Dabigatran increases thrombin generation by inhibiting protein S and FVa degradation

Dabigatran increases thrombin generation by inhibiting protein S and FVa degradation | 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 Dabigatran increases thrombin generation by inhibiting protein S and FVa degradation Chi Zhang, Mianxia Li, Weixiang Chen, Yue Zhang, Bin Jiang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1514109/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Low dose of dabigatran paradoxically increased thrombin generation (TG) through inhibition of protein C (PC) activation. Protein S (PS) is a co-factor in the activation of PC. However, the role of PS in the enhancement of TG has not been addressed. Methods: Firstly, we measured TG by calibrated automated thrombinography (CAT) and prothrombin fragments 1+2 (F 1+2 ) assays. Secondly, we assessed the coagulation and anticoagulation factors activity in normal plasma spiking with dabigatran. Then, free PS and PC activation were measured. Finally, heavy chain of FVa and its degradation products were detected by WB. Results: CAT assay showed that 70-141ng/mL dabigatran paradoxically increased TG in normal plasma. However, dabigatran suppressed TG in a concentration-dependent manner. F 1+2 assay showed the similar results. ELISA assay was not affected by clot methods. Interestingly, results from ELISA showed that dabigatran (2-566ng/mL) suppressed free PS level in normal plasma. Combined with WB results, dabigatran inhibited PS and subsequently suppressed FVa degradation. Conclusions: PS participated in the paradoxical enhancement of TG in normal plasma spiking with low concentrations of dabigatran. dabigatran protein S protein C thrombin generation prothrombin fragments 1+2. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction New oral anticoagulants are widely used to prevent thrombotic events in clinical practice [ 1 ]. Thrombin plays a central role in the clotting process by regulating blood coagulation cascade and platelet activation [ 2 ]. Hence, thrombin acts as an attractive target for anticoagulation. Dabigatran is a direct thrombin inhibitor, and it could overcome the limitations of vitamin K antagonists [ 3 ]. Dabigatran inhibits both thrombin and fibrin-bound thrombin through binding to the active site of thrombin [ 4 ]. Previous studies demonstrated that dabigatran and melagatran paradoxically increased thrombin generation (TG) [ 5 , 6 ]. In rat model, melagatran significantly increased TG leading to rebound hypercoagulability [ 7 ]. Kamisato et al.[ 8 ] demonstrated that paradoxical enhancement of TG might be due to the reduced degradation of FVa and FVIIIa. PC serves as an native serine protease zymogen under normal physiological conditions and can be activated by the thrombin-thrombomodulin (TM) complex [ 9 , 10 ]. Activated Protein C (APC) cleaves factor Va (FVa) at Arg306, Arg506, and Arg679 residues. The Arg306 cleavage requires the presence of protein S [ 11 ]. The fragment of FVa 307–506 indicates the complete loss of FVa activity [ 12 ]. Protein S (PS) is a vitamin K-dependent glycoprotein which plays a vital role in the blood coagulation. PS exists both in a free form (≈ 140nM) and in an inactive form [ 13 ]. PS functions as cofactor to activated protein C in the regulation of FVa and FVIIIa. Furthermore, previous study showed that PS inhibited FVa and FVIIIa activity in APC-independent manner [ 14 ]. Heterozygous PS deficiency increased the risk of thrombosis. PS acts as an important factor of PC system. However, the role of PS in the paradoxical enhancement of TG has not been well described. 2. Methods And Materials 2.1 Agents Dabigatran and dabigatran-d3 were synthesized at Toronto Research Chemicals (Toronto, Canada). Normal plasma was obtained from Boatman Biotech (Shanghai, China). PS- and PC-deficient plasmas and free PS ELISA kit were prepared by HYPHEN BioMed (Oise, France). Recombinant human soluble TM (rhs-TM) was from R&D Systems (North America) and dissolved in 0.9%NaCl. Polyclonal rabbit anti-human protein S antibody was obtained from Dako Cytomation (Carpinteria, CA, USA). Monoclonal mouse anti-human FV antibody and human protein S were from Haematologic Technologies (Essex Junction, VT). Monoclonal mouse anti-human IgG and D-Phe-Pro-Arg-chloromethylketone (PPACK) were purchased from Santa Cruz Biotechnology (Dallas, TX). PPP regent, FluCa, and thrombin calibrator were purchased from Thrombinoscope BV (Maastricht, The Netherlands). Pefabloc FG was from Pentapharm (Basel, Switzerland). Prothrombin fragment 1 + 2 (F 1 + 2 ) ELISA kit was from Dade Behring (Marburg, Germany). Activated PC-PC inhibitor complex (aPC-PCI) ELISA kit was from Affinity Biologicals. 2.2 Preparation of dabigatran Dabigatran was dissolved in 1N HCl and filled up to 1mL with distilled water. Different volumes of distilled water were added to make final concentrations of dabigatran (0.5, 2, 12, 35, 70, 141, 283 and 566nmol/mL). 2.3 Thrombin generation assay Calibrated automated thrombogram (CAT) method (Thrombinoscope, Maastricht, The Netherlands) was used to detect TG according to Morishima’s study [ 15 ]. Normal, PS-, and PC-deficient plasmas (76µL) were spiked with different concentrations (2µL) of dabigatran (n = 9) in the presence of rhs-TM (2µL). In control group, dabigatran was replaced with the same volume of solvent of dabigatran. Then, PPP reagent (20µL) was added to the plasma. After incubation at 37℃ for 5min, plasma with dabigatran was added to the TG wells and plasma without dabigatran was added to the thrombin calibrator wells. Flu-buffer incubated at 37℃ for 5min and Flu-substrate was mixed to generate FluCa. FluCa was automatically added to each well and results were read by a Fluoroskan Ascent fluorometer (Thermo Fisher Scientific, Waltham, MA). The final concentrations of tissue factor, phospholipids, fluorogenic substrate, and CaCl 2 were 5pM, 4µM, 417µM, and 16.7mM. TG curves were generated by the Thrombinoscope software (Thrombinoscope BV). 2.4 Prothrombin fragment 1 + 2 assay F 1 + 2 is a marker of TG. TG was induced according to the method described above with minor modifications. Normal, PS-, and PC-deficient plasmas (76µL) were spiked with different concentrations (2µL) of dabigatran (n = 6) in the presence of rhs-TM (2µL). The control group contained the appropriate vehicles. Plasma was defibrinated using 2µL PefablocFG (final concentration of 6mg/mL). The reaction was started by adding Fluo-buffer containing HEPES, calcium chloride and DMSO. PPACK was used to stop the coagulation reaction after 12min. The mixture (120µL) was transferred to microtiter plates and F 1 + 2 assessed following the manufacturer’s instructions. The intra-assay coefficient of variation (CV) was between 3.6–5.5% and the inter-CV was between 4.4–11.2%. 2.5 Free PS assay Plasma free PS was detected by an ELISA kit. Free PS level was measured following the manufacturer’s instructions. The intra-assay coefficient of variation (CV) was between 3–8% and the inter-CV was between 5–10%. 2.6 PC activation assay PC activation was detected by an aPC-PCI ELISA kit. TG was induced as above and the reaction was stopped 15min after the time to peak in the TG assay by adding 13µL of 0.5M sodium citrate buffer (pH4.3). The assay was performed according to the manufacturer’s instructions. The intra-assay coefficient of variation (CV) was between 3.6–5.5% and the inter-CV was between 4.4–11.2%. 2.7 FV, FVIII, PS, and PC activity Normal and PS-deficient plasmas were spiked with different concentrations of dabigatran in the presence of rhs-TM. Plasma samples were directly loaded in the STA® Compact Max (Diagnostic Stago) using the same batches of reagents. Plasma samples (5µL) were automatically diluted with 45µL Owren-Koller buffer. Coagulation was induced. FV activity was detected by normalization of PT in FV-deficient plasma (50µL). FVIII activity was measured by normalization of APTT in FVIII-deficient plasma (50µL). PC activity in samples (50µL) was detected based on the prolongation of APTT in PC-deficient plasma (50µL). PS activity in samples (50µL) was measured based on the prolongation of clotting time in PS-deficient plasma (50µL). 2.8 Western blot analysis TG was induced as described above in plasma without Fluca. Several reaction time points were set up in PS-deficient plasma and normal plasma spiked with or without 70ng/mL dabigatran according to CAT results. The reaction was stopped with 13µL of 0.5M sodium citrate buffer (pH4.3). FVa HC and its degradation product FVa 307–506 were detected by FV antibody (2.5µg/mL). IgG (dilution 1:1000) was used as the loading control. 2.9 Statistics Statistical analysis was completed with Stata 14.0. All data were expressed as mean ± standard deviation (SD). Unpaired parametric t test or one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was used for data analysis. P < 0.05 indicated a significant difference. 3. Results 3.1 Effect of dabigatran on TG We measured TG by CAT assay in plasma spiking with different concentrations of dabigatran (n = 9). Figure 1 A showed that there was a gradual dose-dependent increase of TG in normal plasma. Interestingly, the paradoxical enhancement of TG was absent in PS- or PC-deficient plasma (Fig. 1 B, C). We analysed important indices including peak thrombin generation (C max ) and endogenous thrombin potential (ETP) from CAT assay. Low concentrations of dabigatran (35-141ng/mL) significantly increased C max in normal plasma (Fig. 1 D). In normal plasma spiking with 70-141ng/mL dabigatran, there was a gradual dose-dependent increase of ETP (Fig. 1 E). Higher concentrations of dabigatran (283ng/mL) suppressed ETP in normal, PS- or PC-deficient plasma (104.28 ± 20.28, 175.86 ± 13.96, 178.6 ± 13.94mmol L − 1 thrombin×min). C max and ETP reached a maximum in the presence of 70ng/mL dabigatran. 3.2 Effect of dabigatran on F1 + 2 assay F 1 + 2 assay is another method to assess TG. F 1 + 2 assay showed results similar to CAT assay (n = 6) (Fig. 2 ). In normal plasma, 70ng/mL dabigatran paradoxically significantly increased F 1 + 2 from 289.4 ± 136.9 to 435.3 ± 115.8nmol/L. There was an enhancement in TG which decreased at higher concentrations. In PS- or PC-deficient plasma, the paradoxical enhancement of TG was absent. Higher concentrations of dabigatran significantly suppressed the generation of F 1 + 2 . F 1 + 2 decreased from 589.3 ± 101.6 to 290.5 ± 127.2nmol/L or from 600.7 ± 209.4 to 305.6 ± 98.1nmol/L in PS- or PC-deficient plasma in the presence of 141ng/mL dabigatran. 3.3 Effect of dabigatran on FV, FVIII, PS, PC activity, free PS, and APC levels We used clot methods to evaluate the effect of dabigatran on FV, FVIII, PS and PC activity (n = 6) There was a gradual concentration-dependent decrease of FV and FVIII activity. FV and FVIII activity significantly decreased at 283 and 70ng/mL dabigatran in normal plasma. PS and PC activity increased with the enhancement of dabigatran concentrations. PS and PC activity increased above 15% at 12 and 141ng/mL dabigatran in normal plasma (Fig. 3 A). In PS-deficient plasma, the activity of FV, FVIII and PC was similar to that in normal plasma. FV and FVIII activity significantly decreased at 283ng/mL and 141ng/mL dabigatran. PC activity increased at 141ng/mL dabigatran (Fig. 3 B). Furthermore, we evaluated free PS and APC levels by ELISA kit. Interestingly, dabigatran could affect free PS levels. Free PS was initially suppressed by 2ng/mL dabigatran (Fig. 3 C). APC was significantly suppressed at 70ng/mL dabigatran in both normal and PS-deficient plasmas (Fig. 3 D-E). In normal plasma, the concentrations of dabigatran for inhibition of free PS and PC activation were almost the same as for enhancement of TG. In PS-deficient plasma, free PS levels can’t be detected. Dabigatran dose-dependently inhibited TG and APC. 3.4 Effect of dabigatran on the generation and degradation FVa HC and FVa 307–506 are indices of FVa generation and inactivation (n = 3). In normal plasma, FVa HC increased with time, peaking around the lag time of CAT assay and then decreased. In contrast, FVa 307–506 increased along with the proteolysis of FVa HC (Fig. 4 A, D). Interestingly, FVa 307–506 was not observed in PS-deficient plasma. FVa HC was sustained during this period (Fig. 4 B, E). In normal plasma spiking with 70ng/mL dabigatran, the lag time was prolonged and FVa HC increased around the lag time. Dabigatran significantly delayed the degradation of FVa (Fig. 4 C, F). 3.5 Effect of anti-PS and PS on CAT assay In normal plasma, CAT assay showed that anti-PS neutralizing antibody could increase TG (Fig. 5 A). In normal plasma spiking 70ng/mL dabigatran, PS concentrations dependently reversed the enhancement of TG (Fig. 5 B). 4. Discussion This study showed that PS participated in the paradoxical enhancement of TG in plasma spiking with low concentrations of dabigatran. Dabigatran increased thrombin generation by inhibiting protein S and FVa degradation. Previous study demonstrated that low concentrations of dabigatran paradoxically increased TG in PC-dependent manner [ 8 ]. Dabigatran inhibited thrombomodulin-bound thrombin more aggressively than thrombin at low concentrations resulting in the decrease of PC activation and FVa degradation. Our previous study speculated that higher concentrations of dabigatran may sufficiently suppress thrombin and coagulation cascade [ 16 ]. PS is a cofactor to activated protein C. PS could cleave FVa and FVIIIa in PC dependent and independent manners [ 11 ]. The effect of PS on the paradoxical enhancement of TG has not been well addressed. It is known that a transient hypercoagulable state occurs within the first 12-60h of warfarin treatment because of inhibition of PC and PS [ 17 ]. Previous study showed that low concentrations of dabigatran paradoxically increased TG. We aimed to disclosure whether the paradoxical enhancement of TG induced by low concentrations of dabigatran depends on PS. PS is a vitamin K-dependent glycoprotein that plays vital role in blood coagulation. PS circulates in plasma at a concentration of around 350nM which exists both in a free form (≈ 140nM) and in an inactive form (~ 60%) combined with the complement regulatory factor C4b-binding protein (C4BP) [ 13 ]. Only the free PS exerts co-factor activity. Hence, the detection of free PS but not total PS was applied in the study [ 18 ]. Firstly, TG was assessed by CAT assay in normal, PS- or PC-deficient plasma containing 10nM rhs-TM. It has been previously shown that 10nM rhs-TM added to the plasma was appropriate to mimic the condition in vivo [ 19 ]. Blood coagulation cascade includes two pathways, one of which is contact factor-dependent intrinsic pathway and the other is TF-induced extrinsic pathway. In most cases, TG was assessed in coagulation reaction induced by extrinsic pathway. In our study, final concentration of tissue factor, phospholipids, fluorogenic substrate, and CaCl 2 were 5pM, 4µM, 417µM, and 16.7mM. Under these circumstances, coagulation cascade process could be induced completely. Wagenvoord et al. [ 20 ] reported that the paradoxical enhancement of TG induced by low concentrations of dabigatran may be caused by the presence of α 2 -macroglobulin-thrombin (α 2 MT) complex. CAT assay uses an algorithm to subtract α 2 MT activity from the total amidolytic activity. The transient enhancement of α 2 MT induced by dabigatran could not be subtracted which leads to a false increase of TG. However, there is study showing different opinion. Study reported α 2 M may not participate in the enhancement of TG [ 21 ]. CAT assay showed that low concentrations of dabigatran paradoxically induced TG in normal plasma. Higher concentrations of dabigatran may sufficiently suppress thrombin and coagulation cascade. Secondly, TG was assessed by F 1 + 2 assay under the same circumstance. F 1 + 2 is also a marker of TG. Our results showed that F 1 + 2 increased in normal plasma spiking with 70ng/mL dabigatran. The paradoxical enhancement of F 1 + 2 was absent in PS deficient plasma. The results are inconsistent between assays. However, the trend of CAT and F 1 + 2 assays is same. Previous study showed that TG increased at 136-545ng/ml dabigatran. But only 273ng/ml dabigatran increased F 1 + 2 [ 22 ]. Our results are similar to other study. Furthermore, FV, FVIII, PS and PC activity was measured to assess the haemostatic situation in clotting blood using the same batches of reagents. Previous study showed different batches of reagents and different coagulometers could lead to highly variable results [ 23 ]. Dabigatran dose-dependent decreased FV and FVIII activity and increased PS and PC activity. It has been reported dabigatran would affect APTT more than PT. FV activity is measured by normalization of PT. Whereas FVIII and PC activity is associated with APTT. Hence, FVIII and PC activity would be more affected than FV. The activity of coagulation and anticoagulation factors using clot methods may be adversely affected by dabigatran [ 24 ]. To avoid the influence by dabigatran, free PS and aPC-PCI ELISA were used. It is well known that dabigatran is a direct thrombin inhibitor. Interestingly, dabigatran could inhibit free PS levels. In normal plasma, the concentrations of dabigatran for inhibition of free PS and PC activation were almost the same as for enhancement of TG. Except for PC, participates in the paradoxical enhancement of TG in plasma spiking with low concentrations of dabigatran. Last but not least, we used WB assay to evaluate the generation and degradation of FVa. In normal plasma, FVa 307–506 increased in line with the decrease of FVa HC . In PS-deficient plasma, the degradation of FVa was absolutely suppressed. PS plays an important role in the degradation of FVa. In normal plasma spiking with 70ng/mL dabigatran, the generation of FVa was not affected. However, the degradation of FVa was inhibited. We speculated low concentrations of dabigatran suppressed PS and APC more than coagulation cascade leading the paradoxical enhancement of TG. This study still has some limitations. In one hand, most parts of our results are based on CAT assay. It has been reported α 2 MT would affect the results. What is more, there may be some confounding factors affect the results. In the other hand, this study performed in vitro . Although the concentrations of dabigatran used in vitro are close to the condition in vivo , the results may not reflect the actually situation in vivo . It would be more interesting to perform the experiments in patients. Declarations Funding This work was supported by Suzhou "Promoting Health through Science and Education" Youth Science and Technology Project (KJXW2019004). Competing Interests The authors state that they have no conflict of interest. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contributions Chi Zhang and Mianxian Li performed the research; Bin Jiang designed the research study; Yue Zhang contributed essential reagents or tools; Weixiang Chen analysed the data；Chi Zhang wrote the paper. Ethics approval This article does not contain any studies with human participants or animals performed by any of the authors. References Tsai CT, Liao JN, Chen SJ et al (2021) Non-vitamin K antagonist oral anticoagulants versus warfarin in AF patients >/= 85 years. Eur J Clin Invest e13488. doi: 10.1111/eci.13488 Wang X, Xu Y, Li L et al (2021) Thrombin Aggravates Hypoxia/Reoxygenation Injury of Cardiomyocytes by Activating an Autophagy Pathway-Mediated by SIRT1. 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Blood transfusion = Trasfusione del sangue 15(6):491–494. doi: 10.2450/2017.0301-16 Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-1514109","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":96019163,"identity":"e81ad005-3a54-4826-81f5-d3cbb3d984ff","order_by":0,"name":"Chi Zhang","email":"","orcid":"","institution":"First Affiliated Hospital of Soochow University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chi","middleName":"","lastName":"Zhang","suffix":""},{"id":96019164,"identity":"7caea5ed-73d8-4d70-a39c-80f53a081b26","order_by":1,"name":"Mianxia Li","email":"","orcid":"","institution":"First Affiliated Hospital of Soochow University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mianxia","middleName":"","lastName":"Li","suffix":""},{"id":96019165,"identity":"e69efa0d-49aa-49bd-a5c2-6c6faccd9a70","order_by":2,"name":"Weixiang Chen","email":"","orcid":"","institution":"First Affiliated Hospital of Soochow University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weixiang","middleName":"","lastName":"Chen","suffix":""},{"id":96019166,"identity":"b7a7ec63-3840-4c25-a180-d1e2dbc241de","order_by":3,"name":"Yue Zhang","email":"","orcid":"","institution":"First Affiliated Hospital of Soochow University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Zhang","suffix":""},{"id":96019167,"identity":"c8474b62-aa88-4fc7-afc5-22c1a1a828af","order_by":4,"name":"Bin Jiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYBACA2YgIWEAJJj5Hxz4UCEhJ0+8FvYexoMzzlgYGzYQ0gJn8ZxhPszbVpHIcICAFnN23gMMFgV2efIRuQcO8M6TSGBsYH746AYeLZbNfAlAhyUXG97ISzgguU0ij52Bzdg4B5/DDvMYALUwJ26ckWBwwHCbRDFjAw+bNBFa6iFaEudIJDYcIE7L4cT5PGcMDhxsIEKLZTNYy/HEDextCQcbjkkYGzYT8Is5/xkDZok/1Ynzm5kPf/5TUycnz9788DE+LUDA/lsC5MIDMD4zfuVgwPgBSMg3EKFyFIyCUTAKRiYAAPrSSp4iJMKgAAAAAElFTkSuQmCC","orcid":"","institution":"First Affiliated Hospital of Soochow University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Jiang","suffix":""}],"badges":[],"createdAt":"2022-04-01 15:20:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1514109/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1514109/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":20032526,"identity":"f5bd605b-986f-420f-be4f-f1b4dbd98766","added_by":"auto","created_at":"2022-04-06 16:44:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":134161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of dabigatran on TG. \u003c/strong\u003eDifferent concentrations of dabigatran (0-566ng/mL) affected TG in normal, PS- or PC deficient plasma (A-C). The expression of C\u003csub\u003emax\u003c/sub\u003e and ETP in normal, PS- or PC deficient plasma spiking with different concentrations of dabigatran (D-E). * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 vs. respective controls\u003c/p\u003e","description":"","filename":"OnlineFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-1514109/v1/1fc975081ad5a62792d89f13.png"},{"id":20032524,"identity":"ad75871c-98f3-4ec9-b301-e62d8291058c","added_by":"auto","created_at":"2022-04-06 16:44:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":49736,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of dabigatran on F\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1+2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e assay\u003c/strong\u003e. Low concentrations of dabigatran dose-dependent increased\u003cstrong\u003e \u003c/strong\u003eF\u003csub\u003e1+2\u003c/sub\u003e in normal plasma. In PS- or PC-deficient plasma, the paradoxical enhancement of TG was absent. * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 vs. respective controls\u003c/p\u003e","description":"","filename":"OnlineFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-1514109/v1/0f8f04e06c70cc24fb194b8e.png"},{"id":20032527,"identity":"fdd17b1c-1771-4ded-9652-1176ed6c53ec","added_by":"auto","created_at":"2022-04-06 16:44:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":113006,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of dabigatran on FV, FVIII, PS, PC activity, free PS, and APC levels.\u003c/strong\u003e Dabigatran dose-dependent decreased FV and FVIII activity and increased PC and PS activity (A-B). ELISA assay showed dabigatran suppressed free PS and PC activation in normal The concentrations of dabigatran for inhibition of free PS and PC activation were almost the same as for enhancement of TG (C-E). * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 vs. respective controls\u003c/p\u003e","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-1514109/v1/42a88fd13f6b3811cc01fc96.png"},{"id":20033504,"identity":"63c09391-ad33-4ca1-ac3a-0ffe905b6510","added_by":"auto","created_at":"2022-04-06 16:54:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":123486,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of dabigatran on the generation and degradation.\u003c/strong\u003e The expression of FVa\u003csup\u003eHC\u003c/sup\u003e and FVa\u003csup\u003e307-506\u003c/sup\u003e levels by WB assay after the activation of the coagulation cascade in normal plasma (A, D), PS-deficient plasma (B, E), and normal plasma spiking with 70ng/mL dabigatran (C, F). * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 vs. respective controls\u003c/p\u003e","description":"","filename":"OnlineFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-1514109/v1/a130f3d3f765d7aa9a46405a.png"},{"id":20033206,"identity":"3d52f841-0886-42c4-a25c-0a8305b37f0f","added_by":"auto","created_at":"2022-04-06 16:49:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":78629,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of anti-PS and PS on CAT assay\u003c/strong\u003e. Anti-PS neutralizing antibody could increase TG (A). In normal plasma spiking 70ng/mL dabigatran, PS concentrations dependently reversed the enhancement of TG (B)\u003c/p\u003e","description":"","filename":"OnlineFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-1514109/v1/15bb7b6b68f5ab7304458b98.png"},{"id":20033505,"identity":"bf23cb19-8d74-4e93-a91c-11733baed7a1","added_by":"auto","created_at":"2022-04-06 16:54:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":993842,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1514109/v1/25e4377a-730d-401d-b730-d6daf9fd7099.pdf"}],"financialInterests":"","formattedTitle":"Dabigatran increases thrombin generation by inhibiting protein S and FVa degradation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNew oral anticoagulants are widely used to prevent thrombotic events in clinical practice [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Thrombin plays a central role in the clotting process by regulating blood coagulation cascade and platelet activation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Hence, thrombin acts as an attractive target for anticoagulation. Dabigatran is a direct thrombin inhibitor, and it could overcome the limitations of vitamin K antagonists [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Dabigatran inhibits both thrombin and fibrin-bound thrombin through binding to the active site of thrombin [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious studies demonstrated that dabigatran and melagatran paradoxically increased thrombin generation (TG) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In rat model, melagatran significantly increased TG leading to rebound hypercoagulability [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Kamisato et al.[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] demonstrated that paradoxical enhancement of TG might be due to the reduced degradation of FVa and FVIIIa. PC serves as an native serine protease zymogen under normal physiological conditions and can be activated by the thrombin-thrombomodulin (TM) complex [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Activated Protein C (APC) cleaves factor Va (FVa) at Arg306, Arg506, and Arg679 residues. The Arg306 cleavage requires the presence of protein S [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The fragment of FVa\u003csup\u003e307\u0026ndash;506\u003c/sup\u003e indicates the complete loss of FVa activity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eProtein S (PS) is a vitamin K-dependent glycoprotein which plays a vital role in the blood coagulation. PS exists both in a free form (\u0026asymp;\u0026thinsp;140nM) and in an inactive form [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. PS functions as cofactor to activated protein C in the regulation of FVa and FVIIIa. Furthermore, previous study showed that PS inhibited FVa and FVIIIa activity in APC-independent manner [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Heterozygous PS deficiency increased the risk of thrombosis. PS acts as an important factor of PC system. However, the role of PS in the paradoxical enhancement of TG has not been well described.\u003c/p\u003e"},{"header":"2. Methods And Materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Agents\u003c/h2\u003e \u003cp\u003eDabigatran and dabigatran-d3 were synthesized at Toronto Research Chemicals (Toronto, Canada). Normal plasma was obtained from Boatman Biotech (Shanghai, China). PS- and PC-deficient plasmas and free PS ELISA kit were prepared by HYPHEN BioMed (Oise, France). Recombinant human soluble TM (rhs-TM) was from R\u0026amp;D Systems (North America) and dissolved in 0.9%NaCl. Polyclonal rabbit anti-human protein S antibody was obtained from Dako Cytomation (Carpinteria, CA, USA). Monoclonal mouse anti-human FV antibody and human protein S were from Haematologic Technologies (Essex Junction, VT). Monoclonal mouse anti-human IgG and D-Phe-Pro-Arg-chloromethylketone (PPACK) were purchased from Santa Cruz Biotechnology (Dallas, TX). PPP regent, FluCa, and thrombin calibrator were purchased from Thrombinoscope BV (Maastricht, The Netherlands). Pefabloc FG was from Pentapharm (Basel, Switzerland). Prothrombin fragment 1\u0026thinsp;+\u0026thinsp;2 (F\u003csub\u003e1\u0026thinsp;+\u0026thinsp;2\u003c/sub\u003e) ELISA kit was from Dade Behring (Marburg, Germany). Activated PC-PC inhibitor complex (aPC-PCI) ELISA kit was from Affinity Biologicals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of dabigatran\u003c/h2\u003e \u003cp\u003eDabigatran was dissolved in 1N HCl and filled up to 1mL with distilled water. Different volumes of distilled water were added to make final concentrations of dabigatran (0.5, 2, 12, 35, 70, 141, 283 and 566nmol/mL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Thrombin generation assay\u003c/h2\u003e \u003cp\u003eCalibrated automated thrombogram (CAT) method (Thrombinoscope, Maastricht, The Netherlands) was used to detect TG according to Morishima\u0026rsquo;s study [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Normal, PS-, and PC-deficient plasmas (76\u0026micro;L) were spiked with different concentrations (2\u0026micro;L) of dabigatran (n\u0026thinsp;=\u0026thinsp;9) in the presence of rhs-TM (2\u0026micro;L). In control group, dabigatran was replaced with the same volume of solvent of dabigatran. Then, PPP reagent (20\u0026micro;L) was added to the plasma. After incubation at 37℃ for 5min, plasma with dabigatran was added to the TG wells and plasma without dabigatran was added to the thrombin calibrator wells. Flu-buffer incubated at 37℃ for 5min and Flu-substrate was mixed to generate FluCa. FluCa was automatically added to each well and results were read by a Fluoroskan Ascent fluorometer (Thermo Fisher Scientific, Waltham, MA). The final concentrations of tissue factor, phospholipids, fluorogenic substrate, and CaCl\u003csub\u003e2\u003c/sub\u003e were 5pM, 4\u0026micro;M, 417\u0026micro;M, and 16.7mM. TG curves were generated by the Thrombinoscope software (Thrombinoscope BV).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Prothrombin fragment 1\u0026thinsp;+\u0026thinsp;2 assay\u003c/h2\u003e \u003cp\u003eF\u003csub\u003e1\u0026thinsp;+\u0026thinsp;2\u003c/sub\u003e is a marker of TG. TG was induced according to the method described above with minor modifications. Normal, PS-, and PC-deficient plasmas (76\u0026micro;L) were spiked with different concentrations (2\u0026micro;L) of dabigatran (n\u0026thinsp;=\u0026thinsp;6) in the presence of rhs-TM (2\u0026micro;L). The control group contained the appropriate vehicles. Plasma was defibrinated using 2\u0026micro;L PefablocFG (final concentration of 6mg/mL). The reaction was started by adding Fluo-buffer containing HEPES, calcium chloride and DMSO. PPACK was used to stop the coagulation reaction after 12min. The mixture (120\u0026micro;L) was transferred to microtiter plates and F\u003csub\u003e1\u0026thinsp;+\u0026thinsp;2\u003c/sub\u003e assessed following the manufacturer\u0026rsquo;s instructions. The intra-assay coefficient of variation (CV) was between 3.6\u0026ndash;5.5% and the inter-CV was between 4.4\u0026ndash;11.2%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Free PS assay\u003c/h2\u003e \u003cp\u003ePlasma free PS was detected by an ELISA kit. Free PS level was measured following the manufacturer\u0026rsquo;s instructions. The intra-assay coefficient of variation (CV) was between 3\u0026ndash;8% and the inter-CV was between 5\u0026ndash;10%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 PC activation assay\u003c/h2\u003e \u003cp\u003ePC activation was detected by an aPC-PCI ELISA kit. TG was induced as above and the reaction was stopped 15min after the time to peak in the TG assay by adding 13\u0026micro;L of 0.5M sodium citrate buffer (pH4.3). The assay was performed according to the manufacturer\u0026rsquo;s instructions. The intra-assay coefficient of variation (CV) was between 3.6\u0026ndash;5.5% and the inter-CV was between 4.4\u0026ndash;11.2%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 FV, FVIII, PS, and PC activity\u003c/h2\u003e \u003cp\u003eNormal and PS-deficient plasmas were spiked with different concentrations of dabigatran in the presence of rhs-TM. Plasma samples were directly loaded in the STA\u0026reg; Compact Max (Diagnostic Stago) using the same batches of reagents. Plasma samples (5\u0026micro;L) were automatically diluted with 45\u0026micro;L Owren-Koller buffer. Coagulation was induced. FV activity was detected by normalization of PT in FV-deficient plasma (50\u0026micro;L). FVIII activity was measured by normalization of APTT in FVIII-deficient plasma (50\u0026micro;L). PC activity in samples (50\u0026micro;L) was detected based on the prolongation of APTT in PC-deficient plasma (50\u0026micro;L). PS activity in samples (50\u0026micro;L) was measured based on the prolongation of clotting time in PS-deficient plasma (50\u0026micro;L).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Western blot analysis\u003c/h2\u003e \u003cp\u003eTG was induced as described above in plasma without Fluca. Several reaction time points were set up in PS-deficient plasma and normal plasma spiked with or without 70ng/mL dabigatran according to CAT results. The reaction was stopped with 13\u0026micro;L of 0.5M sodium citrate buffer (pH4.3). FVa\u003csup\u003eHC\u003c/sup\u003e and its degradation product FVa\u003csup\u003e307\u0026ndash;506\u003c/sup\u003e were detected by FV antibody (2.5\u0026micro;g/mL). IgG (dilution 1:1000) was used as the loading control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistics\u003c/h2\u003e \u003cp\u003eStatistical analysis was completed with Stata 14.0. All data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Unpaired parametric t test or one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s multiple comparison test was used for data analysis. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated a significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effect of dabigatran on TG\u003c/h2\u003e \u003cp\u003eWe measured TG by CAT assay in plasma spiking with different concentrations of dabigatran (n\u0026thinsp;=\u0026thinsp;9). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA showed that there was a gradual dose-dependent increase of TG in normal plasma. Interestingly, the paradoxical enhancement of TG was absent in PS- or PC-deficient plasma (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). We analysed important indices including peak thrombin generation (C\u003csub\u003emax\u003c/sub\u003e) and endogenous thrombin potential (ETP) from CAT assay. Low concentrations of dabigatran (35-141ng/mL) significantly increased C\u003csub\u003emax\u003c/sub\u003e in normal plasma (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). In normal plasma spiking with 70-141ng/mL dabigatran, there was a gradual dose-dependent increase of ETP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Higher concentrations of dabigatran (283ng/mL) suppressed ETP in normal, PS- or PC-deficient plasma (104.28\u0026thinsp;\u0026plusmn;\u0026thinsp;20.28, 175.86\u0026thinsp;\u0026plusmn;\u0026thinsp;13.96, 178.6\u0026thinsp;\u0026plusmn;\u0026thinsp;13.94mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e thrombin\u0026times;min). C\u003csub\u003emax\u003c/sub\u003e and ETP reached a maximum in the presence of 70ng/mL dabigatran.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effect of dabigatran on F1\u0026thinsp;+\u0026thinsp;2 assay\u003c/h2\u003e \u003cp\u003eF\u003csub\u003e1\u0026thinsp;+\u0026thinsp;2\u003c/sub\u003e assay is another method to assess TG. F\u003csub\u003e1\u0026thinsp;+\u0026thinsp;2\u003c/sub\u003e assay showed results similar to CAT assay (n\u0026thinsp;=\u0026thinsp;6) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In normal plasma, 70ng/mL dabigatran paradoxically significantly increased F\u003csub\u003e1\u0026thinsp;+\u0026thinsp;2\u003c/sub\u003e from 289.4\u0026thinsp;\u0026plusmn;\u0026thinsp;136.9 to 435.3\u0026thinsp;\u0026plusmn;\u0026thinsp;115.8nmol/L. There was an enhancement in TG which decreased at higher concentrations. In PS- or PC-deficient plasma, the paradoxical enhancement of TG was absent. Higher concentrations of dabigatran significantly suppressed the generation of F\u003csub\u003e1\u0026thinsp;+\u0026thinsp;2\u003c/sub\u003e. F\u003csub\u003e1\u0026thinsp;+\u0026thinsp;2\u003c/sub\u003e decreased from 589.3\u0026thinsp;\u0026plusmn;\u0026thinsp;101.6 to 290.5\u0026thinsp;\u0026plusmn;\u0026thinsp;127.2nmol/L or from 600.7\u0026thinsp;\u0026plusmn;\u0026thinsp;209.4 to 305.6\u0026thinsp;\u0026plusmn;\u0026thinsp;98.1nmol/L in PS- or PC-deficient plasma in the presence of 141ng/mL dabigatran.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effect of dabigatran on FV, FVIII, PS, PC activity, free PS, and APC levels\u003c/h2\u003e \u003cp\u003eWe used clot methods to evaluate the effect of dabigatran on FV, FVIII, PS and PC activity (n\u0026thinsp;=\u0026thinsp;6) There was a gradual concentration-dependent decrease of FV and FVIII activity. FV and FVIII activity significantly decreased at 283 and 70ng/mL dabigatran in normal plasma. PS and PC activity increased with the enhancement of dabigatran concentrations. PS and PC activity increased above 15% at 12 and 141ng/mL dabigatran in normal plasma (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In PS-deficient plasma, the activity of FV, FVIII and PC was similar to that in normal plasma. FV and FVIII activity significantly decreased at 283ng/mL and 141ng/mL dabigatran. PC activity increased at 141ng/mL dabigatran (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Furthermore, we evaluated free PS and APC levels by ELISA kit. Interestingly, dabigatran could affect free PS levels. Free PS was initially suppressed by 2ng/mL dabigatran (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). APC was significantly suppressed at 70ng/mL dabigatran in both normal and PS-deficient plasmas (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E). In normal plasma, the concentrations of dabigatran for inhibition of free PS and PC activation were almost the same as for enhancement of TG. In PS-deficient plasma, free PS levels can\u0026rsquo;t be detected. Dabigatran dose-dependently inhibited TG and APC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effect of dabigatran on the generation and degradation\u003c/h2\u003e \u003cp\u003eFVa\u003csup\u003eHC\u003c/sup\u003e and FVa\u003csup\u003e307\u0026ndash;506\u003c/sup\u003e are indices of FVa generation and inactivation (n\u0026thinsp;=\u0026thinsp;3). In normal plasma, FVa\u003csup\u003eHC\u003c/sup\u003e increased with time, peaking around the lag time of CAT assay and then decreased. In contrast, FVa\u003csup\u003e307\u0026ndash;506\u003c/sup\u003e increased along with the proteolysis of FVa\u003csup\u003eHC\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, D). Interestingly, FVa\u003csup\u003e307\u0026ndash;506\u003c/sup\u003e was not observed in PS-deficient plasma. FVa\u003csup\u003eHC\u003c/sup\u003e was sustained during this period (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, E). In normal plasma spiking with 70ng/mL dabigatran, the lag time was prolonged and FVa\u003csup\u003eHC\u003c/sup\u003e increased around the lag time. Dabigatran significantly delayed the degradation of FVa (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Effect of anti-PS and PS on CAT assay\u003c/h2\u003e \u003cp\u003eIn normal plasma, CAT assay showed that anti-PS neutralizing antibody could increase TG (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In normal plasma spiking 70ng/mL dabigatran, PS concentrations dependently reversed the enhancement of TG (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study showed that PS participated in the paradoxical enhancement of TG in plasma spiking with low concentrations of dabigatran. Dabigatran increased thrombin generation by inhibiting protein S and FVa degradation. Previous study demonstrated that low concentrations of dabigatran paradoxically increased TG in PC-dependent manner [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Dabigatran inhibited thrombomodulin-bound thrombin more aggressively than thrombin at low concentrations resulting in the decrease of PC activation and FVa degradation. Our previous study speculated that higher concentrations of dabigatran may sufficiently suppress thrombin and coagulation cascade [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. PS is a cofactor to activated protein C. PS could cleave FVa and FVIIIa in PC dependent and independent manners [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The effect of PS on the paradoxical enhancement of TG has not been well addressed.\u003c/p\u003e \u003cp\u003eIt is known that a transient hypercoagulable state occurs within the first 12-60h of warfarin treatment because of inhibition of PC and PS [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Previous study showed that low concentrations of dabigatran paradoxically increased TG. We aimed to disclosure whether the paradoxical enhancement of TG induced by low concentrations of dabigatran depends on PS. PS is a vitamin K-dependent glycoprotein that plays vital role in blood coagulation. PS circulates in plasma at a concentration of around 350nM which exists both in a free form (\u0026asymp;\u0026thinsp;140nM) and in an inactive form (~\u0026thinsp;60%) combined with the complement regulatory factor C4b-binding protein (C4BP) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Only the free PS exerts co-factor activity. Hence, the detection of free PS but not total PS was applied in the study [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFirstly, TG was assessed by CAT assay in normal, PS- or PC-deficient plasma containing 10nM rhs-TM. It has been previously shown that 10nM rhs-TM added to the plasma was appropriate to mimic the condition \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Blood coagulation cascade includes two pathways, one of which is contact factor-dependent intrinsic pathway and the other is TF-induced extrinsic pathway. In most cases, TG was assessed in coagulation reaction induced by extrinsic pathway. In our study, final concentration of tissue factor, phospholipids, fluorogenic substrate, and CaCl\u003csub\u003e2\u003c/sub\u003e were 5pM, 4\u0026micro;M, 417\u0026micro;M, and 16.7mM. Under these circumstances, coagulation cascade process could be induced completely. Wagenvoord et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] reported that the paradoxical enhancement of TG induced by low concentrations of dabigatran may be caused by the presence of α\u003csub\u003e2\u003c/sub\u003e-macroglobulin-thrombin (α\u003csub\u003e2\u003c/sub\u003eMT) complex. CAT assay uses an algorithm to subtract α\u003csub\u003e2\u003c/sub\u003eMT activity from the total amidolytic activity. The transient enhancement of α\u003csub\u003e2\u003c/sub\u003eMT induced by dabigatran could not be subtracted which leads to a false increase of TG. However, there is study showing different opinion. Study reported α\u003csub\u003e2\u003c/sub\u003eM may not participate in the enhancement of TG [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. CAT assay showed that low concentrations of dabigatran paradoxically induced TG in normal plasma. Higher concentrations of dabigatran may sufficiently suppress thrombin and coagulation cascade.\u003c/p\u003e \u003cp\u003eSecondly, TG was assessed by F\u003csub\u003e1\u0026thinsp;+\u0026thinsp;2\u003c/sub\u003e assay under the same circumstance. F\u003csub\u003e1\u0026thinsp;+\u0026thinsp;2\u003c/sub\u003e is also a marker of TG. Our results showed that F\u003csub\u003e1\u0026thinsp;+\u0026thinsp;2\u003c/sub\u003e increased in normal plasma spiking with 70ng/mL dabigatran. The paradoxical enhancement of F\u003csub\u003e1\u0026thinsp;+\u0026thinsp;2\u003c/sub\u003e was absent in PS deficient plasma. The results are inconsistent between assays. However, the trend of CAT and F\u003csub\u003e1\u0026thinsp;+\u0026thinsp;2\u003c/sub\u003e assays is same. Previous study showed that TG increased at 136-545ng/ml dabigatran. But only 273ng/ml dabigatran increased F\u003csub\u003e1\u0026thinsp;+\u0026thinsp;2\u003c/sub\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Our results are similar to other study.\u003c/p\u003e \u003cp\u003eFurthermore, FV, FVIII, PS and PC activity was measured to assess the haemostatic situation in clotting blood using the same batches of reagents. Previous study showed different batches of reagents and different coagulometers could lead to highly variable results [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Dabigatran dose-dependent decreased FV and FVIII activity and increased PS and PC activity. It has been reported dabigatran would affect APTT more than PT. FV activity is measured by normalization of PT. Whereas FVIII and PC activity is associated with APTT. Hence, FVIII and PC activity would be more affected than FV. The activity of coagulation and anticoagulation factors using clot methods may be adversely affected by dabigatran [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. To avoid the influence by dabigatran, free PS and aPC-PCI ELISA were used. It is well known that dabigatran is a direct thrombin inhibitor. Interestingly, dabigatran could inhibit free PS levels. In normal plasma, the concentrations of dabigatran for inhibition of free PS and PC activation were almost the same as for enhancement of TG. Except for PC, participates in the paradoxical enhancement of TG in plasma spiking with low concentrations of dabigatran.\u003c/p\u003e \u003cp\u003eLast but not least, we used WB assay to evaluate the generation and degradation of FVa. In normal plasma, FVa\u003csup\u003e307\u0026ndash;506\u003c/sup\u003e increased in line with the decrease of FVa\u003csup\u003eHC\u003c/sup\u003e. In PS-deficient plasma, the degradation of FVa was absolutely suppressed. PS plays an important role in the degradation of FVa. In normal plasma spiking with 70ng/mL dabigatran, the generation of FVa was not affected. However, the degradation of FVa was inhibited. We speculated low concentrations of dabigatran suppressed PS and APC more than coagulation cascade leading the paradoxical enhancement of TG.\u003c/p\u003e \u003cp\u003eThis study still has some limitations. In one hand, most parts of our results are based on CAT assay. It has been reported α\u003csub\u003e2\u003c/sub\u003eMT would affect the results. What is more, there may be some confounding factors affect the results. In the other hand, this study performed in \u003cem\u003evitro\u003c/em\u003e. Although the concentrations of dabigatran used \u003cem\u003ein vitro\u003c/em\u003e are close to the condition \u003cem\u003ein vivo\u003c/em\u003e, the results may not reflect the actually situation in \u003cem\u003evivo\u003c/em\u003e. It would be more interesting to perform the experiments in patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Suzhou \u0026quot;Promoting Health through Science and Education\u0026quot; Youth Science and Technology Project (KJXW2019004).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors state that they have no conflict of interest. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChi Zhang and Mianxian Li performed the research; Bin Jiang designed the research study; Yue Zhang contributed essential reagents or tools; Weixiang Chen analysed the data；Chi Zhang wrote the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTsai CT, Liao JN, Chen SJ et al (2021) Non-vitamin K antagonist oral anticoagulants versus warfarin in AF patients \u0026gt;/= 85 years. 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Blood transfusion = Trasfusione del sangue 15(6):491\u0026ndash;494. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2450/2017.0301-16\u003c/span\u003e\u003cspan address=\"10.2450/2017.0301-16\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"dabigatran, protein S, protein C, thrombin generation, prothrombin fragments 1+2.","lastPublishedDoi":"10.21203/rs.3.rs-1514109/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1514109/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Low dose of dabigatran paradoxically increased thrombin generation (TG) through inhibition of protein C (PC) activation. Protein S (PS) is a co-factor in the activation of PC. However, the role of PS in the enhancement of TG has not been addressed. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eFirstly, we measured TG by calibrated automated thrombinography (CAT) and prothrombin fragments 1+2 (F\u003csub\u003e1+2\u003c/sub\u003e) assays. Secondly, we assessed the coagulation and anticoagulation factors activity in normal plasma spiking with dabigatran. Then, free PS and PC activation were measured. Finally, heavy chain of FVa and its degradation products were detected by WB. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eCAT assay showed that 70-141ng/mL dabigatran paradoxically increased TG in normal plasma. However, dabigatran suppressed TG in a concentration-dependent manner. F\u003csub\u003e1+2\u003c/sub\u003e assay showed the similar results. ELISA assay was not affected by clot methods. Interestingly, results from ELISA showed that dabigatran (2-566ng/mL) suppressed free PS level in normal plasma. Combined with WB results, dabigatran inhibited PS and subsequently suppressed FVa degradation. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003ePS participated in the paradoxical enhancement of TG in normal plasma spiking with low concentrations of dabigatran.\u003c/p\u003e","manuscriptTitle":"Dabigatran increases thrombin generation by inhibiting protein S and FVa degradation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-04-06 16:44:16","doi":"10.21203/rs.3.rs-1514109/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c43887fd-add0-4a55-a08e-b76160355735","owner":[],"postedDate":"April 6th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-04-06T16:44:17+00:00","versionOfRecord":[],"versionCreatedAt":"2022-04-06 16:44:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1514109","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1514109","identity":"rs-1514109","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}