Revised Manuscript Received June 21, 1993® abstract: A new thrombin inhibitor, bothrojaracin, has been identified and purified to homogeneity from the venom of Bothrops jararaca, the most common venomous snake of South America. Bothrojaracin has an isoelectric point 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. Purified bothrojaracin is devoid of phospholipase A2, amidolytic, or fibrino (geno) lytic activity. Bothrojaracin forms a noncovalent complex with a-thrombin, without changing its catalytic activity on small peptide substrates. Bothrojaracin behaves as a potent and specific antagonist of thrombin-induced platelet aggregation and secretion, characterized by an IC50 ranging from 1 to 20 nM depending on the a-thrombin concentration. Bothrojaracin prolongs fibrinogenclotting time, and this effect is related to a competitive inhibitionof the binding of a-thrombin to fibrin (ogen)(K-, 15 nM). Binding of a-thrombin to thrombomodulin is inhibited upto 87% by bothrojaracin, and the rate of protein C activation by a-thrombin is also decreased. Bothrojaracin antagonizes the inhibition of thrombin amidolytic activity by hirudin. These results indicate that bothrojaracin acts as a very potent ligand of the exosite of a-thrombin.

Venoms from Viperidae snakes alter blood coagulation and platelet functions in a complex manner. Each venom contains several protein components which behave as pro-or anticoagulants or which induce or inhibit platelet aggregation (Brinkhous & Smith, 1988; Kini & Evans, 1990; Stocker, 1990; Longenecker, 1991). Most venoms from Bothrops species induced blood clotting. This activity has been attributed to a fibrinogen clotting enzyme, batroxobin (Funk et al., 1971; Stocker & Egberg, 1973; Stocker & Barlow,