Molecular characterization of anti-platelet aggregating proteins in salivary gland extracts of Hyalomma anatolicum ticks

DOI: 10.18805/ijar.B-3521    | Article Id: B-3521 | Page : 461-468
Citation :- Molecular characterization of anti-platelet aggregating proteins in salivary gland extracts of Hyalomma anatolicum ticks.Indian Journal Of Animal Research.2019.(53):461-468
Surbhi, Nirmal Sangwan and Arun K. Sangwan drsurbhi8@gmail.com
Address : Department of Veterinary Physiology and Biochemistry, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125 004, Haryana, India
Submitted Date : 30-10-2017
Accepted Date : 12-12-2017

Abstract

Tick saliva is a cocktail of potent anti-haemostatic, anti-inflammatory and immunomodulatory molecules that help the ticks to obtain a blood meal from their vertebrate hosts. Thrombin is considered as an activator for stimulation of blood platelets at different receptor sites and thrombin induced platelet aggregation depends on the interaction among thrombin exosites and the receptors on platelet membrane. Thus, the present study was planned to isolate and characterize the salivary anti-platelet aggregating proteins from Hyalomma anatolicum ticks. A hundred pairs of salivary glands were dissected out and extract was prepared by fractionation of proteins by gel filtration chromatography. The fractions so obtained were tested for thrombin induced platelet aggregation and platelet-collagen adhesion inhibition activities. Proteins in fraction nos. 28, 29, 30, 31, 32, 42, 58, 111 and 112 were found to inhibit platelet aggregation. These proteins also showed significant inhibitory effects on platelet adhesion to collagen when compared to agonist thrombin. This indirectly indicates inhibition of glycoprotein 1b (GP1b), glycoprotein VI (GPVI) and integrin a2b1 receptors of platelets as these proteins are involved in aggregation of platelets for formation of platelet plug. On electrophoretic separation of fractions by SDS-PAGE, the proteins/peptides present in initial fractions were having approximate molecular weight in the range of 6.5 to 113.4kDa. However, in the fraction no. 112 only single prominent protein band of approximate 65.4kDa was present and is expected to inhibit platelet aggregation possibly through inhibition of platelet receptors such as proteins platelet activating receptors-1 and 4 playing direct role in platelet aggregation. In conclusion, there are possibilities of exploring these anti-platelet aggregating proteins/peptides for therapeutics against cardio-vascular disorders as well as for raising anti-tick vaccine.  

Keywords

Anti-platelet aggregating proteins/peptides Gel filtration chromatography Hyalomma anatolicum Platelet adhesion Salivary gland Thrombin.

References

  1. Blockmans, D., Deckman, H. and Vermylen, J. (1995). Platelet activation. Platelets. 9: 143–156.
  2. Bowman, A.S. and Sauer, J.R. (2004). Tick salivary glands: function, physiology and future. Parasitol.129(S1): S67-S81.
  3. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
  4. Calvo, E., Tokumasu, F., Marinotti, O., Villeval, J.L., Ribeiro, J.M. and Francischetti I.M.(2007). 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. 282(37): 26928-38.
  5. Ciprandi, A., De Oliveira, S.K., Masuda, A., Horn, F. and Termignoni, C. (2006). Boophilus microplus: its saliva contains microphilin, a small thrombin inhibitor. Exp. Parasitol. 114(1): 40-46.
  6. De la Fuente, J. and Merrino, O. (2013). Vaccinomics, the new road to tick vaccines. Vaccine. 31: 5923-5929.
  7. De Morais, K.B., Vieira, C.O., Hirata, I.Y. and Tanaka-Azevedo, A.M. (2009). Bothrops jararaca antithrombin: isolation,characterization and comparison with other animal antithrombins. Comp. Biochem. Physiol. 152 (2): 171–176. 
  8. Francischetti, I. M., Ribeiro, J. M, Champagne, D. and Andersen, J. (2000). Purification, cloning, expression, and mechanism of action of a novel platelet aggregation inhibitor from the salivary gland of the bloodsucking bug, Rhodnius prolixus. J. Biol.Chem. 275: 12639–12650. 
  9. Francischetti, I.M., Sá-Nunes, A., Mans, B.J., Santos, I.M. and Ribeiro, J.M.C. (2009). The role of saliva in tick feeding. Front. Biosci. 14: 2051–2088.
  10. Gibbins, J.M. (2004). Platelet adhesion signaling and the regulation of thrombus formation. J. Cell. Sci. 117: 3415–3425.
  11. Guerrero, F.D., Miller, R.J. and Pérez de León, A.A. (2012). Cattle tick vaccines: many candidate antigens, but will a commercially viable product emerge. Int. J. Parasitol. 42: 421–427.
  12. Hames, B.D. (1998). Gel Electrophoresis of Proteins A Practical Approach, Third Edition, Oxford University Press.
  13. Harsfalvi, J., Stassen, J.M., Hoylaerts, M.F., Van Houtte, E., Sawyer, R.T., Vermylen, J. and Deckmyn, H. (1995). Calin from Hirudo medicinalis, an inhibitor of von Willebrand factor binding to collagen under static and flow conditions. Blood. 85(3): 705-11.
  14. Hoffmann, A., Walsmann, P., Riesener, G., Paintz, M. and Markwardt, F. (1991). Isolation and characterization of a thrombin inhibitor from the tick Ixodes ricinus. Pharmazie. 46: 209–212.
  15. Horn, F., Coutinho dos Santos, P. and Termignoni, C. (2000). Boophilus microplus anticoagulant protein: an antithrombin inhibitor isolated from the cattle tick saliva. Arch. Biochem. Biophys. 384: 68–73.
  16. Huntigton, J.A. (2005). Molecular recognition mechanisms of thrombin. J. Thromb. Haemost. 3: 1861–1872.
  17. Karczewski, J., Waxman, L., Endris, R.G. and Connolly, T.M. (1995). An inhibitor from the argasid tick Ornithodoros moubata of cell adhesion to collagen. Biochem. Biophys. Res. Comm. 208(2): 532-541.
  18. Kazimírová, M., Jancinová,V., Petríková, M., Takác, P., Labuda, M. and Nosál’, R. (2002). An inhibitor of thrombin-stimulated blood platelet aggregation from the salivary glands of the hard tick Amblyomma variegatum (Acari: Ixodidae). Exp Appl. Acarol. 28(1-4): 97-105.
  19. Miranpuri, G.S. and Gill, H.S. (1983) Ticks of India. Lindsay & Macleod, Edinburgh.
  20. Morrissey, J.H. (1981). Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal. biochem. 117(2):307-310.
  21. Nienaber, J., Gaspar, A.R.M. and Neitz, A.W. (1999). Savignin, a potent thrombin inhibitor isolated from the salivary glands of the tick Ornithodoros savignyi (Acari: Argasidae). Exp. Parasitol. 20: 583–598.
  22. Ofosu, F.A. (2003). Protease activated receptors 1 and 4 govern the responses of human platelets to thrombin. Tranfus. Apheresis. Sci. 28: 265–268.
  23. Ribeiro, J.M.C. (1987). Role of saliva in blood-feeding by arthropods. Annu. Rev. Entomol. 32(1): 463-478.
  24. Snedecor, G.W. and Cochran, W.G. (1994). Statistical Methods. 1st East West Press ed., Affiliated East West Private Ltd., New Delhi.
  25. Sonenshine, D.E (1993) Biology of Ticks (II). Oxford University Press, New York and Oxford.
  26. Steen, N.A., Barker, S.C. and Alewood, P.F. (2006). Proteins in the saliva of the Ixodida (ticks): pharmacological features and biological significance. Toxicon. 47(1):1-20.
  27. Tanaka-Azevedo, A.M., Morais-Zani, K., Torquato, R.J.S. and Tanaka, A.S. (2010). Thrombin inhibitors from different animals. J. Biomed. Biotech. 2010: 9. 
  28. Tang, J., Fang, Y., Han, Y., Bai, X., Yan, X., Zhang, Y., Lai, R. and Zhang, Z. (2015). YY-39, a tick anti-thrombosis peptide containing RGD domain. Peptides. 68: 99-104.
  29. Titus, R.G., Bishop, J.V. and Mejia, J.S. (2006). The immunomodulatory factors of arthropod saliva and the potential for these factors to serve as vaccine targets to prevent pathogen transmission. Parasite immunol. 28(4): 131-141.
  30. Valenzuela, J.G. (2004). Exploring tick saliva: from biochemistry to ‘sialomes’ and functional genomics. Parasitol. 129(S1):S83-S94 

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