Thrombocytin, A Novel Platelet Activating Enzyme from Bothrops Atrox (MARAJOENSIS) Venom

Thrombocytin (TCN) was purified from Bothrops atrox (BA) venom by precipitation with 1.2% Na-salicylate and chromatography on heparin-agarose column using increasing concentrations of lysine as eluent. It was homogeneous on SDS electrophoresis and had an apparent MW of 36,000. Immunoelectrophoresis with polyvalent anti-BA venom serum gave one cathodic arc indicating an isoelectric point higher than pH 8.6.TCN at a concentration of 1 yg/ml caused aggregation of human platelets, release of low affinity platelet factor 4 and serotonin, and stimulated platelets to retract fibrin.TCN was essentially free of fibrinogen clotting and fibrinolytic activities.TCN action on platelets was not mediated by the formation of thrombin since TCN did not activate Factor X or prothrombin and its action was not inhibited by hirudin.TCN is a serine protease since it was inhibited by DFP and it hydrolyzed a synthetic peptide, chromozyme UK (BZ-Val-Gly-Arg-pNA·HCl).TCN-induced aggregation of human platelets was completely inhibited by soy bean trypsin inhibitor, heparin, prostaglandin E₁ and apyrase. Washed human platelets were 2-4 times less sensitive to TCN as compared to platelets in freshly prepared platelet rich plasma (PRP); their sensitivity to TCN gradually deteriorated during incubation of PRP at room temperature for 3 hours. Electron microscopic observations revealed formation of platelet aggregates characterized by pseudopod formation, centralization and partial loss of platelet granules. Infusion of TCN (3 yg) into the main artery of bat wing resulted in the formation of platelet aggregates seen on arterial and venous side which occasionally occluded small vessels.