Pharmacogenetics Research On Antiplatelet Drugs (Aspirin And Clopidogrel)

Pharmacogenetics is the newly rising study of the role ofinheritance in individual variation in clinical drug treatment. Lots ofstudies have shown that inherited variations in activities ofdrug-metabolizing enzymes, transporters or drug targets are involved ininter-individual differences in drug efficacy and toxicity. Recently, withthe rapid development of the Human Genome Project (HGP) and newexperimental techniques, pharmacogenetics has made great progress andhas developed to a more broad subject-"pharmacogenomics". Thedeepgoing study of pharmacogenetics will help us how to choose drugand how to adjust drug dose in order to avoid drug side effect and reachmore drug efficacy.Cardiovascular disease is the first of death cause. Large scalerandomized clinical trials have consistently demonstrated thatantiplatelet drugs (Aspirin and Clopidogrel) have a significant reductionof risk of cardiovascular (CV) events. The concept of aspirin orclopidogrel resistance is increasingly emerging in the literature in lightof the newly acknowledged variability in the antiplatelet response.The process of platelet aggregation: A2 (TXA2), adenosinediphosphate (ADP), thrombin can activate platelet. Platelet activatedrelease ADP and TXA2, which sequentially activate other platelets, andexpose glycoprotein (GP)Ⅱb/Ⅲa receptors at their surface, allowingfibrinogen binding, which forma bridges between adjacent activatedplatelets causing platelet aggregation. Aspirin irreversibly inhibitscyclooxygenasel (COX1) and effectively blocks the TXA2 formation;Clopidogrel inhibits ADP binding to ADP receptor (P2Y) and blocks platelet activation. The genetic variations of these genes coding proteinsinvolving in platelet aggregation pathway are likely to be associated toantiplatelet drugs response.The human COX-1 gene has been reported that there are more than20 variants existing, of which the haplotype including -842G mutation isrelated to aspirin resistance. The human P2Y1 gene has just been found 2variants in coding region, A1622G and C893T. It has been reported thatthe two polymorphisms are likely to be associated with antiplatelet drugsresponse in retrospective study. Many studies in Caucasian indicated thatthe GPⅡa/Ⅲb PLA1/PLA2 polymorphisms are common and have a greatrelationship with antiplatelet response. So far, it has not been reportedthat the genetic variations of COX1, P2Y1 and GPⅡa/Ⅲb gene and thatthe association between these variations and antiplatelet drugs response inChinese.Firstly, we determined 14 variants of COX1 gene, 2 variants ofP2Y1 gene and GPⅡa/Ⅲb PLA1/A2 polymorphism by polymerase chainreaction and restriction fragment length polymorphism analysis andsequence, of which most genotype methods is the first to be established inour lab, COX1 C22T, G639A, G123A and P2Y1 A1622G, C893Tvariants is common in Chinese population. The frequencies of them are1.0％, 4.1％, 5.1％, 30.5％and 3.5％, respectively. The other variants werenot detected in Chinese. Secondly, A 4-panel P2Y1 genotype-stratifiedsample (CT893/AG1622, CC893/GG1622, CC893/AG1622 andCC893/AA1622) of healthy volunteers (N=25 in total) were recruited andprospectively received a 100 mg daily oral dose of aspirin for seven days.We measured platelet aggregation induced with 1.6mM AA and 10uMADP by aggregometry before and after aspirin treatment. After aspirin treatment, the net decrease in arachidonic acid induced plateletaggregation was significantly larger in the P2Y1 CT893/AG1622genotype panel compared to CC893/GG1622, CC893/AG1622 andCC893/AA1622 genotypic groups. Thirdly, A 4-panel P2Y1genotype-stratified sample (CT893/AG1622, CC893/GG1622,CC893/AG1622 and CC893/AA1622) of healthy volunteers (N=25 intotal) were recruited and prospectively received a 300 mg daily oral doseof clopidlgrel. We measured platelet aggregation induced with 2uM 6uMand 10uM ADP by aggregometry before and after clopidogrel treatment.Haplotype combinations and genotypes for the two polymorphisms werenot associated with parameters of different concentration ADP-inducedplatelet aggregation. Fourthly, we create the luciferase reporter constructcontaining COX1 -842A wild-type promoter (-974, -27) and also createthe -842G mutation luciferase reporter construct using the site-directedmutagenesis technology. The two constructs were cotransfected to COS-7cell line with pRL-TK control vector. We found the luciferase activity of-842G mutation construct was significantly lower than that of -842Awild-type constructs.In conclusion, (1) the distribution of COX1, P2Y1 and GPⅢagenetic variations have great ethnic difference among African, Caucasianand Chinese; (2) P2Y1 1622A＞G and 893C＞T polymorphisms have aneffect on individual variation of aspirin response, the individuals with1622AG/893CT genotype are more sensitive to aspirin than those withother genotypes; (3) P2Y1 1622A＞G and 893C＞T polymorphisms is notlikely to be associated with clopidogrel response; (4) COX1 -842Gmutation down regulates COX1 gene transcription and decreases theactivity of COX1, which provides some molecular evidence for the relationship between the mutation and aspirin response from clinicalstudies. The present studies provide new clinically and molecularlyrelevant information regarding the genetic determinants modulatingantiplatelet drugs response and the potential underlying mechanisms. Forgene-directed rationalization and individualization of antiplatelet drugs inpatients with vascular disease provided an important foundation andtheoretical evidence.