Studies Of W1 On ADP Receptor Signaling Pathways Of Plaelates And Some Related Drugs

W1,as a new synthetic compound,contains 2-O-clopidogrel and aspirin,the former being the first step metabolite of clopidogrel.The idea of W1 synthesis is based on the prodrug property of clopidogrel,and the instability of its metabolites,in order to reduce or overcome the main problems in the clinical use of clopidogrel.Clopidogrel is widely used in the treatment of antithrombotic therapy in clinic,and the activation process in vivo needs two-step oxidative metabolism by drug metabolizing enzymes of liver,and the major subtypes of metabolic enzymes are CYP2C19 and CYP3A4.Therefore,the pharmacodynamics of clopidogrel is usually affected by CYP450 enzyme activity in the liver,such as the patients with the above enzymes activity congenitally deficient or the situations of combination administration,the latter including proton pump inhibitors(such as omeprazole)or cholesterol lowering drugs(such as statins)and other clinical common drugs.These factors can induce less antiplatelets of clopidogrel to achieve the desired effect,namely called "clopidogrel resistance" phenomenon,which has become the most worrying problem in clopidogrelusage.Our previous studies have shown that W1 has a stronger antiplatelet effect than clopidogrel,and can significantly reduce clopidogrel resistance.This study focus on the mechanism of W1 action,mainly observing its effect on platelet P2Y12 signaling pathway of ADP receptor,in addition,omeprazole and simvastatin which might affect pharmacodynamics of clopidogrel,was also investigated in some aspects.Part one: effects of W1 on platelet ADP receptor signaling pathway.Wistar rats were used experimental objects,the animals were divided into three groups:normal control group,clopidogrel group(10mg/kg)and W1 group(3mg/kg).Clopidogrel and W1 were respectively full suspended with 0.5% methyl cellulose(MC)solution.The two drug-treated groups of rats were single dose with the method of gavage administration,the normal control group of rats take only an equal volume of MC suspension.After 4 hours of administration,the rats were anesthetized and blood was collected through abdominal aorta blood,Then the washed platelets were prepared and used for the protein expression of ADP receptor signaling pathway and platelet spreading test respectively.In the detection of protein expression of preceptor signal molecules,the platelets of rats were treated with ADP(50 M)for 5 minutes.After ADP treatment,platelet protein was extracted and then Akt,Erk proteins with phosphorylation and Rap1 protein were detected by Western blot method.In the platelet spreading experiment,observation was carried out under confocal laser scanning microscope,single platelet area was measured to reflect the spreading situation using FITC-phalloidin staining platelets.The results of Western blot showed that compared with that of the normal control group of rats,the expression of Akt,Erk protein and Rap1 protein in platelets of W1 or clopidogrel group wereinhibited.Laser scanning confocal microscopy showed that compared with that of normal control group of rats,the platelet area of W1 or clopidogreladministrated group of rats decreased significantly,indicating that W1 can inhibit platelet spreading on fibrinogen,blocking the activation of fibrinogen receptor.Part two,the effect of simvastatin on clopidogrel.Wistar rats were divided into three groups: clopidogrel(10 mg/kg)group,clopidogrel group(10 mg/kg)combined with simvastatin(40 mg/kg)group and clopidogrel group(10 mg/kg)combined with simvastatin(80 mg/kg)group,every group was take drugs with single dose and oral administration.According to the main methods of the first part,the effect of simvastatin on clopidogrel was observed.The results showed that there was no significant effect of simvastatin on the expression of Akt phosphorylation protein and the change of the area in the platelets in platelet spreading experiment.In order to further analyze the reasons,on the one hand,simvastatin(10-9,10-8,10-7mol/L)were take to culture Hep G2 cells,Q-PCR and Western blot were detected in mRNA and CYP3A4 protein expression level in HepG2 cell respectively;on the other hand,simvastatin(10-9,10-8,10-7,10-6,10-5 mol/L)and microsomes the expression of CYP450 s and CYP450 s reductase isozymes were incubated to study the effect of simvastatin on CYP3A4 enzyme activity by analysis the changes of fluorescence intensity.The results showed that simvastatin did not affect the expression of CYP3A4 protein and mRNA in the HepG2 cells,and also had no significant effect on the activity of CYP3A4 in vitro.Part three,the effect of omeprazole on the expression of CYP2C19 in HepG2 cells.Omeprazole(0,0.1,1,10 mg/L)was used for HepG2 cells for 24 hours.The mRNA and protein of CYP2C19 in HepG2 cells was detected byQ-PCR and Western.Omeprazole(0,0.1,1,10,100 mg/L)and microsomes the expression of CYP450 s and CYP450 s reductase isozymes were incubated to study the effect of omeprazole on CYP2C19 enzyme activity by analysis the changes of fluorescence intensity.The results showed that the expression of mRNA and protein of CYP2C19 was inhibited by the concentration of omeprazole.The enzyme activity test showed that when the concentration of omeprazole reached 10mg/L and 100 mg/L,the fluorescence intensity was significantly decreased,which indicated that the high concentration of omeprazole inhibited the expression of CYP2C19.Conclusions: W1 can inhibit the protein expression of Akt,Erk in phosphorylation and Rap1 in rat platelets,which mediate ADP receptor signaling pathway,suggesting that W1 plays its antiplatelet effect by inhibiting ADP receptors signaling pathways.Effect of simvastatin on clopidogrel have not detected significantly,including the expression of Akt and the area change of platelet,and the expression of CYP3A4 of HepG2 cell and the microsomal CYP3A4 activity has no effect;omeprazole can reduce expression of CYP2C19 in HepG2 and inhibit the CYP2C19 activity of liver microsomes in vitro.