Poster 189 - Antithrombotic effect of bothrojaracin, a Bothrops jararaca snake venom-derived (pro)thrombin

Poster 189. Congresso da Sociedade Brasileira de Toxinologia, 8., Symposium of the Pan American Section of the International Society on Toxinology, 8., 2004, Angra dos Reis, Brasil. Abstracts... J. Venom. Anim. Toxins incl.Trop. Dis., 2004, 10, 3, p.548.

Antithrombotic effect of bothrojaracin, a Bothrops jararaca snake venom-derived (pro)thrombin inhibitor

Ferreira, M. S.; Assafim, M.; Mendes-Silva, W.; Frattani F. S.; Monteiro, R. Q.; Zingali, R. B.*

Departamento de Bioquímica Médica, ICB, CCS, Universidade Federal do Rio de Janeiro.

Bothrojaracin (BJC), a 27-kDa C-type lectin like protein from Bothrops jararaca venom, is a selective and potent thrombin inhibitor (K_D = 0.6 nM) which interacts with both thrombin anion binding exosites (I and II) but not with the enzyme’s catalytic site. BJC also binds with high affinity (K_D = 175 nM) to proexosite I, a recently characterized factor Va binding site on prothrombin. This ability would confer to BJC a new mechanism of action for an antithrombotic drug. In order to determine whether BJC could bind prothrombin in rat plasma, we performed a western blotting assay using anti BJC polyclonal antibodies. Incubation of BJC with plasma produced a band with identical migration pattern to that observed upon incubation of the protein with purified prothrombin, thus suggesting that BJC specifically interacts with prothrombin in rat plasma. We further analyzed the in vivo antithrombotic effect of BJC on a venous thrombosis model in rats that combines stasis and hypercoagulability. It was observed that intravenous administration of 3 mg/kg of thromboplastin combined with stasis caused 100% of thrombus incidence (9.1 ± 2.0 mg). In contrast, co-administration of 1 mg/kg of BJC decreased thrombus weight by ~95% (0.5 ± 0.1 mg). Accordingly, ex vivo APTT was enhanced by ~1.7-fold for BJC doses of 1 mg/kg after 30 min of drug administration. In addition, we observed that these BJC doses caused significant hemorrhagic effect as compared to control animals. Experiments are now in progress in order to evaluate the in vivo effect of BJC in mice using a thromboembolism model induced by thrombin. Altogether, our data show that BJC is a potent antithrombotic agent that could further help the development of new dual mechanistic drugs directed to prothrombin and thrombin inhibition.