[22] Snake venom proteases that activate blood-coagulation factor V

This chapter presents the procedure for purification and assaying of Russell's viper venom (RVV). The procedures for the isolation of RVV-V and thrombocytin used in this laboratory are virtually identical and include an initial gel filtration of the crude venom followed by final purification of the enzyme by SPSephadex ion-exchange chromatography. Because both of these enzymes are highly cationic, plasticware is recommended in the collection and storage of these proteins. In the assay venom protease is incubated with factor V substrate for a defined period. Following the incubation period, factor V is assayed by its ability to correct the clotting time of human factor V-deficient plasma following addition of tissue factor and calcium ions. The clotting times in this assay are proportional to the concentration of factor Va present in the incubation mixture, which in turn is directly related to the concentration of the venom factor V activator. In the assay of RVV-V, barium sulfateadsorbed plasma is a satisfactory source of factor V. In the assay of thrombocytin, however, partially purified factor V is required in that B. atrox venom contains a thrombin-like protease (batroxabin) that coelutes with thrombocytin in gel-filtration columns.