Studies on the Development of a Thrombolytic Agent from Korean Snake Venom II. Characterization and Thrombolytic Activity of a Pretense from the Venom of a Protease from the Venom of A. bromhoffi brevicaudus

한국 독사독으로부터의 혈전 용해제 개발에 관한 연구 II. 살모사(A. bromhoffi brevicaudus) 사독 Protease의 특성과 혈전 용해능에 관한 연구

The biochemical properties of the fibrinolytic protease of 50,800 Da isolated from the venom of Kgdistrodon blomhoffi brevicaudus were characterized. The enzyme hydrolyzed the carboxyl side of arginine in the synthetic chromogenic peptides, N-Benzoyl-Phe-Val-Arg-pNA and N-p-Tosyl-Gly-Pro-Arg-pNA, and the enzyme activity was inhibited by phenylmethylsulfonylfluoride indicating that the enzyme belongs to the serine protease family. The pretense showed maximum activity at pH 7.5 and inhibited by ZnCl$_2$, CuSO$_4$, but not by soybean trypsin inhibitor, pepstatin A, 2-mercaptoethanol and EDTA. The fm value determined with N-p-Tosyl-Gly-Pro-Arg-pNA was 0.2 mM. The thrombolytic activity of the purified enzyme was evaluated by platelet aggregation test in rabbits. While the platelet count ratio in blood of the rabbits injected with thrombin alone declined from 1.0 to 0.6 within 7 min and maintained around 0.6 for 24 hours thereafter, the ratio rapidly recovered from around 0.6 to 0.8 in 1 hr, to 1.0 in 24 hrs when the rabbits were sequentially treated with thrombin and the purified enzyme. The result showed that the serine protease from A. blomhoffi brevicoudus of 50,800 Da had a thrombolytic activity in vivo and the enzyme might be developed as a therapuetic agent for the treatment of thrombic disease.