The Mechanism of Activation of Bovine Prothrombin by an Activator Isolated from Echis carinatus Venom and Characterization of the New Active Intermediates1

Abstract

Bovine prothrombin was activated, in both the absence and presence of diisopropyl-phosphofluoridate (DFP) and benzamidine, by an activator which was highly purified from the venom of Echis carinatus (saw-scaled viper, ECV). The process of activation was monitored by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, and the reaction products were isolated and chemically characterized. In the absence of the inhibitors, prothrombin yielded two fragments with molecular weights of 28, 000 and 57, 000, of which the former was the N-terminal fragment of the zymogen and the latter was intermediate 1, consisting of a single polypeptide chain. Intermediate 1 was subsequently converted to an active intermediate, named intermediate ECV, without decrease of molecular weight. This new intermediate ECV, which showed little clotting activity but a strong α-N-tosyl-L-arginine methyl ester (TAME)-esterolytic activity and which bound with hirudin or antithrombin III, consisted of two polypeptide chains with molecular weights of 35, 000 and 27, 000 daltons. The former was identified as the thrombin B chain with the N-terminal sequence Ile-Val-Glu-Gly and C-terminal serine, and the latter was a fragment with N-terminal Ser-Gly-Gly-, linked to the thrombin A chain. On prolonged incubation, intermediate ECV autocatalytically yielded a fragment (inner fragment) of 14, 000 daltons with N-terminal serine and the clotting enzyme α-thrombin [EC 3.4. 21.5], which consists of A and B chains. In the presence of the inhibitors, intermediate ECV and the N-terminal fragment were accumulated in the activation mixture. On the other hand, when prothrombin was activated by the venom activator in the presence of hirudin, antithrombin HI, or p-nitrophenyl p'-guanidinobenzoate, it did not yield any fragments but was converted to a derivative with two polypeptide chains having molecular weights of 51, 000 and 34, 000 daltons, of which the former consisted of the N-terminal fragment, the inner fragment, and thrombin A chain, and the latter was thrombin B chain. This new prothrombin derivative, named prothrombin ECV, formed a high-molecular-weight complex, associating with anti-thrombin III. The complex was not dissociable even in the presence of SDS. Moreover, prothrombin ECV reacted with pniitrophenyl p'-guanidinobenzoate. On the basis of the results described above, the mechanism of activation of prothrombin by Echis carinatus venom activator can be summarized as follows: The venom activator first cleaves an Arg-Ile bond linking thrombin A and B chains in the zymogen molecule, forming an active derivative, prothrombin ECV. This active derivative converts autocatalytically to intermediate ECV, liberating the N-terminal fragment, and active intermediate ECV generates α-thrombin, releasing the inner fragment. Thus, only a single peptide bond cleavage along the polypeptide chain of prothrombin is associated with activation by the venom activator. Intermediate 1, formed temporarily in both the absence and presence of DFP and benzamidine, could be a by-product directly derived from prothrombin by the action of prothrombin ECV or active intermediate ECV developed in the reaction mixture. Thus, the overall process of zymogen activation by the venom activator differs significantly from that by Factor Xa [EC 3.4.21.6].