| Objectives:Currently, a large number of clinical trials and basic researches ascribed "anti-platelet drug resistance" to interindividual variation in platelet reactivity. Therefore it is necessary to monitor the platelet reactivity of patients who are taking anti-platelet drugs. However in clinical practice, the methods for determining the reactivity of platelet do not have a regular standard. MicroRNA (miRNA) is a class of non-coding RNA whose final product is a～22nt functional RNA molecule. Recent data indicated that human platelets contain miRNA, and their expression profile is closely related to platelet reactivity. Meanwhile this variance in miRNA expression profile could impact diseases’susceptibility, especially the heterogenicity of platelet reactivity. Therefore, the purpose of the present work is to investigate the relationship between platelet miRNA (miR-223, miR-96) expression and clopidogrel response in paticnts with coronary artery disease, which could provide novel molecular mechanism underlying inter-individual variability of platelet reactivity.Materials and Methods:Thirty-six patients with unstable angina pectoris were enrolled in the study. 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, ankle-brachial index and brachial-ankle artery pulse wave velocity measurement to all patients. 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.Because platelet preparations are often contaminated by leukocytes, and small amounts of residual leukocytes, which have much higher levels of RNA than platelets, may cause false detection of miRNA. We therefore carried out leukocyte depletion with anti-CD45conjugated magnetic beads according to the manufacturer’s recommendations. Then we extracted miRNA in platelet. The expression analysis of miRNA-223which is band to3’UTR of platelet P2Y12receptor and miR-96which is related to platelet vesicle-associated microtubule protein8(VAMP8) expression regulation was performed using real time Polymerase Chain Reaction (real time PCR).Results:①All patients were divided into2groups by platelet reactivity index (PRI) median level (56.55%) which was assessed with VASP phosphorylation assay by flow cytometric analysis. Group A with PRI<56.55%, consisted of9male and9female. Group B with PRI>56.55%, consisted of7male,11female.②There was no statistical differences (P>0.05) in age, gender, body mass index and medications between two groups. There was no between-group statistical differences (P>0.05) in cardiovascular disease history, complications, blood pressure and heart rate when they were admitted to hospital. There was no statistical differences (P>0.05) interms of cardiac function, ABI, BaPWV, blood cell parameters and bio-chemical parameters. There was statistical differences (36.64±15.24vs52.19±13.17, P=0.002) in platelet aggregation rate between two groups.③There was no statistical differences (P>0.05) in TIMI grading, Syntax score and Gensim score. There is statistical differences (28.22±19.65vs12.39±15.47, P=0.011) in stents’length between two groups.④There was a positive correlation between PRI and platelet (r=0.400, P=0.016). There was a positive correlation between stents’length and Syntax score(r=0.457,P=0.005). There was a positive correlation between stents’length and Genism score (r=0.497, P=0.002). There was a positive correlation between Syntax score and Genism score (r=0.660,P=0.000).⑤Compared with A group, the relative expression of miR-223was down regulated with statistical differences (P=0.0398), while the expression of miR-96presented with down-regulated trend, no statistical difference was observed (P=0.073).⑥There was a negative correlation between the relative expression quantity of miR-223and PRI (r=-0.4045, P=0.0328). There was no correlation between the relative expression quantity of miR-96and PRI (r=-0.0186, P=0.9251)Conclusions:The present work for the first time demonstrated that heterogenicity of human platelet response to clopidogrel is correlated with expression pattern of platelet microRNA. Sepcifially, down-regulation of miR-223is associated with a blunted response to clopidogrel (increased platelet reactivity index measured by VASP phosphorylation) in CHD patients. Our results may provide novel molecular mechanism underlying inter-individual response to anti-platetlet drugs, and would be helpful for individualized drug dosing in clinical practice. |
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