Degradation of coagulation proteins by an enzyme from Malayan pit viper (Akistrodon rhodostoma) venom

Abstract

Three hydrolases from the crude venom of the Malayan pit viper (Akistrodon rhodostoma) can be differentiated. The first, which we designate ARH.alpha., is the well-known fibrinogenolytic enzyme ancrod. The second, ARH.beta., which has not been described previously, is identified by its electrophoretic mobility after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), by its ability to hydrolyze H-D-phenylalanyl-L-piperyl-L-arginyl-p-nitroanilide, and by inhibition of its activity by diisopropyl phosphorofluoridate. The third, ARH.gamma., also previously not described, has been purified by using gel permeation and ion-exchange chromatography and preparative PAGE. Chemical, electrophoretic, and hydrodynamic data indicate that it is a single-chain, nonglobular glycoprotein with a molecular weight of 25 600. ARH.gamma. catalyzes the degradation of several plasma vitamin K dependent coagulation factors, including factor IX, factor X, prothrombin, and protein C. The products are electrophoretically similar to factor IXa.beta., factor Xa, thrombin, and activted protein C, respectively. However, these products contain little or no enzymatic activity. ARH.gamma.-degraded factor IX, factor X, prothrombin, and protein C can be subsequently activated by factor XIa, Russell''s viper venom X coagulant protein, crude taipan snake venom, and thrombin, respectively. The N-terminal sequence of the peptides resulting from the ARH.gamma. digest of porcine factor IX shows that at least three bonds are hydrolyzed: (1) at position 152, seven residues from the Arg145-Ala146 factor XIa cleavage site; (2) at position 167 within the factor IX activation peptide; and (3) at position 177, three residues from the Arg180-Val181 factor XIa cleavage site. The degradation of factor IX by ARH.gamma. is not affected by several serine protease inhibitors. ARH.gamma. catalyzes the degradation of both the heavy and light chains of porcine factor VIII which results in the inability of thrombin to activate factor VIII. ARH.gamma. also catalyzes the degradation of porcine antithrombin III which abolishes its ability to inhibit thrombin. These findings may have relevance to studies of hemostatic derangements following envenomation by this snake. Additionally, several novel coagulation factor derivatives have been generated for structure-function studies.