Pharmacoepigenetic Study Of Variability Of Antiplatelet Effect Of Clopidogrel,a P2Y12 Receptor Antagonist

P2Y12 receptor activation is a key pathway that promote platelet activation and aggregation in ACS patients or after PCI.Therefore,a dual antiplatelet treatment(DAPT)consisting of aspirin and a P2Y12 receptor antagonist,has been the standard of care for ACS patients.Although the new P2Y12 receptor antagonist(such as ticagrelor)has been used in clinical practice,clopidogrel-the classic P2Y12 receptor antagonist is still the first-line antiplatelet agent for patients with ACS or undergoing PCI.However,due to the individual difference of antithrombotic effectof clopidogrel,how to individualize the treatment of clopidogrel safely and effectively remains a challenge.Previous epigenetic study showed that platelet microRNAs might play a role in the platelet activation and antiplatelet responsiveness.However,the association between microRNAs and clopidogrel anti-platelet response,as well as the mechanism is still unclear.The present study aimed to analysis the association between platelet-derived microRNA and clopidogrel anti-platelet responsiveness;and then to elucidate the association between platelet-derived miR-126 functional genotype and clopidogrel antiplatelet responsiveness in ACS patients;finally to elucidate the pharmacoepigenetic mechanism of miR-126 for the regulation of clopidogrel antiplatelet responsiveness.Part 1:To analysis the association between platelet-derived microRNA and clopidogrel anti-platelet responsivenessObjective:To investigate the association between platelet-derived miRNAs and P2Y12 receptor antagonist antiplatelet responsiveness in clopidogrel treated patients with acute coronary syndrome(ACS).Methods:Consecutive hospitalized ACS patients with the measurement of clopidogrel on-treatment platelet reactivity(OTPR),as well as the healthy volunteers with the measurement of the adenosine-diphosphatediphosphate(ADP)induced platelet reactivity(PR)by thromboelastogram were recruited,respectively.The expression of platelet-derived microRNAs targeting the genes on the pathway of P2Y12 inhibitors was firstly analyzed for the association with PR in the healthy.Then,the related microRNAs were validated in the patients with ACS for the association with clopidogrel OTPR.Results:A total of214 healthy subjects and 430 clopidogrel treated ACS patients were recruited.The expression of miR-223,miR-130,miR-126,and miR-150 was significantly associated with the PR in the healthy extreme cases for PR(p<0.03).When validated in the patients with ACS,the expression of miR-223,miR-126,and miR-150 was found associated with the extreme clopidogrel OTPR[miR-223:0.68 ± 0.10 in low on-treatment platelet reactivity(LTPR)vs.0.34 ± 0.10 in high on-treatment platelet reactivity(HTPR),p?0.04;miR-126:5.94 ± 1.13 in LTPR vs.1.27 ±023 in HTPR,P<0.01;miR-150:0.99 ±0.14 in LTPR vs.1.60 ± 0.18 in HTPR,p = 0.02].Conclusions:Platelet-derived miR-223,miR-126,and miR-150 could influence clopidogrel antiplatelet responsiveness in patients with ACS.Part 2:Association between platelet-derived miR-126 functonal single nucleotide polymorphisms(SNP)and clopidogrel antiplatelet responsiveness in ACS patientsObjective:We aimed to analysis the contribution of platelet miR-126 functional SNP variants to clopidogrel antiplatelet responsiveness in ACS patients.Methods:Consecutive hospitalized ACS patients with the measurement of clopidogrel on-treatment platelet reactivity by TEG were recruited.Patients were allocated for high on treatment platelet reactivity(HTPR)according to the calculated of TEG-MAADP>47 and ADP inhibition<3 0%,as low on treatment platelet reactivity(LTPR)with the TEG-MAADP<31mm and ADP inhibition>90%.The peripheral blood DNA of all recruited patients was genotyped for miR-126 rs4636297 by the method of Snapshot.The association between genotypes and clopidogrel antiplatelet responsiveness was analyzed.Results:A consecutive 364 ACS patients treated by clopidogrel were recruited from November 2015 to February 2017.A total of 193 patients were allocated as the extreme cases for HTPR,and 171 as LTPR.The univariate analysis showed that the frequency of the miR-126 rs4636297AG+AA genotypeswere significantly higher in HTPR than in LTPR group(37.3%vs.23.4%,HR:1.949,95%CI:1.232-3.083,P=0.004).With the adjustment of the covariates including gender,age,smoking,hypertension,diabetes,and CYP2C19*2/*3 genotypes by logistic regression analysis,significantly higher frequency of miR-126 rs4636297 AG+AA genotype could still be found in HTPR group(HR:1.581,95%CI:1.012-2.470,P=0.04).Conclusions:Functional genotypes of miR-126 rs4636297 could contribute to the clopidogrel antiplatelet responsiveness in patients with ACS.It indicated that miR-126 might regulate the clopidogrel antiplatelet responsiveness by involving in the platelet activation.Part 3:pharmacoepigenetic mechanism of miR-126 for the regulation of clopidogrel antiplatelet responsivenessObjective:We aimed to explore the pharmacoepigenetic mechanism of miR-126 for the regulation of the downstream target gene PI3KR2on the pathway of clopidogrel antiplatelet effect.Methods:By cell transfection,miR-126 was over-expressed in bone marrow megakaryocytes cell line(MEG-01).The expression of PI3KR2 was measured in the transfected MEG-01 by the fluorescent quantitative PCR technique.Constructed wild and mutant plasmid of PI3KR2 3'UTR were sub-cloned into the psiCheck2 vector immediate downstream of the stop codon of the luciferase gene,and then trafected into the 293T cell lines.The interaction between miR-126 and PI3KR2 was observed according to the luciferase activity by luciferase reporter gene system.Results:In MEG-01 cells,the over-expression of miR-126 could significantly reduce the expression of PI3KR2;and when the expression level of miR-126 reduced,the PI3KR2 expression level increased significantly(P<0.05).In 293T cells,luciferase activity of miR-126 and PI3KR2 wild-type group was decreased significantly compare to miR-126 and PI3KR2 mutant-type group(P<0.01).The luciferase assay showed that the translational activity of PI3KR2 3'UTR was inhibited by miR-126.Conclusions:miR-126 might influence clopidogrel antiplatelet responsiveness by regulating the expression of the target gene of PI3KR2.