Hirudins and ornatins in five species of the genus Placobdella (Annelida: Hirudinea: Glossiphoniidae)

Hirudins and ornatins in five species of the genus Placobdella (Annelida: Hirudinea: Glossiphoniidae) | 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 Hirudins and ornatins in five species of the genus Placobdella (Annelida: Hirudinea: Glossiphoniidae) Raja Ben Ahmed, Robert Wolf, Gabriele Jedlitschky, Céline Tolksdorf, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8807632/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 Leeches of the genus Placobdella Blanchard, 1893 (Annelida: Rhynchobdellida: Glossiphoniidae) are mainly distributed across North and Central America, but at least two species are also present in the palearctic region: the European turtle leech Placobdella costata Müller, 1846, and the North African turtle leech Placobdella nabeulensis Ben Ahmed et al., 2023. All species of the genus are hematophagous and feed on vertebrates, but are known to preferentially target aquatic and semi-aquatic reptiles like turtles and snakes. Placobdella ornata Verrill, 1872, is the original source of ornatins, a class of potent platelet aggregation inhibitors. However, the whole repertoire of bioactive peptides, including anticoagulation factors remains largely underexplored for Placobdella , and functional characterizations of putative antithrombotics are scarce. Here we describe the genes and cDNAs that encode putative hirudins and ornatins in both of the palearctic and three American species of the genus Placobdella . A selection of putative hirudins and ornatins was recombinantly expressed and functionally characterized using appropriate coagulation and platelet aggregation assays, and our data confirm the expression of functional hirudins and ornatins in the studied leeches. In addition, our genetic analyses strongly support the hypothesis that blood feeding evolved only once in the evolutionary history of leeches. Placobdella hirudin ornatin blood coagulation platelet aggregation hematophagous leeches Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Leeches are segmented worms that form the subclass Hirudinea within the phylum Annelida (Phillips et al. 2019). Crudely, leeches are traditionally divided into two major clades based on the morphology of their feeding apparatus: Rhynchobdellida Blanchard, 1894, whose members possess a proboscis, and Arhynchobdellida Blanchard, 1894, without a proboscis. The former group includes the Glossiphoniidae Vaillant, 1890, jawless dorsoventrally flattened leeches that inhabit freshwater ecosystems. The latter group includes the Hirudiniformes Caballero, 1952, in which the feeding apparatus is usually characterized by the presence of jaws (Kuo and Lai 2019). Many leeches of both groups are hematophagous, whereas others are macrophagous or liquidosomatophagous (Lynggaard et al. 2022). The medicinal use of hematophagous leeches, consequently also termed medicinal leeches, traces far back in human history and was common in many civilizations throughout time (Elliot and Kutschera 2011; Abdualkader et al. 2013; Marchiori et al. 2024). However, mainly representatives of jawed leeches were and are regularly used for medical purposes, including the European medicinal leech Hirudo medicinalis Linnaeus, 1758, the Mediterranean medicinal leech Hirudo verbana Carena, 1820, the African medicinal leech Hirudo troctina Johnson, 1816, the Asian medicinal leeches Hirudo nipponia Whitman, 1886, and Hirudinaria manillensis Lesson, 1842, the North-American medicinal leech Macrobdella decora Say, 1824, or the Australian medicinal leech Richardsonianus australis Bosisto, 1859 (Kvist et al. 2013). By contrast, proboscis-bearing leeches are much less frequently considered for the direct use in medical practice, yet members of the Central and South American genus Haementeria de Filippi, 1849, may represent an exception (Oceguera-Figueroa 2012). As a consequence, only few studies have specifically addressed the antithrombotic repertoire of rhynchobdellid leeches. Again, the genus Haementeria represents a remarkable exception (Sawyer et al. 1991; Seale et al. 1997; Faria et al. 1999; Oliveira et al. 2012; Amorim et al. 2015; Pfordt et al. 2022). Antistasin, a factor Xa inhibitor that was isolated from the Mexican leech Haementeria officinalis de Filippi, 1849 (Tuszynski et al. 1987), is probably the most well-known Haementeria -derived anticoagulant (Iwama et al. 2020). Factor Xa is a crucial component of the blood coagulation-cascade and facilitates the cleavage of prothrombin to thrombin (Núñez-Navarro et al. 2019). The activity of thrombin in turn is inhibited by direct thrombin-inhibitors like hirudin (Markwardt 1956). Both antistasin and hirudin are hence inhibitors of the secondary hemostasis. In contrast, the primary hemostasis (the process of platelet aggregation) is inhibited by leech-derived factors like decorsin and ornatin. Whereas decorsin was first described from M. decora (Seymour et al. 1990), ornatin originates from Placobdella ornata Verrill, 1872 (Mazur et al. 1991). Both factors are potent glycoprotein IIb/IIIa antagonists and contain a highly conserved RGD motif (Lazarus and McDowell 1993). Like the genus Haementeria , the genus Placobdella Blanchard, 1893, also belongs to the order of Rhynchobdellida Blanchard, 1894, and the family of Glossiphoniidae Vaillant, 1890. The genus Placobdella currently comprises about 25 different species that inhabit large areas of North America (de Carle et al. 2017). Members of the genus are hematophagous and primarily ectoparasitic on turtles (Bielecki et al. 2012), but occasionally also feed on amphibians, birds and mammals including humans (Moser et al. 2014; Cichocka et al. 2021). Placobdella costata Müller, 1846, represents the only European member of the genus. The species is widely distributed across Europe, and the distribution range largely overlaps with the distribution range of its natural host, the European mud turtle Emys orbicularis Linnaeus, 1758 (Bielecki et al. 2012). A contemporary study showed that the genetic diversity between the various populations of P. costata may suggest up to seven separately evolving European lineages throughout its range, five of which exhibit degrees of separation at the molecular level to warrant species level status (Kvist et al. 2022). One of these lineages was recently confirmed as a second Palaearctic member of the genus Placobdella , namely Placobdella nabeulensis (Ben Ahmed et al. 2023). This species occurs in distinct regions of Tunisia and Algeria in northern Africa. So far, investigations into members of the genus Placobdella have mainly focused on the description and analysis of morphological (Richardson et al. 2017; Sawyer 2022) and genetic (de Carle et al. 2017; Ben Ahmed et al. 2023) markers or on the composition of their bacterial symbionts (Manglicmot et al. 2020). Besidess the historical identification of ornatins, investigations into the presence and diversity of anticoagulants in the salivary glands of these animals are rare. In 2016, Mark Siddall et al. conducted a comparative transcriptomic analysis of three members of the genus Placobdella , namely Placobdella ali Hughes & Siddall, 2007, Placobdella kwetlumye Oceguera-Figueroa, Kvist, Watson, Sankar, Overstreet & Siddall, 2010 and Placobdella parasitica Say, 1824, and the authors identified a broad variety of putative anticoagulants including hirudins and ornatins (Siddall et al. 2016). Functional investigations of the respective factors, however, are still missing. In 2024, the complete genome sequence data of P. costata were deposited in the nucleotide database of GenBank by the Wellcome Sanger Darwin Tree of Life Programme including the sequences of 18 chromosomes (accession numbers OZ194472.1 - OZ194489.1) and the mitochondrial genome (accession number OZ194490.1). Strikingly, the information on the genome was neither published nor is it available on the website of the Darwin Tree of Life Programme. Over the last years, we have successfully applied the approach to conduct in-depth analyses and re-analyses of genome and transcriptome sequence data of several leech species in order to further explore the diversity of putative anticoagulants (Khan et al. 2025). Whenever feasible, factors of interest were expressed as recombinant proteins and subsequently functionally characterized (Pfordt et al. 2022; Müller et al. 2024; Schulz et al. 2025; Tolksdorf et al. 2025). The availability of both transcriptome and genome sequence data has enabled such analyses also for P. costata . The primary aim of the present study was hence to identify, and functionally characterize putative anticoagulants in five species of Placobdella , with a special focus on hirudins and ornatins. The second aim, however, was to eventually answer the question whether or not the hirudins and ornatins of glossiphoniid leeches also belong to the hirudin superfamily whose members are characterized not only by similar structural features of the respective molecules, but also by a common gene structure (Müller et al. 2019). Materials and Methods GenBank transcriptome and genome data Transcriptome data for members of the genus Placobdella were obtained from the following GenBank sequence read archives (SRAs): SRX3734179 ( P. ali ), SRX3734173 ( P. kwetlumye ) and SRX1335044 ( P. parasitica ), respectively. Chromosome-level genome data of P. costata were obtained from GenBank accession numbers OZ194472.1 - OZ194489.1. Animal collection, tissue preparation, genomic DNA isolation and sequencing Individuals of Placobdella costata were collected in November 2018 from the Kapengraben in Saxony-Anhalt, Germany (51.81488 N, 12.40539 E) (collection codes 695 and 696, Kvist et al. 2022), whereas individuals of Placobdella nabeulensis were collected in June 2021 from El Malaabi Dam (36.81666 N, 10.98333 E) in Menzel Temime located in the North-East of Tunisia (Ben Ahmed et al. 2023). The caudal suckers of two individuals of P. costata and one individual of P. nabeulensis were dissected and placed in 2 ml microcentrifuge tubes each. Prior to extraction of genomic DNA, 350 µl of the lysis buffer ML 1 and 25 µl of proteinase K solution (E.Z.N.A. Mollusc DNA kit, Omega Bio-tek Inc., Norcross, GA) were added to the samples and the tissues were manually homogenized using scissors. DNA extractions were performed according to the manufacturer’s protocol. Purified genomic DNA was eluted with sterile double distilled water and stored at -20°C. For whole genome sequencing, the samples were sent to the Competence Centre for Genomic Analysis (Kiel, Germany). The genomic DNA was quality-controlled and quantified via fluorometric measurement. Library preparation and sequencing were carried out at the Competence Centre for Genomic Analysis (Kiel, Germany). Library preparations were done using the Illumina DNA Prep Kit (Illumina, Berlin, Germany) according to the manufacturer’s protocol. Sequencing of the libraries was performed on an Illumina NovaSeq 6000 S4 device, using 2×150 bp reads on v1.5 Flowcells. The raw genome sequence data of both the two individuals of P. costata and the single individual of P. nabeulensis were deposited in BioStudies (The European Bioinformatics Institute, EMBL-EBI, Sarkans et al. 2018) and received the following accession number: S-BSST2339 (DOI: 10.6019/S-BSST2339 ). The data can be freely accessed and used. During the course of the present study a high-quality, chromosome-level reference genome of P. costata was generated by the Wellcome Sanger Institute and deposited in GenBank (GCA_964276725.1). All P. costata -related sequence information in the manuscript hence refer to the reference genome. Bioinformatics and graphical tools Raw genome sequence data were imported into Geneious v9.1.8 (Biomatters Ltd., Auckland, New Zealand; Kearse et al. 2012) for further analyses. Basic Local Alignment Search Tool (BLAST) searches were performed using the NCBI web portal ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ), BioEdit v7.2.5 (Hall 1999) or Geneious v9.1.8 with adjusted parameter settings for both word size and the expected threshold values. Multiple sequence alignments (MSA) were generated using ClustalX 2.1 (Larkin et al. 2007) or the CLC Sequence Viewer software package v8.0 (QIAGEN, Aarhus, Denmark) using default settings. Particular attention was paid to the conservation of disulfide bond-forming cysteines. Alignments were exported as msf-files and further processed using Gene-Doc v2.7 (Nicholas and Nicholas 1997). Signal peptide sequences were predicted using the SignalP6.0 server (Teufel et al. 2022) and the Phobius web portal (Käll et al. 2007). Graphs were generated and analyzed using GraphPad Prism V5.01 (GraphPad Software, Boston, MA, USA). Gene synthesis The cDNA fragments of putative hirudins, hirudin-like factors (HLFs) and ornatins of P. costata were synthesized using the GeneArt gene synthesis service of Thermo Fisher Scientific (Darmstadt, Germany). Amplification and cloning of putative hirudin and decorsin cDNAs For the amplification of putative hirudin and decorsin cDNAs, primers were derived from the respective transcriptome or genome sequences. A list of all primers that were used in the study is provided in Supplementary Information File Table S1 . PCR reactions were performed using Q5 high-fidelity DNA polymerase (New England Biolabs, Frankfurt a. M., Germany), fragments of relevant sizes were purified and cloned into the expression vector pQE30Xa (QIAGEN, Hilden, Germany). Successfully cloned cDNAs were sequenced for control purposes by Biosearch Technologies (LGC, Berlin, Germany) or by Eurofins Genomics (Cologne, Germany). Expression, purification, processing, and quantification of putative hirudins and decorsins The detailed procedure to express, purify, process and quantify the respective recombinant hirudins and decorsins of P. costata was described in numerous recent publications (e.g. Müller et al. 2016; Wang et al. 2025; Tolksdorf et al. 2025). Briefly, all factors were either expressed in Escherichia coli laboratory strains DH5α at a cultivation temperature of 37°C (Hanahan 1985) or SHuffle® T7 at a cultivation temperature of 30°C (New England Biolabs, Frankfurt a. M., Germany; Lobstein et al. 2012). To obtain the recombinant proteins, we applied an expression and purification system that was developed by QIAGEN (Hilden, Germany). The pQE30Xa vector encodes a factor Xa protease recognition site that is located between the His-tag coding region at the 5′ side and the multiple cloning site at the 3′ side. A subsequent factor Xa protease-treatment cleaves off the His-tag and results in a recombinant protein that is devoid of any vector-derived amino acid residues at the N-terminus. The successful expression, purification and factor Xa treatment of all factors was controlled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses. Molar concentrations of final protein solutions were calculated by dividing the absorbance at 280 nm by the molar absorption coefficient according to the equation ε = (nW × 5,500) + (nY × 1,490) + ( nC × 125) (Gill and von Hippel 1989; Pace et al. 1995). Blood coagulation assays To verify the thrombin-inhibitory potencies of putative hirudins of P. costata , we performed a thrombin time test (TTT; reference range 16.8–21.4 s) using a BFT II analyzer (Siemens Healthcare, Erlangen, Germany). All steps were carried out according to the manufacturer’s instructions. Protein samples were diluted with buffer to reach final concentrations in the reaction assays of 3.2 µmol/l or 0.32 µmol/l, respectively. The desired amount of substrate was directly transferred into the test cuvette immediately before the plasma was added. Dade® Ci-Trol® 1 (Siemens Healthcare, Erlangen, Germany) was used as standardized human plasma. The incubation of reaction mixtures was carried out at 37.4°C. Measurements that reached 300 s before any coagulation was detected were stopped and considered as complete inhibition of coagulation. Blood coagulation tests were performed in three to four technical replicates. Platelet aggregation assays Platelet aggregation was analyzed by light transmission aggregometry (LTA) with human blood samples that were obtained from healthy human volunteers after written informed consent and approval from the institutional ethics committee. Blood collection, sample preparation, and the subsequent experimental procedures were performed as described in Müller et al. (2024) and Tolksdorf et al. (2025). Briefly, 10 ml of venous blood was taken from the antecubital vein using an S-Monovette® (Sarstedt, Nürnbrecht, Germany) prefilled with citrate buffer. The first centrifugation step of the blood collection tube was performed at 200 g for 20 min at room temperature. After centrifugation, the supernatant (platelet-rich plasma, PRP) was transferred, and the remaining blood was centrifuged again for 10 min at 2000 g at room temperature. The supernatant was dedicated as platelet-poor plasma (PPP), transferred, and used as a reference value for maximal platelet aggregation. Measurements were performed using either an APACT-4004 aggregometer (LABiTec, Ahrensburg, Germany) or a TA-8 V aggregometer (Diagnostica Stago S.A.S., Asnièressur-Seine, France). The snake venom-derived platelet aggregation inhibitors eptifibatide and tirofiban (Sigma-Aldrich, Taufkirchen, Germany) were used as positive controls for complete inhibition of platelet aggregation. PRP was pre-incubated with the respective test and control compounds (final concentration 3.2 µmol/l) or buffer for 1 min at 37°C. For the measurement, PRP was subsequently transferred into the test cuvettes and stimulated with ADP (200 µmol/l; Hart Biologicals, Hartlepool, UK; final concentration 5 µmol/l) after 1 min of runtime. The final volume in each test cuvette was 250 µL of diluted PRP. All experiments were performed at 37°C over a time period of 400 s. Maximal aggregation in percentage and the area under the curve were calculated as quantitative output parameters (Zhou and Schmaier 2005). Platelet aggregation tests were performed in two to four technical replicates. Results Identification of putative hirudin and ornatin cDNAs in P. ali , P. kwetlumye and P. parasitica Whereas ornatins have already been described from P. ornata many years ago (Mazur et al. 1991), the investigations of Siddall et al. (2016) for the first time revealed the presence of putative hirudins in members of the genus Placobdella . Altogether, the authors identified putative cDNAs of hirudin in P. kwetlumye and P. parasitica and putative cDNAs of ornatin in P. ali and P. parasitica , respectively. However, none of the putative hirudins or ornatins was functionally verified so far. Our in-depth re-analysis of the transcriptomic data confirmed the results of Siddall et al. (2016), but we were able to identify additional putative hirudin and ornatin-encoding cDNAs. In total, we identified two putative hirudins in P. ali (Pali_HV1 and Pali_HV2), three putative hirudins in P. kwetlumye (Pkwe_HV1, Pkwe_HV2 and Pkwe_HV3) and one putative hirudin in P. parasitica (Ppar_HV1). Pkwe_HV1 and Ppar_HV1 were already described by Siddall et al. (2016). The corresponding contigs of all putative hirudins-encoding cDNAs are provided in Supplementary Information Files S1-S3. The identification of putative ornatin-encoding contigs turned out to be a challenging task and required some manual sequence editing. However, we eventually identified three putative ornatin-encoding cDNAs in P. ali (Pali_OV1-3) and one cDNA in both P. kwetlumye (Pkwe_OV1) and P. parasitica (Ppar_OV1), respectively. The corresponding contigs are provided in Supplementary Information Files S1-S3. Identification of putative hirudin and ornatin genes in P. costata and P. nabeulensis Based on the cDNA sequences of putative hirudins and ornatins of P. ali , P. kwetlumye and P. parasitica we subsequently screened the genome of P. costata to identify the respective gene sequences. Based on our analyses, the genome of P. costata contains three genes that encode putative hirudin variants (HV), namely Pcos_HV1-3, and three genes that encode putative ornatin variants (OV), namely Pcos_OV1a, _OV1b and _OV2. All genes are located on chromosome 18, the exact position and orientation of each gene are provided in Table 1 . Table 1 Position and orientation of putative hirudin (HV) and ornatin (OV) genes on chromosome 18 in P. costata . rev+comp indicates reverse and complementary orientation gene position Pcos_HV3 3772705–3773531 rev+comp Pcos_HV2 5750243–5752539 rev+comp Pcos_HV1 5755993–5756621 rev+comp Pcos_OV1a 9984659–9985686 rev+comp Pcos_OV1b 9989819–9991063 rev+comp Pcos_OV2 10001584–10002179 rev+comp All hirudin genes are composed of four exons and three introns, whereas the ornatin genes are composed of three exons and two introns and apparently lack the third intron and the fourth exon of the hirudin genes which encode the elongated acidic C-terminal tail of hirudin (see Fig. 1 ). For each gene, the exon/intron composition, the location of the introns and the characteristics of the exon boundaries (boundary-overlapping versus non-overlapping codons) exactly match the structures of all hirudin and decorsin/ornatin genes analyzed so far. A schematic representation of the gene structures of Pcos_HV1 and Pcos_OV2 is provided in Fig. 1 . Table 2 lists the exon and intron lengths, and the annotated sequences for each of the genes are provided in Supplementary Information Files S4 (hirudin genes) and S5 (ornatin genes). Table 2 Comparison of putative hirudin (HV) and ornatin (OV) gene structures in P. costata (Pcos) and P. nabeulensis (Pnab) Gene Exon1 Intron1 Exon2 Intron2 Exon3 Intron3 Exon4 Pcos_HV1 61 bp 256 bp 50 bp 58 bp 58 bp 84 bp 62 bp Pcos_HV2 61 bp 1540 bp 50 bp 85 bp 58 bp 429 bp 80 bp Pcos_HV3 55 bp 344 bp 62 bp 71 bp 73 bp 151 bp 35 bp Pnab_HV1 61 bp 255 bp 50 bp 58 bp 58 bp 84 bp 62 bp Pnab_HV2 61 bp 50 bp 85 bp 58 bp 80 bp Pnab_HV3 55 bp 335 bp 62 bp 75 bp 73 bp 151 bp 77 bp Pcos_OV1a 70 bp 663 bp 65 bp 116 bp 114 bp Pcos_OV1b 70 bp 880 bp 65 bp 116 bp 114 bp Pcos_OV2 70 bp 183 bp 74 bp 140 bp 129 bp Pnab_OV1 70 bp 65 bp 116 bp 105 bp Pnab_OV2 70 bp 194 bp 74 bp 140 bp 138 bp To verify the data for P. costata we generated and analyzed raw genome sequence data of P. nabeulensis , the second Palearctic representative of the genus Placobdella . The raw Illumina reads were manually assembled, and all putative hirudin and ornatin genes of P. costata could successfully be identified and reconstructed in P. nabeulensis with the exception of intron1 of both the Pnab_HV2 and Pnab_OV1 genes and intron 3 of the Pnab_HV2 gene. Exon and intron lengths of all putative hirudin and ornatin genes in P. nabeulensis are in close proximity to the values of the respective genes of P. costata (see Table 2 for details). The annotated sequences for all genes are provided in Supplementary Information Files S6 (hirudin genes) and S7 (ornatin genes). Structural and functional characterization of putative hirudins The amino acid sequences of all putative hirudins of both the three North American and the two Palearctic Placobdella species were derived from the respective cDNA or gene sequences. A multiple sequence alignment (MSA) of all putative hirudins is provided in Fig. 2 , their biochemical features are summarized in Table 3 . Table 3 Structural and biochemical features of putative hirudin variants HV1-3 of P. costata (Pcos_HV1-3), HV1-3 of P. nabeulensis (Pnab_HV1-3), HV1 and HV2 of P. ali (Pali_HV1-2), HV1-3 of P. kwetlumye (Pkwe_HV1-3) and HV1 of P. parasitica (Ppar_HV1). The length is without the signal peptide sequence. aa = amino acids; MW = molecular weight; kDa = kilodalton; pI = isoelectric point Factor Length in aa MW in kDa pI value Pcos_HV1 56 6382.99 4.25 Pcos_HV2 60 7149.79 4.49 Pcos_HV3 58 6532.12 4.12 Pnab_HV1 56 6378.01 4.09 Pnab_HV2 62 7304.85 4.27 Pnab_HV3 72 7979.53 3.94 Pali_HV1 56 6292.01 4.29 Pali_HV2 49 4970.41 4.34 Pkwe_HV1 56 6313.03 4.13 Pkwe_HV2 64 7426.18 4.38 Pkwe_HV3 54 6017.56 4.64 Ppar_HV1 56 6266.02 5.23 All putative hirudins contain the six highly conserved cysteine residues, and both the lengths (49–72 amino acid residues without the signal peptides) and the acidic pI values (3.94–5.23) are in the range of functional hirudins. However, all HV3 variants apparently lack the essential phenylalanine (or tyrosin) residue at position 3 of the mature protein (see Fig. 2 ; Lazar et al. 1991). For the functional characterization, we focused on the putative hirudins of P. costata . All three hirudin variants, namely Pcos_HV1-3, were expressed as recombinant proteins in E. coli strain DH5α and further processed as described in the Materials and Methods section. The thrombin time tests revealed that Pcos_HV1 displayed a high thrombin-inhibitory potency, but neither Pcos_HV2 nor Pcos_HV3 did negatively influence the activity of thrombin (Fig. 3 ). Pcos_HV1 can hence be termed a true hirudin, whereas Pcos_HV2 and Pcos_HV3 are rather HLFs. Gain-of-function analyses of Pcos_HV2 and HV3 The N-terminus of hirudin is of special importance for its interaction with hirudin, and a replacement of the first hydrophobic valyl residue in hirudin variant HV1 of H. medicinals with polar amino acids results in a dramatic increase in the inhibition constant K i (Wallace et al. 1989). Whereas Pcos_HV1 contains a valyl residue at position 1, both Pcos_HV2 and HV3 contain polar amino acid residues at the respective position: a glutamine residue (Q1) in Pcos_HV2 and a glutamyl residue (E1) in Pcos_HV3. Strikingly, within the N-terminal five amino acid residues of Pcos_HV2 only the Q1 residue differs from the N-terminus of Pcos_HV1 (see Fig. 2 ). A closer look at the conjunction of exons 1 and 2 of the Pcos_HV2 and Pnab_HV2 genes (see Fig. 1 and Supplementary Material Files S4 and S6) revealed that with only one nucleotide substitution the codons that encodes the polar glutamine residue Q1 (CAA) can be changed to codons that encode a hydrophobic leucine residue (CTA) and with two substitutions to codons that encode a hydrophobic valyl residue (GTA). To evaluate the effects of variations of the N-terminal amino acid sequences on the thrombin-inhibitory potency of Pcos_HV2 we hence constructed two variants that contain either a valyl residue (Pcos_HV2V) or a leucine residue (Pcos_HV2L) at position 1. In addition we constructed respective variants of Pcos_HV3 that contain either the N-terminal five amino acid residues of Pcos_HV2 (Pcos_HV3Q), Pcos_HV2V (Pcos_HV3V) or Pcos_HV2L (Pcos_HV3L), respectively. Table 4 lists all Pcos_HV2 and HV3 variants and their N-terminal amino acid sequences. Table 4 Nomenclature and N-terminal amino acid sequences of wildtype and mutant variants of Pcos_HV2 and HV3 Factor N-terminal sequence Pcos_HV1 VHFPPC Pcos_HV2 QHFPPC Pcos_HV2V VHFPPC Pcos_HV2L LHFPPC Pcos_HV3 EDIPEC Pcos_HV3Q QHFPKC Pcos_HV3V VHFPPC Pcos_HV3L LHFPPC All variants of Pcos_HV2 and HV3 were expressed as recombinant proteins in E. coli strains DH5α and SHuffle T7® and further processed as described in the Materials and Methods section. The thrombin time tests revealed that both Pcos_HV2V and HV2L displayed moderate, but clearly detectable thrombin-inhibitory potencies, whereas none of the Pcos_HV3 variants negatively influenced the activity of thrombin. The inhibitory potencies of recombinant Pcos_HV2V and HV2L did not differ between the two E. coli strains (see Fig. 4 for the recombinant factors expressed in E. coli strain DH5α and Supplementary Information File Figure S1 for the recombinant factors expressed in E. coli strain SHuffle® T7). Interestingly, in both bacterial expression systems the leucine residue at position 1 conferred higher thrombin-inhibitory potencies of the respective recombinant Pcos_HV2 variants compared to the variants containing a valyl residue at position 1. It is worth to note in that context that lepirudin, a commercially available recombinant hirudin (rec-Hirudin, HBW 023, Refludan®), is a variant of hirudin HV2 (hirudin-IT) of H. medicinalis (Harvey et al. 1986) and contains a leucine residue at position 1 instead the wildtype isoleucine residue (Marckwardt 1994). Structural and functional characterization of putative ornatins The amino acid sequences of the putative ornatins of all five Placobdella species were derived from the respective cDNA or gene sequences. A multiple sequence alignment (MSA) of the putative ornatins is provided in Fig. 5 , their biochemical features are summarized in Table 5 . Table 5 Structural and biochemical features of pitative ornatin variants OV1a, 1b and 2 of P. costata (Pcos_OV1a, 1b and 2), OV1 and 2 of P. nabeulensis (Pnab_OV1 and 2), OV1-3 of P. ali (Pali_OV1-3), OV1 of P. kwetlumye (Pkwe_OV1) and OV1 of P. parasitica (Ppar_OV1). The length is without the signal peptide sequence. aa = amino acids; MW = molecular weight; kDa = kilodalton; pI = isoelectric point Factor Length in aa MW in kDa pI value Pcos_OV1a 62 7073.28 9.44 Pcos_OV1b 62 7104.33 9.41 Pcos_OV2 70 8358.83 9.50 Pnab_OV1 59 6733.86 9.25 Pnab_OV2 73 8560.07 9.15 Pali_OV1 50 5738.61 9.04 Pali_OV2 57 6404.56 9.54 Pali_OV3 56 6052.96 8.64 Pkwe_OV1 68 7634.84 9.80 Ppar_OV1 52 5722.23 4.90 All putative ornatins except Pnab_OV1 contain the six highly conserved cysteine residues, however, the essential RGD/KGD motif (Lazarus and McDowell 1993) is absent in Pnab_OV1, Pali_OV3 and Ppar_OV1 (see Fig. 5 ). The basic pI value of most putative ornatins except for Ppar_OV1 (see Table 5 ) is often observed in functional ornatins (Mazur et al. 1991). However, a basic pI does not seem to be an essential biochemical feature (Pfordt et al. 2022). For the functional characterization we focused on the putative ornatins of P. costata . Again, Pcos_OV1a and OV2 were expressed as recombinant proteins in E. coli strain DH5α and further processed as described in the Materials and Methods section. Platelet function analyzed by light transmission aggregometry, however, revealed that none of the factors exhibited an inhibitory effect on platelet aggregation (Fig. 6 A und C). To evaluate whether or not the genetic background of the bacterial host strain may have negatively influenced the correct folding and hence the activity of the recombinant ornatins Pcos_OV1a and 2, we repeated their expression and purification, but used the E. coli strain SHuffle® T7 as an alternative host. The strain was specifically developed to improve the correct formation of disulfide bonds in recombinant proteins (Lobstein et al. 2012). Indeed, the recombinant ornatins Pcos_OV1a and 2 that were produced by E. coli strain SHuffle® T7 negatively affected ADP-induced platelet aggregation, both in terms of shape of the curve and the maximal aggregation value (Fig. 6 B). A statistical analyses revealed that the area under the curve (AUC) values of both putative ornatins Pcos_OV1a and Pcos_OV2 significantly differed from the negative control samples (buffer) with p-values ≤ 0.01 (Pcos_OV1a) and ≤ 0.001 (Pcos_OV2), respectively (Fig. 6 D). Both factors can hence be considered as active ornatins. Discussion Leeches are frequently used for medical purposes, but the term “medicinal leeches” does not refer to a specific taxon of leeches and is rather a description of their anthropogenic use. So far, almost exclusively jawed (or arhynchobdellid) leeches (Hirudiniformes) have been used by humans to treat various maladies including cardiovascular disorders, wound healing and problems associated with plastic reconstructive surgery (Singh 2010; Hackenberger and Janis 2019; Nair et al. 2020). Consequently, in-depth investigations of the therapeutic potential of proboscis-bearing leeches (especially Glossiphoniidae) are rare. Notably, leeches of the glossiphoniid genus Placobdella comprise a broad variety of anticoagulants including putative hirudins and ornatins (Siddall et al. 2016), yet detailed analyses of their biochemical properties and functional characterizations are still pending. In a series of previous studies we have already determined the structures of hirudin, hirudin-like factor and decorsin/ornatin genes in representatives of various arhynchobdellid leech species including the European medicinal leeches H. medicinalis , H. verbana , H. orientalis and H. troctina (Müller et al. 2016; Ben Ahmed et al. 2024), the Asian medicinal leeches Hirudinaria manillensis Lesson, 1842, Whitmania pigra Whitman, 1884, and Hirudo nipponia (Müller et al. 2017; Müller et al. 2022; Tolksdorf et al. 2025; Kalathejari et al. 2026) and the North American medicinal leech Macrobdella decora (Müller et al. 2019). The generation of genome sequence data of two Palearctic species of the genus Placobdella enabled us to conduct respective analyses in representatives of glossiphoniid leeches. The primary aim of the present study was to take a step forward on the track to close the "analytical gap" between glossiphoniid and arhynchobdellid leech species. Transcriptomic sequence data of three North American species and genome sequence data of the two Palearctic representatives of the genus Placobdella were analyzed for the presence of putative hirudin and ornatin cDNAs or genes. In all five leech species, respective coding sequences for both factors could be identified (see Figs. 2 and 5 ). Whereas hirudins are present in all hematophagous leech species analyzed so far and may even occur in predatory leeches (Müller et al. 2022), decorsins/ornatins are apparently absent in European members of the genus Hirudo (Babenko et al. 2020; Kvist et al. 2020) and in the African medicinal leech Asiaticobdella fenestrata (Schulz et al. 2025) and may hence have been lost during evolution. However, the verification of hirudin and ornatin genes in representatives of the glossiphoniid leech genus Placobdella indicates that independent genes of both types of anticoagulants were probably present already in the last common ancestor (LCA) of Hirudinida (or true leeches) (Tessler et al. 2018; Kuo and Lai 2019; de Carle et al. 2025). Strong support for this assumption comes from the observation that the gene structures of both the hirudin and the decorsin/ornatin genes are highly conserved in glossiphoniid and arhynchobdellid leeches including the exact position of introns and the presence of exon-boundary overlapping and non-overlapping codons (see Fig. 1 ; Müller et al. 2016; Müller et al. 2019; Lukas et al. 2022). Whether or not the LCA of the hirudin and ornatin genes dates back even further in time and is already present in Hirudinea Lamarck, 1818, a group that also includes the family Piscicolidae Johnston, 1865, and the two orders Acanthobdellida Livanow, 1905, and Branchiobdellida Holt, 1965 (de Carle et al. 2025), still remains to be defined. A recurring feature in hematophagous leeches is the presence of multiple copies of anticoagulant genes including genes that encode hirudins and ornatins/decorsins (Liu et al. 2023; Zhao et al. 2024; Khan et al. 2025; Kalathejari et al. 2026), very likely to prevent deleterious effects of occasional gene loss events. The genome sequence data of P. costata only partially confirm this observation with the presence of just a single gene that encodes a functional hirudin (Pcos_HV1)and three genes that encode functional ornatins (Pcos_OV1a/b and OV2) (see Fig. 2 + 3 and 5 + 6). However, with only a single amino acid substitution (Q1 to either V1 or L1) Pcos_HV2 can gain a moderate thrombin-inhibitory potency (see Fig. 4 ). We can only speculate whether the current situation of Pcos_HV2 is the consequence of a past loss-of-function event (GTA ◊ CTA ◊ CAA) or primes the leech for a future gain-of-function event (CAA ◊ CTA ◊ GTA), but each scenario is not mutually exclusive. Unfortunately, the biological targets of both Pcos_HV2 and HV3 remain obscure and are difficult to identify. In P. nabeulensis the Pnab_OV1 gene encodes a factor that lacks the fourth conserved cysteine residue and may hence be inaccurately folded (see Fig. 5 ). With that. P. nabeulensis may contain only single genes that encode a functional hirudin (Pnab_HV1) and a functional ornatin (Pnab_OV2). Interestingly, both Pnab_OV1 and Ppar_OV1 of P. parasitica comprise RSD motifs instead of the canonical RGD/KGD motif that facilitates integrin binding (Lazarus and McDowell 1993; Reiss et al. 2006). An RSD motif may be functionally equivalent to an RGD motif (Van de Walle et al. 2008), but this remains to be tested for ornatins. Based on the current status of investigations, neither P. costata nor P. nabeulensis contain genes that encode putative multimeric hirudins/HLFs or decorsins. The recombinant expression of putatve hirudins and decorsins/ornatins in bacterial hosts for subsequent functional analyses is well established in our lab. In a recent publication we compared two E. coli strains with different characteristics, namely DH5α (Hanahan 1985) and SHuffle® T7 (Lobstein et al. 2012). The strain SHuffle® T7 supports the formation of disulfide bonds, generally a crucial step in correct folding of cystein-containing proteins (Feige et al. 2018) and in correct folding of members of the hirudin superfamily in particular (Dodt et al. 1985; Mazur et al. 1993; Micheletti et al. 2003; Wu et al. 2013). We deciphered that a preparation of recombinant hirudin HV1 of H. medicinalis (Hmed_HV1) that was expressed in SHuffle® T7 at its optimal cultivation temperature of 30°C revealed a higher thrombin inhibitory potency compared to a preparation of Hmed_HV1 that was expressed in DH5α at its optimal cultivation temperature of 37°C (Wang et al. 2025). The current study partially supports this observation: the putative ornatins Pcos_OV1a and OV2 did not exhibit platelet aggregation inhibitory potencies when both factors were expressed as recombinant proteins in E. coli strain DH5α at 37°C. However, platelet aggregation was negatively affected when both factors were expressed in E. coli strain SHuffle® T7 at 30°C (see Fig. 6 ). In contrast, the thrombin-inhibitory potencies of the recombinant Pcos_HV2V and HV2L variants did not differ between the two bacterial expression systems (see Fig. 4 and Supplementary Information Figure S1 ). Nevertheless, it might be a reasonable option to consequently use the strain SHuffle® T7 for the expression of recombinant putative hirudins, ornatins and other cysteine-rich proteins. Declarations Acknowledgements The authors are thankful to the colleagues who generated and provided the genome and transcriptome datasets explored in the present study. In addition, we would like to thank all members of the Animal Physiology Working Group at the University of Greifswald for their help and support. Special thanks go to Jan-Peter Hildebrandt and Undine Lauf for their critical reading of the manuscript. Author contribution C.M. conceived the ideas, designed the methodology and performed the sequence-based investigations; R.B.A. and C.G. collected the animals; R.B.A., C.M., C.T. and R.W. performed the experiments; C.M.,R.B.A. and S.K. analyzed the data and drafted the manuscript; C.M., B.H.R. and G.J. supervised the experimental work. Funding Open Access funding enabled and organized by Projekt DEAL. Data availability Reference genome chromosome-level sequence data of P. costata are deposited in GenBank under the accession numbers OZ194472.1 - OZ194489.1. Information on specific gene locations are provided either within the manuscript or as Supplementary Information Files. The raw genome sequence data of P. costata and P. nabeulensis generated in the present study were deposited in BioStudies under the following accession number: S-BSST2339 (DOI: 10.6019/S-BSST2339). Images that illustrate the expression, purification and processing of all recombinant factors are available on request from the corresponding author. Declarations Ethical approval The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of the University Medicine Greifswald (protocol code BB 20/12, approval date 25 July 2018) and by the Institutional Review Board (or Ethics Committee) of the University Medicine Oldenburg, Carl von Ossietzky University Oldenburg (ethics vote 2024-034, approval date 27 March 2024). Consent to participate and consent for publication Written informed consent was obtained from all subjects involved in the study. Competing interests The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. References Abdualkader AM, Ghawi AM, Alaama M, Awang M, Merzouk A (2013) Leech Therapeutic Applications. Indian J Pharm Sci 75(2):127–137. Amorim AMXP, de Oliveira UC, Faria F, Pasqualoto KFM, Junqueira-de-Azevedo IdLM, Chudzinski-Tavassi AM (2015) Transcripts involved in hemostasis: Exploring salivary complexes from Haementeria vizottoi leeches through transcriptomics, phylogenetic studies and structural features. Toxicon 106:20–29. https://doi.org/10.1016/j.toxicon.2015.09.002 Babenko VV, Podgorny OV, Manuvera VA, Kasianov AS, Manolov AI, Grafskaia EN, Shirokov DA, Kurdyumov AS, Vinogradov DV, Nikitina AS, Kovalchuk SI, Anikanov NA, Butenko IO, Pobeguts OV, Matyushkina DS, Rakitina DV, Kostryukova ES, Zgoda VG, Baskova IP, Trukhan VM, Gelfand MS, Govorun VM, Schiöth HB, Lazarev VN (2020) Draft genome sequences of Hirudo medicinalis and salivary transcriptome of three closely related medicinal leeches. BMC Genomics 21(1):331. https://doi.org/10.1186/s12864-020-6748-0 Ben Ahmed R, Gajda Ł, Utevsky S, Kvist S, Świątek P (2023) Placobdella nabeulensis sp. nov. (Hirudinea: Glossiphoniidae), a new glossiphoniiform leech from Palearctic North Africa. Mol Biol Rep 50(8):6753–6767. https://doi.org/10.1007/s11033-023-08594-z Ben Ahmed R, Abilov A, Müller C (2024) Diversity of hirudin and hirudin-like factor genes in the North-African medicinal leech, Hirudo troctina . Parasitol Res 123(11):382. https://doi.org/10.1007/s00436-024-08411-x Bielecki A, Cichocka JM, Jabłoński A, Jeleń I, Ropelewska E, Biedunkiewicz A, Terlecki J, Nowakowski JJ, Pakulnicka J, Szlachciak J (2012) Coexistence of Placobdella costata (Fr. Müller, 1846) (Hirudinida: Glossiphoniidae) and mud turtle Emys orbicularis . Biologia 67:731–738. https://doi.org/10.2478/s11756-012-0069-y de Carle D, Oceguera-Figueroa A, Tessler M, Siddall ME (2017) Phylogenetic analysis of Placobdella (Hirudinea: Rhynchobdellida: Glossiphoniidae) with consideration of COI variation. Mol Phylogenet Evol 234–248. https://doi.org/10.1016/j.ympev.2017.06.017 de Carle D, Iwama RE, Wendruff AJ, Babcock LE, Nanglu K (2025) The first leech body fossil predates estimated hirudinidan origins by 200 million years. PeerJ 13:e19962. https://doi.org/10.7717/peerj.19962 Cichocka JM, Bielecki A, Jabłońska-Barna I, Krajewski Ł, Topolska K, Hildebrand J, Dmitryjuk M, Biedunkiewicz A, Abramchuk A (2021) Sucking of human blood by Placobdella costata (O. F. Müller, 1846) (Hirudinida: Glossiphoniidae): Case study with notes on body form. Ecol Evol 11(24):17593–17603. https://doi.org/10.1002/ece3.8261 Dodt J, Seemüller U, Maschler R, Fritz H (1985) The complete covalent structure of hirudin. Localization of the disulfide bonds. Biol Chem Hoppe Seyler 366(4):379–385. https://doi.org/10.1515/bchm3.1985.366.1.379 Elliott JM, Kutschera U (2011) Medicinal leeches: Historical use, ecology, genetics and conservation. Freshwater Rev 4(1):21–41. https://doi.org/10.1608/FRJ-4.1.417 Faria F, Kelen EM, Sampaio CA, Bon C, Duval N, Chudzinski-Tavassi AM (1999) A new factor Xa inhibitor (lefaxin) from the Haementeria depressa leech. Thromb Haemost 82(5):1469–1473. https://doi.org/10.1055/s-0037-1614857 Feige MJ, Braakman I, Hendershot LM (2018) Disulfide bonds in protein folding and stability. In Oxidative Folding of Proteins: Basic Principles, Cellular Regulation and Engineering, ed. M. J. Feige, The Royal Society of Chemistry, pp. 1–33. https://doi.org/10.1039/9781788013253-00001 Gill SC, von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182:319–326. https://doi.org/10.1016/0003-2697(89)90602-7 Hackenberger PN, Janis JE (2019) A comprehensive review of medicinal leeches in plastic and reconstructive surgery. Plast Reconstr Surg Glob Open 7(12):e2555. https://doi.org/10.1097/GOX.0000000000002555 Hall TA (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41(2):95–98 Hanahan D (1985) Techniques for transformation of E. coli . In: Glover DM (ed.) DNA cloning: a practical approach. Vol.1:109–135. IRL Press, Oxford, UK Harvey RP, Degryse E, Stefani L, Schamber F, Cazenave JP, Courtney M, Tolstoshev P, Lecocq JP (1986) Cloning and expression of a cDNA coding for the anticoagulant hirudin from the bloodsucking leech, Hirudo medicinalis . Proc Nat Acad Sci USA 83(4):1084–1088. https://doi.org/10.1073/pnas.83.4.1084 Iwama RE, Tessler M, Siddall ME, Kvist S (2021) The origin and evolution of antistasin-like proteins in leeches (Hirudinida, Clitellata). Genome Biol Evol 13(1):evaa242. https://doi.org/10.1093/gbe/evaa242 Kalatehjari P, Wolf R, Jedlitschky G, Tolksdorf C, Rauch BH, Müller C (2026) The great diversity: Monomeric and oligomeric hirudins, hirudin-like factors and decorsins in the Asian medicinal leeches Hirudo nipponia and Hirudo tianjinensis . Parasitol Res accepted for publication https://doi.org/10.21203/rs.3.rs-8307310/v1 Käll L, Krogh A, Sonnhammer ELL (2007) Advantages of combined transmembrane topology and signal peptide prediction - the Phobius web server. Nucleic Acids Res 35(Web Server issue):W429-432. https://doi.org/10.1093/nar/gkm256 Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28(12):1647–1649. https://doi.org/10.1093/bioinformatics/bts199 Khan MS, Müller C, Zhou B (2025) Chromosome-level genome assembly and anticoagulant protein annotation of the buffalo leech Hirudinaria bpling (Hirudinea: Hirudinidae). BMC Genomics 26(1):558. https://doi.org/10.1186/s12864-025-11690-y Kuo D-H, Lai Y-T (2019) On the origin of leeches by evolution of development. Dev Growth Differ 61(1):43–57. https://doi.org/10.1111/dgd.12573 Kvist S, Min G-S, Siddall ME (2013) Diversity and selective pressures of anticoagulants in three medicinal leeches (Hirudinida: Hirudinidae, Macrobdellidae). Ecol Evol 3(4):918–933. https://doi.org/10.1002/ece3.480 Kvist S, Manzano-Marín A, de Carle D, Trontelj P, Siddall ME (2020) Draft genome of the European medicinal leech Hirudo medicinalis (Annelida, Clitellata, Hirudiniformes) with emphasis on anticoagulants. Sci Rep 10:9885. https://doi.org/10.1038/s41598-020-66749-5 Kvist S, Utevsky S, Marrone F, Ben Ahmed R, Gajda Ł, Grosser C, Huseynov M, Jueg U, Khomenko A, Oceguera-Figueroa A, Vladimir Pešić, Pupins M, Rouag R, Sağlam N, Świątek P, Trontelj P, Vecchioni L, Müller C (2022) Extensive sampling sheds light on species-level diversity in Palearctic Placobdella (Annelida: Clitellata: Glossiphoniiformes). Hydrobiologia 849:1239–1259. https://doi.org/10.1007/s10750-021-04786-5 Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21):2947–2948. https://doi.org/10.1093/bioinformatics/btm404 Lazar JB, Winant RC, Johnson PH (1991) Hirudin: amino-terminal residues play a major role in the interaction with thrombin. J Biol Chem 266(2):685–688. https://doi.org/10.1016/S0021-9258(17)35224-9 Lazarus RA, McDowell RS (1993) Structural and functional aspects of RGD-containing protein antagonists of glycoprotein IIb-IIIa. Curr Opin Biotechnol 4(4):438–445. https://doi.org/10.1016/0958-1669(93)90009-l Liu Z, Zhao F, Huang Z, Hu Q, Meng R, Lin Y, Qi H, Lin G (2023) Revisiting the Asian buffalo leech ( Hirudinaria manillensis ) genome: Focus on antithrombotic genes and their corresponding proteins. Genes (Basel) 14(11):2068. https://doi.org/10.3390/genes14112068 Lobstein J, Emrich CA, Jeans C, Faulkner M, Riggs P, Berkmen M (2012) SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microb Cell Fact 11:56. https://doi.org/10.1186/1475-2859-11-56 Lukas P, Melikian G, Hildebrandt J-P, Müller C (2022) Make it double: identification and characterization of a Tandem-Hirudin from the Asian medicinal leech Hirudinaria manillensis . Parasitol Res 121(10):2995–3006. https://doi.org/10.1007/s00436-022-07634-0 Lynggaard C, Oceguera-Figueroa A, Kvist S, Gilbert MTP, Bohmann K (2022) The potential of aquatic bloodfeeding and nonbloodfeeding leeches as a tool for iDNA characterisation. Mol Ecol Resour 22(2):539–553. https://doi.org/10.1111/1755-0998.13486 Manglicmot C, Oceguera-Figueroa A, Kvist S (2020) Bacterial endosymbionts of Placobdella (Annelida: Hirudinea: Glossiphoniidae): phylogeny, genetic distance, and vertical transmission. Hydrobiologia 847:1177–1194. https://doi.org/10.1007/s10750-019-04175-z Marchiori C, Santana M, Malheiros K (2024) Leeches and their use in medicine (Annellida: Hirudinea: Rhynchobdelliformes). Middle East Res J Biol Sci 4(03):72–81. https://doi.org/10.36348/merjbs.2024.v04i03.003 Markwardt F (1956) Untersuchungen über den Mechanismus der blutgerinnungshemmenden Wirkung des Hirudins. Naunyn - Schmiedebergs Arch 229:389–399 (1956). https://doi.org/10.1007/BF00245864 Markwardt F (1994) The development of hirudin as an antithrombotic drug. Thromb Res 74(1):1–23. https://doi.org/10.1016/0049-3848(94)90032-9 Mazur P, Henzel WJ, Seymour JL, Lazarus RA (1991) Ornatins: potent glycoprotein IIb-IIIa antagonists and platelet aggregation inhibitors from the leech Placobdella ornata . Eur J Biochem 202(3):1073–1082. https://doi.org/10.1111/j.1432-1033.1991.tb16472.x Mazur P, Dennis MS, Seymour JL, Lazarus RA (1993) Expression, purification, and characterization of recombinant ornatin E, a potent glycoprotein IIb-IIIa antagonist. Protein Expr Purif 4(4):282–289. https://doi.org/10.1006/prep.1993.1036 Micheletti C, De Filippis V, Maritan A, Seno F (2003) Elucidation of the disulfide-folding pathway of hirudin by a topology-based approach. Proteins 53(3):720–730. https://doi.org/10.1002/prot.10463. Moser WE, Bowerman J, Hovingh P, Pearl CA, Oceguera-Figueroa A (2014) New host and distribution records of the leech Placobdella sophieae Oceguera-Figueroa et al., 2010 (Hirudinida: Glossiphoniidae). Comp Parasitol 81(2):199–202. https://doi.org/10.1654/4678.1 Müller C, Mescke K, Liebig S, Mahfoud H, Lemke S, Hildebrandt J-P (2016) More than just one: multiplicity of hirudins and hirudin-like factors in the medicinal leech, Hirudo medicinalis . Mol Genet Genomics 291:227–240. https://doi.org/10.1007/s00438-015-1100-0 Müller C, Haase M, Lemke S, Hildebrandt J-P (2017) Hirudins and hirudin-like factors in Hirudinidae: implications for function and phylogenetic relationships. Parasitol Res 116(1):313–325. https://doi.org/10.1007/s00436-016-5294-9 Müller C, Lukas P, Lemke S, Hildebrandt J-P (2019) Hirudin and decorsins of the North American medicinal leech Macrobdella decora : gene structure reveals homology to hirudins and hirudin-like factors of Eurasian medicinal leeches. J Parasitol 105(3):423–431. https://doi.org/10.1645/18-117 Müller C, Wang Z, Hamann M, Sponholz D, Hildebrandt J-P (2022) Life without blood: Molecular and functional analysis of hirudins and hirudin-like factors of the Asian non-hematophagous leech Whitmania pigra . J Thromb Haemost 20(8):1808–1817. https://doi.org/10.1111/jth.15762 Müller C, Sponholz D, Tolksdorf C, Rauch BH, Kvist S (2024) Identification and functional characterization of multiple haemadins and an oligomeric decorsin in the Asian land leech Haemadipsa interrupta . Parasitol Res 123(11):390. https://doi.org/10.1007/s00436-024-08404-w Nair HKR, Ahmad NW, Lee HL, Ahmad N, Othamn S, Mokhtar NSHM, Chong SSY (2022) Hirudotherapy in wound healing. Int J Low Extrem Wounds 21(4):425–431. https://doi.org/10.1177/1534734620948299 Nicholas KB, Nicholas Jr HB (1997) GeneDoc: a tool for editing and annotating multiple sequence alignments. Distributed by the author Núñez-Navarro NE, Santana FM, Parra LP, Zacconi FC (2019) Surfing the blood coagulation cascade: Insight into the vital factor Xa. Curr Med Chem 26(17):3175–3200. https://doi.org/10.2174/0929867325666180125165340 Oceguera-Figueroa A (2012) Molecular phylogeny of the New World bloodfeeding leeches of the genus Haementeria and reconsideration of the biannulate genus Oligobdella . Mol Phylogenet Evol 62(1):508–514. https://doi.org/10.1016/j.ympev.2011.10.020 Oliveira DGL Alvarez-Flores MP, Lopes AR, Chudzinski-Tavassi AN (2012) Functional characterisation of vizottin, the first factor Xa inhibitor purified from the leech Haementeria vizottoi . Thromb Haemost 108(3):570–578. https://doi.org/10.1160/TH12-04-0235 Pace CN, Vajdos F, Fee L, Grimsley G, Gray T (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci 4:2411–2423. https://doi.org/10.1002/pro.5560041120 Pfordt V, Kalatehjari P, Tolksdorf C, Rauch BH, Müller C (2022) Go West: Hirudins and decorsin/ ornatin-like platelet aggregation inhibitors in two representatives of American hematophagous leeches. Parasitologia 2:313–325. https://doi.org/10.3390/parasitologia2040026 Phillips J, Dornburg A, Zapfe KL, Anderson FE, James SW, Erséus C, Lemmon EM, Lemmon AR, Williams BW (2019) Phylogenomic analysis of a putative missing link sparks reinterpretation of leech evolution. Genome Biol Evol 11(11):3082–3093. https://doi.org/10.1093/gbe/evz120 Reiss S, Sieber M, Oberle V, Wentzel A, Spangenberg P, Claus R, Kolmar H, Lösche W (2006) Inhibition of platelet aggregation by grafting RGD and KGD sequences on the structural scaffold of small disulfide-rich proteins. Platelets 17(3):153–157. https://doi.org/10.1080/09537100500436663 Richardson DJ, Moser WE, Hammond CI, Lazo-Wasem EA, McAllister CT, Pulis EE (2017) A new species of leech of the genus Placobdella (Hirudinida, Glossiphoniidae) from the American alligator ( Alligator mississippiensis ) in Mississippi, USA. Zookeys 667:39–49. https://doi.org/10.3897/zookeys.667.10680 Sarkans U, Gostev M, Athar A, Behrangi E, Melnichuk O, Ali A, Minguet J, Rada JC, Snow C, Tikhonov A, Brazma A, McEntyre J (2018) The BioStudies database-one stop shop for all data supporting a life sciences study. Nucleic Acids Res 46(D1):D1266-D1270. doi: 10.1093/nar/gkx965 Sawyer RT, Casellas M, Munro R, Powell Jones C (1991) Secretion of hementin and other antihaemostatic factors in the salivary gland complex of the giant amazon leech Haementeria ghilianii . Comp Haematol Int 1:35–41. https://doi.org/10.1007/BF00422691 Sawyer RT (2022) Comparative morphological analysis of two species of turtle leeches coexisting in North America (Hirudinea:Glossiphoniidae): Embryological evidence for character displacement. Taxonomy 2:160–179. https://doi.org/10.3390/taxonomy2020013 Schulz L, Tolksdorf C, Rauch BH, Kvist S, Müller C (2025) Hirudins and fenestrins of the African medicinal leech Asiaticobdella fenestrata . Parasitol Res 124(11):124. https://doi.org/10.1007/s00436-025-08578-x Seale L, Finney S, Sawyer RT, Wallis RB (1997) Tridegin, a novel peptidic inhibitor of factor XIIIa from the leech, Haementeria ghilianii , enhances fibrinolysis in vitro . Thromb Haemost 77(5):959–963. https://doi.org/10.1055/s-0038-1656085 Seymour JL, Henzel WJ, Nevins B, Stults JT, Lazarus RA (1990) Decorsin. A potent glycoprotein IIb-IIIa antagonist and platelet aggregation inhibitor from the leech Macrobdella decora . J Biol Chem 265(17):10143–10147. https://doi.org/10.1016/S0021-9258(19)38791-5 Shakouri A, Wollina U (2021) Time to change theory; medical leech from a molecular medicine perspective leech salivary proteins playing a potential role in medicine. Adv Pharm Bull 11(2):261–266. https://doi.org/10.34172/apb.2021.038 Siddall ME, Brugler MR, Kvist S (2016) Comparative transcriptomic analyses of three species of Placobdella (Rhynchobdellida: Glossiphoniidae) confirms a single origin of blood feeding in leeches. J Parasitol 102(1):143–150. https://doi.org/10.1645/15-802 Singh AP (201) Medicinal leech therapy (hirudotherapy): a brief overview. Complement Ther Clin Pract 16(4):213–215. https://doi.org/10.1016/j.ctcp.2009.11.005 Tessler M, de Carle D, Voiklis ML, Gresham OA, Neumann JS, Cios S, Siddall ME (2018) Worms that suck: Phylogenetic analysis of Hirudinea solidifies the position of Acanthobdellida and necessitates the dissolution of Rhynchobdellida. Mol Phylogenet Evol 127:129–134. https://doi.org/10.1016/j.ympev.2018.05.001 Teufel F, Armenteros JJA, Johansen AR, Gíslason MH, Pihl SI, Tsirigos KD, Winther O, Brunak S, von Heijne G, Nielsen H (2022) SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat Biotechnol 40(7):1023–1025. https://doi.org/10.1038/s41587-021-01156-3 Tolksdorf C, Wolf R, Rauch BH, Jedlitschky G, Müller C (2025) Monomeric and oligomeric decorsins of the Asian medicinal leech Hirudinaria manillensis . Int J Mol Sci 26, 11017. https://doi.org/10.3390/ijms262211017 Tuszynski GP, Gasic TB, Gasic GJ (1987) Isolation and characterization of antistasin. An inhibitor of metastasis and coagulation. J Biol Chem 262(20):9718–9723. https://doi.org/10.1016/S0021-9258(18)47993-8 Van de Walle GR, Peters ST, VanderVen BC, O'Callaghan DJ, Osterrieder N (2008) Equine herpesvirus 1 entry via endocytosis is facilitated by alphaV integrins and an RSD motif in glycoprotein D. J Virol 82(23):11859–11868. https://doi.org/10.1128/JVI.00868-08 Wallace A, Dennis S, Hofsteenge J, Stone SR (1989) Contribution of the N-terminal region of hirudin to its interaction with thrombin. Biochemistry 28(26):10079–10084. https://doi.org/10.1021/bi00452a030 Wang Z, Böttcher D, Bornscheuer UT, Müller C (2025) Expression of recombinant hirudin in bacteria and yeast: A comparative approach. Methods Protoc 8(4):89. https://doi.org/10.3390/mps8040089 Wu L, Li Y, Yang Y, Qin M (2013) Deciphering structural and functional roles of disulfide bonds in decorsin. Sci China Chem 56:1485–1492. https://doi.org/10.1007/s11426-013-4954-1 Zhao F, Huang Z, He B, Liu K, Li J, Liu Z, Lin G (2024) Comparative genomics of two Asian medicinal leeches Hirudo nipponia and Hirudo tianjinensis : With emphasis on antithrombotic genes and their corresponding proteins. Int J Biol Macromol 270(Pt 1):132278. https://doi.org/10.1016/j.ijbiomac.2024.132278 Zhou L, Schmaier AH (2005) Platelet aggregation testing in platelet rich plasma: description of procedures with the aim to develop standards in the field. Am J Clin Pathol 123(2):172–183. https://doi.org/10.1309/y9ec-63rw-3xg1-v313 Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.zip 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. 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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-8807632","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":591151292,"identity":"6a737bca-e665-4ba4-831d-5c8548c542de","order_by":0,"name":"Raja Ben Ahmed","email":"","orcid":"","institution":"University of Tunis El Manar","correspondingAuthor":false,"prefix":"","firstName":"Raja","middleName":"Ben","lastName":"Ahmed","suffix":""},{"id":591151294,"identity":"001eec79-7b3b-4466-b239-a2596bfb5082","order_by":1,"name":"Robert Wolf","email":"","orcid":"","institution":"University Medicine Greifswald","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Wolf","suffix":""},{"id":591151301,"identity":"5fa0f939-0aac-4408-b1cd-0c1cbf9ddb35","order_by":2,"name":"Gabriele Jedlitschky","email":"","orcid":"","institution":"University Medicine Greifswald","correspondingAuthor":false,"prefix":"","firstName":"Gabriele","middleName":"","lastName":"Jedlitschky","suffix":""},{"id":591151304,"identity":"e564277b-dab2-4e02-8a84-3db769f0226d","order_by":3,"name":"Céline Tolksdorf","email":"","orcid":"","institution":"University Medicine Oldenburg, Carl von Ossietzky University","correspondingAuthor":false,"prefix":"","firstName":"Céline","middleName":"","lastName":"Tolksdorf","suffix":""},{"id":591151306,"identity":"d415079f-d0e8-4b8f-af6a-6e1fcbefad53","order_by":4,"name":"Bernhard H. Rauch","email":"","orcid":"","institution":"University Medicine Oldenburg, Carl von Ossietzky University","correspondingAuthor":false,"prefix":"","firstName":"Bernhard","middleName":"H.","lastName":"Rauch","suffix":""},{"id":591151315,"identity":"c77e2e5c-5c6d-4d7c-8e4c-8e1395ce6a12","order_by":5,"name":"Clemens Grosser","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Clemens","middleName":"","lastName":"Grosser","suffix":""},{"id":591151320,"identity":"bdedc74d-e0da-434e-ab4c-d9d17e68ea37","order_by":6,"name":"Sebastian Kvist","email":"","orcid":"","institution":"Swedish Museum of Natural History","correspondingAuthor":false,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Kvist","suffix":""},{"id":591151321,"identity":"50771179-e4d6-41e1-89ab-ed9871fdd989","order_by":7,"name":"Christian Müller","email":"data:image/png;base64,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","orcid":"","institution":"University of Greifswald","correspondingAuthor":true,"prefix":"","firstName":"Christian","middleName":"","lastName":"Müller","suffix":""}],"badges":[],"createdAt":"2026-02-06 13:27:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8807632/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8807632/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102984846,"identity":"4cdb0d09-b533-4f5b-81fa-b4a9e8161f84","added_by":"auto","created_at":"2026-02-19 09:57:34","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":102551,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of hirudin Pcos_HV1 and ornatin Pcos_OV2 gene and cDNA structures in \u003cem\u003eP. costata\u003c/em\u003e. Exons are labeled in green, introns are labeled in red. Exon boundary-overlapping codons are labeled in yellow, non-overlapping codons are labeled in blue. Sizes are adjusted relative to the size of exon 1 of the Pcos_HV1 gene (see Table 2 for details)\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8807632/v1/0d60aa454c1ba49c6af72fd2.jpeg"},{"id":103049671,"identity":"5bcae709-4030-4179-a52b-91783fe68ce7","added_by":"auto","created_at":"2026-02-20 07:44:34","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":228095,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple amino acid sequence alignments of putative hirudin variants HV1-3 of \u003cem\u003eP. costata\u003c/em\u003e (Pcos_HV1-3), HV1-3 of \u003cem\u003eP. nabeulensis \u003c/em\u003e(Pnab_HV1-3), HV1 and HV2 of \u003cem\u003eP. ali\u003c/em\u003e (Pali_HV1 and 2), HV1-3 of \u003cem\u003eP. kwetlumye\u003c/em\u003e (Pkwe_HV1-3) and HV1 of \u003cem\u003eP. parasitica\u003c/em\u003e (Ppar_HV1). A black background indicates fully conserved residues; a gray background indicates partially conserved residues. The six conserved cysteine residues giving rise to the three-dimensional structure of the central globular domain are marked in bold and red. The essential phenylalanine residue at position 3 is marked with an arrow. The signal peptide sequence is underlined. Abbreviations are used according to the IUPAC code\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8807632/v1/b1af12ec92ba1904ad8981b8.jpeg"},{"id":103049575,"identity":"030217ca-e568-4c08-b607-cae3fe28f3e9","added_by":"auto","created_at":"2026-02-20 07:42:50","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":43813,"visible":true,"origin":"","legend":"\u003cp\u003eStandard blood coagulation assays of putative hirudins Pcos_HV1-3 of \u003cem\u003eP. costata\u003c/em\u003e using the thrombin time test (TT). All test compounds were used at final concentrations of 3.2 or 0.32 μmol/l, respectively. Results are the mean of three to four independent measurements, bars indicate standard deviation (SD)\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8807632/v1/88ebdc1c2de4f5460b29be32.jpeg"},{"id":103049675,"identity":"9f729dd1-b7d6-4cbc-aedf-c98a1f065c19","added_by":"auto","created_at":"2026-02-20 07:44:39","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":62759,"visible":true,"origin":"","legend":"\u003cp\u003eStandard blood coagulation assays of Pcos_HV2 and HV3 variants of \u003cem\u003eP. costata\u003c/em\u003e using the thrombin time test (TT). All factors were expressed in \u003cem\u003eE. coli\u003c/em\u003e strain DH5a. All test compounds were used at final concentrations of 3.2 or 0.32 μmol/l, respectively. Results are the mean of three independent measurements, bars indicate standard deviation (SD)\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8807632/v1/31273a5378c0aef41152db7a.jpeg"},{"id":102984849,"identity":"d0db9c17-f18a-4918-a768-77e574028d04","added_by":"auto","created_at":"2026-02-19 09:57:34","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":210904,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple amino acid sequence alignments of putative ornatin variants OV1a, 1b and 2 of \u003cem\u003eP. costata\u003c/em\u003e (Pcos_OV1a, 1b and 2), OV1 and 2 of \u003cem\u003eP. nabeulensis \u003c/em\u003e(Pnab_OV1 and 2), OV1-3 of \u003cem\u003eP. ali\u003c/em\u003e (Pali_OV1-3), OV1 of \u003cem\u003eP. kwetlumye\u003c/em\u003e (Pkwe_OV1) and OV1 of \u003cem\u003eP. parasitica\u003c/em\u003e (Ppar_OV1). A black background indicates fully conserved residues; a gray background indicates partially conserved residues. The six conserved cysteine residues giving rise to the three-dimensional structure of the central globular domain and the RGD motif are marked in bold and red, divergent residues are labeled in yellow. The signal peptide sequence is underlined. Abbreviations are used according to the IUPAC code\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8807632/v1/8cc82cd392074166debc65b6.jpeg"},{"id":102984852,"identity":"ec1481d3-3a94-4f62-9f4f-f870323fedb7","added_by":"auto","created_at":"2026-02-19 09:57:34","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":124007,"visible":true,"origin":"","legend":"\u003cp\u003eStandard platelet aggregation assays of putative decorsin variants Pcos_OV1a and Pcos_OV2 of \u003cem\u003eP. costata\u003c/em\u003e expressed in \u003cem\u003eE. coli\u003c/em\u003e strains DH5a (A+C) or SHuffle® T7 (B+D). A and B display the aggregation curves, B and D the derived AUC values. Tirofiban and eptifibatide were used as positive control substances for the complete inhibition of aggregation; buffer was used as a negative control. All test compounds and the control substances were used at a final concentration of 3.2 μmol/l. Platelet aggregation was induced by the addition of adenosine diphosphate (ADP) in a final concentration of 5 μmol/l. Results are the mean of two to four independent measurements, bars indicate SD. ** indicates a p-value ≤ 0.01, *** indicates p-values ≤ 0.001\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8807632/v1/b778239235a0ba901c309009.jpeg"},{"id":103056496,"identity":"5fe31c92-bcd9-4ba1-80a1-d0aa97139ed2","added_by":"auto","created_at":"2026-02-20 09:12:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1900602,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8807632/v1/d148a40a-10e7-40ff-a4b6-974040c9217e.pdf"},{"id":102984850,"identity":"718128f3-6dcf-4253-a476-6db714ffb8f1","added_by":"auto","created_at":"2026-02-19 09:57:34","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":384132,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.zip","url":"https://assets-eu.researchsquare.com/files/rs-8807632/v1/fcdfe1003d0468bd10680c24.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hirudins and ornatins in five species of the genus Placobdella (Annelida: Hirudinea: Glossiphoniidae)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLeeches are segmented worms that form the subclass Hirudinea within the phylum Annelida (Phillips et al. 2019). Crudely, leeches are traditionally divided into two major clades based on the morphology of their feeding apparatus: Rhynchobdellida Blanchard, 1894, whose members possess a proboscis, and Arhynchobdellida Blanchard, 1894, without a proboscis. The former group includes the Glossiphoniidae Vaillant, 1890, jawless dorsoventrally flattened leeches that inhabit freshwater ecosystems. The latter group includes the Hirudiniformes Caballero, 1952, in which the feeding apparatus is usually characterized by the presence of jaws (Kuo and Lai 2019). Many leeches of both groups are hematophagous, whereas others are macrophagous or liquidosomatophagous (Lynggaard et al. 2022). The medicinal use of hematophagous leeches, consequently also termed medicinal leeches, traces far back in human history and was common in many civilizations throughout time (Elliot and Kutschera 2011; Abdualkader et al. 2013; Marchiori et al. 2024). However, mainly representatives of jawed leeches were and are regularly used for medical purposes, including the European medicinal leech \u003cem\u003eHirudo medicinalis\u003c/em\u003e Linnaeus, 1758, the Mediterranean medicinal leech \u003cem\u003eHirudo verbana\u003c/em\u003e Carena, 1820, the African medicinal leech \u003cem\u003eHirudo troctina\u003c/em\u003e Johnson, 1816, the Asian medicinal leeches \u003cem\u003eHirudo nipponia\u003c/em\u003e Whitman, 1886, and \u003cem\u003eHirudinaria manillensis\u003c/em\u003e Lesson, 1842, the North-American medicinal leech \u003cem\u003eMacrobdella decora\u003c/em\u003e Say, 1824, or the Australian medicinal leech \u003cem\u003eRichardsonianus australis\u003c/em\u003e Bosisto, 1859 (Kvist et al. 2013). By contrast, proboscis-bearing leeches are much less frequently considered for the direct use in medical practice, yet members of the Central and South American genus \u003cem\u003eHaementeria\u003c/em\u003e de Filippi, 1849, may represent an exception (Oceguera-Figueroa 2012). As a consequence, only few studies have specifically addressed the antithrombotic repertoire of rhynchobdellid leeches. Again, the genus \u003cem\u003eHaementeria\u003c/em\u003e represents a remarkable exception (Sawyer et al. 1991; Seale et al. 1997; Faria et al. 1999; Oliveira et al. 2012; Amorim et al. 2015; Pfordt et al. 2022). Antistasin, a factor Xa inhibitor that was isolated from the Mexican leech \u003cem\u003eHaementeria officinalis\u003c/em\u003e de Filippi, 1849 (Tuszynski et al. 1987), is probably the most well-known \u003cem\u003eHaementeria\u003c/em\u003e-derived anticoagulant (Iwama et al. 2020). Factor Xa is a crucial component of the blood coagulation-cascade and facilitates the cleavage of prothrombin to thrombin (N\u0026uacute;\u0026ntilde;ez-Navarro et al. 2019). The activity of thrombin in turn is inhibited by direct thrombin-inhibitors like hirudin (Markwardt 1956). Both antistasin and hirudin are hence inhibitors of the secondary hemostasis. In contrast, the primary hemostasis (the process of platelet aggregation) is inhibited by leech-derived factors like decorsin and ornatin. Whereas decorsin was first described from \u003cem\u003eM. decora\u003c/em\u003e (Seymour et al. 1990), ornatin originates from \u003cem\u003ePlacobdella ornata\u003c/em\u003e Verrill, 1872 (Mazur et al. 1991). Both factors are potent glycoprotein IIb/IIIa antagonists and contain a highly conserved RGD motif (Lazarus and McDowell 1993).\u003c/p\u003e \u003cp\u003eLike the genus \u003cem\u003eHaementeria\u003c/em\u003e, the genus \u003cem\u003ePlacobdella\u003c/em\u003e Blanchard, 1893, also belongs to the order of \u003cem\u003eRhynchobdellida\u003c/em\u003e Blanchard, 1894, and the family of \u003cem\u003eGlossiphoniidae\u003c/em\u003e Vaillant, 1890. The genus \u003cem\u003ePlacobdella\u003c/em\u003e currently comprises about 25 different species that inhabit large areas of North America (de Carle et al. 2017). Members of the genus are hematophagous and primarily ectoparasitic on turtles (Bielecki et al. 2012), but occasionally also feed on amphibians, birds and mammals including humans (Moser et al. 2014; Cichocka et al. 2021). \u003cem\u003ePlacobdella costata\u003c/em\u003e M\u0026uuml;ller, 1846, represents the only European member of the genus. The species is widely distributed across Europe, and the distribution range largely overlaps with the distribution range of its natural host, the European mud turtle \u003cem\u003eEmys orbicularis\u003c/em\u003e Linnaeus, 1758 (Bielecki et al. 2012). A contemporary study showed that the genetic diversity between the various populations of \u003cem\u003eP. costata\u003c/em\u003e may suggest up to seven separately evolving European lineages throughout its range, five of which exhibit degrees of separation at the molecular level to warrant species level status (Kvist et al. 2022). One of these lineages was recently confirmed as a second Palaearctic member of the genus \u003cem\u003ePlacobdella\u003c/em\u003e, namely \u003cem\u003ePlacobdella nabeulensis\u003c/em\u003e (Ben Ahmed et al. 2023). This species occurs in distinct regions of Tunisia and Algeria in northern Africa.\u003c/p\u003e \u003cp\u003eSo far, investigations into members of the genus \u003cem\u003ePlacobdella\u003c/em\u003e have mainly focused on the description and analysis of morphological (Richardson et al. 2017; Sawyer 2022) and genetic (de Carle et al. 2017; Ben Ahmed et al. 2023) markers or on the composition of their bacterial symbionts (Manglicmot et al. 2020). Besidess the historical identification of ornatins, investigations into the presence and diversity of anticoagulants in the salivary glands of these animals are rare. In 2016, Mark Siddall et al. conducted a comparative transcriptomic analysis of three members of the genus \u003cem\u003ePlacobdella\u003c/em\u003e, namely \u003cem\u003ePlacobdella ali\u003c/em\u003e Hughes \u0026amp; Siddall, 2007, \u003cem\u003ePlacobdella kwetlumye\u003c/em\u003e Oceguera-Figueroa, Kvist, Watson, Sankar, Overstreet \u0026amp; Siddall, 2010 and \u003cem\u003ePlacobdella parasitica\u003c/em\u003e Say, 1824, and the authors identified a broad variety of putative anticoagulants including hirudins and ornatins (Siddall et al. 2016). Functional investigations of the respective factors, however, are still missing. In 2024, the complete genome sequence data of \u003cem\u003eP. costata\u003c/em\u003e were deposited in the nucleotide database of GenBank by the Wellcome Sanger Darwin Tree of Life Programme including the sequences of 18 chromosomes (accession numbers OZ194472.1 - OZ194489.1) and the mitochondrial genome (accession number OZ194490.1). Strikingly, the information on the genome was neither published nor is it available on the website of the Darwin Tree of Life Programme.\u003c/p\u003e \u003cp\u003eOver the last years, we have successfully applied the approach to conduct in-depth analyses and re-analyses of genome and transcriptome sequence data of several leech species in order to further explore the diversity of putative anticoagulants (Khan et al. 2025). Whenever feasible, factors of interest were expressed as recombinant proteins and subsequently functionally characterized (Pfordt et al. 2022; M\u0026uuml;ller et al. 2024; Schulz et al. 2025; Tolksdorf et al. 2025). The availability of both transcriptome and genome sequence data has enabled such analyses also for \u003cem\u003eP. costata\u003c/em\u003e. The primary aim of the present study was hence to identify, and functionally characterize putative anticoagulants in five species of \u003cem\u003ePlacobdella\u003c/em\u003e, with a special focus on hirudins and ornatins. The second aim, however, was to eventually answer the question whether or not the hirudins and ornatins of glossiphoniid leeches also belong to the hirudin superfamily whose members are characterized not only by similar structural features of the respective molecules, but also by a common gene structure (M\u0026uuml;ller et al. 2019).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGenBank transcriptome and genome data\u003c/h2\u003e \u003cp\u003eTranscriptome data for members of the genus \u003cem\u003ePlacobdella\u003c/em\u003e were obtained from the following GenBank sequence read archives (SRAs): SRX3734179 (\u003cem\u003eP. ali\u003c/em\u003e), SRX3734173 (\u003cem\u003eP. kwetlumye\u003c/em\u003e) and SRX1335044 (\u003cem\u003eP. parasitica\u003c/em\u003e), respectively. Chromosome-level genome data of \u003cem\u003eP. costata\u003c/em\u003e were obtained from GenBank accession numbers OZ194472.1 - OZ194489.1.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimal collection, tissue preparation, genomic DNA isolation and sequencing\u003c/h3\u003e\n\u003cp\u003eIndividuals of \u003cem\u003ePlacobdella costata\u003c/em\u003e were collected in November 2018 from the Kapengraben in Saxony-Anhalt, Germany (51.81488 N, 12.40539 E) (collection codes 695 and 696, Kvist et al. 2022), whereas individuals of \u003cem\u003ePlacobdella nabeulensis\u003c/em\u003e were collected in June 2021 from El Malaabi Dam (36.81666 N, 10.98333 E) in Menzel Temime located in the North-East of Tunisia (Ben Ahmed et al. 2023). The caudal suckers of two individuals of \u003cem\u003eP. costata\u003c/em\u003e and one individual of \u003cem\u003eP. nabeulensis\u003c/em\u003e were dissected and placed in 2 ml microcentrifuge tubes each. Prior to extraction of genomic DNA, 350 \u0026micro;l of the lysis buffer ML 1 and 25 \u0026micro;l of proteinase K solution (E.Z.N.A. Mollusc DNA kit, Omega Bio-tek Inc., Norcross, GA) were added to the samples and the tissues were manually homogenized using scissors. DNA extractions were performed according to the manufacturer\u0026rsquo;s protocol. Purified genomic DNA was eluted with sterile double distilled water and stored at -20\u0026deg;C. For whole genome sequencing, the samples were sent to the Competence Centre for Genomic Analysis (Kiel, Germany). The genomic DNA was quality-controlled and quantified via fluorometric measurement. Library preparation and sequencing were carried out at the Competence Centre for Genomic Analysis (Kiel, Germany). Library preparations were done using the Illumina DNA Prep Kit (Illumina, Berlin, Germany) according to the manufacturer\u0026rsquo;s protocol. Sequencing of the libraries was performed on an Illumina NovaSeq 6000 S4 device, using 2\u0026times;150 bp reads on v1.5 Flowcells. The raw genome sequence data of both the two individuals of \u003cem\u003eP. costata\u003c/em\u003e and the single individual of \u003cem\u003eP. nabeulensis\u003c/em\u003e were deposited in BioStudies (The European Bioinformatics Institute, EMBL-EBI, Sarkans et al. 2018) and received the following accession number: S-BSST2339 (DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.6019/S-BSST2339\u003c/span\u003e\u003cspan address=\"10.6019/S-BSST2339\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The data can be freely accessed and used. During the course of the present study a high-quality, chromosome-level reference genome of \u003cem\u003eP. costata\u003c/em\u003e was generated by the Wellcome Sanger Institute and deposited in GenBank (GCA_964276725.1). All \u003cem\u003eP. costata\u003c/em\u003e-related sequence information in the manuscript hence refer to the reference genome.\u003c/p\u003e\n\u003ch3\u003eBioinformatics and graphical tools\u003c/h3\u003e\n\u003cp\u003eRaw genome sequence data were imported into Geneious v9.1.8 (Biomatters Ltd., Auckland, New Zealand; Kearse et al. 2012) for further analyses. Basic Local Alignment Search Tool (BLAST) searches were performed using the NCBI web portal (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), BioEdit v7.2.5 (Hall 1999) or Geneious v9.1.8 with adjusted parameter settings for both word size and the expected threshold values. Multiple sequence alignments (MSA) were generated using ClustalX 2.1 (Larkin et al. 2007) or the CLC Sequence Viewer software package v8.0 (QIAGEN, Aarhus, Denmark) using default settings. Particular attention was paid to the conservation of disulfide bond-forming cysteines. Alignments were exported as msf-files and further processed using Gene-Doc v2.7 (Nicholas and Nicholas 1997). Signal peptide sequences were predicted using the SignalP6.0 server (Teufel et al. 2022) and the Phobius web portal (K\u0026auml;ll et al. 2007). Graphs were generated and analyzed using GraphPad Prism V5.01 (GraphPad Software, Boston, MA, USA).\u003c/p\u003e\n\u003ch3\u003eGene synthesis\u003c/h3\u003e\n\u003cp\u003eThe cDNA fragments of putative hirudins, hirudin-like factors (HLFs) and ornatins of \u003cem\u003eP. costata\u003c/em\u003e were synthesized using the GeneArt gene synthesis service of Thermo Fisher Scientific (Darmstadt, Germany).\u003c/p\u003e\n\u003ch3\u003eAmplification and cloning of putative hirudin and decorsin cDNAs\u003c/h3\u003e\n\u003cp\u003eFor the amplification of putative hirudin and decorsin cDNAs, primers were derived from the respective transcriptome or genome sequences. A list of all primers that were used in the study is provided in Supplementary Information File Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. PCR reactions were performed using Q5 high-fidelity DNA polymerase (New England Biolabs, Frankfurt a. M., Germany), fragments of relevant sizes were purified and cloned into the expression vector pQE30Xa (QIAGEN, Hilden, Germany). Successfully cloned cDNAs were sequenced for control purposes by Biosearch Technologies (LGC, Berlin, Germany) or by Eurofins Genomics (Cologne, Germany).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eExpression, purification, processing, and quantification of putative hirudins and decorsins\u003c/h2\u003e \u003cp\u003eThe detailed procedure to express, purify, process and quantify the respective recombinant hirudins and decorsins of \u003cem\u003eP. costata\u003c/em\u003e was described in numerous recent publications (e.g. M\u0026uuml;ller et al. 2016; Wang et al. 2025; Tolksdorf et al. 2025). Briefly, all factors were either expressed in \u003cem\u003eEscherichia coli\u003c/em\u003e laboratory strains DH5α at a cultivation temperature of 37\u0026deg;C (Hanahan 1985) or SHuffle\u0026reg; T7 at a cultivation temperature of 30\u0026deg;C (New England Biolabs, Frankfurt a. M., Germany; Lobstein et al. 2012). To obtain the recombinant proteins, we applied an expression and purification system that was developed by QIAGEN (Hilden, Germany). The pQE30Xa vector encodes a factor Xa protease recognition site that is located between the His-tag coding region at the 5\u0026prime; side and the multiple cloning site at the 3\u0026prime; side. A subsequent factor Xa protease-treatment cleaves off the His-tag and results in a recombinant protein that is devoid of any vector-derived amino acid residues at the N-terminus. The successful expression, purification and factor Xa treatment of all factors was controlled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses. Molar concentrations of final protein solutions were calculated by dividing the absorbance at 280 nm by the molar absorption coefficient according to the equation ε = (nW \u0026times; 5,500) + (nY \u0026times; 1,490) + ( nC \u0026times; 125) (Gill and von Hippel 1989; Pace et al. 1995).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBlood coagulation assays\u003c/h3\u003e\n\u003cp\u003eTo verify the thrombin-inhibitory potencies of putative hirudins of \u003cem\u003eP. costata\u003c/em\u003e, we performed a thrombin time test (TTT; reference range 16.8\u0026ndash;21.4 s) using a BFT II analyzer (Siemens Healthcare, Erlangen, Germany). All steps were carried out according to the manufacturer\u0026rsquo;s instructions. Protein samples were diluted with buffer to reach final concentrations in the reaction assays of 3.2 \u0026micro;mol/l or 0.32 \u0026micro;mol/l, respectively. The desired amount of substrate was directly transferred into the test cuvette immediately before the plasma was added. Dade\u0026reg; Ci-Trol\u0026reg; 1 (Siemens Healthcare, Erlangen, Germany) was used as standardized human plasma. The incubation of reaction mixtures was carried out at 37.4\u0026deg;C. Measurements that reached 300 s before any coagulation was detected were stopped and considered as complete inhibition of coagulation. Blood coagulation tests were performed in three to four technical replicates.\u003c/p\u003e\n\u003ch3\u003ePlatelet aggregation assays\u003c/h3\u003e\n\u003cp\u003ePlatelet aggregation was analyzed by light transmission aggregometry (LTA) with human blood samples that were obtained from healthy human volunteers after written informed consent and approval from the institutional ethics committee. Blood collection, sample preparation, and the subsequent experimental procedures were performed as described in M\u0026uuml;ller et al. (2024) and Tolksdorf et al. (2025). Briefly, 10 ml of venous blood was taken from the antecubital vein using an S-Monovette\u0026reg; (Sarstedt, N\u0026uuml;rnbrecht, Germany) prefilled with citrate buffer. The first centrifugation step of the blood collection tube was performed at 200 g for 20 min at room temperature. After centrifugation, the supernatant (platelet-rich plasma, PRP) was transferred, and the remaining blood was centrifuged again for 10 min at 2000 g at room temperature. The supernatant was dedicated as platelet-poor plasma (PPP), transferred, and used as a reference value for maximal platelet aggregation. Measurements were performed using either an APACT-4004 aggregometer (LABiTec, Ahrensburg, Germany) or a TA-8 V aggregometer (Diagnostica Stago S.A.S., Asni\u0026egrave;ressur-Seine, France). The snake venom-derived platelet aggregation inhibitors eptifibatide and tirofiban (Sigma-Aldrich, Taufkirchen, Germany) were used as positive controls for complete inhibition of platelet aggregation. PRP was pre-incubated with the respective test and control compounds (final concentration 3.2 \u0026micro;mol/l) or buffer for 1 min at 37\u0026deg;C. For the measurement, PRP was subsequently transferred into the test cuvettes and stimulated with ADP (200 \u0026micro;mol/l; Hart Biologicals, Hartlepool, UK; final concentration 5 \u0026micro;mol/l) after 1 min of runtime. The final volume in each test cuvette was 250 \u0026micro;L of diluted PRP. All experiments were performed at 37\u0026deg;C over a time period of 400 s. Maximal aggregation in percentage and the area under the curve were calculated as quantitative output parameters (Zhou and Schmaier 2005). Platelet aggregation tests were performed in two to four technical replicates.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIdentification of putative hirudin and ornatin cDNAs in\u003c/b\u003e \u003cb\u003eP. ali\u003c/b\u003e, \u003cb\u003eP. kwetlumye\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eP. parasitica\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWhereas ornatins have already been described from \u003cem\u003eP. ornata\u003c/em\u003e many years ago (Mazur et al. 1991), the investigations of Siddall et al. (2016) for the first time revealed the presence of putative hirudins in members of the genus \u003cem\u003ePlacobdella\u003c/em\u003e. Altogether, the authors identified putative cDNAs of hirudin in \u003cem\u003eP. kwetlumye\u003c/em\u003e and \u003cem\u003eP. parasitica\u003c/em\u003e and putative cDNAs of ornatin in \u003cem\u003eP. ali\u003c/em\u003e and \u003cem\u003eP. parasitica\u003c/em\u003e, respectively. However, none of the putative hirudins or ornatins was functionally verified so far. Our in-depth re-analysis of the transcriptomic data confirmed the results of Siddall et al. (2016), but we were able to identify additional putative hirudin and ornatin-encoding cDNAs. In total, we identified two putative hirudins in \u003cem\u003eP. ali\u003c/em\u003e (Pali_HV1 and Pali_HV2), three putative hirudins in \u003cem\u003eP. kwetlumye\u003c/em\u003e (Pkwe_HV1, Pkwe_HV2 and Pkwe_HV3) and one putative hirudin in \u003cem\u003eP. parasitica\u003c/em\u003e (Ppar_HV1). Pkwe_HV1 and Ppar_HV1 were already described by Siddall et al. (2016). The corresponding contigs of all putative hirudins-encoding cDNAs are provided in Supplementary Information Files S1-S3.\u003c/p\u003e \u003cp\u003eThe identification of putative ornatin-encoding contigs turned out to be a challenging task and required some manual sequence editing. However, we eventually identified three putative ornatin-encoding cDNAs in \u003cem\u003eP. ali\u003c/em\u003e (Pali_OV1-3) and one cDNA in both \u003cem\u003eP. kwetlumye\u003c/em\u003e (Pkwe_OV1) and \u003cem\u003eP. parasitica\u003c/em\u003e (Ppar_OV1), respectively. The corresponding contigs are provided in Supplementary Information Files S1-S3.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of putative hirudin and ornatin genes in\u003c/b\u003e \u003cb\u003eP. costata\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eP. nabeulensis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBased on the cDNA sequences of putative hirudins and ornatins of \u003cem\u003eP. ali\u003c/em\u003e, \u003cem\u003eP. kwetlumye\u003c/em\u003e and \u003cem\u003eP. parasitica\u003c/em\u003e we subsequently screened the genome of \u003cem\u003eP. costata\u003c/em\u003e to identify the respective gene sequences. Based on our analyses, the genome of \u003cem\u003eP. costata\u003c/em\u003e contains three genes that encode putative hirudin variants (HV), namely Pcos_HV1-3, and three genes that encode putative ornatin variants (OV), namely Pcos_OV1a, _OV1b and _OV2. All genes are located on chromosome 18, the exact position and orientation of each gene are provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePosition and orientation of putative hirudin (HV) and ornatin (OV) genes on chromosome 18 in \u003cem\u003eP. costata\u003c/em\u003e. rev+comp indicates reverse and complementary orientation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003egene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eposition\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_HV3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3772705\u0026ndash;3773531 rev+comp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_HV2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5750243\u0026ndash;5752539 rev+comp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_HV1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5755993\u0026ndash;5756621 rev+comp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_OV1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9984659\u0026ndash;9985686 rev+comp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_OV1b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9989819\u0026ndash;9991063 rev+comp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_OV2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10001584\u0026ndash;10002179 rev+comp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAll hirudin genes are composed of four exons and three introns, whereas the ornatin genes are composed of three exons and two introns and apparently lack the third intron and the fourth exon of the hirudin genes which encode the elongated acidic C-terminal tail of hirudin (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For each gene, the exon/intron composition, the location of the introns and the characteristics of the exon boundaries (boundary-overlapping versus non-overlapping codons) exactly match the structures of all hirudin and decorsin/ornatin genes analyzed so far. A schematic representation of the gene structures of Pcos_HV1 and Pcos_OV2 is provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e lists the exon and intron lengths, and the annotated sequences for each of the genes are provided in Supplementary Information Files S4 (hirudin genes) and S5 (ornatin genes).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of putative hirudin (HV) and ornatin (OV) gene structures in \u003cem\u003eP. costata\u003c/em\u003e (Pcos) and \u003cem\u003eP. nabeulensis\u003c/em\u003e (Pnab)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExon1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIntron1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eExon2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIntron2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eExon3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eIntron3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eExon4\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_HV1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e61 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e256 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e58 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e58 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e84 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e62 bp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_HV2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e61 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1540 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e85 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e58 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e429 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e80 bp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_HV3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e344 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e71 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e73 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e151 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e35 bp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePnab_HV1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e61 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e255 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e58 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e58 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e84 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e62 bp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePnab_HV2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e61 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e85 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e58 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e80 bp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePnab_HV3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e335 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e75 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e73 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e151 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e77 bp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_OV1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e663 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e65 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e116 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e114 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_OV1b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e880 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e65 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e116 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e114 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_OV2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e183 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e140 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e129 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePnab_OV1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e65 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e116 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e105 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePnab_OV2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e194 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e140 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e138 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo verify the data for \u003cem\u003eP. costata\u003c/em\u003e we generated and analyzed raw genome sequence data of \u003cem\u003eP. nabeulensis\u003c/em\u003e, the second Palearctic representative of the genus \u003cem\u003ePlacobdella\u003c/em\u003e. The raw Illumina reads were manually assembled, and all putative hirudin and ornatin genes of \u003cem\u003eP. costata\u003c/em\u003e could successfully be identified and reconstructed in \u003cem\u003eP. nabeulensis\u003c/em\u003e with the exception of intron1 of both the Pnab_HV2 and Pnab_OV1 genes and intron 3 of the Pnab_HV2 gene. Exon and intron lengths of all putative hirudin and ornatin genes in \u003cem\u003eP. nabeulensis\u003c/em\u003e are in close proximity to the values of the respective genes of \u003cem\u003eP. costata\u003c/em\u003e (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e for details). The annotated sequences for all genes are provided in Supplementary Information Files S6 (hirudin genes) and S7 (ornatin genes).\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStructural and functional characterization of putative hirudins\u003c/h2\u003e \u003cp\u003eThe amino acid sequences of all putative hirudins of both the three North American and the two Palearctic \u003cem\u003ePlacobdella\u003c/em\u003e species were derived from the respective cDNA or gene sequences. A multiple sequence alignment (MSA) of all putative hirudins is provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, their biochemical features are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStructural and biochemical features of putative hirudin variants HV1-3 of \u003cem\u003eP. costata\u003c/em\u003e (Pcos_HV1-3), HV1-3 of \u003cem\u003eP. nabeulensis\u003c/em\u003e (Pnab_HV1-3), HV1 and HV2 of \u003cem\u003eP. ali\u003c/em\u003e (Pali_HV1-2), HV1-3 of \u003cem\u003eP. kwetlumye\u003c/em\u003e (Pkwe_HV1-3) and HV1 of \u003cem\u003eP. parasitica\u003c/em\u003e (Ppar_HV1). The length is without the signal peptide sequence. aa\u0026thinsp;=\u0026thinsp;amino acids; MW\u0026thinsp;=\u0026thinsp;molecular weight; kDa\u0026thinsp;=\u0026thinsp;kilodalton; pI\u0026thinsp;=\u0026thinsp;isoelectric point\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFactor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLength in aa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMW in kDa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003epI value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_HV1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6382.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_HV2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7149.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_HV3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6532.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePnab_HV1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6378.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePnab_HV2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7304.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePnab_HV3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7979.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePali_HV1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6292.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePali_HV2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4970.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePkwe_HV1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6313.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePkwe_HV2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7426.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePkwe_HV3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6017.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePpar_HV1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6266.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAll putative hirudins contain the six highly conserved cysteine residues, and both the lengths (49\u0026ndash;72 amino acid residues without the signal peptides) and the acidic pI values (3.94\u0026ndash;5.23) are in the range of functional hirudins. However, all HV3 variants apparently lack the essential phenylalanine (or tyrosin) residue at position 3 of the mature protein (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Lazar et al. 1991).\u003c/p\u003e \u003cp\u003eFor the functional characterization, we focused on the putative hirudins of \u003cem\u003eP. costata\u003c/em\u003e. All three hirudin variants, namely Pcos_HV1-3, were expressed as recombinant proteins in \u003cem\u003eE. coli\u003c/em\u003e strain DH5α and further processed as described in the Materials and Methods section. The thrombin time tests revealed that Pcos_HV1 displayed a high thrombin-inhibitory potency, but neither Pcos_HV2 nor Pcos_HV3 did negatively influence the activity of thrombin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Pcos_HV1 can hence be termed a true hirudin, whereas Pcos_HV2 and Pcos_HV3 are rather HLFs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGain-of-function analyses of Pcos_HV2 and HV3\u003c/h2\u003e \u003cp\u003eThe N-terminus of hirudin is of special importance for its interaction with hirudin, and a replacement of the first hydrophobic valyl residue in hirudin variant HV1 of \u003cem\u003eH. medicinals\u003c/em\u003e with polar amino acids results in a dramatic increase in the inhibition constant K\u003csub\u003ei\u003c/sub\u003e (Wallace et al. 1989). Whereas Pcos_HV1 contains a valyl residue at position 1, both Pcos_HV2 and HV3 contain polar amino acid residues at the respective position: a glutamine residue (Q1) in Pcos_HV2 and a glutamyl residue (E1) in Pcos_HV3. Strikingly, within the N-terminal five amino acid residues of Pcos_HV2 only the Q1 residue differs from the N-terminus of Pcos_HV1 (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A closer look at the conjunction of exons 1 and 2 of the Pcos_HV2 and Pnab_HV2 genes (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Material Files S4 and S6) revealed that with only one nucleotide substitution the codons that encodes the polar glutamine residue Q1 (CAA) can be changed to codons that encode a hydrophobic leucine residue (CTA) and with two substitutions to codons that encode a hydrophobic valyl residue (GTA). To evaluate the effects of variations of the N-terminal amino acid sequences on the thrombin-inhibitory potency of Pcos_HV2 we hence constructed two variants that contain either a valyl residue (Pcos_HV2V) or a leucine residue (Pcos_HV2L) at position 1. In addition we constructed respective variants of Pcos_HV3 that contain either the N-terminal five amino acid residues of Pcos_HV2 (Pcos_HV3Q), Pcos_HV2V (Pcos_HV3V) or Pcos_HV2L (Pcos_HV3L), respectively. Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e lists all Pcos_HV2 and HV3 variants and their N-terminal amino acid sequences.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNomenclature and N-terminal amino acid sequences of wildtype and mutant variants of Pcos_HV2 and HV3\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFactor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN-terminal sequence\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_HV1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVHFPPC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_HV2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQHFPPC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_HV2V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVHFPPC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_HV2L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLHFPPC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_HV3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEDIPEC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_HV3Q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQHFPKC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_HV3V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVHFPPC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_HV3L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLHFPPC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAll variants of Pcos_HV2 and HV3 were expressed as recombinant proteins in \u003cem\u003eE. coli\u003c/em\u003e strains DH5α and SHuffle T7\u0026reg; and further processed as described in the Materials and Methods section. The thrombin time tests revealed that both Pcos_HV2V and HV2L displayed moderate, but clearly detectable thrombin-inhibitory potencies, whereas none of the Pcos_HV3 variants negatively influenced the activity of thrombin. The inhibitory potencies of recombinant Pcos_HV2V and HV2L did not differ between the two \u003cem\u003eE. coli\u003c/em\u003e strains (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e for the recombinant factors expressed in \u003cem\u003eE. coli\u003c/em\u003e strain DH5α and Supplementary Information File Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e for the recombinant factors expressed in \u003cem\u003eE. coli\u003c/em\u003e strain SHuffle\u0026reg; T7). Interestingly, in both bacterial expression systems the leucine residue at position 1 conferred higher thrombin-inhibitory potencies of the respective recombinant Pcos_HV2 variants compared to the variants containing a valyl residue at position 1. It is worth to note in that context that lepirudin, a commercially available recombinant hirudin (rec-Hirudin, HBW 023, Refludan\u0026reg;), is a variant of hirudin HV2 (hirudin-IT) of \u003cem\u003eH. medicinalis\u003c/em\u003e (Harvey et al. 1986) and contains a leucine residue at position 1 instead the wildtype isoleucine residue (Marckwardt 1994).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStructural and functional characterization of putative ornatins\u003c/h2\u003e \u003cp\u003eThe amino acid sequences of the putative ornatins of all five \u003cem\u003ePlacobdella\u003c/em\u003e species were derived from the respective cDNA or gene sequences. A multiple sequence alignment (MSA) of the putative ornatins is provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e, their biochemical features are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStructural and biochemical features of pitative ornatin variants OV1a, 1b and 2 of \u003cem\u003eP. costata\u003c/em\u003e (Pcos_OV1a, 1b and 2), OV1 and 2 of \u003cem\u003eP. nabeulensis\u003c/em\u003e (Pnab_OV1 and 2), OV1-3 of \u003cem\u003eP. ali\u003c/em\u003e (Pali_OV1-3), OV1 of \u003cem\u003eP. kwetlumye\u003c/em\u003e (Pkwe_OV1) and OV1 of \u003cem\u003eP. parasitica\u003c/em\u003e (Ppar_OV1). The length is without the signal peptide sequence. aa\u0026thinsp;=\u0026thinsp;amino acids; MW\u0026thinsp;=\u0026thinsp;molecular weight; kDa\u0026thinsp;=\u0026thinsp;kilodalton; pI\u0026thinsp;=\u0026thinsp;isoelectric point\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFactor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLength in aa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMW in kDa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003epI value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_OV1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7073.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_OV1b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7104.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePcos_OV2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8358.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePnab_OV1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6733.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePnab_OV2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8560.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePali_OV1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5738.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePali_OV2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6404.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePali_OV3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6052.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePkwe_OV1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7634.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePpar_OV1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5722.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAll putative ornatins except Pnab_OV1 contain the six highly conserved cysteine residues, however, the essential RGD/KGD motif (Lazarus and McDowell 1993) is absent in Pnab_OV1, Pali_OV3 and Ppar_OV1 (see Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The basic pI value of most putative ornatins except for Ppar_OV1 (see Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) is often observed in functional ornatins (Mazur et al. 1991). However, a basic pI does not seem to be an essential biochemical feature (Pfordt et al. 2022).\u003c/p\u003e \u003cp\u003eFor the functional characterization we focused on the putative ornatins of \u003cem\u003eP. costata\u003c/em\u003e. Again, Pcos_OV1a and OV2 were expressed as recombinant proteins in \u003cem\u003eE. coli\u003c/em\u003e strain DH5α and further processed as described in the Materials and Methods section. Platelet function analyzed by light transmission aggregometry, however, revealed that none of the factors exhibited an inhibitory effect on platelet aggregation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA und C). To evaluate whether or not the genetic background of the bacterial host strain may have negatively influenced the correct folding and hence the activity of the recombinant ornatins Pcos_OV1a and 2, we repeated their expression and purification, but used the \u003cem\u003eE. coli\u003c/em\u003e strain SHuffle\u0026reg; T7 as an alternative host. The strain was specifically developed to improve the correct formation of disulfide bonds in recombinant proteins (Lobstein et al. 2012). Indeed, the recombinant ornatins Pcos_OV1a and 2 that were produced by \u003cem\u003eE. coli\u003c/em\u003e strain SHuffle\u0026reg; T7 negatively affected ADP-induced platelet aggregation, both in terms of shape of the curve and the maximal aggregation value (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). A statistical analyses revealed that the area under the curve (AUC) values of both putative ornatins Pcos_OV1a and Pcos_OV2 significantly differed from the negative control samples (buffer) with p-values\u0026thinsp;\u0026le;\u0026thinsp;0.01 (Pcos_OV1a) and \u0026le;\u0026thinsp;0.001 (Pcos_OV2), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Both factors can hence be considered as active ornatins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eLeeches are frequently used for medical purposes, but the term \u0026ldquo;medicinal leeches\u0026rdquo; does not refer to a specific taxon of leeches and is rather a description of their anthropogenic use. So far, almost exclusively jawed (or arhynchobdellid) leeches (Hirudiniformes) have been used by humans to treat various maladies including cardiovascular disorders, wound healing and problems associated with plastic reconstructive surgery (Singh 2010; Hackenberger and Janis 2019; Nair et al. 2020). Consequently, in-depth investigations of the therapeutic potential of proboscis-bearing leeches (especially Glossiphoniidae) are rare. Notably, leeches of the glossiphoniid genus \u003cem\u003ePlacobdella\u003c/em\u003e comprise a broad variety of anticoagulants including putative hirudins and ornatins (Siddall et al. 2016), yet detailed analyses of their biochemical properties and functional characterizations are still pending. In a series of previous studies we have already determined the structures of hirudin, hirudin-like factor and decorsin/ornatin genes in representatives of various arhynchobdellid leech species including the European medicinal leeches \u003cem\u003eH. medicinalis\u003c/em\u003e, \u003cem\u003eH. verbana\u003c/em\u003e, \u003cem\u003eH. orientalis\u003c/em\u003e and \u003cem\u003eH. troctina\u003c/em\u003e (M\u0026uuml;ller et al. 2016; Ben Ahmed et al. 2024), the Asian medicinal leeches \u003cem\u003eHirudinaria manillensis\u003c/em\u003e Lesson, 1842, \u003cem\u003eWhitmania pigra\u003c/em\u003e Whitman, 1884, and \u003cem\u003eHirudo nipponia\u003c/em\u003e (M\u0026uuml;ller et al. 2017; M\u0026uuml;ller et al. 2022; Tolksdorf et al. 2025; Kalathejari et al. 2026) and the North American medicinal leech \u003cem\u003eMacrobdella decora\u003c/em\u003e (M\u0026uuml;ller et al. 2019). The generation of genome sequence data of two Palearctic species of the genus \u003cem\u003ePlacobdella\u003c/em\u003e enabled us to conduct respective analyses in representatives of glossiphoniid leeches. The primary aim of the present study was to take a step forward on the track to close the \"analytical gap\" between glossiphoniid and arhynchobdellid leech species. Transcriptomic sequence data of three North American species and genome sequence data of the two Palearctic representatives of the genus \u003cem\u003ePlacobdella\u003c/em\u003e were analyzed for the presence of putative hirudin and ornatin cDNAs or genes. In all five leech species, respective coding sequences for both factors could be identified (see Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Whereas hirudins are present in all hematophagous leech species analyzed so far and may even occur in predatory leeches (M\u0026uuml;ller et al. 2022), decorsins/ornatins are apparently absent in European members of the genus \u003cem\u003eHirudo\u003c/em\u003e (Babenko et al. 2020; Kvist et al. 2020) and in the African medicinal leech \u003cem\u003eAsiaticobdella fenestrata\u003c/em\u003e (Schulz et al. 2025) and may hence have been lost during evolution. However, the verification of hirudin and ornatin genes in representatives of the glossiphoniid leech genus \u003cem\u003ePlacobdella\u003c/em\u003e indicates that independent genes of both types of anticoagulants were probably present already in the last common ancestor (LCA) of Hirudinida (or true leeches) (Tessler et al. 2018; Kuo and Lai 2019; de Carle et al. 2025). Strong support for this assumption comes from the observation that the gene structures of both the hirudin and the decorsin/ornatin genes are highly conserved in glossiphoniid and arhynchobdellid leeches including the exact position of introns and the presence of exon-boundary overlapping and non-overlapping codons (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; M\u0026uuml;ller et al. 2016; M\u0026uuml;ller et al. 2019; Lukas et al. 2022). Whether or not the LCA of the hirudin and ornatin genes dates back even further in time and is already present in Hirudinea Lamarck, 1818, a group that also includes the family Piscicolidae Johnston, 1865, and the two orders Acanthobdellida Livanow, 1905, and Branchiobdellida Holt, 1965 (de Carle et al. 2025), still remains to be defined.\u003c/p\u003e \u003cp\u003eA recurring feature in hematophagous leeches is the presence of multiple copies of anticoagulant genes including genes that encode hirudins and ornatins/decorsins (Liu et al. 2023; Zhao et al. 2024; Khan et al. 2025; Kalathejari et al. 2026), very likely to prevent deleterious effects of occasional gene loss events. The genome sequence data of \u003cem\u003eP. costata\u003c/em\u003e only partially confirm this observation with the presence of just a single gene that encodes a functional hirudin (Pcos_HV1)and three genes that encode functional ornatins (Pcos_OV1a/b and OV2) (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026thinsp;+\u0026thinsp;3 and 5\u0026thinsp;+\u0026thinsp;6). However, with only a single amino acid substitution (Q1 to either V1 or L1) Pcos_HV2 can gain a moderate thrombin-inhibitory potency (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We can only speculate whether the current situation of Pcos_HV2 is the consequence of a past loss-of-function event (GTA \u0026loz; CTA \u0026loz; CAA) or primes the leech for a future gain-of-function event (CAA \u0026loz; CTA \u0026loz; GTA), but each scenario is not mutually exclusive. Unfortunately, the biological targets of both Pcos_HV2 and HV3 remain obscure and are difficult to identify.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eP. nabeulensis\u003c/em\u003e the Pnab_OV1 gene encodes a factor that lacks the fourth conserved cysteine residue and may hence be inaccurately folded (see Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). With that. \u003cem\u003eP. nabeulensis\u003c/em\u003e may contain only single genes that encode a functional hirudin (Pnab_HV1) and a functional ornatin (Pnab_OV2). Interestingly, both Pnab_OV1 and Ppar_OV1 of \u003cem\u003eP. parasitica\u003c/em\u003e comprise RSD motifs instead of the canonical RGD/KGD motif that facilitates integrin binding (Lazarus and McDowell 1993; Reiss et al. 2006). An RSD motif may be functionally equivalent to an RGD motif (Van de Walle et al. 2008), but this remains to be tested for ornatins. Based on the current status of investigations, neither \u003cem\u003eP. costata\u003c/em\u003e nor \u003cem\u003eP. nabeulensis\u003c/em\u003e contain genes that encode putative multimeric hirudins/HLFs or decorsins.\u003c/p\u003e \u003cp\u003eThe recombinant expression of putatve hirudins and decorsins/ornatins in bacterial hosts for subsequent functional analyses is well established in our lab. In a recent publication we compared two \u003cem\u003eE. coli\u003c/em\u003e strains with different characteristics, namely DH5α (Hanahan 1985) and SHuffle\u0026reg; T7 (Lobstein et al. 2012). The strain SHuffle\u0026reg; T7 supports the formation of disulfide bonds, generally a crucial step in correct folding of cystein-containing proteins (Feige et al. 2018) and in correct folding of members of the hirudin superfamily in particular (Dodt et al. 1985; Mazur et al. 1993; Micheletti et al. 2003; Wu et al. 2013). We deciphered that a preparation of recombinant hirudin HV1 of \u003cem\u003eH. medicinalis\u003c/em\u003e (Hmed_HV1) that was expressed in SHuffle\u0026reg; T7 at its optimal cultivation temperature of 30\u0026deg;C revealed a higher thrombin inhibitory potency compared to a preparation of Hmed_HV1 that was expressed in DH5α at its optimal cultivation temperature of 37\u0026deg;C (Wang et al. 2025). The current study partially supports this observation: the putative ornatins Pcos_OV1a and OV2 did not exhibit platelet aggregation inhibitory potencies when both factors were expressed as recombinant proteins in \u003cem\u003eE. coli\u003c/em\u003e strain DH5α at 37\u0026deg;C. However, platelet aggregation was negatively affected when both factors were expressed in \u003cem\u003eE. coli\u003c/em\u003e strain SHuffle\u0026reg; T7 at 30\u0026deg;C (see Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In contrast, the thrombin-inhibitory potencies of the recombinant Pcos_HV2V and HV2L variants did not differ between the two bacterial expression systems (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Supplementary Information Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Nevertheless, it might be a reasonable option to consequently use the strain SHuffle\u0026reg; T7 for the expression of recombinant putative hirudins, ornatins and other cysteine-rich proteins.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors are thankful to the colleagues who generated and provided the genome and transcriptome datasets explored in the present study. In addition, we would like to thank all members of the Animal Physiology Working Group at the University of Greifswald for their help and support. Special thanks go to Jan-Peter Hildebrandt and Undine Lauf for their critical reading of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.M. conceived the ideas, designed the methodology and performed the sequence-based investigations; R.B.A. and C.G. collected the animals; R.B.A., C.M., C.T. and R.W. performed the experiments; C.M.,R.B.A. and S.K. analyzed the data and drafted the manuscript; C.M., B.H.R. and G.J. supervised the experimental work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOpen Access funding enabled and organized by Projekt DEAL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReference genome chromosome-level sequence data of \u003cem\u003eP. costata\u003c/em\u003e are deposited in GenBank under the accession numbers OZ194472.1 - OZ194489.1. Information on specific gene locations are provided either within the manuscript or as Supplementary Information Files. The raw genome sequence data of \u003cem\u003eP. costata\u003c/em\u003e and \u003cem\u003eP. nabeulensis\u003c/em\u003e generated in the present study were deposited in BioStudies under the following accession number: S-BSST2339 (DOI: 10.6019/S-BSST2339). Images that illustrate the expression, purification and processing of all recombinant factors are available on request from the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations Ethical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of the University Medicine Greifswald (protocol code BB 20/12, approval date 25 July 2018) and by the Institutional Review Board (or Ethics Committee) of the University Medicine Oldenburg, Carl von Ossietzky University Oldenburg (ethics vote 2024-034, approval date 27 March 2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate and consent for publication\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from all subjects involved in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen Access\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article\u0026rsquo;s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article\u0026rsquo;s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdualkader AM, Ghawi AM, Alaama M, Awang M, Merzouk A (2013) Leech Therapeutic Applications. Indian J Pharm Sci 75(2):127\u0026ndash;137.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmorim AMXP, de Oliveira UC, Faria F, Pasqualoto KFM, Junqueira-de-Azevedo IdLM, Chudzinski-Tavassi AM (2015) Transcripts involved in hemostasis: Exploring salivary complexes from \u003cem\u003eHaementeria vizottoi\u003c/em\u003e leeches through transcriptomics, phylogenetic studies and structural features. Toxicon 106:20\u0026ndash;29. https://doi.org/10.1016/j.toxicon.2015.09.002\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBabenko VV, Podgorny OV, Manuvera VA, Kasianov AS, Manolov AI, Grafskaia EN, Shirokov DA, Kurdyumov AS, Vinogradov DV, Nikitina AS, Kovalchuk SI, Anikanov NA, Butenko IO, Pobeguts OV, Matyushkina DS, Rakitina DV, Kostryukova ES, Zgoda VG, Baskova IP, Trukhan VM, Gelfand MS, Govorun VM, Schi\u0026ouml;th HB, Lazarev VN (2020) Draft genome sequences of \u003cem\u003eHirudo medicinalis\u003c/em\u003e and salivary transcriptome of three closely related medicinal leeches. BMC Genomics 21(1):331. https://doi.org/10.1186/s12864-020-6748-0\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBen Ahmed R, Gajda Ł, Utevsky S, Kvist S, Świątek P (2023) \u003cem\u003ePlacobdella nabeulensis\u003c/em\u003e sp. nov. (Hirudinea: Glossiphoniidae), a new glossiphoniiform leech from Palearctic North Africa. Mol Biol Rep 50(8):6753\u0026ndash;6767. https://doi.org/10.1007/s11033-023-08594-z\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBen Ahmed R, Abilov A, M\u0026uuml;ller C (2024) Diversity of hirudin and hirudin-like factor genes in the North-African medicinal leech, \u003cem\u003eHirudo troctina\u003c/em\u003e. Parasitol Res 123(11):382. https://doi.org/10.1007/s00436-024-08411-x\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBielecki A, Cichocka JM, Jabłoński A, Jeleń I, Ropelewska E, Biedunkiewicz A, Terlecki J, Nowakowski JJ, Pakulnicka J, Szlachciak J (2012) Coexistence of \u003cem\u003ePlacobdella costata\u003c/em\u003e (Fr. M\u0026uuml;ller, 1846) (Hirudinida: Glossiphoniidae) and mud turtle \u003cem\u003eEmys orbicularis\u003c/em\u003e. Biologia 67:731\u0026ndash;738. https://doi.org/10.2478/s11756-012-0069-y\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Carle D, Oceguera-Figueroa A, Tessler M, Siddall ME (2017) Phylogenetic analysis of \u003cem\u003ePlacobdella\u003c/em\u003e (Hirudinea: Rhynchobdellida: Glossiphoniidae) with consideration of COI variation. Mol Phylogenet Evol 234\u0026ndash;248. https://doi.org/10.1016/j.ympev.2017.06.017\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Carle D, Iwama RE, Wendruff AJ, Babcock LE, Nanglu K (2025) The first leech body fossil predates estimated hirudinidan origins by 200\u0026nbsp;million years. PeerJ 13:e19962. https://doi.org/10.7717/peerj.19962\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCichocka JM, Bielecki A, Jabłońska-Barna I, Krajewski Ł, Topolska K, Hildebrand J, Dmitryjuk M, Biedunkiewicz A, Abramchuk A (2021) Sucking of human blood by \u003cem\u003ePlacobdella costata\u003c/em\u003e (O. F. M\u0026uuml;ller, 1846) (Hirudinida: Glossiphoniidae): Case study with notes on body form. Ecol Evol 11(24):17593\u0026ndash;17603. https://doi.org/10.1002/ece3.8261\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDodt J, Seem\u0026uuml;ller U, Maschler R, Fritz H (1985) The complete covalent structure of hirudin. Localization of the disulfide bonds. Biol Chem Hoppe Seyler 366(4):379\u0026ndash;385. https://doi.org/10.1515/bchm3.1985.366.1.379\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElliott JM, Kutschera U (2011) Medicinal leeches: Historical use, ecology, genetics and conservation. Freshwater Rev 4(1):21\u0026ndash;41. https://doi.org/10.1608/FRJ-4.1.417\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFaria F, Kelen EM, Sampaio CA, Bon C, Duval N, Chudzinski-Tavassi AM (1999) A new factor Xa inhibitor (lefaxin) from the \u003cem\u003eHaementeria depressa\u003c/em\u003e leech. Thromb Haemost 82(5):1469\u0026ndash;1473. https://doi.org/10.1055/s-0037-1614857\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeige MJ, Braakman I, Hendershot LM (2018) Disulfide bonds in protein folding and stability. In Oxidative Folding of Proteins: Basic Principles, Cellular Regulation and Engineering, ed. M. J. Feige, The Royal Society of Chemistry, pp. 1\u0026ndash;33. https://doi.org/10.1039/9781788013253-00001\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGill SC, von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182:319\u0026ndash;326. https://doi.org/10.1016/0003-2697(89)90602-7\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHackenberger PN, Janis JE (2019) A comprehensive review of medicinal leeches in plastic and reconstructive surgery. Plast Reconstr Surg Glob Open 7(12):e2555. https://doi.org/10.1097/GOX.0000000000002555\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHall TA (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41(2):95\u0026ndash;98\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHanahan D (1985) Techniques for transformation of \u003cem\u003eE. coli\u003c/em\u003e. In: Glover DM (ed.) DNA cloning: a practical approach. Vol.1:109\u0026ndash;135. IRL Press, Oxford, UK\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarvey RP, Degryse E, Stefani L, Schamber F, Cazenave JP, Courtney M, Tolstoshev P, Lecocq JP (1986) Cloning and expression of a cDNA coding for the anticoagulant hirudin from the bloodsucking leech, \u003cem\u003eHirudo medicinalis\u003c/em\u003e. Proc Nat Acad Sci USA 83(4):1084\u0026ndash;1088. https://doi.org/10.1073/pnas.83.4.1084\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIwama RE, Tessler M, Siddall ME, Kvist S (2021) The origin and evolution of antistasin-like proteins in leeches (Hirudinida, Clitellata). Genome Biol Evol 13(1):evaa242. https://doi.org/10.1093/gbe/evaa242\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalatehjari P, Wolf R, Jedlitschky G, Tolksdorf C, Rauch BH, M\u0026uuml;ller C (2026) The great diversity: Monomeric and oligomeric hirudins, hirudin-like factors and decorsins in the Asian medicinal leeches \u003cem\u003eHirudo nipponia\u003c/em\u003e and \u003cem\u003eHirudo tianjinensis\u003c/em\u003e. Parasitol Res accepted for publication https://doi.org/10.21203/rs.3.rs-8307310/v1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK\u0026auml;ll L, Krogh A, Sonnhammer ELL (2007) Advantages of combined transmembrane topology and signal peptide prediction - the Phobius web server. Nucleic Acids Res 35(Web Server issue):W429-432. https://doi.org/10.1093/nar/gkm256\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28(12):1647\u0026ndash;1649. https://doi.org/10.1093/bioinformatics/bts199\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan MS, M\u0026uuml;ller C, Zhou B (2025) Chromosome-level genome assembly and anticoagulant protein annotation of the buffalo leech \u003cem\u003eHirudinaria bpling\u003c/em\u003e (Hirudinea: Hirudinidae). BMC Genomics 26(1):558. https://doi.org/10.1186/s12864-025-11690-y\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuo D-H, Lai Y-T (2019) On the origin of leeches by evolution of development. Dev Growth Differ 61(1):43\u0026ndash;57. https://doi.org/10.1111/dgd.12573\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKvist S, Min G-S, Siddall ME (2013) Diversity and selective pressures of anticoagulants in three medicinal leeches (Hirudinida: Hirudinidae, Macrobdellidae). Ecol Evol 3(4):918\u0026ndash;933. https://doi.org/10.1002/ece3.480\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKvist S, Manzano-Mar\u0026iacute;n A, de Carle D, Trontelj P, Siddall ME (2020) Draft genome of the European medicinal leech \u003cem\u003eHirudo medicinalis\u003c/em\u003e (Annelida, Clitellata, Hirudiniformes) with emphasis on anticoagulants. Sci Rep 10:9885. https://doi.org/10.1038/s41598-020-66749-5\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKvist S, Utevsky S, Marrone F, Ben Ahmed R, Gajda Ł, Grosser C, Huseynov M, Jueg U, Khomenko A, Oceguera-Figueroa A, Vladimir Pešić, Pupins M, Rouag R, Sağlam N, Świątek P, Trontelj P, Vecchioni L, M\u0026uuml;ller C (2022) Extensive sampling sheds light on species-level diversity in Palearctic \u003cem\u003ePlacobdella\u003c/em\u003e (Annelida: Clitellata: Glossiphoniiformes). Hydrobiologia 849:1239\u0026ndash;1259. https://doi.org/10.1007/s10750-021-04786-5\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLarkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21):2947\u0026ndash;2948. https://doi.org/10.1093/bioinformatics/btm404\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLazar JB, Winant RC, Johnson PH (1991) Hirudin: amino-terminal residues play a major role in the interaction with thrombin. J Biol Chem 266(2):685\u0026ndash;688. https://doi.org/10.1016/S0021-9258(17)35224-9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLazarus RA, McDowell RS (1993) Structural and functional aspects of RGD-containing protein antagonists of glycoprotein IIb-IIIa. Curr Opin Biotechnol 4(4):438\u0026ndash;445. https://doi.org/10.1016/0958-1669(93)90009-l\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Z, Zhao F, Huang Z, Hu Q, Meng R, Lin Y, Qi H, Lin G (2023) Revisiting the Asian buffalo leech (\u003cem\u003eHirudinaria manillensis\u003c/em\u003e) genome: Focus on antithrombotic genes and their corresponding proteins. Genes (Basel) 14(11):2068. https://doi.org/10.3390/genes14112068\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLobstein J, Emrich CA, Jeans C, Faulkner M, Riggs P, Berkmen M (2012) SHuffle, a novel \u003cem\u003eEscherichia coli\u003c/em\u003e protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microb Cell Fact 11:56. https://doi.org/10.1186/1475-2859-11-56\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLukas P, Melikian G, Hildebrandt J-P, M\u0026uuml;ller C (2022) Make it double: identification and characterization of a Tandem-Hirudin from the Asian medicinal leech \u003cem\u003eHirudinaria manillensis\u003c/em\u003e. Parasitol Res 121(10):2995\u0026ndash;3006. https://doi.org/10.1007/s00436-022-07634-0\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLynggaard C, Oceguera-Figueroa A, Kvist S, Gilbert MTP, Bohmann K (2022) The potential of aquatic bloodfeeding and nonbloodfeeding leeches as a tool for iDNA characterisation. Mol Ecol Resour 22(2):539\u0026ndash;553. https://doi.org/10.1111/1755-0998.13486\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManglicmot C, Oceguera-Figueroa A, Kvist S (2020) Bacterial endosymbionts of \u003cem\u003ePlacobdella\u003c/em\u003e (Annelida: Hirudinea: Glossiphoniidae): phylogeny, genetic distance, and vertical transmission. Hydrobiologia 847:1177\u0026ndash;1194. https://doi.org/10.1007/s10750-019-04175-z\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarchiori C, Santana M, Malheiros K (2024) Leeches and their use in medicine (Annellida: Hirudinea: Rhynchobdelliformes). Middle East Res J Biol Sci 4(03):72\u0026ndash;81. https://doi.org/10.36348/merjbs.2024.v04i03.003\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarkwardt F (1956) Untersuchungen \u0026uuml;ber den Mechanismus der blutgerinnungshemmenden Wirkung des Hirudins. Naunyn - Schmiedebergs Arch 229:389\u0026ndash;399 (1956). https://doi.org/10.1007/BF00245864\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarkwardt F (1994) The development of hirudin as an antithrombotic drug. Thromb Res 74(1):1\u0026ndash;23. https://doi.org/10.1016/0049-3848(94)90032-9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMazur P, Henzel WJ, Seymour JL, Lazarus RA (1991) Ornatins: potent glycoprotein IIb-IIIa antagonists and platelet aggregation inhibitors from the leech \u003cem\u003ePlacobdella ornata\u003c/em\u003e. Eur J Biochem 202(3):1073\u0026ndash;1082. https://doi.org/10.1111/j.1432-1033.1991.tb16472.x\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMazur P, Dennis MS, Seymour JL, Lazarus RA (1993) Expression, purification, and characterization of recombinant ornatin E, a potent glycoprotein IIb-IIIa antagonist. Protein Expr Purif 4(4):282\u0026ndash;289. https://doi.org/10.1006/prep.1993.1036\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMicheletti C, De Filippis V, Maritan A, Seno F (2003) Elucidation of the disulfide-folding pathway of hirudin by a topology-based approach. Proteins 53(3):720\u0026ndash;730. https://doi.org/10.1002/prot.10463.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoser WE, Bowerman J, Hovingh P, Pearl CA, Oceguera-Figueroa A (2014) New host and distribution records of the leech \u003cem\u003ePlacobdella sophieae\u003c/em\u003e Oceguera-Figueroa et al., 2010 (Hirudinida: Glossiphoniidae). Comp Parasitol 81(2):199\u0026ndash;202. https://doi.org/10.1654/4678.1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM\u0026uuml;ller C, Mescke K, Liebig S, Mahfoud H, Lemke S, Hildebrandt J-P (2016) More than just one: multiplicity of hirudins and hirudin-like factors in the medicinal leech, \u003cem\u003eHirudo medicinalis\u003c/em\u003e. Mol Genet Genomics 291:227\u0026ndash;240. https://doi.org/10.1007/s00438-015-1100-0\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM\u0026uuml;ller C, Haase M, Lemke S, Hildebrandt J-P (2017) Hirudins and hirudin-like factors in Hirudinidae: implications for function and phylogenetic relationships. Parasitol Res 116(1):313\u0026ndash;325. https://doi.org/10.1007/s00436-016-5294-9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM\u0026uuml;ller C, Lukas P, Lemke S, Hildebrandt J-P (2019) Hirudin and decorsins of the North American medicinal leech \u003cem\u003eMacrobdella decora\u003c/em\u003e: gene structure reveals homology to hirudins and hirudin-like factors of Eurasian medicinal leeches. J Parasitol 105(3):423\u0026ndash;431. https://doi.org/10.1645/18-117\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM\u0026uuml;ller C, Wang Z, Hamann M, Sponholz D, Hildebrandt J-P (2022) Life without blood: Molecular and functional analysis of hirudins and hirudin-like factors of the Asian non-hematophagous leech \u003cem\u003eWhitmania pigra\u003c/em\u003e. J Thromb Haemost 20(8):1808\u0026ndash;1817. https://doi.org/10.1111/jth.15762\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM\u0026uuml;ller C, Sponholz D, Tolksdorf C, Rauch BH, Kvist S (2024) Identification and functional characterization of multiple haemadins and an oligomeric decorsin in the Asian land leech \u003cem\u003eHaemadipsa interrupta\u003c/em\u003e. Parasitol Res 123(11):390. https://doi.org/10.1007/s00436-024-08404-w\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNair HKR, Ahmad NW, Lee HL, Ahmad N, Othamn S, Mokhtar NSHM, Chong SSY (2022) Hirudotherapy in wound healing. Int J Low Extrem Wounds 21(4):425\u0026ndash;431. https://doi.org/10.1177/1534734620948299\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNicholas KB, Nicholas Jr HB (1997) GeneDoc: a tool for editing and annotating multiple sequence alignments. Distributed by the author\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eN\u0026uacute;\u0026ntilde;ez-Navarro NE, Santana FM, Parra LP, Zacconi FC (2019) Surfing the blood coagulation cascade: Insight into the vital factor Xa. Curr Med Chem 26(17):3175\u0026ndash;3200. https://doi.org/10.2174/0929867325666180125165340\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOceguera-Figueroa A (2012) Molecular phylogeny of the New World bloodfeeding leeches of the genus \u003cem\u003eHaementeria\u003c/em\u003e and reconsideration of the biannulate genus \u003cem\u003eOligobdella\u003c/em\u003e. Mol Phylogenet Evol 62(1):508\u0026ndash;514. https://doi.org/10.1016/j.ympev.2011.10.020\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOliveira DGL Alvarez-Flores MP, Lopes AR, Chudzinski-Tavassi AN (2012) Functional characterisation of vizottin, the first factor Xa inhibitor purified from the leech \u003cem\u003eHaementeria vizottoi\u003c/em\u003e. Thromb Haemost 108(3):570\u0026ndash;578. https://doi.org/10.1160/TH12-04-0235\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePace CN, Vajdos F, Fee L, Grimsley G, Gray T (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci 4:2411\u0026ndash;2423. https://doi.org/10.1002/pro.5560041120\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePfordt V, Kalatehjari P, Tolksdorf C, Rauch BH, M\u0026uuml;ller C (2022) Go West: Hirudins and decorsin/ ornatin-like platelet aggregation inhibitors in two representatives of American hematophagous leeches. Parasitologia 2:313\u0026ndash;325. https://doi.org/10.3390/parasitologia2040026\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePhillips J, Dornburg A, Zapfe KL, Anderson FE, James SW, Ers\u0026eacute;us C, Lemmon EM, Lemmon AR, Williams BW (2019) Phylogenomic analysis of a putative missing link sparks reinterpretation of leech evolution. Genome Biol Evol 11(11):3082\u0026ndash;3093. https://doi.org/10.1093/gbe/evz120\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReiss S, Sieber M, Oberle V, Wentzel A, Spangenberg P, Claus R, Kolmar H, L\u0026ouml;sche W (2006) Inhibition of platelet aggregation by grafting RGD and KGD sequences on the structural scaffold of small disulfide-rich proteins. Platelets 17(3):153\u0026ndash;157. https://doi.org/10.1080/09537100500436663\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRichardson DJ, Moser WE, Hammond CI, Lazo-Wasem EA, McAllister CT, Pulis EE (2017) A new species of leech of the genus \u003cem\u003ePlacobdella\u003c/em\u003e (Hirudinida, Glossiphoniidae) from the American alligator (\u003cem\u003eAlligator mississippiensis\u003c/em\u003e) in Mississippi, USA. Zookeys 667:39\u0026ndash;49. https://doi.org/10.3897/zookeys.667.10680\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarkans U, Gostev M, Athar A, Behrangi E, Melnichuk O, Ali A, Minguet J, Rada JC, Snow C, Tikhonov A, Brazma A, McEntyre J (2018) The BioStudies database-one stop shop for all data supporting a life sciences study. Nucleic Acids Res 46(D1):D1266-D1270. doi: 10.1093/nar/gkx965\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSawyer RT, Casellas M, Munro R, Powell Jones C (1991) Secretion of hementin and other antihaemostatic factors in the salivary gland complex of the giant amazon leech \u003cem\u003eHaementeria ghilianii\u003c/em\u003e. Comp Haematol Int 1:35\u0026ndash;41. https://doi.org/10.1007/BF00422691\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSawyer RT (2022) Comparative morphological analysis of two species of turtle leeches coexisting in North America (Hirudinea:Glossiphoniidae): Embryological evidence for character displacement. Taxonomy 2:160\u0026ndash;179. https://doi.org/10.3390/taxonomy2020013\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchulz L, Tolksdorf C, Rauch BH, Kvist S, M\u0026uuml;ller C (2025) Hirudins and fenestrins of the African medicinal leech \u003cem\u003eAsiaticobdella fenestrata\u003c/em\u003e. Parasitol Res 124(11):124. https://doi.org/10.1007/s00436-025-08578-x\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeale L, Finney S, Sawyer RT, Wallis RB (1997) Tridegin, a novel peptidic inhibitor of factor XIIIa from the leech, \u003cem\u003eHaementeria ghilianii\u003c/em\u003e, enhances fibrinolysis \u003cem\u003ein vitro\u003c/em\u003e. Thromb Haemost 77(5):959\u0026ndash;963. https://doi.org/10.1055/s-0038-1656085\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeymour JL, Henzel WJ, Nevins B, Stults JT, Lazarus RA (1990) Decorsin. A potent glycoprotein IIb-IIIa antagonist and platelet aggregation inhibitor from the leech \u003cem\u003eMacrobdella decora\u003c/em\u003e. J Biol Chem 265(17):10143\u0026ndash;10147. https://doi.org/10.1016/S0021-9258(19)38791-5\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShakouri A, Wollina U (2021) Time to change theory; medical leech from a molecular medicine perspective leech salivary proteins playing a potential role in medicine. Adv Pharm Bull 11(2):261\u0026ndash;266. https://doi.org/10.34172/apb.2021.038\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiddall ME, Brugler MR, Kvist S (2016) Comparative transcriptomic analyses of three species of \u003cem\u003ePlacobdella\u003c/em\u003e (Rhynchobdellida: Glossiphoniidae) confirms a single origin of blood feeding in leeches. J Parasitol 102(1):143\u0026ndash;150. https://doi.org/10.1645/15-802\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh AP (201) Medicinal leech therapy (hirudotherapy): a brief overview. Complement Ther Clin Pract 16(4):213\u0026ndash;215. https://doi.org/10.1016/j.ctcp.2009.11.005\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTessler M, de Carle D, Voiklis ML, Gresham OA, Neumann JS, Cios S, Siddall ME (2018) Worms that suck: Phylogenetic analysis of Hirudinea solidifies the position of Acanthobdellida and necessitates the dissolution of Rhynchobdellida. Mol Phylogenet Evol 127:129\u0026ndash;134. https://doi.org/10.1016/j.ympev.2018.05.001\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeufel F, Armenteros JJA, Johansen AR, G\u0026iacute;slason MH, Pihl SI, Tsirigos KD, Winther O, Brunak S, von Heijne G, Nielsen H (2022) SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat Biotechnol 40(7):1023\u0026ndash;1025. https://doi.org/10.1038/s41587-021-01156-3\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTolksdorf C, Wolf R, Rauch BH, Jedlitschky G, M\u0026uuml;ller C (2025) Monomeric and oligomeric decorsins of the Asian medicinal leech \u003cem\u003eHirudinaria manillensis\u003c/em\u003e. Int J Mol Sci 26, 11017. https://doi.org/10.3390/ijms262211017\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTuszynski GP, Gasic TB, Gasic GJ (1987) Isolation and characterization of antistasin. An inhibitor of metastasis and coagulation. J Biol Chem 262(20):9718\u0026ndash;9723. https://doi.org/10.1016/S0021-9258(18)47993-8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan de Walle GR, Peters ST, VanderVen BC, O'Callaghan DJ, Osterrieder N (2008) Equine herpesvirus 1 entry via endocytosis is facilitated by alphaV integrins and an RSD motif in glycoprotein D. J Virol 82(23):11859\u0026ndash;11868. https://doi.org/10.1128/JVI.00868-08\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWallace A, Dennis S, Hofsteenge J, Stone SR (1989) Contribution of the N-terminal region of hirudin to its interaction with thrombin. Biochemistry 28(26):10079\u0026ndash;10084. https://doi.org/10.1021/bi00452a030\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Z, B\u0026ouml;ttcher D, Bornscheuer UT, M\u0026uuml;ller C (2025) Expression of recombinant hirudin in bacteria and yeast: A comparative approach. Methods Protoc 8(4):89. https://doi.org/10.3390/mps8040089\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu L, Li Y, Yang Y, Qin M (2013) Deciphering structural and functional roles of disulfide bonds in decorsin. Sci China Chem 56:1485\u0026ndash;1492. https://doi.org/10.1007/s11426-013-4954-1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao F, Huang Z, He B, Liu K, Li J, Liu Z, Lin G (2024) Comparative genomics of two Asian medicinal leeches \u003cem\u003eHirudo nipponia\u003c/em\u003e and \u003cem\u003eHirudo tianjinensis\u003c/em\u003e: With emphasis on antithrombotic genes and their corresponding proteins. Int J Biol Macromol 270(Pt 1):132278. https://doi.org/10.1016/j.ijbiomac.2024.132278\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou L, Schmaier AH (2005) Platelet aggregation testing in platelet rich plasma: description of procedures with the aim to develop standards in the field. Am J Clin Pathol 123(2):172\u0026ndash;183. https://doi.org/10.1309/y9ec-63rw-3xg1-v313\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":"Placobdella, hirudin, ornatin, blood coagulation, platelet aggregation, hematophagous leeches","lastPublishedDoi":"10.21203/rs.3.rs-8807632/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8807632/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLeeches of the genus \u003cem\u003ePlacobdella\u003c/em\u003e Blanchard, 1893 (Annelida: Rhynchobdellida: Glossiphoniidae) are mainly distributed across North and Central America, but at least two species are also present in the palearctic region: the European turtle leech \u003cem\u003ePlacobdella costata\u003c/em\u003e M\u0026uuml;ller, 1846, and the North African turtle leech \u003cem\u003ePlacobdella nabeulensis\u003c/em\u003e Ben Ahmed et al., 2023. All species of the genus are hematophagous and feed on vertebrates, but are known to preferentially target aquatic and semi-aquatic reptiles like turtles and snakes. \u003cem\u003ePlacobdella ornata\u003c/em\u003e Verrill, 1872, is the original source of ornatins, a class of potent platelet aggregation inhibitors. However, the whole repertoire of bioactive peptides, including anticoagulation factors remains largely underexplored for \u003cem\u003ePlacobdella\u003c/em\u003e, and functional characterizations of putative antithrombotics are scarce. Here we describe the genes and cDNAs that encode putative hirudins and ornatins in both of the palearctic and three American species of the genus \u003cem\u003ePlacobdella\u003c/em\u003e. A selection of putative hirudins and ornatins was recombinantly expressed and functionally characterized using appropriate coagulation and platelet aggregation assays, and our data confirm the expression of functional hirudins and ornatins in the studied leeches. In addition, our genetic analyses strongly support the hypothesis that blood feeding evolved only once in the evolutionary history of leeches.\u003c/p\u003e","manuscriptTitle":"Hirudins and ornatins in five species of the genus Placobdella (Annelida: Hirudinea: Glossiphoniidae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-19 09:57:29","doi":"10.21203/rs.3.rs-8807632/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":"2d53e2e0-307b-4e4e-bb12-06ae17d7f968","owner":[],"postedDate":"February 19th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-13T07:18:05+00:00","index":38,"fulltext":""},{"type":"reviewerAgreed","content":"115334977746062117091678740833103579106","date":"2026-05-07T09:37:16+00:00","index":37,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-19T09:57:29+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-19 09:57:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8807632","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8807632","identity":"rs-8807632","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}