Purification and Characterization of a Ca²⁺-Dependent Prothrombin Activator, Multactivase, from the Venom of Echis multisquamatus

Abstract

We previously found a novel Ca²⁺-dependent prothrombin activator, designated as carinactivase-1, in Echis carinatus leucogaster venom [D. Yamada, F. Sekiya, and T. Morita (1996) J. Biol. Chem. 271, 5200-5207]. Of the Viperidae snake venoms examined, the Echis multisquamatus venom had the strongest carinactivase-like activity. We isolated and characterized the carinactivase-like prothrombin activator in E. multisquamatus venom. From 50mg of E. multisquamatus venom, we isolated 2.3mg of a Ca²⁺-dependent prothrombin activator designated as multactivase. Unlike other Echis snake venoms, the E. multisquamatus venom contained no ecarin-like Ca²⁺-independent prothrombin activator. The structure and function of multactivase are similar to those of carinactivase. Multactivase is composed of a catalytic subunit with metalloprotease activity and a regulatory subunit comprising two homologous polypeptides bound by S-S bridge (s), and it activates prothrombin via recognition of the Ca²⁺-bound conformation of its Gla domain. We developed a chromogenic assay involving multactivase for normal prothrombin activity in plasma from individuals orally administered anticoagulants. The normal prothrombin activity, as a percentage, measured with multactivase was highly correlated with the prothrombin time. Multactivase is useful for the simple quantification of normal prothrombin in plasma from warfarin-treated individuals.