BOTHROJARACIN - A POTENT 2-SITE-DIRECTED THROMBIN INHIBITOR

The thrombin inhibitor, bothrojaracin [Zingali, R. B., Jandrot-Perrus, M., Guillin, M. C., & Bon, C. (1993) Biochemistry 32, 10794-10802], i s a 27 kDa protein isolated from the venom of Bothrops jararaca that b locks several thrombin functions, including fibrinogen clotting, plate let activation, and fibrin and thrombomodulin binding, but does not in teract with the catalytic site. In the present report, we show that th e high affinity binding of a-thrombin to immobilized bothrojaracin (K- d = 0.6 nM) is inhibited by the C-terminal peptide of hirudin and that the gamma-cleavage within exosite 1 reduces the affinity of bothrojar acin for thrombin (K-d = 0.3 mu M), indicating that bothrojaracin bind ing to exosite 1 is a major determinant of the thrombin-bothrojaracin interaction. In addition, we show that bothrojaracin decreases the rat e of inhibition of alpha- and gamma-thrombin by the antithrombin III-h eparin complex. Competition of bothrojaracin with heparin or prothromb in fragment 2 for binding to thrombin indicates that bothrojaracin not only binds exosite 1 but also binds exosite 2 or in close proximity. Bothrojaracin binds to the thrombin precursor, prothrombin. This inter action is calcium-independent and is prevented by heparin, suggesting that it is mediated by exosite 2. Bothrojaracin inhibits platelet acti vation induced by clot-bound thrombin and slowly dissociates thrombin from the fibrin clots. Altogether, our results indicate that the high affinity of bothrojaracin for thrombin is supported by a double-site i nteraction and results in an efficient inhibition of both soluble and clot-bound thrombin.