Hemostatic disorders are caused either by platelet-related dysfunctions, defective blood coagulation, or by a combination of both, leading to an increased susceptibility to cardiovascular diseases (CVD) and other related illnesses. The unique specificity of anticoagulants from hematophagous arthropods, such as ticks, suggests that tick saliva holds great promise for discovering new treatments for these life-threatening diseases. In this study, we combined in silico and in vitro analyses to characterize the first recombinant serpin, herein called Dromaserpin, from the sialotranscriptome of the Hyalomma dromedarii tick. Our in silico data described Dromaserpin as a secreted protein of ~43 kDa with high similarities to previously characterized inhibitory serpins. The recombinant protein (rDromaserpin) was obtained as a well-structured monomer, which was tested using global blood coagulation and platelet aggregation assays. With this approach, we confirmed rDromaserpin anticoagulant activity as it significantly delayed plasma clotting in activated partial thromboplastin time and thrombin time assays. The profiling of proteolytic activity shows its capacity to inhibit thrombin in the micromolar range (0.2 to 1 μM) and in the presence of heparin this inhibition was clearly increased. It was also able to inhibit Kallikrein, FXIa and slightly FXIIa, with no significant effect on other factors. In addition, the rDromaserpin inhibited thrombin-induced platelet aggregation. Taken together, our data suggest that rDromaserpin deserves to be further investigated as a potential candidate for developing therapeutic compounds targeting disorders related to blood clotting and/or platelet aggregation.

Centre of Excellence in New Target Discovery Butantan Institute Butantã São Paulo 05503 900 Brazil

Innovation and Development Laboratory Innovation and Development Center Instituto Butantan São Paulo 05503 900 Brazil

Institute of Parasitology Biology Centre Czech Academy of Sciences 37005 Ceske Budejovice Czech Republic

Laboratory of Bacteriology Butantan Institute São Paulo 05503 900 Brazil

Laboratory of Viruses Vectors and Hosts Pasteur Institute of Tunis University of Tunis El Manar Tunis 1002 Tunisia

Zobrazit více v PubMed

The World Health Organization Web Site. [(accessed on 17 September 2020)]. Available online: https://www.who.int/health-topics/cardiovascular-diseases/#tab=tab_1.

Stoney C.M., Kaufmann P.G., Czajkowski S.M. Cardiovascular disease: Psychological, social, and behavioral influences: Introduction to the special issue. Am. Psychol. 2018;73:949–954. doi: 10.1037/amp0000359. PubMed DOI

Chang J.C. Hemostasis based on a bovel “two-path unifying theory” and classification of hemostatic disorders. Blood Coagul. Fibrinolysis. 2018;29:573–584. doi: 10.1097/MBC.0000000000000765. PubMed DOI

Ogedegbe H.O. An overview of hemostasis. Lab. Med. 2002;33:948–953. doi: 10.1309/50UQ-GUPF-W6XW-1X7B. DOI

Versteeg H.H., Heemskerk J.W.M., Levi M., Reitsma P.H. New fundamentals in hemostasis. Physiol. Rev. 2013;93:327–358. doi: 10.1152/physrev.00016.2011. PubMed DOI

Mussbacher M., Kral-Pointner J.B., Salzmann M., Schrottmaier W.C., Assinger A. Mechanisms of hemostasis: Contributions of platelets, coagulation factors, and the vessel wall. In: Geiger M., editor. Fundamentals of Vascular Biology. Springer International Publishing; Cham, Switzerland: 2019. pp. 145–169. (Learning Materials in Biosciences).

Page M.J., Macgillivray R.T.A., Di Cera E. Determinants of specificity in coagulation proteases. J. Thromb. Haemost. 2005;3:2401–2408. doi: 10.1111/j.1538-7836.2005.01456.x. PubMed DOI

Gettins P.G.W. Serpin structure, mechanism, and function. Chem. Rev. 2002;102:4751–4804. doi: 10.1021/cr010170+. PubMed DOI

Crawley J.T.B., Zanardelli S., Chion C.K.N.K., Lane D.A. The central role of thrombin in hemostasis. J. Thromb. Haemost. 2007;5((Suppl. S1)):95–101. doi: 10.1111/j.1538-7836.2007.02500.x. PubMed DOI

Siller-Matula J.M., Schwameis M., Blann A., Mannhalter C., Jilma B. Thrombin as a multi-functional enzyme. Focus on in vitro and in vivo effects. Thromb. Haemost. 2011;106:1020–1033. doi: 10.1160/TH10-11-0711. PubMed DOI

Mega J.L., Simon T. Pharmacology of antithrombotic drugs: An assessment of oral antiplatelet and anticoagulant treatments. Lancet. 2015;386:281–291. doi: 10.1016/S0140-6736(15)60243-4. PubMed DOI

Tang N., Bai H., Chen X., Gong J., Li D., Sun Z. Anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy. J. Thromb. Haemost. 2020;18:1094–1099. doi: 10.1111/jth.14817. PubMed DOI PMC

Koehn F.E., Carter G.T. The evolving role of natural products in drug discovery. Nat. Rev. Drug Discov. 2005;4:206–220. doi: 10.1038/nrd1657. PubMed DOI

Carvalhal F., Cristelo R.R., Resende D.I.S.P., Pinto M.M.M., Sousa E., Correia-da-Silva M. Antithrombotics from the sea: Polysaccharides and beyond. Mar. Drugs. 2019;17:170. doi: 10.3390/md17030170. PubMed DOI PMC

Lucas A., Yaron J.R., Zhang L., Ambadapadi S. Overview of serpins and their roles in biological systems. Methods Mol. Biol. 2018;1826:1–7. doi: 10.1007/978-1-4939-8645-3_1. PubMed DOI

Lucas A., Yaron J.R., Zhang L., Macaulay C., McFadden G. Serpins: Development for therapeutic applications. Methods Mol. Biol. 2018;1826:255–265. doi: 10.1007/978-1-4939-8645-3_17. PubMed DOI

Van Gent D., Sharp P., Morgan K., Kalsheker N. Serpins: Structure, Function and Molecular Evolution. Int. J. Biochem. Cell Biol. 2003;35:1536–1547. doi: 10.1016/S1357-2725(03)00134-1. PubMed DOI

Stein P.E., Leslie A.G., Finch J.T., Carrell R.W. Crystal structure of uncleaved ovalbumin at 1.95 A Resolution. J. Mol. Biol. 1991;221:941–959. doi: 10.1016/0022-2836(91)80185-W. PubMed DOI

Engh R., Löbermann H., Schneider M., Wiegand G., Huber R., Laurell C.B. The S variant of human alpha 1-antitrypsin, structure and implications for function and metabolism. Protein Eng. 1989;2:407–415. doi: 10.1093/protein/2.6.407. PubMed DOI

Skinner R., Abrahams J.P., Whisstock J.C., Lesk A.M., Carrell R.W., Wardell M.R. The 2.6 A structure of antithrombin indicates a conformational change at the heparin binding site. J. Mol. Biol. 1997;266:601–609. doi: 10.1006/jmbi.1996.0798. PubMed DOI

Tucker H.M., Mottonen J., Goldsmith E.J., Gerard R.D. Engineering of plasminogen activator inhibitor-1 to reduce the rate of latency tansition. Nat. Struct. Biol. 1995;2:442–445. doi: 10.1038/nsb0695-442. PubMed DOI

Chen H., Davids J.A., Zheng D., Bryant M., Bot I., Van Berckel T.J.C., Biessen E., Pepine C., Ryman K., Progulski-Fox A., et al. The serpin solution; targeting thrombotic and thrombolytic serine proteases in inflammation. Cardiovasc. Hematol. Disord. Drug Targets. 2013;13:99–110. doi: 10.2174/1871529X11313020003. PubMed DOI

Irving J.A., Ekeowa U.I., Belorgey D., Haq I., Gooptu B., Miranda E., Pérez J., Roussel B.D., Ordóñez A., Dalton L.E., et al. The serpinopathies studying serpin polymerization in vivo. Meth. Enzymol. 2011;501:421–466. doi: 10.1016/B978-0-12-385950-1.00018-3. PubMed DOI

Rau J.C., Beaulieu L.M., Huntington J.A., Church F.C. Serpins in thrombosis, hemostasis and fibrinolysis. J. Thromb. Haemost. 2007;5((Suppl. S1)):102–115. doi: 10.1111/j.1538-7836.2007.02516.x. PubMed DOI PMC

Bang N.U. Leeches, snakes, ticks, and vampire bats in today’s cardiovascular drug development. Circulation. 1991;84:436–438. doi: 10.1161/01.CIR.84.1.436. PubMed DOI

Jmel M.A., Aounallah H., Bensaoud C., Mekki I., Chmelař J., Faria F., M’ghirbi Y., Kotsyfakis M. Insights into the role of tick salivary protease inhibitors during ectoparasite-host crosstalk. Int. J. Mol. Sci. 2021;22:892. doi: 10.3390/ijms22020892. PubMed DOI PMC

Chmelař J., Kotál J., Kovaříková A., Kotsyfakis M. The use of tick salivary proteins as novel therapeutics. Front. Physiol. 2019;10:812. doi: 10.3389/fphys.2019.00812. PubMed DOI PMC

Chmelař J., Kotál J., Langhansová H., Kotsyfakis M. Protease inhibitors in tick saliva: The role of serpins and cystatins in tick-host-pathogen interaction. Front. Cell Infect. Microbiol. 2017;7:216. doi: 10.3389/fcimb.2017.00216. PubMed DOI PMC

Irving J.A., Pike R.N., Lesk A.M., Whisstock J.C. Phylogeny of the serpin superfamily: Implications of patterns of amino acid conservation for structure and function. Genome Res. 2000;10:1845–1864. doi: 10.1101/gr.147800. PubMed DOI

Schechter I., Berger A. On the size of the active site in proteases. I. Papain. 1967. Biochem. Biophys. Res. Commun. 2012;425:497–502. doi: 10.1016/j.bbrc.2012.08.015. PubMed DOI

Nguyen K.D., Pan Y. A knowledge-based multiple-sequence alignment algorithm. IEEE/ACM Trans. Comput. Biol. Bioinform. 2013;10:884–896. doi: 10.1109/TCBB.2013.102. PubMed DOI

Yu Y., Cao J., Zhou Y., Zhang H., Zhou J. Isolation and characterization of two novel serpins from the tick Rhipicephalus haemaphysaloides. Ticks Tick Borne Dis. 2013;4:297–303. doi: 10.1016/j.ttbdis.2013.02.001. PubMed DOI

Kumar S., Stecher G., Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 2016;33:1870–1874. doi: 10.1093/molbev/msw054. PubMed DOI PMC

Tirloni L., Seixas A., Mulenga A., Vaz I.d.S., Termignoni C. A Family of serine protease inhibitors (serpins) in the cattle tick Rhipicephalus (boophilus) microplus. Exp. Parasitol. 2014;137:25–34. doi: 10.1016/j.exppara.2013.12.001. PubMed DOI

Martin S.R., Schilstra M.J. Circular dichroism and its application to the study of biomolecules. Methods Cell Biol. 2008;84:263–293. doi: 10.1016/S0091-679X(07)84010-6. PubMed DOI

Sanrattana W., Maas C., De Maat S. SERPINs—From trap to treatment. Front. Med. 2019;6:25. doi: 10.3389/fmed.2019.00025. PubMed DOI PMC

Ng V.L. Prothrombin time and partial thromboplastin time assay considerations. Clin. Lab. Med. 2009;29:253–263. doi: 10.1016/j.cll.2009.05.002. PubMed DOI

Higgins W.J., Fox D.M., Kowalski P.S., Nielsen J.E., Worrall D.M. Heparin enhances serpin inhibition of the cysteine protease cathepsin L. J. Biol. Chem. 2010;285:3722–3729. doi: 10.1074/jbc.M109.037358. PubMed DOI PMC

Chmelar J., Calvo E., Pedra J.H.F., Francischetti I.M.B., Kotsyfakis M. Tick salivary secretion as a source of antihemostatics. J. Proteom. 2012;75:3842–3854. doi: 10.1016/j.jprot.2012.04.026. PubMed DOI PMC

Bensaoud C., Aounallah H., Sciani J.M., Faria F., Chudzinski-Tavassi A.M., Bouattour A., M’ghirbi Y. Proteomic informed by transcriptomic for salivary glands components of the camel tick Hyalomma dromedarii. BMC Genom. 2019;20:675. doi: 10.1186/s12864-019-6042-1. PubMed DOI PMC

Bensaoud C., Nishiyama M.Y., Ben Hamda C., Lichtenstein F., Castro de Oliveira U., Faria F., Loiola Meirelles Junqueira-de-Azevedo I., Ghedira K., Bouattour A., M’Ghirbi Y., et al. De novo assembly and annotation of Hyalomma dromedarii Tick (Acari: Ixodidae) sialotranscriptome with regard to gender differences in gene expression. Parasit. Vectors. 2018;11:314. doi: 10.1186/s13071-018-2874-9. PubMed DOI PMC

Schussler G.C. The thyroxine-binding proteins. Thyroid. 2000;10:141–149. doi: 10.1089/thy.2000.10.141. PubMed DOI

Simard M., Underhill C., Hammond G.L. Functional implications of corticosteroid-binding globulin N-glycosylation. J. Mol. Endocrinol. 2018;60:71–84. doi: 10.1530/JME-17-0234. PubMed DOI PMC

Polderdijk S.G.I., Huntington J.A. Identification of serpins specific for activated protein C using a lysate-based screening assay. Sci. Rep. 2018;8:8793. doi: 10.1038/s41598-018-27067-z. PubMed DOI PMC

Roberts T.H., Hejgaard J., Saunders N.F.W., Cavicchioli R., Curmi P.M.G. Serpins in unicellular eukarya, archaea, and bacteria: Sequence analysis and evolution. J. Mol. Evol. 2004;59:437–447. doi: 10.1007/s00239-004-2635-6. PubMed DOI

Kim T.K., Radulovic Z., Mulenga A. Target validation of highly conserved Amblyomma americanum tick saliva serine protease inhibitor 19. Ticks Tick Borne Dis. 2016;7:405–414. doi: 10.1016/j.ttbdis.2015.12.017. PubMed DOI PMC

Wang F., Song Z., Chen J., Wu Q., Zhou X., Ni X., Dai J. The immunosuppressive functions of two novel tick serpins, HlSerpin-a and HlSerpin-b, from Haemaphysalis longicornis. Immunology. 2019;159:109–120. doi: 10.1111/imm.13130. PubMed DOI PMC

Mahon B.P., Ambadapadi S., Yaron J.R., Lomelino C.L., Pinard M.A., Keinan S., Kurnikov I., Macaulay C., Zhang L., Reeves W., et al. Crystal structure of cleaved Serp-1, a Myxomavirus-derived immune modulating serpin: Structural design of serpin reactive center loop peptides with improved therapeutic function. Biochemistry. 2018;57:1096–1107. doi: 10.1021/acs.biochem.7b01171. PubMed DOI

Pongprayoon P., Niramitranon J., Kaewhom P., Kaewmongkol S., Suwan E., Stich R.W., Jittapalapong S. Dynamic and structural insights into tick serpin from Ixodes ricinus. J. Biomol. Struct. Dyn. 2019;38:2296–2303. doi: 10.1080/07391102.2019.1630003. PubMed DOI

Chmelar J., Oliveira C.J., Rezacova P., Francischetti I.M.B., Kovarova Z., Pejler G., Kopacek P., Ribeiro J.M.C., Mares M., Kopecky J., et al. A Tick salivary protein targets cathepsin G and chymase and inhibits host inflammation and platelet aggregation. Blood. 2011;117:736–744. doi: 10.1182/blood-2010-06-293241. PubMed DOI PMC

Kovářová Z., Chmelař J., Sanda M., Brynda J., Mareš M., Rezáčová P. Crystallization and diffraction analysis of the serpin IRS-2 from the hard tick Ixodes ricinus. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2010;66:1453–1457. doi: 10.1107/S1744309110032343. PubMed DOI PMC

Meekins D.A., Kanost M.R., Michel K. Serpins in arthropod biology. Semin. Cell Dev. Biol. 2017;62:105–119. doi: 10.1016/j.semcdb.2016.09.001. PubMed DOI PMC

Smith S.A. The Cell-based model of coagulation. J. Vet. Emerg. Crit. Care. 2009;19:3–10. doi: 10.1111/j.1476-4431.2009.00389.x. PubMed DOI

Ibrahim M.A., Masoud H.M.M. Thrombin inhibitor from the salivary gland of the camel Tick Hyalomma dromedarii. Exp. Appl. Acarol. 2018;74:85–97. doi: 10.1007/s10493-017-0196-9. PubMed DOI

Francischetti I.M.B., Sa-Nunes A., Mans B.J., Santos I.M., Ribeiro J.M.C. The role of saliva in tick feeding. Front. Biosci. 2009;14:2051–2088. doi: 10.2741/3363. PubMed DOI PMC

Rumbaut R.E., Thiagarajan P. Platelet Aggregation. Morgan & Claypool Life Sciences, Texas Medical Center; Houston, TX, USA: 2010. PubMed

Davì G., Patrono C. Platelet activation and atherothrombosis. N. Engl. J. Med. 2007;357:2482–2494. doi: 10.1056/NEJMra071014. PubMed DOI

Furman M.I., Liu L., Benoit S.E., Becker R.C., Barnard M.R., Michelson A.D. The cleaved peptide of the thrombin receptor is a strong platelet agonist. Proc. Natl. Acad. Sci. USA. 1998;95:3082–3087. doi: 10.1073/pnas.95.6.3082. PubMed DOI PMC

Al Ghumlas A.K., Gader A.G.M.A. Characterization of the aggregation responses of camel platelets. Vet. Clin. Pathol. 2013;42:307–313. doi: 10.1111/vcp.12062. PubMed DOI

Abdel Gader A.G.M., Al Momen A.K.M., Alhaider A., Brooks M.B., Catalfamo J.L., Al Haidary A.A., Hussain M.F. Clotting factor VIII (FVIII) and thrombin generation in camel plasma: A comparative study with humans. Can. J. Vet. Res. 2013;77:150–157. PubMed PMC

Schechter N.M., Plotnick M.I. Measurement of the kinetic parameters mediating protease-serpin inhibition. Methods. 2004;32:159–168. doi: 10.1016/S1046-2023(03)00207-X. PubMed DOI

Gray E., Hogwood J., Mulloy B. Heparin—A Century of Progress. Springer; Berlin/Heidelberg, Germany: 2012. The anticoagulant and antithrombotic mechanisms of heparin; pp. 43–61. PubMed DOI

Varki A., Cummings R.D., Esko J.D., Freeze H.H., Stanley P., Bertozzi C.R., Hart G.W., Etzler M.E. Essentials of Glycobiology. 2nd ed. Cold Spring Harbor Laboratory Press; Woodbury, NY, USA: 2009. 784p PubMed

Forster M., Mulloy B. Computational approaches to the identification of heparin-binding sites on the surfaces of proteins. Biochem. Soc. Trans. 2006;34:431–434. doi: 10.1042/BST0340431. PubMed DOI

Handel T.M., Johnson Z., Crown S.E., Lau E.K., Sweeney M., Proudfoot A.E. Regulation of protein function by glycosaminoglycans—As exemplified by chemokines. Annu. Rev. Biochem. 2005;74:385–410. doi: 10.1146/annurev.biochem.72.121801.161747. PubMed DOI

Fromm J.R., Hileman R.E., Caldwell E.E.O., Weiler J.M., Linhardt R.J. Pattern and spacing of basic amino acids in heparin binding sites. Arch. Biochem. Biophys. 1997;343:92–100. doi: 10.1006/abbi.1997.0147. PubMed DOI

Li W., Huntington J.A. Crystal structures of protease Nexin-1 in complex with heparin and thrombin suggest a 2-Step recognition mechanism. Blood. 2012;120:459–467. doi: 10.1182/blood-2012-03-415869. PubMed DOI

Li W., Johnson D.J.D., Esmon C.T., Huntington J.A. Structure of the antithrombin-thrombin-heparin ternary complex reveals the antithrombotic mechanism of heparin. Nat. Struct. Mol. Biol. 2004;11:857–862. doi: 10.1038/nsmb811. PubMed DOI

Jones D.T., Taylor W.R., Thornton J.M. The Rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 1992;8:275–282. doi: 10.1093/bioinformatics/8.3.275. PubMed DOI

Batista I.F.C., Ramos O.H.P., Ventura J.S., Junqueira-de-Azevedo I.L.M., Ho P.L., Chudzinski-Tavassi A.M. A new Factor Xa inhibitor from Amblyomma cajennense with a Unique Domain Composition. Arch. Biochem. Biophys. 2010;493:151–156. doi: 10.1016/j.abb.2009.10.009. PubMed DOI

Bock S.C., Wion K.L., Vehar G.A., Lawn R.M. Cloning and Expression of the cDNA for Human Antithrombin III. Nucleic Acids Res. 1982;10:8113–8125. doi: 10.1093/nar/10.24.8113. PubMed DOI PMC

Mulenga A., Khumthong R., Blandon M.A. Molecular and Expression Analysis of a Family of the Amblyomma americanum Tick Lospins. J. Exp. Biol. 2007;210:3188–3198. doi: 10.1242/jeb.006494. PubMed DOI

Porter L., Radulović Ž., Kim T., Braz G.R.C., Da Silva Vaz I., Mulenga A. Bioinformatic Analyses of Male and Female Amblyomma americanum Tick Expressed Serine Protease Inhibitors (Serpins) Ticks Tick Borne Dis. 2015;6:16–30. doi: 10.1016/j.ttbdis.2014.08.002. PubMed DOI PMC

Imamura S., Da Silva Vaz Junior I., Sugino M., Ohashi K., Onuma M. A Serine Protease Inhibitor (Serpin) from Haemaphysalis longicornis as an Anti-Tick Vaccine. Vaccine. 2005;23:1301–1311. doi: 10.1016/j.vaccine.2004.08.041. PubMed DOI

Chlastáková A., Kotál J., Beránková Z., Kaščáková B., Martins L.A., Langhansová H., Prudnikova T., Ederová M., Kutá Smatanová I., Kotsyfakis M., et al. Iripin-3, a New Salivary Protein Isolated From Ixodes ricinus Ticks, Displays Immunomodulatory and Anti-Hemostatic Properties In Vitro. Front. Immunol. 2021;12:626200. doi: 10.3389/fimmu.2021.626200. PubMed DOI PMC

Schwarz A., Von Reumont B.M., Erhart J., Chagas A.C., Ribeiro J.M.C., Kotsyfakis M. De Novo Ixodes ricinus Salivary Gland Transcriptome Analysis Using Two Next-Generation Sequencing Methodologies. FASEB J. 2013;27:4745–4756. doi: 10.1096/fj.13-232140. PubMed DOI PMC

Ibelli A.M.G., Kim T.K., Hill C.C., Lewis L.A., Bakshi M., Miller S., Porter L., Mulenga A. A Blood Meal-Induced Ixodes scapularis Tick Saliva Serpin Inhibits Trypsin and Thrombin, and Interferes with Platelet Aggregation and Blood Clotting. Int. J. Parasitol. 2014;44:369–379. doi: 10.1016/j.ijpara.2014.01.010. PubMed DOI PMC

Mulenga A., Tsuda A., Onuma M., Sugimoto C. Four Serine Proteinase Inhibitors (Serpin) from the Brown Ear Tick, Rhiphicephalus appendiculatus; cDNA Cloning and Preliminary Characterization. Insect Biochem. Mol. Biol. 2003;33:267–276. doi: 10.1016/S0965-1748(02)00240-0. PubMed DOI