Circulating MiR-223and High On-Treatment Platelet Reactivity In Patients With Unstable Angina

Objective:Platelet activation and aggregation directly contribute to atherosclerotic thrombosis. Even if the application of standard dual antiplatelet therapy of aspirin combined with clopidogrel, there are still some patients suffered from thrombotic events. This phenomenon may be related to high platelet reactivity after treatment (high on-treatment platelet reactivity, HTPR). With the wide used of clopidogrel, the low response to clopidogrel has been reported constantly. At present, there are many laboratory methods for detection the platelet reactivity after the antiplatelet theropy. Our previous study has confirmed that the platelet microRNA-223(miR-223) expression levels is negatively correlated with platelet reactivity after taking standard clopidogrel in patients with coronary heart disease(CHD). This study sought to explore the relationship between plasma circulating miRNA-223levels, and clopidogrel responsiveness in patients with unstable angina.Materials and Methods:We enrolled consecutive patients with unstable angina pectoris confirmed by coronary angiography during November2012to January2013in the cardiovascular disease center, Pingin hospital, Logistics univisity of Chinese people’s armed police forces. All the enrolled patients prescribed300mg asprin plus300mg clopidogrel24hours before the blood sample collected, or300mg asprin plus75mg clopidogrel more than5days. Main demographic and clinical characteristics of the study population were recorded. Patients’ blood cell parameters and bio-chemical parameters were tested. We performed cardiac ultrasound, and all patients underwent coronary artery angiography (CAG) and received appropriate coronary artery interventional treatment. Gensini score and Syntax score were calculated according to quantitative CAG. Platelet reactivity after loading doses of clopidogrel was assessed with10μmol/L adenosine diphosphate (ADP)-induced light transmittance aggregometry and with vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay by flow cytometric analysis. Total miRNA was extracted from plasma, and the miR-223level was assessed by the real-time quantitative Polymerase Chain Reaction (Real-time PCR). The genomic DNA was also extracted from where. CYP2C19*2/*3gene segments were amplification by PCR and analyzed by the restriction fragment length polymorphism (RFLP).Results:1.62cases of patients with unstable angina were enrolled, which was confirmed by coronary angiography. The patients was divided into2groups according to the median level of platelet response index, which was calculated by the flow cytometry VASP phosphorylation level, the normal responses group (PRI≤56.3%, n=31, mean age=60.52±8.88,14male,17female) and the low responses group (PRI>56.3%, the average age=57.68±10.60,19male,12female).①The patients’ age, gender, blood pressure, heart rate and other basic data between the two groups have no statistical difference (P>0.05).②There are no statistical differences about the history of cardiovascular disease, concomitant diseases and the major medications of the cardiovascular disease between the2groups (P>0.05).③There are no statistical differences about the patients’ left ventricular ejection fraction, the left ventricular end-diastolic dimension (LVDED), the blood cells and the biochemical parameters between the2groups (P>0.05).④There is no statistical significance about CYP2C19*2/*3genes carries ratio between the2groups according to the CYP2C19gene polymorphism (P>0.05).⑤There is no statistical significance about the coronary artery Syntax score and Gensim score between the2groups (P>0.05).⑥Compared with the normal responses group, the plasma level of miR-223was decreased in the low responses group (P=0.008*).⑦The plasma level of miR-223was negatively correlated with PRI (Spearman r=0.379, P=0.002*).2. According to the median level of platelet aggregation (74.5%) the enrolled patients were divided into2groups:normal response group (PAG≤74.5%) and low reaction group (PAG>74.5%).①The patients’ age, gender, blood pressure, heart rate and other basic data between the two groups have no statistical difference (P>0.05).②There are no statistical differences about the history of cardiovascular disease, concomitant diseases and the major medications of the cardiovascular disease between the2groups (P>0.05).③There are no statistical differences about the patients’ left ventricular ejection fraction, the left ventricular end-diastolic dimension (LVDED), the blood cells and the biochemical parameters between the2groups (P>0.05).④There is no statistical significance about CYP2C19*2/*3genes carries ratio between the2groups according to the CYP2C19gene polymorphism (P>0.05).⑤There is no statistical significance about the coronary artery Syntax score and Gensim score between the2groups (P>0.05).⑥There is no statistical difference about the plasma level of miR-223between the two groups, despite the plasma level of miR-223is decreased in the low responses groups compared with the normal one (P>0.05).3、①Stepwise binary logistic regression analysis revealed that among factors that potentially influence platelet reactivity (CYP2C19*2/*3loss-of-function genotypes, use of calcium channel blockers/proton-pump inhibitors, age, diabetes and smoking), decreased circulating miR-223level was the only independent predictor for the presence of PRI-determined lower responders②ROC curve analysis revealed that miR-223expression had statistical discrininative value for PRI-determined normal and low responders (AUC=0.700, P=0.008,95%CI:0.562～0.833).Conclusion:Dcreased circulating microRNA-2231evel predicts high on-treatmant platelet reactivity in CHD patients. Our data suggest that circulating miR-223may serve as a novel biomarker for assessment of clopidogrel responsiveness in CHD patients.