Isolation And Purification Of Protein From Agkistrodon Acutus Venom And Its Inhibition On Platelet Aggregation

Snake venom was known to possess components affecting platelet function in various ways. Screening anti-platelet aggregation protein from snake venom becomes an intriguing work because of their potent clinical use in treating abnormal platelet activation and arterial thrombus. This study was designed to isolate and purify anti-platelet aggregation protein from Agkistrodon acutus venom(AAV) and analyse its characterization and biological activities.1. Purification and characterization of the anti-platelet aggregation protein from Agkistrodon acutus VenomSix fractions(Ⅰ～Ⅵ) were isolated from AAV by Gel filtration chromatography on Superdex 75 .The fractionⅣpossess the best anti-platelet aggregation activity, so it suggested as a contained the anti-platelet aggregation component. An anti-platelet aggregation protein named AAV-IV3-2 was purified from the fraction by sephadex G-25 gel filtration, DEAE Sepharose Fast Flow ion-exchange and Lichrospher C18 reverse chromatography. It was homogeneous as a single band showed on SDS-polyacrylmide gel electrophoresis (SDS-PAGE) with molecular weight 15.3kDa as calculated by Image Master VDS system.2. The effects of AAV-IV3-2 on the human platelet aggregation.According to the Born's method, the effects of AAV-IV3-2 on platelet aggregation induced by ADP and thrombin were assayed. The results showed that AAV-IV3-2 dose-dependently inhibited ADP- and thrombin-induced platelet aggregation with IC50 1.78 and 5.06μg/ml respectively.3. The effect of AAV-IV3-2 on the time of respiratory distress of acute pulmonary thrombosis in mice induced by ADP-Na. The time of respiratory distress of acute pulmonary thrombosis in mice of middle, high dose groups(114.2±31.53,101.9±42.19,S)respectively were remarkably shorter than that of NS group (176.6±57.52,S)(P<0.05).CONCLUSIONAAV-IV3-2 was purified from Agkistrodon acutus venom with molecular weight 15.3kDa. It was a potent platelet aggregation inhibitor.