To Investigate The Effect On Platelet Signaling Pathway And Its Enzyme Activity Of Platelet Inhibitor From Agkistrodon Halys Venom

Objective: To explore the possible mechanism on inhibiting platelet aggregation of platelet inhibitor from Agkistrodon halys venom.Methods: Thirty rabbits were divided into blank control group, artery thrombosis model group, positive control group(Three phosphatidylinositol protein kinase inhibitor at dose of 20μM), low dose(0.05mg/kg), medium dose(0.1mg/kg) and high dose(0.2mg/kg) of AHV-PI experiment groups(n=5) randomly. The model of rabbits carotid artery thrombosis model was induced by infiltering 70%Fe Cl3. In the blank control group, the thrombosis model was not copied and equal volume 0.9% saline solution was injected only. The blood was taken from another carotid artery after 1 hour. Application of dual channel blood coagulation instrument was used to determined the activation of blood coagulation time( APTT),prothrombin time( PT), thrombin time(TT) and the content of fibrinogen(FIB).The plasma content of plasmin, 5-nucleotidase(5-NT),platelet membrane glycoprotein Ib(GPIb) was determined by Enzyme-Linked Immunosorbnent Assay(ELISA). XS-1000 I blood cell counter was used for platelet count. Observed the effect of AHV-PI on binding rate between the fluorescence labeled monoclonal antibody CD61(FITC-CD61) and platelet membrae glycoprotein IIb/IIIa(GPIIb/IIIa) by flow cytometry(FCM). Western bloting measured protein kinase Akt phosphorylation levels in platelet.Results: Pathological section showed that carotid artery intima isrelatively smooth, no intraluminal thrombosis in medium-dose and high-dose group. Platelet count and protein kinase Akt phosphorylation levels in positive control group, medium-dose and high-dose group were decreased compared with model group(P<0.05), had no significant changes compared with the blank control group(P>0.05). Among them, there was no significant differences between the model group and low-dose group, medium-dose group and positive control group(P>0.05). Compared with the blank control group, significantly shortened APTT, PT, TT, reduced plasmin level,increased FIB and GPIb content(P<0.05), no significant change in 5-NT(P>0.05)were observed in artery thrombosis model group and low-dose group. In medium-dose and high-dose groups, except PT and TT, APTT and FIB had no significant changes(P>0.05), the levels of GPIb and plasmin were reduced while 5-NT increased(P<0.05). Compared with the model group, APTT, TT were prolonged(P<0.05), PT had no obvious change(P>0.05), the content of FIB, plasmin and GPIb were decreased(P<0.05), while 5-NT was creased(P>0.05). Except for PT, no statistical significance compared with the above indicators differences between model group and low-dose group, medium-dose and high-dose group( P>0.05). Flow cytometry displayed AHV-PI did not affect the rate of combination between platelet GPIIb/IIIa and FITC-CD61(P<0.05). Conclusion: AHV-PI can improve blood hypercoagulable state, inhibit platelet aggregation, it has obvious effect of anticoagulation, the effect was dose-dependent, high dose effect. The mechanism of inhibition of platelet aggregation may be through inhibitng protein kinase Akt phosphorylation to block the signal transduction pathway of Akt. Limit cell grouth and reduce platelet number to prevent the formation of platelet thrombus. Also may be related to its various enzyme activity, such as plasmin and 5-NT that can decompose aggregating components correspondently.