BOTHROJARACIN, A NEW THROMBIN INHIBITOR ISOLATED FROM BOTHROPS-JARARACA VENOM - CHARACTERIZATION AND MECHANISM OF THROMBIN INHIBITION

A new thrombin inhibitor, bothrojaracin, has been identified and purif ied to homogeneity from the venom of Bothrops jararaca, the most commo n venomous snake of South America. Bothrojaracin has an isoelectric po int of 4.2 and a molecular mass of 27 kDa and is made of two distinct polypeptide chains of 15 and 13 kDa, linked by disulfide bridges. Puri fied bothrojaracin is devoid of phospholipase A2, amidolytic, or fibri no(geno)lytic activity. Bothrojaracin forms a noncovalent complex with alpha-thrombin, without changing its catalytic activity on small pept ide substrates. Bothrojaracin behaves as a potent and specific antagon ist of thrombin-induced platelet aggregation and secretion, characteri zed by an IC50 ranging from 1 to 20 nM depending on the alpha-thrombin concentration. Bothrojaracin prolongs fibrinogen clotting time, and t his effect is related to a competitive inhibition of the binding of al pha-thrombin to fibrin(ogen) (K(i) 15 nM). Binding of alpha-thrombin t o thrombomodulin is inhibited up to 87% by bothrojaracin, and the rate of protein C activation by alpha-thrombin is also decreased. Bothroja racin antagonizes the inhibition of thrombin amidolytic activity by hi rudin. These results indicate that bothrojaracin acts as a very potent ligand of the exosite of alpha-thrombin.