Isolation and characterization of multiple forms of human thrombin

Abstract

Established procedures for the isolation of human prothrombin (1) and thrombin (2) have been modified so that large quantities of human thrombin could be isolated in a rapid, yet facile, manner. Thrombins derived from the activation of human prothrombin with thromboplastin or venom from the Australian Taipan (Oxyuranus Scutellatus Scutellatus) have been isolated and characterized with respect to their molecular weights and isoelectric points. Multiple active human thrombin components were found. Molecular weights of proteins found in these preparations conformed to the molecular weights reported for bovine thrombins. Activity studies with the synthetic substrate Benzoyl-Phe-Val-Arg-p-nitro-anilide and inhibitors fibrinopeptides A and B, have been performed. The majority of coagulant and esterase activity of the human thrombin preparations was associated with a protein component with an isoelectric point of 7,1. Coagulant, but no esterase activity, was found at isoelectric point 6, 7. Additional minor peaks of both activities had isoelectric points of 5,6 and 6,2, which are the sole isoelectric points reported previously for bovine thrombin. Results of the characterization studies show that thromboplastin and taipan venom activation produce very similar thrombins.