Purification And Characterization Of A Disintegrin From Agkitrodon Acutus Venom

AIM To purify a disintegrin from Agkistrodon Acutus snake venom , and to study the physical and chemical properties of the peptide, as well as its effects on the platelet aggregation stimulated by ADP, collagen, and thrombin. Method To extract snake venom through Superdex 75 gel filtration, ultrafiltration and DEAE -Sepharose CL-6B ionexchange. The purified product was identified by HPLC C18. The molecular weight was determined by SDS- polyacrylamid gel electrphoresis. The isoelectric point was estimated by the isoelectric focusing electrophoresis. Platelet aggragation was measured by nephelometry. The effect of fraction K on GPâ…¡b/â…¢a was detected by flow eytometry. The level of GMP-140 in plasma was measured by ELISA. The ultrastructures of platelet were observed as well. The antiangiogenesis effect in vivo of fraction K on CAM assay was observed. Result The molecular weight of a small peptide purified from Agkistrodon Acutus snake venom was 7862D. Its isoelectric point was pH4.29. This peptide could dose-dependently inhibit the platelet aggregation induced by ADP, collagen and thrombin,and the binding reaction of fluorescein isothiocyanate (FITC)-conjugated CD41a.The peptide could lighten the disruption of platelets ultrastructure and inhibit the spontaneous angiogenesis of CAM. And it had no effect on GMP-140 in plasma. Conclusion The method has been proved to be successful for the purification of disintegrin that inhibits platelet aggregation.