Biochemical properties and comparative pharmacology of a coagulant from Deinagkistrodon acutus snake venom

Abstract

A number of snake venom thrombin-like enzymes (TLEs) have already been characterized. Some TLEs play significant roles in vessel injury hemostasis. A novel TLE (Agacutase) was purified from Deinagkistrodon acutus snake venom by the means of Sephadex G-75, DEAE-Sepharose FF, and Sephadex G-25 column chromatography. Structural analysis indicated that Agacutase is a single-chain glycoprotein with a molecular mass of 31,084Da, isoelectric point of 4.38, optimal activity at 37°C and pH 6.6, sugar content of 7.6%. Its N-terminal 44 amino acid sequence was determined to be VIGGNECDTNEHRFLAAFFTSRPWIFQCAGTLIHEEWVLAAAHC, showing maximum identity of 80% with that of Dav-X protease. The Agacutase-induced clotting activity was not influenced by heparin, hirudin, or Dextran 40, but activated by Ca2+ and inhibited by PMSF or lactose, which suggests that Agacutase is a serine protease and the coagulation activity is independent of Thrombin. Agacutase with arginine esterase activity specifically cleaves the α-chain of fibrinogen. Agacutase iv (0.03–0.12U/kg) shortened 16–68% of the rabbit blood clotting time. No significant influence was indicated on platelet, Factor II and XIII, or fibrinolytic system. It converts fibrinogen into the soluble fibrin that accelerates hemostasis at wound. Pharmacological comparison showed the hemostatic effect of Agacutase lasted 24h while Reptilase did 8h. Its maximum tolerated, abnormal toxicity, allergic, and hemorrhagin doses were 80U/kg, 1U, 2U, and 50U, respectively, whereas those of Reptilase or Agacutin were 35U/kg, 0.25U, 0.25U, and 0.2U, respectively. The results indicated that Agacutase may be a predominant coagulant.