Purification Of Thrombin-like Enzyme From Agkistrodon Acutus Venom

Thrombotic disease with high incidence,high morbidity and high mortality rate,serious harms to human health.Thrombolysis is considered the most effectivetreatment for thrombotic diseases;Usually antithrombotic drugs have anticoagulant,antiplatelet,thrombolytic drugs,preparation of snake venom and others.In the preparat-ion of snake venom thrombin-like enzymes in selective effect on fibrinogen A alphachain,degrades fibrinogen into fibrin monomer,and then decomposed into fibrindegradation products(FDP).FDP was cleared up by the reticuloendothelial syste-m,urine excretion.So it can reduce the content of fibrinogen and decrease the bloodviscosity,then exert antithrombotic effect.Acutase injection (a thrombin-like enzyme-e)was purified from Agkistrodon acutus venom.The preparation is the first venompreparationtime approved by State food and drug administration.Acutase injection isnot a single protein component,but2-3isozyme because of the separation and purific-ation technology.Acutase injection is a safe and effective drug in thrombotic disease’spatients for more than ten years clinical ues.But it has some adverse effects in aminority of patients,such as bleeding,allergic reaction and thrombocytopenia.Thisstudy was designed to separate and purify single component of thrombin-like enzymefrom Agkistrodon acutus venom by the new technology and observes its effect onplatelet.1. Purification and characterization of the thrombin-like enzyme from Agkistrodonacutus venom.Five fractions(I～Ⅴ) were isolated from AAV by Gel filtration chromatograph-y on Superdex75.The protein peak II named AAV-II in vitro thrombin activity wasthe strongest.Six fractions(1～6) were purified from AAV-II by DEAE-SepharoseFast Flow ion-exchange.The protein peak3named AAV-II3in vitro thrombin activitywas the strongest.Finally a thrombin-like enzyme protein named AAV-Ⅱ3-1purifiedfrom AAV-II3by Superdex30gel chromatography.It was homogeneous as a single band showed on Electrophoresis.2. Purity analysisElectrophoresis was analysed each peak of proteins separating and purifying bySuperdex75,DEAE-Sepharose Fast Flow,Superdex30.Twelve sodium dodecyl sulfa-te polyacrylamide gel is the best separation gel.AAV-Ⅱ3-1was homogeneous as asingle band showed on SDS-polyacrylmide gel electrophoresis(SDS-PAGE) withmolecular weight about25kD as estimated by contrasting with markers.3. The influence of AAV-Ⅱ3-1on peripheral blood in rat, platelet especiallyThe SD rats were divided into AAV-Ⅱ3-1group and normal saline group.Eachobservation index had no significant change before drug and After administration(P>0.05).Testing dose of AAV-Ⅱ3-1without influences of platelet,leukocyte,neutrophil and hemoglobin count in rat.CONCLUSIONAAV-Ⅱ3-1was purified from Agkistrodon acutus venom with molecular weightabout25kD.It was a thrombin-like enzyme protein.AAV-Ⅱ3-1had no significanteffect of platelet,leukocyte, neutrophil and hemoglobin count in rat.