The Study Of Influencing Factors On Individual Variability Of Clopidogrel Antiplatelet Activity In Patients With Coronary Heart Disease

Objectives: To investigate the effect of genetic and non-genetic factors on the inter-individual variability of clopidogrel anti-platelet activity in patients with coronary heart disease, and to analyses the relationship between genetic factors and stent-related adverse prognosis in PCI patients with stent implantation.Methods:(1) Blood samples were collected from119Chinese patients with coronary heart disease and patient’s clinical characteristics were registered. Plasma concentration of clopidogrel active metabolite was determined by UPLC-MS/MS. PCR-RFLP method was used for the detection of2C19*2/*3, PON1Q192R, ABCB1C3435T genotype. Mutation-sensitive molecular switch method was used to detect the polymorphism of CES1A2A(-816)C. Pharmacologic specific index of clopidogrel anti^latelet (Vasodilator Stimulated Phosphoprotein Phosphorylation) was determined by flow cytometer. Multiple linear regression was performed to model the relationships of platelet reactivity index with genetic and non-genetic factors.(2)22patients who developed stent-related adverse cardiovascular events within one year after PCI were collected, matched with86patients with no adverse event. Blood samples were available for genotyping and clinical characteristics were also collected. Chi-square test was used to analyze the genetic risk of adverse prognosis.Results:(1) The methods for detection of2C19*2/*3, P0N1Q192R, ABCB1C3435T, CES1A2A(-816)C were established, and were well validated respectively by direct sequencing of20samples randomly selected. The gene distribution in healthy individuals and CHD patients was investigated. In183～204healthy individuals, the frequencies of mutant allele were33.88%,6.01%,61.52%,36.34%and23.77%respectively,34.69%,5.63%,67.81%,39.24%and27.12%in153-160CHD patients. There was no significantly difference between the two populations (P=0.21~0.82).(2) Method by UPLC-MS/MS for determination of clopidogrel active metabolite was developed and validated. There was no interference from endogenous substances, and calibration curves were well linear during the range of1～200ng·mL-1(R2=0.9977). The method including accuracy, precision, recovery, matrix effect and stability met the requirements of biological sample analysis. The samples at1hour after300mg loading dose from19patients were collected and determined, the mean plasma concentration was26.15±24.77ng·mL-1, there was marked correlation between the concentration and PRI (R2=0.33, P=0.01).(3) In119CHD patients, CYP2C19genotyping showed that49patients were homozygous*1/*1,61patients were heterozygous*1/*2,9patients were homozygous*2/*2. The PRI of the three genotype groups were43.79±18.22,53.72±18.14,61.14±20.86respectively. CYP2C19*2carriers showed significantly increased residual platelet reactivity compared with noncarriers. CYP2C19*3genotyping showed that106patients were homozygous*1/*1,13patients were heterozygous*1/*3. The PRI of the two genotype groups were49.64±19.68and56.11±12.32, variant allele2019*3attenuated the response to clopidogrel with no significant difference. We further evaluated the effect of CYP2C19phenotype on the clopidogrel antiplatelet activity, there were44extensive metabolizers,59intermediate metabolizers and16poor metabolizers. The PRI of three phenotype groups were42.54±18.75,53.10±17.80and60.49±17.78(P=0.001), poor metabolizers showed significantly increased residual platelet reactivity.(4) CES1A2A(-816)C genotyping showed that61patients were AA genotype,49patients were AC genotype,5patients were CC genotype. The PRI of AA genotype group and mutant group were46.28±18.69and55.31±18.61respectively (P=0.01), variant allele C attenuated the effectiveness of clopidogrel antiplatelet.(5) PONl Q192R genotyping showed that19patients were QQ genotype,40patients were QR genotype,60patients were RR genotype. The PRI of the three genotype groups were50.58±13.98,50.58±19.73and49.81±20.19respectively. ABCB1C3435T genotyping showed that48patients were CC genotype,54patients were CT genotype,17patients were TT genotype. The PRI of the three genotype groups were51.55±19.36,50.46±20.32and45.48±13.79respectively. The two SNPs had no influence on the effectiveness of clopidogrel.(6) The multiple linear regression model indicated that CYP2C19phenotype, CES1A2A(-816)C SNP, weight, HDL-C and TG could explain the inter-individual variation in clopidogrel antiplatelet activity about10%,6.4%,5.1%,3.6%,3%respectively. The total factors together could explain about26%.(7) The results of case-control study indicated that the presence of CYP2C19*2variant allele was significantly associated with stenWelated adverse events (OR=3.25,95%CI:1.10-9.59, P=0.03). No significant associations of other genetic variations and adverse prognosis were found, but the results showed the higher tendency in case group than that in control group for CES1A2C allele and PON1Q192R polymorphism. Nongenetic independent correlate factor that closely related to stenMelated adverse events was multivessel disease (OR=1.44,95%CI:1.07-1.95, P=0.02).Conclusion: Genetic factors including CYP2C19and CES1A2A(-816)C polymorphism and nongenetic factors including weight, HDL-C and TG played an important role in the inter-individual variability of clopidogrel anti-platelet activity, the total factors could explain about26%of variability in PRI. CYP2C19*2polymorphism and multivessel disease were the potent predictors of adverse prognosis. The results of this study could provide basis and guide in clinical use and personalized medicine of clopidogrel.