWXIAQ8Mo3yvehFOd8tAV8tKOqaLy3LwESXxplVu5qXHWHmpSqaHWbOpJjPdG25rC/VygxZb7uqv6z2TnV2uU6GRbJcqq95LKuyS7wfZ8nk1CkP6O3tyHR7u/MZUz3OYz4vMl4ui3Z1OkP7+2MczM+8HFy65GBd1v5w8GkfUMt1dn03aH9OlJ+J8fK7Ksdrat9H+ZmzXaynxMGX2hwOkPleIl3ak5ixPEhnnbwnUv6y7kxnaH8PeT7vx/by/uRcyjojn6E9v43fYl7wBD5PWatFKoZ24E4tH791tj3ltkQwwraCddbSA1Ce7M9HWzeJoC+ozJIuSDfik51uM00X4XQNzkC37OD59jjPp5vxqC2HdDuO8v1YR5ZjmnG6K2c6y/MeDOWymZ9leE1em/XwPnkun60t68r7ZRpZJ++babDedGnPY+azLNNZV7aD6cxjXfkcvK4cLz8/Q96Dz1m+X9bTxntkHVkerKv2G+f7KcfL77T2ObO9PJbPl68rx7N8vteMl98Hsn28lvEsX8preORzZN3ZhvI1+Uz5Pnhk+xnnMetifjkOxjPNI+tBtpHXt7U/Zxc+3yLlMvnOwfqDz5f34rFrvfO2mc+Xzxb5zG1d86W2WjmxxkSXRnZfakK+fPnSnKnljDFJ9SY7xabZozyrQ9dywTRSvfz169fmMajCxtWrV5v3nZTnZprrYLhWYdl2bs4AJzvco6dPn87OJGtnhTxHdThnnGmOKpsKcmbL2Smfje3Cr7/+2pw58zoeyyR0nGVn23nPfIZFzV2c7efzc5bLwLrZRmpycjbPbzMP7zk5SM1qFjiDbzddsE6ev379ejPNuvlMbbXPmQSKV65caWqaatc2zLPo+2CdDLWmvbI5h++K8sfvzLZRC3Lr1q3m+Xw+fv+UuXy385Tlke+wXWZSbmvYFj5bbbuD7bpz5850qo7frGzqoVy0/2fg8+W9+F4oI10mgUtTnnj/UrvJSNoWAxNdGjs8dnzszNKcsksERCUOOuysJ2eO0zmLpdqaavAEFG3smDmYZCgP6MhBngM021QeIDjg5XXlBZPtgxnfH9XlHByC9eRgQbDFe5QHtMnZ7Wzd7fUtIwe2XNR7GV2fsx1ULqv2fZTmfV6eY3v4jRIMJljgu2wfwAlasu3t4Izvm++d9REksT21AADtA/q6KG8JnrqUTT2UwwTODOCR7WG7s135Xtr4rgk8+KyUqfZ/ud1kJG2LgYkujTNodnYEAhyUs/Pi7A0cGGpnfssul4MKZ92l7JBzVwln++x8qS1hm9oByzxsf4KKrp1v++6VHNDLO3zYfj5HPhO4XiMBDNvXdV0Bz3HWTs1E+9oQpnPQpEYI7c+fAwmfIzUT7fXU5CDMe9cOhDmQZV2su3ZRZ+1zZn0JetoHO3TVosz7PpaVGqMEJPnO+I4in4/3AoFtgs0Sy+U3KAPDdfDZCBrmXWPC77ooEKCGke0Cy2b7GMAjz/M9EHSDz1f7/dim1HDV8Ly0E5OCe0FlljQz2aE3ZSQD7dlpl2ZI23Ta0Bl4TUzONGfz0bXc5EAxm8/A61C+/+QgeGF97WXK7Srfh/nl+oNtzzrasny2sVwfA+sE28Q0z5eyHOtBe9si8/I875d1lkPer/z8+e7L5dvbHeX7szzTWSbfXwa2r71OtL9TtJdB+V7ZRh7LZXnks5TvXb5n1sHztfftkmVZV/B+7d+5ts5sF0O5veW88rOV4+3vLJ878n5d5Q3tz8b2lL8j65/3etZfynfb/m3KdWZ7GcrvjO1tf4by8zFE13ypplZG7JJevZRrJFa9JuEyOEumViZnoH3CHR7t6wL8n+5WmkdickCf1V7tCjUsl62tkfqkFnPYlKPRo3mBgWrsPgYlaVbgz5thctbbzNNuEBCcnZ3Nvv+PH3ffTwfBaS4Ul4bM7MKSJGlvWmGI2YUlSdJ+2JQjSZJ6zcBE6gnOHGpD7RbbLiybC4d3bd3tLT9run9fVvn6Rbez8r2Uy3DNRl4rqT8MTKSeoDqTi1oZGGdY5iJLDubb6mOCC29rfXrUrHNBKJ2wcVEp3r59+4eOzeZhu+h3hu/pvNIhWInvqNy+BEC8lvdfJZiStF0GJlKPcZdQ2YNqGwEJvZgGy27yFus+p6+nA710FEbHafM+N530lXcy0QFeOvOjEzg7D5P6w8BE6qlk0w1uWU3TQ3oMzcGWx//7f/9v81yacsplM17Oz8GYx8wr52c9BCepYWAey5Q1DGkSSc+ibeX68/qse5G8tlajQTBCb63UnNB9e9kDb4nXtoM7ghG6b2f93CZOb8GS+sHAROoZmhw4GLc7VCMZHc0dDOCAfHJy0ozzmn//93+/UCtA8wbyOrDeNGm8fPmyeSSg4HWZzzQH7NRGpImFAISAguaPBAQELGwnyyTJXxu1PmmuIXhgWd4rwdW6WC+fkcCJGpFaMxDbWEumR/ftbFO+r0Vdv0vaHQMTqWc4WHLwp2fRUjuL87IH07ImIQECkn+G2gSu1SiDGg767UCDAISB4AbUNJR5jJLNdx4SJGa7l8l/w3bwXXQ1Z/H+BDoESqnVKbGNXdet8P4ENtScEMBI6gcDE6mnyu7OqcEgGKGmYdNZnDkolzUm8xAsESgw7LtrdAIRgicCnXwvJb6zMtsuy/A5+bzU1lCTQtBCYJOEjJL2z8BE6jmCEGoOaEbhkQMpNRybQo0KB+yvX79O59QRlFBjwgE/zThpJuG1i14fqZ3oavppI6ioBWJcwEp23S6pbcmQ4ItAhtqS1PZI6pnJH/aCyixJO8B/rzZMDqjN82X22mR65THz/uM//mM2flZkHSZ7LAPjrKtcz+RAPZtmGZ7P/EkANFuOceZlmmWjXHeeZ9lSuT1ZFuW2MORzRd6T5Wry3uVrea+sv8S8crvK7eXzSdo9/n9tdkkvaetoOqFZhdoeLzSVFLWYw6YcSZLUG9aYSNoqrkm5Udzx4/5FUtRiDgMTSZK0FzblSJKkXjMwkSRJvVFtypEkSdqFdlOO15hIkqS98BoTSZLUawYmGjU6/iJirw3c5roslr9sttx1lNu/zPbSjXyWHyO+o3x+Brr5p4v8ch5ZlCXtj4GJRo1EdGSYBY9UKTKcnJw08+bZZiCy7LrZ/mW2NUhad9zKWjwmyZ8DvocPHz7MkgCC77JMnihp9wxMpArS7HMQ60JSuTJtPwe7TWXbJSiZl5xOkobMwEQqJCV+qazmB88/ffq0Gaj2Z+A55jMwThMBwUt7fpklN8+X82lqIacMWXxZR+ZluTTXlM0P8+R9GRgvldsXvGeW532R5XjM+pDPXQ4oty2ZhNvarym/C94303l9nsu2ZjtZNr9BKa+vfZ5VdX3+jDPk/TOP9833k/dtNyMxsE6U2yuN3uRM74LKLGnQygy9DGSnDZ5LJt08ZvlkvE0W3rwuWWvz2jzH8owjWXNZJvOZl3XlvTLNI+vIfN4j4zyf15fK1/IevAbZpmxf5udzsR7mZT6YzzTbwHgyGbPuvI7x8nMxlOsI3jPbXo63vx/WB9bBc1l3nmM821R7nzwP3iPrBuO1gfep4bm8F+PZFqYzr9zeTDNeftZyezKe14P5TEtjkbJfssZEmpocSJvstyWuyUjtRa49uHnzZvM4z+Sg07w2ymYezpxzrcObN2+ampdoZ9599+5d8/jdd9/NalJ4/eTAOGtqmhxMm8e2vPbKlStN0xTXU5TK7QPTbNPdu3dn11yU+A74HCzzv//3/57O/d2XL1+O3r9/34zfv3+/GVhPanmCz5Bt55Fp3L59u3mkhuHly5fNd5HPynfEd4q8B3744Ydmm9qfLVIDcefOneax3JZJENB8Foba520rP39+P/C7tLGti7Io5z3TbEeNCd+FzXgaOwMTqcDBJEFEDmIcgDhYc+BoH2Qvg6r+69evL3XxKgFTDqKr+vr163Ssrjwo8xmfP39+IeleJJgAQQzbTcBEAMJ4GegQ5GV7y9fNk9cTrF27dq0ZD9af9ZXv015um9qfg+YXrjM6OzubzqlL0ERgy3edAKQdBOfzefGtxs7ARKogAOGsnbN3rhugxoEz7PJs/TK4doKDEwfWecHOrVu3mscXL140jxwMOUASOOR1Xa+vvXaeXL9BDcsyNQjIwZTvB6lNIrhB7T35HsttZzoYp3bk6tWrzXQ+a2qV+D26rluZJ7UQywZJi7DdbBOBZXkR9DxsdxmwpUYltTm5FmXR7yQN3uQPckFlljRYad+vDblOYnKwbKYnB8jpq36/jmByJj97nuHx48ez8XLd5TivmRz4L0znEVkfj8jzDLmOgcfMK7elrXxtPk+my+fYvtpzzFv0/hlYB8rPyutryteVsg2l8r3yGfOZ28uW8nwG1l1+7wysp/1Z8r1H7fMj8/I8r2v/zhlvf48Z2B4s83mkIaqVebukl7QWrrkoa1YmB9fOaz32gX3ZJFjoTdMINSY0fZUmgcyF64+ksanFHDblSFoZzQ5cd8MOJYPmOz09vXCt0Pm0Yz9JF5ldWJIk7U37xMamHKmHPEHYDfd10n7VYg4DE6mH+vo/HNL+wX2dtH+1/6HXmEiSpN4wMJEGjn4xOCvJwIWryeOSYZ2+QbrQx0d73fSRUs5Pnx2S1GZgIg0cnZ9xWyq4K4SeU7mF9mTa4yy3/C7qPn1ZBBzcNsy6qZ5l4G6UdNvunSiSFjEwkbQxdE9PYJKeYFEGQZK0iIGJNABlMwkdn+Vxl9I8Q/8mbQQqZXfw5MNpbyPNPfkMNDWBZqDMy2fkfcr5GdKlfj6/XbtLh8nARBoADvpprklHXjTR5GAd9DyaA3mZ1XgTPn/+3DySP2YRggyaddjGXN9Ccw+fgYE8QsyniSm1LQQajPM+Dx8+bHp1pamIGhrG6UGVgIbAhPl8vqxb0uEwMJEGhgR4uWaknWCu7Hl0080ryfS7TFI7Aos2tgm5HqWNWheGWhfu6QqfgIaBwAvv3r1rHiUdDgMTSRuRzMK//vpr81iaV3Px5cuX5pEaEYKa1Py0lU1BvEcCEGpdyvdMTQqDeWikw2NgIg1QAoFlmlU2Jc1J7SYktuXFixfTqTqadmh6YXuXqXF5//79hdqfBC0EJQQsrI/39bZk6QBN/tQXVGZJ2rF1/oeToKB5XQZS6eOklW6fFPyTA/iFeZODfLPsIiy7COsv1817RTmP7WM825nnsr0sw3aV86O9/QyYBEWz6fJ9a/IaSftT+x/aJb3UQ+v8D6ml4PoMahI21S9JW1/2D9SEcGtyiYtp6aNlWe7rpP2r/Q9typF0cH766aemyYgdGkPXdSmSDo81JlIP8T/U9rmvk/arFnMYmEg91Nf/4ZD2D+7rpP2r/Q9typEkSb1hYCINBBe/tnt6jbLLesaDW2ozfx/SfXy6oO/CNu+6i31J+2FgIg1EV4+pIH8NF4sylLls6KKealTu5FkUHGwaHarRfT7vTz8kXUEVnjx5Mh2TNHQGJtIAcJDvujOFWhFqGzj4MxCYpPOxdEuf24vL2pRto7fW3N776NGjo2fPnjXjbWzrLjuKk7RfBibSgePAffv27enUH5Ev5s6dO9Opb73BMo8Mv+XrCFroUXVXqL1JIMR7M11Dr7H37t2bTkkaOgMT6cBx4F6lY7G+OD4+Pnr58mUzToBCluA2mneoTZE0HgYm0gGjOeZQD9xkCua6GC5+JSgpr30pUZsiaTwMTKQD9vz58+agzsGdgzwD15uUbt26dfT69evp1FEzzjyacWjOCa75SIbgXSDg4MJXBmpPHjx4MH3mG4KuMnChqWdfdw9J2h07WJN6aJ3/Ye5qqaX65+JXAg9QM/Hhw4dmPO/DdSrcIfPq1atmfpdt7B+4G4hrYGrbHTT1lNu9Ce7rpP2r/Q+tMZEGiAM5f3gCDqRmhSEBCkh8x3LcNrwoKNm09GFC3psyKKHGZ9e3LkvqD2tMpB7q6/9wSPsH93XS/tX+h9aYSJKk3jAwkSRJvWFTjtRD/A+1fe7rpP2qxRwGJlIP9fV/OKT9g/s6af9q/0ObciRJUm8YmEgDQT8m8zL05vZchuB24va8XWFb894M7Y7hwLxymXmfT9IwGJhIA0EvqV3oRZX+S6gyLatN6b+E6bdv3+6875Br167NtqcrgR+902YZtlHS8BmYSANAzcLZ2dl06o/oxIzO1dLhGghWTk5OmvG//OUvzWOy/e5CmXiQrMbtLulRLkNGZLMMS8NnYCIdOIINahbmobaEWglqSAhIQJ6c8nXkriFA2Ae2JcFRl0+fPpnQTxoBAxPpwL148eJCzUINB3QGghNqT/pmUU3NLmtyJO2XgYl0wKj9ePTo0XRqMYITLoLtE2p8Hj58OJ2q62rqkTQ8BibSAUtyPu5Y4eJXhtrdLaU0h9CMQxNK0Nxz8+bN6dTucO3IovddpqlH0jDYwZrUQ+v8D3MrbZmpty3Xl6TpJ+9DrcXp6enCDMPb2D9wN9Ci911mmVW5r5P2r/Y/tMZEGiCuyeAPT8BBMMI4AzUP5fUo5+fnzXwuit30gX8ZbOedO3emU99Q41PeurxMU4+k4bDGROqhvv4Ph7R/cF8n7V/tf2iNiSRJ6g1rTKQe4n+o7XNfJ+1XLeYwMJF6qK//wyHtH9zXSftX+x/alCNJknrDwEQagFrm4FLu0mEoe1Hljpd5r9uFbEOt47fyjqLc6ixp2AxMpAPHAbuWObh09+7dpjt6BsZjn9mFQd8rbAPb9eHDh+ncbwiguL0523f//v0LQZWkYTIwkQ5cLXNwifnURiRfDoEJ8who9pldmG2gp1oCj/RGW2Lezz//3IyzfcfHx3tLMihpdwxMpANXyxxcosv3shOz69evN/OojdhndmGSDxIYpalmUVDE9l29enU6JWmoDEykA8cBm6GvmYO7EFARJFFjcnZ2tjDHD4GL+XKk4TMwkQaC4KRvmYPnIZBKXp979+4d/fLLL814DTVBhxR0SVqfgYk0IAQnbbdu3Tp6/fr1dOqoGWfevrMLc11Mmm/YbqZrWMbswtJ4GJhIA0GtQnnNSHBA544XDvAMjDOPZH5Pnz5tlikvkN2VH3744ejJkyfNONte3i1UYn4ugl3U3CPp8BmYSAeMA3ouHi0zBxOAMC936jx//rypkWCgZiT2mV2YZpxsJ800ZfDBrctsO8/R5MMjA9ekSBo2u6SXeqiv/8Mh7R/c10n7V/sfWmMiSZJ6wxoTqYf4H2r73NdJ+1WLOQxMpB7q6/9wSPsH93XS/tX+hzblSJKk3jAwkQZgUXZh1JbJnS/zXrdN5V1FJPRry107Xc9LGh4DE+nAcXBflF24a5l9Zxf+/PnzbJvSC2yJfk54jluGSfgnafgMTKQDtyi7MGrLEKzsM7sw70+wMS8gevToUfNIx29kF5Y0fAYm0oGjJmRedmHUltl3dmG6v6c2hPftyvGTnmhpxkkHbJKGzcBEOnAcvBkIPLoS3S2zzK4l6CDgoNv5rqCKGhVqVrq6rJc0LAYm0kBwoF+UXXiZZfbhwYMHzfUmNXSVT0DFMK+5StIwGJhIA5JaiHmyzL6zC7ddu3ZtOvZHbHOuh5E0bHawJvXQOv/DNIUkkV9Ne5m8DzURp6enCxP5bWv/QHPNovempofMyJvivk7av9r/0BoT6YARaPDHZujKLty1DPaZXbjsV6W8sDXZhcs+TBio0ZE0fNaYSD3U1//hkPYP7uuk/av9D60xkSRJvWFgIkmSesOmHKmH+B9q+9zXSftVizkMTKQe6uv/cEj7B/d10v7V/oc25UiSpN4wMJEGgltsOfuode1e3npbJurjduLM36d575/t4zZiScNnYCINAAduekelSrTWwRp5ZtKte5lzhv5LeM3bt2/nZvndJhL0dWGb2Da2kYCqFnRJGhYDE+nAcfA+OzvrzL5LrQidmRG4MBCYpOO1dPP+l7/8pXksa1N2YdH70dNrto3kg8+fP2/GJQ2XgYl0wDiwc/B+/fp1Z3PHu3fvju7cuTOdOjq6fv16M49eYMmXEwQt79+/n07txpMnT45+/PHH6dRFfDZqeOLKlSsb7ZJeUj8ZmEgHjECC2hBqE2juePr0aVMbcgiosSGrcBcCpRs3bsyab75+/drLzMiSNsvARDpgnz9/bmpD0txB0wy1IYeAbc92d6Hp5v79+01tENfDlDU/kobJwEQ6YNeuXTv69OnTdOpbMw3zSrdu3WqaeoJx5tGMQ3NOkCTv5s2b06ntolbn8ePHsztuwGP7mhMCF2qCGKg9uXfv3vQZSUNlB2tSD63yP2RZrsWg6YOmDgIMxkuZDy5+zbUaeR8ChdPT04UZhre1f1i0Xraf7avdcbSubX0WScur/Q+tMZEOHLfTUpvAH5yDN0EJNQ9M53oTmkRYhiEBCs7Pz5vlaCZZFJTsChfw5tZlto2Bbd5kUCKpv6wxkXqor//DIe0f3NdJ+1f7H1pjIkmSesMaE6mH+B9q+9zXSftVizkMTKQe6uv/cEj7B/d10v7V/oc25UiSpN4wMJEGYl52YXDLLc8zBHfttOftw7z3J8lfttEkftLwGZhIA8BBm9uEqRKt3VbLAZ1bbnm+rDbte3bhyHZ7y7A0fAYm0oFblF0Y5NKhD5Myjw7BSt+zCxO00ENsLTmhpGEyMJEOGAf2RdmFQW0JvcNSQ5LmkL5nFwZd0FNTwufcV42OpN0yMJEO2LLZhQk6GAhOWLYPCJDmZRcG24z0Slv7bJKGxcBEOmCrZhfmQE8g0wfLZBcuPXz48OjLly/TKUlDZWAiHbBlsgu3pRbiELILt129enU6Jmmo7GBN6qFV/ocsuyi7cOT6ktzdkvchUOhzdmFwjckmEw1u67NIWl7tf2iNiXTgFmUXJhhhnIEakvKW20PJLszQl+2TtF3WmEg91Nf/4ZD2D+7rpP2r/Q+tMZEkSb1hjYnUQ/wPtX3u66T9qsUcBiZSD/X1fzik/YP7Omn/av9Dm3IkSVJvGJhIB44zjnKo9QWSu3Taz3PXTubv07z35w4dnl8m2Z+kw2dgIh0wbgWmGjRDkvK13b17t+nrhIHx6Ht2YZ6jZ1u2kXxAdkkvDZ+BiXTAyj5Jot25GgdzOl5jPgOBSfo36Xt24WfPnjWJ/ECOnxcvXjTjkobLwEQaCA7ydEnfRu4cah2CZZjX9+zCCVoSaF25cqXp1VbSsBmYSAPx8uXLo1u3bk2n+m+Z7MKSxsfARBoIkvmtkq1331bNLixpHAxMpIGjFoULR4Nx5vU9u3CacDKPZqbywl1Jw2QHa1IPrfo/bGcNbkvWYXBw//DhQzOe9yFQ6GN24dyxw3Uo3DXEBbCbqmVxXyftX+1/aI2JNADtrMHUMvCHz+21z58/bzIQM5QXkPY9uzABCXfmsI1cwGvTjzR81phIPdTX/+GQ9g/u66T9q/0PrTGRJEm9YWAiSZJ6w6YcqYf4H2r73NdJ+1WLOQxMpB7q6/9wSPsH93XS/tX+hzblSJKk3jAwkQ4cZxzlUHZSVqIvkywT3E7cnrdLeW9uEa7Jbc8M87IQSxoOAxPpgNGxGtWgGZItuI3l6L8kywX9lzD99u3bWd8hu0Iwku15+vRpNaAiyR/Pf/z4sekpVtLwGZhIB6zW02u6ci/RYyqdq6XDNRCsJJBJx2VdtS3b8OjRo+nY0dHx8XF1u7MMz7GMpOEzMJEGgqDi+vXr06mLqC2h1oEaknRfT2+x5MsJDv7ko9mVBCJsD4FTTZahGefnn39uxiUNm4GJNBAvX75skvPVcIBnIDjpCgL2geac+/fvNwFTF5qYaMYxgZ80DgYm0kB8+vRpYS4ZghMugu0LakFyzUtqctrI4UNAxVA2RUkaJgMTaWTSPEIzDs05QXPPzZs3p1O7dXZ2Nh2rY5u7LuyVNCwGJtIAUNtQXi/SpVyOC2e5GwbURFCTkqBl116/fr0wKCJwMruwNHwGJtIAUPNR3qGT/j8IOAhG0hdIe7nz8/NmPtd40GSyS9kmBq57SVDEdSdcV1L2YcJAYCJp+OySXuqhvv4Ph7R/cF8n7V/tf2iNiSRJ6g1rTKQe4n+o7XNfJ+1XLeYwMJF6qK//wyHtH9zXSftX+x/alCNJknrDwEQ6cOVdN12dlJV3uJT5cPaZXbh873mZg7lDZ9EykobDwEQ6cHTpnp5RGa+hO/csU3btvs/swqenp817s010OV/r1ZVg5M6dO81y9HVSW0bSsBiYSAeM2g+yBtMHCAPjZY0IOJin8zQGAhPmUbuyz+zC6TeFbWI73r1710yXnj17dnTv3r1mnL5OXrx40YxLGi4DE+mAcVAHNQsEFQQdmRcc8Kl1CDIQM2/f2YVLbNO1a9emU98kSMrnuXLlip2sSSNgYCIdOA7Wyb5LUrxDRDNN2SOtpPEyMJEOHLUcXCPCtRp0535oaFZ6+PDhdErS2BmYSAeM5o7nz58314hwgSi1J+0LRG/dutXUSATjzOtLdmEugq3VlqQJJ006BGDlhbuShsnARDpgX79+Pfrll1+mU3UELR8+fGgO8AyMM68P2YV5z1wEW6vt+eGHH45evnzZjBOAPXjwoBmXNFwGJtIBI8A4Ozub9QdCjQLzCECYTu0JB3Xu2GEoLyDdV3bhbB/NT9n2SHZh/Pjjj82dOTzPBbx8NknDZpf0Ug/19X84pP2D+zpp/2r/Q2tMJElSb1hjIvUQ/0Ntn/s6ab9qMYeBidRDff0fDmn/4L5O2r/a/9CmHEmS1BsGJtKBWya7MLg1N8sFd+205+0Dd+KwDfM6iMu2Sho2AxPpwC2TXZiAhduEqTItq033mV04Ekxlu2rBFUEJ2ypp+AxMpANGfyCLsguDzLw8l35NQACwz+zCUXac9ujRo2a6je0j8JI0fAYm0gFLT63zsguD2hIO7NQ6pEaiL9mF6bk2gRHbsKgnW0nDZmAiHTiCjkXZhTngMxCcUHsiSX1lYCIduFWyCxOccBGsJPWVgYl0wGi+WZRduI3gBPvILsz25i6g3GFzfHw822YemZY0XgYm0gFbJrtwietLcl3JPrILs/7cGZS7cB4+fHj04sWLZpxHpiWNl4GJdMCoKVmUXZhgJM9TQ0JAEvvKLlxie7K9mQa3L6dpiue5qwhZTtIw2SW91EN9/R8Oaf/gvk7av9r/0BoTSZLUGwYmkiSpN2zKkXqI/6G2z32dtF+1mMPAROqhvv4Ph7R/cF8n7V/tf2hTjiRJ6g0DE+nAlbcDd3WuVnZsxniwfObvS7kN5PyZh22351pp2AxMpAPGgZrcN1SFJklfDf2b8DwD48HyvJYu7ek3ZB9OT09n20/On67gCk+ePJmOSRoqAxPpgJEn54cffmjG6VX15ORklj04ONCnV1cGAhPmsRzLI9l9y9qUXUnHbtn+d+/eNdNtbPP169enU5KGysBEOmCfP38++vTp03TqqDlwM6/Egf7OnTvTqW/LMI9eYNM9PQgMCHT2iW27du3adOoiuqu/d+/edErSUBmYSAfs1q1bs3w3IEjpOrAfgtevX1/oMj+49uTRo0fTKUlDZmAiHTCaYGj+yMWjBCnbzhC8LTTVzEvgR42OpOEzMJEO3M8//zy7gPX4+PgPB3BqVaiJCMaZRzMOzTnx66+/bj2oKe8OYihxEWyttoRrYbgoluVJ5MdFsu3XShoOO1iTemjV/yG1DbnDpoaLXwk8wMWvHz58aMbzPryewGBRhuFt7R/YvmwTGYUJtmoIbMrtvwz3ddL+1f6H1phIByx9mOSW20jNBAEHnj9/3tQ2MCRAwfn5ebMcQc2ioGQbsp2pBWEIbl8mSJE0LtaYSD3U1//hkPYP7uuk/av9D60xkSRJvWGNidRD/A+1fe7rpP2qxRwGJlIP9fV/OKT9g/s6af9q/0ObciRJUm8YmEgHbpnswuCW3CwXLN+et0/ztiPbua9kg5J2w8BEOmDcbrtMdmGCF24TZrmy2rQP2YWDbue7cNvw2dnZbNv5PJKGycBEOmDLZBcGwQt9mJQ1KizXh+zCWPS+PE9vtSAhYTtRoaThMDCRDtgy2YVBbUlqVBK49Cm78JMnT45+/PHH6dQfEYwkjw5d6s9bVtJhMzCRDtiy2YUJOhgITqg96RMCpQcPHkyn6ghEco1M37Zf0mYZmEgHbNXswgQnHOD7hBqeNCXNk6aqrutoJA2DgYl04BZlF27L833ILsw1L8kczAAe29eccGHuo0ePms/KRbDm0JGGy8BEGgAO8Msk4qPZJNeVfP/997NmIF5PTcqioOayWH/uDGKgpqScBo/t7SCb8L6uf5G0W/b8KvXQsv9DAo379+83NSVlUEKNA3fhUIvy5cuXZhnQFEKtQ+T1WOb9tr1/KNdPLQkBCtubzxOb2Ab3ddL+1f6HBiZSD/X1fzik/YP7Omn/av9Dm3IkSVJvWGMi9RD/Q22f+zppv2oxh4GJ1EN9/R8Oaf/gvk7av9r/0KYcSZLUGwYm0oGiL492fx5lPyHtvkCiK6Nw1/xtI3lf3pehq4+SRc9LGgYDE+kAcXAuu6KPu3fvNt3OMzBe05VReF+ZhulCn/dlYLtr2B62i2UIuJLvR9LwGJhIByg9oJbKTtIYCEyYV+KAXsso3DV/F+joLehErZY3hw7Wsl3kynn+/HkzLml4DEykgXj37l2ThTfINMy8UldG4b5kGmY72nlzCJDKmpQrV640gYqkYTIwkdQbtVoagiR6fU3zzdevX3uXiFDS5hiYSOoFmp0ePnw4nbqIphu6zufiV66FKWuGJA2LgYk0ELdu3Tp6/fr1dOqoGWdeqSuj8K4yDVMjkrtrGEo0O3W9Z5nsj9qTe/fuTZ+RNDQGJtJAcPDm2gsO/gzlBaPRlVF4V5mGWWcCDIYSgdSi92S7Tk9Pt7JtkvrBwEQ6QPT98fjx4yaYKPv1oMmDGgUGaj2QWorcoXN+fj5rEikzEnfN3wW2sd08wy3C+WypYeEzlXfxSBoeu6SXeqiv/8Mh7R/c10n7V/sfWmMiSZJ6o1pjIkmStAvtGpM/BCaSJEn7YlOOJEnqDQMTSZLUGwYmkiSpNwxMJElSTxwd/f8QaAyrnos71QAAAABJRU5ErkJggg==\" width=\"550\" height=\"693\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5\u003c/strong\u003e : Summarized data retrieved from the different molecular dockings performed in order to determine the interactions between Cerastokunin and coagulation factors and trypsin compared to Apixaban \u0026nbsp;and Rivaroxaban.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein- Protein docking bonds on position 0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCerastokunin-FXa interaction\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFXa involved residues\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCerastokunin involved residues\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eGlu97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eAsn17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eThr98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eAsn17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eTyr99 (double bonds)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eTyr19 (double bonds)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eGlu39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eTyr23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eArg143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThr34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eGln192\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003ePhe35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eGln192 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eTyr19\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eGln61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eLys48\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCerastokunin-Thrombin interaction\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eThrombin involved residues\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCerastokunin involved residues\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eLys372\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eAsn17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003ePro369\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eTyr13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eTrp412\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eTyr19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eArg413\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eThr34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eGlu466\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eGly41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eGlu522\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eAsn17 (2 bonds)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eGlu522\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eAla18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eGlu 549(double bonds)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eLys48 (2 bonds)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eArg553 (double bonds)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eTyr37 (2 bonds)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCerastokunin-Trypsin interaction\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTrypsin involved residues\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCerastokunin involved residues\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eTyr39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003ePro21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eTyr151\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eTyr19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eGly216\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eAsn17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eGln192\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eCys16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eGly219\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003ePro15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eSer190 (2 involved bonds)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eAsn17 (double bonds)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eAsp189\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.145695364238414%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eAsn17\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein-Ligand docking\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein-Ligand docking of FX-Apixaban bond\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFX involved residue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.437086092715234%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBond length\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.70860927152318%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cstrong\u003eG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eGln192\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.437086092715234%\" valign=\"top\"\u003e\n \u003cp\u003e84\u0026Aring;\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.70860927152318%\" valign=\"top\"\u003e\n \u003cp\u003e-8.35 Kcal/mol\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein- ligand docking of FX-Rivaroxaban bond\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFX involved residue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.437086092715234%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBond length\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.70860927152318%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cstrong\u003eG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eGly 193\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.437086092715234%\" valign=\"top\"\u003e\n \u003cp\u003e2.5\u0026Aring;\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.70860927152318%\" valign=\"top\"\u003e\n \u003cp\u003e-7.69 Kcal/mol\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein- ligand docking of Thrombin-Apixaban bond\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eThrombin involved residues\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.437086092715234%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBonds length\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.70860927152318%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cstrong\u003eG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eArg310\u0026nbsp;(with H21 of Apixaban)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.437086092715234%\" valign=\"top\"\u003e\n \u003cp\u003e2.3 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.70860927152318%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e-7.86 Kcal/mol\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"57.63747454175153%\" valign=\"top\"\u003e\n \u003cp\u003eAsn537 (with the N6 of Apixaban)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.36252545824847%\" valign=\"top\"\u003e\n \u003cp\u003e2.75 \u0026Aring;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein- ligand docking of Thrombin-Rivaroxaban bond\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eThrombin involved residue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.437086092715234%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBond length\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.70860927152318%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cstrong\u003eG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eTRP 370 (with the O13 of Rivaroxaban)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.437086092715234%\" valign=\"top\"\u003e\n \u003cp\u003e2.37 \u0026Aring;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.70860927152318%\" valign=\"top\"\u003e\n \u003cp\u003e-7.43 Kcal/mol\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein- ligand docking of Thrombin-Apixaban bond\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTrypsin involved residues\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.437086092715234%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBonds length\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.70860927152318%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cstrong\u003eG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eSer96\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.437086092715234%\" valign=\"top\"\u003e\n \u003cp\u003e2.75 \u0026Aring;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.70860927152318%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e-8,96\u0026nbsp;Kcal/mol\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"57.63747454175153%\" valign=\"top\"\u003e\n \u003cp\u003eGln192 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.36252545824847%\" valign=\"top\"\u003e\n \u003cp\u003e2.28 \u0026Aring;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein- ligand docking of Trypsin-Rivaroxaban bond\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTrypsin involved residue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.437086092715234%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBond length\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.70860927152318%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cstrong\u003eG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46.854304635761586%\" valign=\"top\"\u003e\n \u003cp\u003eGln175\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.437086092715234%\" valign=\"top\"\u003e\n \u003cp\u003e2.95 \u0026Aring;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.70860927152318%\" valign=\"top\"\u003e\n \u003cp\u003e-8,18 Kcal/mol\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"the-protein-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jopc","sideBox":"Learn more about [The Protein Journal](http://link.springer.com/journal/10930)","snPcode":"10930","submissionUrl":"https://submission.nature.com/new-submission/10930/3","title":"The Protein Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Cerastokunin, Kunitz-Type peptide, Antithrombotic, Anti-platelet, FXa inhibitor, Thrombin inhibitor","lastPublishedDoi":"10.21203/rs.3.rs-4354127/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4354127/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe current investigation focused on separating \u003cem\u003eCerastes cerastes\u003c/em\u003e venom to produce the first Kunitz-type peptide. Three stages of chromatography were used to purify a 7.75 kDa peptide called Cerastokunin with pI 8.48 till homogeneity based on antitrypsin activity. Cerastokunin was found to include 67 amino acid residues that were obtained by de novo sequencing using LC-MALDI-MSMS. Upon alignment with kunitz-type peptides, there was a high degree of similarity. Cerastokunin's 3D structure had 12% α-helices and 21% β-strands. Cerastokunin showed a strong anticoagulant potential by completely eliminating the protease activity of thrombin and trypsin as well as blocking the intrinsic and extrinsic coagulation pathways. In both PT and aPPT, Cerastokunin increased the blood clotting time in a dose-dependent way. Using Lys48 and Gln192 for direct binding, Cerastokunin inhibited thrombin, Factor Xa and trypsin as shown by molecular docking. Cerastokunin exhibited a dose-response blockade of PARs-dependent pathway platelet once stimulated by thrombin. In vivo study showed a substantial reduction in tail thrombus of mice-carrageenan model; in contrast to antithrombotic medications, this antithrombosis was boosted by a greater dose of Cerastokunin. Throughout the trial course, no in vivo toxicity was observed in challenged mice at any of Cerastokunin doses up to 6 mg/kg.\u003c/p\u003e","manuscriptTitle":"Structural, biochemical characterization and molecular mechanism of Cerastokunin: A new Kunitz-type peptide with potential inhibition of thrombin, factor Xa and platelets","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-08 05:16:26","doi":"10.21203/rs.3.rs-4354127/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-05T09:39:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-22T06:54:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-20T11:28:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"167434359476854296163878411684307705596","date":"2024-05-08T07:36:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305826929527774608414764256565910396049","date":"2024-05-06T19:47:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282625725143604493207188109582451958328","date":"2024-05-06T19:07:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-05T19:10:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-02T13:37:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-02T13:37:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"The Protein Journal","date":"2024-05-01T11:54:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"the-protein-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jopc","sideBox":"Learn more about [The Protein Journal](http://link.springer.com/journal/10930)","snPcode":"10930","submissionUrl":"https://submission.nature.com/new-submission/10930/3","title":"The Protein Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f8dabad0-1584-4bd6-9add-8b8f4fa4bc1b","owner":[],"postedDate":"May 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-05T15:59:12+00:00","versionOfRecord":{"articleIdentity":"rs-4354127","link":"https://doi.org/10.1007/s10930-024-10226-9","journal":{"identity":"the-protein-journal","isVorOnly":false,"title":"The Protein Journal"},"publishedOn":"2024-08-02 15:56:55","publishedOnDateReadable":"August 2nd, 2024"},"versionCreatedAt":"2024-05-08 05:16:26","video":"","vorDoi":"10.1007/s10930-024-10226-9","vorDoiUrl":"https://doi.org/10.1007/s10930-024-10226-9","workflowStages":[]},"version":"v1","identity":"rs-4354127","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4354127","identity":"rs-4354127","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}