Prothrombin activation induced by ecarin - A prothrombin converting enzyme from echis carinatus venom

Abstract

Purified prothrombin was activated by a physiological activator of a thromboplastin type (AC) and by Ecarin, a procoagulant enzyme derived from Echis carinatus venom. Intermediate products obtained during 2 min. to 2 hours of activation were analyzed by SDS poly-acrylamide gel electrophoresis and NH 2-terminal analysis. Thrombin activity was measured on fibrinogen as substrate. It was found that Ecarin cleaves prothrombin at a bond, which results in release of the NH 2 -terminal portion of the molecule (A-fragment; intermediate 3). A second cleavage site appears to be at the ArgIle bond linking the A and B chain in thrombin precursors. The latter cleavage was considered to be responsible for the appearance of the biological activity. The resulting Ecarin thrombin in constrast to thrombin produced by physiological activation appears to consist of a B-chain and an extended A-chain (A-chain + intermediate 4). This is believed to be the reason for the lower specific activity of Ecarin thrombin on fibrinogen. Our results do not exclude the possibility that prothrombin is cleaved by Ecarin as well as by physiological activator at other bonds than already identified.