Bothrojaractivase, a prothrombin activating enzyme isolated from Bothrops jararaca venom
Berger, M.1 ; Pinto, A.F.M.1; de Amorim, H. L. N.1,2 and Guimarães, J. A.1
1Centro de Biotecnologia - UFRGS - Porto Alegre, RS.
2Departamento de Química – ULBRA - Canoas, RS.
Introduction: Bleeding at the site of bite or systemic hemorrhage are symptoms frequently observed in human envenomation by Bothrops jararaca snakes. Unclottable blood, due to the consumption of clotting factors is another common feature of envenoming. There are known toxins involved in these clinical manifestations. Prothrombin activators are procoagulant toxins that acts by generation of intravascular thrombin causing decrease of plasma fibrinogen levels. In this work, we isolated a procoagulant factor from Bothrops jararaca venom (BjV), named bothrojaractivase, that is able to activate specifically prothrombin. The study can contribute for our knowledge on the envenomation process.
Methods: Prothrombin activator from BjV was purified by chromatography on gel filtration and ion exchange. The chromatographic fractions or crude venom were assayed by their amidolytic activity upon chromogenic substrate, fibrinocoagulation, coagulation of human plasma and prothrombin activating assays. SDS-PAGE was also used to analyze the electrophoretic pattern of the prothrombin activation.
Results and conclusions: Kinetic assays with crude BjV demonstrated that the venom was able to directly convert prothrombin into thrombin in a dose-dependent manner. Furthermore, thrombin (36 kDa) and others prothrombin fragments were confirmed by electhrophoresis. The purified activator showed, by itself, no fibrinogen clotting and amidolytic activities. On the other hand a procoagulant activity on human citrated plasma was observed. Specific action upon prothrombin was independent of the factor Va, calcium ions and phospholipids. This activity was completely inhibited by chelating agents (EDTA and EGTA) and in SDS-PAGE analysis the activator exhibited an apparent molecular weight of 20,8 kDa. The results indicate that the highly specific prothrombin activator from BjV is a metalloproteinase belonging to group A of activators. Complete biochemical characterization of bothrojaractivase is under way.
Supported by CNPq.
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