Introduction: Thrombosis and other diseases concerning abnormal hemostasis are the major cause of mortality and morbidity in Western societies. For that reason, it is of significant importance the identification and characterization of naturally occurring molecules able to interfere with blood coagulation in order to create models to design new inhibitors. Since hirudin, a potent thrombin inhibitor isolated from the leech Hirudo medicinalis in 1967, hematophagous species have been targets in the research for new anticoagulant agents. It is assumed that the production of anticoagulant factors by these animals is important during feeding, since they maintain the host blood in a fluid state, then facilitating digestion. At this time, two different thrombin inhibitors have already been described by our group in the saliva of the bovine tick Boophilus microplus and a new one was described last year in gut extract. In the present study, a new purification procedure was developed to isolate this inhibitor from gut extract.
Methodology: The purification protocol consisted of an ion exchange cromatography in HiTrapDEAE followed by affinity chromatography in a Sepharose resin bound to bovine thrombin and finally a gel filtration in Superose 12. Fractions were tested in recalcification time and thrombin induced fibrinogen coagulation assays, as well as platelet aggregation. The inhibitor activity was also assayed upon coagulation factors using specific chromogenic substrates.
Results and Conclusions: The partially purified inhibitor is able to retard coagulation in recalcification assays and to inhibit both thrombin induced fibrinogen coagulation and platelet aggregation, but it not interfers with the thrombin activity upon specific synthetic substrate S-2238, indicating an exosite binding. When tested against other coagulation enzymes, the inhibitor was also able to enhance the protein C activity upon specific chromogenic substrate. The inhibitor showed an apparent molecular mass of 28 kDa in SDS-PAGE.
In view of this, the present study indicates that this B. microplus gut protein is a thrombin exosite inhibitor and also suggests it may possess anticoagulant activity by modulating protein C. Purification to homogeneity and complete biochemical and structural characterization of this new inhibitor, as well as in vivo studies are under way.
Supported by CNPq and Pronex