Purification Of Platelet Inhibitor From Wannan Agkistrodon Halys Venom And Its Mechanism On Antiplatelet

Objective: To isolate and purificate of platelet inhibitor from Wannan Agkistrodonhalys venom (AHV-PI) and to explore the effect and mechanism of AHV-PI on plateletin rabbit.Methods: Isolation and purification of AHV-PI were carried out by a combination ofCellulose DEAE-52, CM-Sephadex C-25and Sephadex G-75column chromatography.Homogenicity and molecular weight of AHV-PI were determined in Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Thirty-six New Zealandrabbits were divided into sham operation group, artery thrombosis model group,positive control group (sodium ozagrel at dose of5mg/kg), low dose (0.05mg/kg),medium dose (0.1mg/kg) and high dose (0.2mg/kg) of AHV-PI experiment groupsrandomly (n=6). The model of rabbits carotid artery thrombosis was induced byinfiltering70%FeCl3. In the sham group, the thrombosis model was not copied and0.9%saline solution was injected only. The blood was taken from femoral artery after1h. The content of plasma fibrinogen (FIB) was determined by the dual-channelcoagulation. Thrombelastogram was obtained by thromboelastography instrumentsystem. The experiment of platelet aggregation was determined by the turbidimetricmethod. The content of vorl Willebran Factor (vWF), α granule membrane protein(GMP-140), thromboxane B2(TXB2) and platelet membrane glycoprotein IIb/IIIa(GPIIb/IIIa) in plasma were determined by Enzyme-Linked Immunosorbnent Assay(ELISA). The structural changes of the rabbits carotid artery thrombosis and plateletwere observed under microscope and transmission electron microscope respectively.Results: There were seven chromatography peaks (I～VII peak) through Cellulose DEAE-52column. IV peak had the most obviously degree of inhibition of plateletaggregation. Then AHV-PI was separated from CM-Sephadex C-25and Sephadex G-75column chromatography. IC50of AHV-PI was221μg/ml. The purified AHV-PI fractionfrom Wannan Agkistrodon halys venom showed a single band in SDS-PAGE and themolecular mass was about31.0kDa. In the rabbits experiment, compared with modelgroup, clotting time (R) and clot formation time (K) significantly prolonged inmedium-dose and high-dose AHV-PI experiment groups(P<0.01). The alpha angle,maximum amplitude (MA), coagulation (CI) and the indices of platelet aggregationwere decreased (P<0.05and P<0.01). while the levels of FIB, GMP-140, TXB2andvWF in plasma were decreased (P<0.01), which had no difference as compared withsham operation and positive control group (P>0.05). The level of GPIIb/IIIa was notdecrease obviously (P>0.05). Carotid artery thrombosis histological observation showedthat thrombosis in medium-dose and high-dose AHV-PI experiment groups were notfound. The carotid artery endometrial was relatively complete. Platelet electronmicroscope showed that the platelet morphology and ultrastructure approach shamgroup. The platelet was smooth. AHV-PI can inhibit producing pseudopods process andincreasing in the number of the granules associated with enhanced intensities of theirelectron densities compared with model group.Conclusion: The purified AHV-PI fraction from Agkistrodon halys venom thoughCellulose DEAE-52, CM-Sephadex C-25and Sephadex G-75column chromatographyshowed a single band in SDS-PAGE and the molecular mass was about31.0kDa.AHV-PI may be a class of proteins with plamin activity. In the rabbits experiment,AHV-PI can inhibit platelet aggregation and provent the information of carotid arterythrombosis. The mechanism may be attributed to protecting platelet ultrastructure andreducing the release of platelet lipid metabolism and particulate contents.