BACKGROUND: Salivary glands from blood-feeding arthropods secrete several molecules that inhibit mammalian hemostasis and facilitate blood feeding and pathogen transmission. The salivary functions from Simulium guianense, the main vector of Onchocerciasis in South America, remain largely understudied. Here, we have characterized a salivary protease inhibitor (Guianensin) from the blackfly Simulium guianense. MATERIALS AND METHODS: A combination of bioinformatic and biophysical analyses, recombinant protein production, in vitro and in vivo experiments were utilized to characterize the molecula mechanism of action of Guianensin. Kinetics of Guianensin interaction with proteases involved in vertebrate inflammation and coagulation were carried out by surface plasmon resonance and isothermal titration calorimetry. Plasma recalcification and coagulometry and tail bleeding assays were performed to understand the role of Guianensin in coagulation. RESULTS: Guianensin was identified in the sialotranscriptome of adult S. guianense flies and belongs to the Kunitz domain of protease inhibitors. It targets various serine proteases involved in hemostasis and inflammation. Binding to these enzymes is highly specific to the catalytic site and is not detectable for their zymogens, the catalytic site-blocked human coagulation factor Xa (FXa), or thrombin. Accordingly, Guianensin significantly increased both PT (Prothrombin time) and aPTT (Activated partial thromboplastin time) in human plasma and consequently increased blood clotting time ex vivo. Guianensin also inhibited prothrombinase activity on endothelial cells. We show that Guianensin acts as a potent anti-inflammatory molecule on FXa-induced paw edema formation in mice. CONCLUSION: The information generated by this work highlights the biological functionality of Guianensin as an antithrombotic and anti-inflammatory protein that may play significant roles in blood feeding and pathogen transmission.

Institute of Molecular Biology and Biotechnology Foundation for Research and Technology Hellas Heraklion Greece

Laboratory of Genomics and Proteomics of Disease Vectors Institute of Parasitology Biology Centre Czech Academy of Sciences Ceske Budejovice Czechia

Laboratory of Malaria and Vector Research National Institutes of Health Bethesda MD United States

Tick Pathogen Transmission Unit Laboratory of Bacteriology National Institute of Allergy and Infectious Diseases Hamilton MT United States

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Tsujimoto H, Gray EW, Champagne DE. Black fly salivary gland extract inhibits proliferation and induces apoptosis in murine splenocytes. Parasite Immunol (2010) 32(4):275–84. doi: 10.1111/j.1365-3024.2009.01186.x PubMed DOI

Abebe M, Cupp MS, Ramberg FB, Cupp EW. Anticoagulant activity in salivary gland extracts of black flies (Diptera: simuliidae). J Med entomol (1994) 31(6):908–11. doi: 10.1093/jmedent/31.6.908 PubMed DOI

Andersen JF, Pham VM, Meng Z, Champagne DE, Ribeiro JM. Insight into the sialome of the black fly, simulium vittatum. J Proteome Res (2009) 8(3):1474–88. doi: 10.1021/pr8008429 PubMed DOI PMC

Cross ML, Cupp MS, Cupp EW, Ramberg FB, Enriquez FJ. Antibody responses of BALB/c mice to salivary antigens of hematophagous black flies (Diptera: simuliidae). J Med Entomol (1993) 30(4):725–34. doi: 10.1093/jmedent/30.4.725 PubMed DOI

Sainas S, Dosio F, Boschi D, Lolli ML. Targeting human onchocerciasis: recent advances beyond ivermectin. Annu Rep Med Chem (2018) 51:1–38. doi: 10.1016/bs.armc.2018.08.001 DOI

Gustavsen K, Hopkins A, Sauerbrey M. Onchocerciasis in the americas: from arrival to (near) elimination. Parasit Vectors (2011) 4:205. doi: 10.1186/1756-3305-4-205 PubMed DOI PMC

Cao J, Shi L, Zhou Y, Gao X, Zhang H, Gong H, et al. . Characterization of a new kunitz-type serine protease inhibitor from the hard tick Rhipicephalus hemaphysaloides. Arch Insect Biochem Physiol (2013) 84:104–13. doi: 10.1002/arch.211 PubMed DOI

Bernardo MJ, Cupp EW, Kiszewski AE. Rearing black flies (Diptera, simuliidae) in the laboratory - colonization and life table statistics for simulium-vittatum. Ann Entomol Soc America (1986) 79(4):610–21. doi: 10.1093/aesa/79.4.610 PubMed DOI

Chagas AC, Medeiros JF, Astolfi-Filho S, Py-Daniel V. Anticoagulant activity in salivary gland homogenates of Thyrsopelma guianense (Diptera: simuliidae), the primary vector of onchocerciasis in the Brazilian Amazon. Mem Inst Oswaldo Cruz. (2010) 105(2):174–8. doi: 10.1590/S0074-02762010000200011 PubMed DOI

Cupp EW, Cupp MS. Black fly (Diptera: simuliidae) salivary secretions: importance in vector competence and disease. J Med entomol (1997) 34(2):87–94. doi: 10.1093/jmedent/34.2.87 PubMed DOI

Rau JC, Beaulieu LM, Huntington JA, Church FC. Serpins in thrombosis, hemostasis and fibrinolysis. J Thromb Haemost (2007) 5(Suppl 1):102–15. doi: 10.1111/j.1538-7836.2007.02516.x PubMed DOI PMC

Nienaber J, Gaspar AR, Neitz AW. Savignin, a potent thrombin inhibitor isolated from the salivary glands of the tick Ornithodoros savignyi (Acari: argasidae). Exp Parasitol (1999) 93(2):82–91. doi: 10.1006/expr.1999.4448 PubMed DOI

Oliveira F, Lawyer PG, Kamhawi S, Valenzuela JG. Immunity to distinct sand fly salivary proteins primes the anti-leishmania immune response towards protection or exacerbation of disease. PloS Negl Trop Dis (2008) 2(4):e226. doi: 10.1371/journal.pntd.0000226 PubMed DOI PMC

Borah S, Naglot A, Goswami S, Rahman I, Deka M. Anticoagulation activity of salivary gland extract of oriental blackfly simulium indicum. Asian Pacific J Trop Biomed (2014) 4(Suppl 1):S252–7. doi: 10.12980/APJTB.4.2014C265 PubMed DOI PMC

Assumpcao TC, Mizurini DM, Ma D, Monteiro RQ, Ahlstedt S, Reyes M, et al. . Ixonnexin from tick saliva promotes fibrinolysis by interacting with plasminogen and tissue-type plasminogen activator, and prevents arterial thrombosis. Sci Rep (2018) 8(1):4806. doi: 10.1038/s41598-018-22780-1 PubMed DOI PMC

Ribeiro JMC, Francischetti IMB. Role of arthropod saliva in blood feeding: sialome and post-sialome perspectives. Annu Rev Entomol (2003) 48:73–88. doi: 10.1146/annurev.ento.48.060402.102812 PubMed DOI

Cupp MS, Ribeiro JM, Champagne DE, Cupp EW. Analyses of cDNA and recombinant protein for a potent vasoactive protein in saliva of a blood-feeding black fly, Simulium vittatum . J Exp Biol (1998) 201(Pt 10):1553–61. doi: 10.1242/jeb.201.10.1553 PubMed DOI

Ribeiro JM, Mather TN. Ixodes scapularis: salivary kininase activity is a metallo dipeptidyl carboxypeptidase. Exp Parasitol (1998) 89(2):213–21. doi: 10.1006/expr.1998.4296 PubMed DOI

Levi M, van der Poll T. Inflammation and coagulation. Crit Care Med (2010) 38(2 Suppl):S26–34. doi: 10.1097/CCM.0b013e3181c98d21 PubMed DOI

Kato N, Iwanaga S, Okayama T, Isawa H, Yuda M, Chinzei Y. Identification and characterization of the plasma kallikrein-kinin system inhibitor, haemaphysalin, from hard tick, haemaphysalis longicornis. Thromb Haemost (2005) 93(2):359–67. doi: 10.1160/TH04-05-0319 PubMed DOI

Paterson KJ, Zambreanu L, Bennett DL, McMahon SB. Characterisation and mechanisms of bradykinin-evoked pain in man using iontophoresis. Pain (2013) 154(6):782–92. doi: 10.1016/j.pain.2013.01.003 PubMed DOI PMC

Vassalli JD, Sappino AP, Belin D. The plasminogen activator/plasmin system. J Clin Invest (1991) 88(4):1067–72. doi: 10.1172/JCI115405 PubMed DOI PMC

Macedo-Ribeiro S, Almeida C, Calisto BM, Friedrich T, Mentele R, Sturzebecher J, et al. . Isolation, cloning and structural characterisation of boophilin, a multifunctional kunitz-type proteinase inhibitor from the cattle tick. PloS One (2008) 3(2):e1624. doi: 10.1371/journal.pone.0001624 PubMed DOI PMC

Ascenzi P, Bocedi A, Bolognesi M, Spallarossa A, Coletta M, De Cristofaro R, et al. . The bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor): a milestone protein. Curr Protein Pept Sci (2003) 4(3):231–51. doi: 10.2174/1389203033487180 PubMed DOI

Tsujimoto H, Kotsyfakis M, Francischetti IM, Eum JH, Strand MR, Champagne DE. Simukunin from the salivary glands of the black fly Simulium vittatum inhibits enzymes that regulate clotting and inflammatory responses. PloS One (2012) 7(2):e29964. doi: 10.1371/journal.pone.0029964 PubMed DOI PMC

Ranasinghe S, McManus DP. Structure and function of invertebrate kunitz serine protease inhibitors. Dev Comp Immunol (2013) 39(3):219–27. doi: 10.1016/j.dci.2012.10.005 PubMed DOI

Parizi LF, Ali A, Tirloni L, Oldiges DP, Sabadin GA, Coutinho ML, et al. . Peptidase inhibitors in tick physiology. Med Vet Entomol (2018) 32(2):129–44. doi: 10.1111/mve.12276 PubMed DOI

Corral-Rodriguez MA, Macedo-Ribeiro S, Barbosa Pereira PJ, Fuentes-Prior P. Tick-derived kunitz-type inhibitors as antihemostatic factors. Insect Biochem Mol Biol (2009) 39(9):579–95. doi: 10.1016/j.ibmb.2009.07.003 PubMed DOI

Chagas AC, Calvo E, Pimenta PF, Ribeiro JM. An insight into the sialome of Simulium guianense (Diptera : simuliidae), the main vector of river blindness disease in Brazil. BMC Genomics (2011) 12:612. doi: 10.1186/1471-2164-12-612 PubMed DOI PMC

Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y. The I-TASSER suite: protein structure and function prediction. Nat Methods (2015) 12(1):7–8. doi: 10.1038/nmeth.3213 PubMed DOI PMC

Martin-Martin I, Chagas AC, Guimaraes-Costa AB, Amo L, Oliveira F, Moore IN, et al. . Immunity to LuloHya and lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection. PloS pathogens (2018) 14(5):e1007006. doi: 10.1371/journal.ppat.1007006 PubMed DOI PMC

Wiedemann C, Bellstedt P, Gorlach M. CAPITO–a web server-based analysis and plotting tool for circular dichroism data. Bioinformatics (2013) 29(14):1750–7. doi: 10.1093/bioinformatics/btt278 PubMed DOI

Smith LB, Duge E, Valenzuela-Leon PC, Brooks S, Martin-Martin I, Ackerman H, et al. . Novel salivary antihemostatic activities of long-form D7 proteins from the malaria vector Anopheles gambiae facilitate hematophagy. J Biol Chem (2022) 298(6):101971. doi: 10.1016/j.jbc.2022.101971 PubMed DOI PMC

Calvo E, Mizurini DM, Sa-Nunes A, Ribeiro JM, Andersen JF, Mans BJ, et al. . Alboserpin, a factor xa inhibitor from the mosquito vector of yellow fever, binds heparin and membrane phospholipids and exhibits antithrombotic activity. J Biol Chem (2011) 286(32):27998–8010. doi: 10.1074/jbc.M111.247924 PubMed DOI PMC

Chmelar J, Oliveira CJ, Rezacova P, Francischetti IM, Kovarova Z, Pejler G, et al. . A tick salivary protein targets cathepsin G and chymase and inhibits host inflammation and platelet aggregation. Blood (2011) 117(2):736–44. doi: 10.1182/blood-2010-06-293241 PubMed DOI PMC

Shrivastava G, Valenzuela-Leon PC, Chagas AC, Kern O, Botello K, Zhang Y, et al. . Alboserpin, the main salivary anticoagulant from the disease vector Aedes albopictus, displays anti-FXa-PAR signaling In vitro and in vivo. Immunohorizons (2022) 6(6):373–83. doi: 10.4049/immunohorizons.2200045 PubMed DOI PMC

Nielsen H, Engelbrecht J, Brunak S, von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng (1997) 10(1):1–6. doi: 10.1093/protein/10.1.1 PubMed DOI

Xu Y, Carr PD, Guss JM, Ollis DL. The crystal structure of bikunin from the inter-alpha-inhibitor complex: a serine protease inhibitor with two kunitz domains. J Mol Biol (1998) 276(5):955–66. doi: 10.1006/jmbi.1997.1582 PubMed DOI

Kotsyfakis M, Sa-Nunes A, Francischetti IM, Mather TN, Andersen JF, Ribeiro JM. Antiinflammatory and immunosuppressive activity of sialostatin l, a salivary cystatin from the tick ixodes scapularis. J Biol Chem (2006) 281(36):26298–307. doi: 10.1074/jbc.M513010200 PubMed DOI

Cirino G, Cicala C, Bucci M, Sorrentino L, Ambrosini G, DeDominicis G, et al. . Factor xa as an interface between coagulation and inflammation. molecular mimicry of factor xa association with effector cell protease receptor-1 induces acute inflammation in vivo . J Clin Invest (1997) 99(10):2446–51. doi: 10.1172/JCI119428 PubMed DOI PMC

Fallis AM. Feeding and related behavior of female simuliidae (Diptera). Exp Parasitol (1964) 15:439–70. doi: 10.1016/0014-4894(64)90038-4 PubMed DOI

Arca B, Ribeiro JMC. Saliva of hematophagous insects: a multifaceted toolkit. Curr Opin Insect Sci (2018) 29:102–9. doi: 10.1016/j.cois.2018.07.012 PubMed DOI

Chagas AC, McPhie P, San H, Narum D, Reiter K, Tokomasu F, et al. . Simplagrin, a platelet aggregation inhibitor from simulium nigrimanum salivary glands specifically binds to the Von willebrand factor receptor in collagen and inhibits carotid thrombus formation in vivo. PloS Negl Trop Dis (2014) 8(6):e2947. doi: 10.1371/journal.pntd.0002947 PubMed DOI PMC

Wiedow O, Meyer-Hoffert U. Neutrophil serine proteases: potential key regulators of cell signalling during inflammation. J Intern Med (2005) 257(4):319–28. doi: 10.1111/j.1365-2796.2005.01476.x PubMed DOI

Caughey GH. Mast cell tryptases and chymases in inflammation and host defense. Immunol Rev (2007) 217:141–54. doi: 10.1111/j.1600-065X.2007.00509.x PubMed DOI PMC

Kuravi SJ, Bevins A, Satchell SC, Harper L, Williams JM, Rainger GE, et al. . Neutrophil serine proteases mediate inflammatory cell recruitment by glomerular endothelium and progression towards dysfunction. Nephrol Dial Transplant (2012) 27(12):4331–8. doi: 10.1093/ndt/gfs180 PubMed DOI

van Hinsbergh VW. Endothelium–role in regulation of coagulation and inflammation. Semin Immunopathol (2012) 34(1):93–106. doi: 10.1007/s00281-011-0285-5 PubMed DOI PMC

Bombeli T, Karsan A, Tait JF, Harlan JM. Apoptotic vascular endothelial cells become procoagulant. Blood (1997) 89(7):2429–42. doi: 10.1182/blood.V89.7.2429 PubMed DOI

Ahmad SS, London FS, Walsh PN. The assembly of the factor X-activating complex on activated human platelets. J Thromb Haemost (2003) 1(1):48–59. doi: 10.1046/j.1538-7836.2003.00020.x PubMed DOI

Fredenburgh JC, Weitz JI. Chapter 122 - overview of hemostasis and thrombosis. In: Hoffman R, Benz EJ, Silberstein LE, Heslop HE, Weitz JI, Anastasi J, et al.. editors. Hematology, (7th edition). (2018). p. 1831–42. doi: 10.1016/B978-0-323-35762-3.00122-0 DOI

Bukowska A, Zacharias I, Weinert S, Skopp K, Hartmann C, Huth C, et al. . Coagulation factor xa induces an inflammatory signalling by activation of protease-activated receptors in human atrial tissue. Eur J Pharmacol (2013) 718(1-3):114–23. doi: 10.1016/j.ejphar.2013.09.006 PubMed DOI

McNamara CA, Sarembock IJ, Gimple LW, Fenton JW, 2nd, Coughlin SR, Owens GK. Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor. J Clin Invest (1993) 91(1):94–8. doi: 10.1172/JCI116206 PubMed DOI PMC

Griffin CT, Srinivasan Y, Zheng YW, Huang W, Coughlin SR. A role for thrombin receptor signaling in endothelial cells during embryonic development. Science (2001) 293(5535):1666–70. doi: 10.1126/science.1061259 PubMed DOI

Shinohara T, Suzuki K, Takada K, Okada M, Ohsuzu F. Regulation of proteinase-activated receptor 1 by inflammatory mediators in human vascular endothelial cells. Cytokine (2002) 19(2):66–75. doi: 10.1006/cyto.2002.1946 PubMed DOI

Hirano K. The roles of proteinase-activated receptors in the vascular physiology and pathophysiology. Arterioscler Thromb Vasc Biol (2007) 27(1):27–36. doi: 10.1161/01.ATV.0000251995.73307.2d PubMed DOI

Francischetti IM, Sa-Nunes A, Mans BJ, Santos IM, Ribeiro JM. The role of saliva in tick feeding. Front Biosci (Landmark Ed) (2009) 14(6):2051–88. doi: 10.2741/3363 PubMed DOI PMC

Calvo E, Tokumasu F, Marinotti O, Villeval JL, Ribeiro JM, Francischetti IM. Aegyptin, a novel mosquito salivary gland protein, specifically binds to collagen and prevents its interaction with platelet glycoprotein VI, integrin alpha2beta1, and von willebrand factor. J Biol Chem (2007) 282(37):26928–38. doi: 10.1074/jbc.M705669200 PubMed DOI PMC

Martin-Martin I, Valenzuela Leon PC, Amo L, Shrivastava G, Iniguez E, Aryan A, et al. . Aedes aegypti sialokinin facilitates mosquito blood feeding and modulates host immunity and vascular biology. Cell Rep (2022) 39(2):110648. doi: 10.1016/j.celrep.2022.110648 PubMed DOI PMC

Chagas AC, Ramirez JL, Jasinskiene N, James AA, Ribeiro JM, Marinotti O, et al. . Collagen-binding protein, aegyptin, regulates probing time and blood feeding success in the dengue vector mosquito, aedes aegypti. Proc Natl Acad Sci USA (2014) 111(19):6946–51. doi: 10.1073/pnas.1404179111 PubMed DOI PMC