The Contribution Of Platelet MicroRNA Expression And Its Interaction With CYP2C19~*2 To Clopidogrel Antiplatelet Responsiveness In Acute Coronary Syndrome Patients

Objective: Our study aimed to investigate relationship between platelet miRNA (miR-223、miR-221、miR-21) expression and clopidogrel antiplatelet responsiveness, as well as the contribution of platelet microRNA expression and its interaction with CYP2C19 *2 allele on clopidogrel antiplatelet responsiveness in acute coronary syndrome (ACS) patients.Methods:We enrolled consecutive patients with ACS from December 2013 to December 2014 in the Department of Cardiology, PLA General Hospital, who finally enrolled by calculating the difference between the four quantile in order to screen out qualified extreme cases, all the cases were one-year follow-up. Recorded all the basic demographic information, various clinical characteristics, laboratory results, and the results of coronary angiography results of the study population.20umol/L adenosine diphosphate (ADP)-induced light transmittance aggregation (LTA) was applied to detect the platelet aggregation, Bioinformatic analysis was used to investigate the candidate microRNA. To avoid the false detection of microRNA from leukocytes, we therefore extracted pure platelet microRNA by using the anti-CD45 conjugated magnetic beads to remove the leukocytes according to the manufacturer’s recommendations. Real time polymerase chain reaction (real time PCR) was performed to analyse the expression levels of all miRNAs which is band to platelet P2Y12 receptor, as well as to genotype candidate single nucleotide polymorphisms (SNP) including CYP2C19*2 and CYP2C19*3. The occurrence of clinical adverse cardiovascular events was followed for 1 year. Univariate and multivariate analysis and receiver operating characteristic curve (ROC curve) were utilized to assess the relationship between miRNA expression levels and clopidogrel platelet reactivity, the non-conditional Logistic regression was used to evaluate the interaction between expression of miRNA and CYP2C19 * 2 allelic variant.Results:A total of 272 cases with ACS were enrolled in this study, with 165 cases of them testing the platelet aggregation befored and after clopidogrel, and then screened out qualified 39 extreme cases, all the 39 cases were completed one-year follow-up. Then 39 cases were divided into two groups by the four quantiles:high platelet reactivity group of extreme values (high response group),21 cases; and low platelet extreme value group (low response group),18 cases.①There was no significant differences in clinical characteristics between the two groups (p> 0.05), but there was statistical differences in the on-treatment platelet aggregation (OTPA) and the platelet aggregation change (PA change) (OTPA:24.14 ± 24.35 vs 6.757 ± 3.23,p<0.001; PA change:39.78 ± 61.43 vs 90.07 ± 4.09,p<0.001) between the two groups.② According to bioinformatic analysis and forecast, we finally confirmed three candidates microRNA, including miR-223, miR-221 and miR-21.③Compared with the low response group, the levels of three miRNAs expression were up regulated with statistical differences (miR-223:p=0.002; miR-221:p=0.004; miR-21:p=0.007) in the high response group. According to the univariate correlation analysis, there was a negative correlation between miR-221 expression level and OTPA (r=-0.440, p =0.034), as well as a negative correlation between miR-21 expression level and OTPA (r=-0.341,p=0.005). There was a positive correlation between miR-221 expression level and PA change (r= 0.358, p=0.025), also a positive correlation between miR-21 expression level and PA change (r = 0.411,p=0.009), but there was no significant correlation between the miR-223 expression level and OTPA or PA change (p> 0.05). In addition, our study also found that the miR-223 expression and cTnT levels were positively correlated (r = 0.424,p=0.034).④R0C curve analysis showed that the relative expression levels of the three miRNAs can effectively identify the high platelet response (miR-223:AUC = 0.704, the sensitivity and specificity was 70% and 100%, p=0.030; miR-221:AUC=0.762, the sensitivity and specificity was 80% and 62.5%,p=0.005; miR-21:AUC=0.796, the sensitivity and specificity was 88.9% and 76.3%, p=0.002).⑤After multivariate adjustment (such as CYP2C19*2/*3 loss-of- function genotypes, age, smoking, diabetes, using of proton-pump inhibitors and so no), we found that the three microRNAs were independent predictors of high platelet reactivity (miR-223:adjusted OR 2.133,95% CI:1.191-3.820,p= 0.011; miR-221: adjusted OR 1.133,95% CI:1.027-1.250, p= 0.013 and miR-21:adjusted OR 1.012, 95% CI:1.001-1.023, p = 0.030).⑥Interaction analysis revealed significantly higher expression levels of miRNAs in CYP2C19 * 2 allele carriers who were high platelet responders (miR-223:p=0.037; miR-221:p=0.001; miR-21:p<0.001),but not in CYP2C19 * 2 non-carriers (p>0.05).Conclusion:Our study find that up-regulation of three kinds of microRNAs(miR-223, miR-221 and miR-21) expression is associated with clopidogrel high platelet reactivity. What’s more, we find that the interaction between the expression levels of miRNAs and the CYP2C19 * 2 variant carriers may have an impact on clopidogrel platelet reactivity. Our results suggest that miR-223, miR-221 and miR-21 may serve as the novel biomarkers for assessment clopidogrel antiplatelet responsiveness in ACS patients.