Plasma MicroRNAs Predict Major Adverse Cardiovascular Events As Intermediate Biomarkers Of Clopidogrel Antiplatelet Efficacy

Aims :The circulating micro RNAs(mi RN As) own many characteristics of biomarkers and can be released from platelet. We hypothesize plasma mi RN A level can reflect the effect of antiplatelet treatment coronary atherosclerotic heart disease(CAD). O ur published data suggested that plasma mi R-126 has the potential as a biomarker for predicting major adverse cardiac events in dual antiplatelet-treated patients after percutaneous coronary intervention(PCI). In this paper, we aimed to explore the association between plasma mi RN As and clopidogrel pharmacokinetics / pharmacodynamics(PK / PD), and to investigate whether plasma mi RNAs were associated with clopidogrel antiplatelet efficacy by evaluating the risk of major adverse cardiovascular events(MACE) and occurrence of bleeding events in C AD patients after PCI.Methods :(1) Screening of mi RNAs associated with clopidogrel efficacy: The series of preliminary experiments had been designed to establish and optimize a stable method for extraction of plasma total RNA and quantification of the mi RN As expression levels. A total of 153 CAD patients received loading doses of clopidogrel 300 mg and aspirin 100 mg, platelet aggregation of asipirin response unit(ARU) and P2Y12 response unit(PRU) were detected by Verify Now assay. The patients were divided into 16 groups according to the high or low ARU or PRU. The plasma from each subgroup was pooled and then total RNA were extracted. Pooled plasma mi RNAs levels were detected by a high-throughput illumina sequencing method. Technique validation by stem loop q RT-PCR was designed to validate and screen plasma mi RNAs levels in all plasma pools. The levels of plasma mi RN A were further verified in patients individually by q RT-PCR. The association between clopidogrel PD indexes PRU and plasma mi RN As levels were analysed by Spearman correlation method.(2) Predicting of the selected mi RNAs for clopidogrel pharmacokinetics: Further to measure and analyse the correlation between expression levels of relative plasma mi RN As and clopidogrel and MP-H4 PK parameters(Cmax, AUC0-4h, Tmax) in another independent group of 31 CAD patients who received 300 mg clopidogrel and aspirin 100 mg.(3) Validating the effects of mi RNAs on adverse cardiovascular events: The screened mi RN As were confirmed by q RT-PCR and validated in an prospective cohort of 1230 CAD patients who followed up more than 3 years. The risk factors of MACE or bleeding events were explored through the Cox and Logistic regression model combined clinical characteristics of follow up and the mi RN As expression levels. Results:(1) The q RT-PCR quantification method of plasma mi RN As is available and effective. Seventeen mi RN As were differentially expressed among all subgoups screened by illumina sequencing. Among which, six plasma mi RNAs(mi R-126,-130 a,-27 a,-106 a,-21,-142) were technically validated by a stem loop q PT-PCR method in the same pools and individual patients. The expression levels of mi R-126,-130 a,-27 a,-142 were further validated to be higher in resistant to clopidogrel treatment subgroup(all P < 0.05). However, when patients grouped by resistance to aspirin, the levels of mi R-126,-130 a,-142,-21,-27 a were significantly increased in patients resistant to clopidogrel than sensitive to clopidogrel(all P < 0.05).(2) After correcting multiple comparisons of P value by false discovery(FDR) control, we found that plasma mi R-142 level was negatively associated with MP-H4 Cmax and AUC0-4h after 300 mg loading dose of clopidogrel in 31 CAD patients, respectively(P < 0.05).(3) Moreover, a higher plasma mi R-142 and mi R-126 levels were associated with a higher risk for MACE in 1230 CAD patients(P = 0.0006, 0.0353, respectively). Multivariable Cox regression analysis revealed that a higher plasma mi R-142 level, diabetes mellitus, heart failure, and the use of calcium channel blockers(CCBs) were independent risk factors for MAC E(all P < 0.05). There was no significant associatio n between the levels of six plasma mi RNAs and bleeding events during follow- up. Conclusions:Among coronary atherosclerotic heart disease patients who received clopidogrel for anti-platelet therapy after percutaneous coronary intervention, plasma mi R-126, mi R-130 a, mi R-142, mi R-21 and mi R-27 a could be potential intermediate biomarkers of clopidogrel antiplatelet efficacy. Plasma mi R-142 level, diabetes mellitus, heart failure, and the use of CCBs were independent risk factors for MACE. Based on the correlation study of mi R-142 and clopidogrel pharmacokinetics / pharmacodynamics, we suggested that plasma mi R-142 could serve as a potential biomarker for evaluating the inter-patient variability of clopidogrel antiplatelet efficacy and predicting clinical endpoints MAC E.