Circulating MicroRNAs as Novel Biomarkers for Platelet Activation

Abstract

Rationale: MicroRNA (miRNA) biomarkers are attracting considerable interest. Effects of medication, however, have not been investigated thus far. Objective: To analyze changes in plasma miRNAs in response to antiplatelet therapy. Methods and Results: Profiling for 377 miRNAs was performed in platelets, platelet microparticles, platelet-rich plasma, platelet-poor plasma, and serum. Platelet-rich plasma showed markedly higher levels of miRNAs than serum and platelet-poor plasma. Few abundant platelet miRNAs, such as miR-24, miR-197, miR-191, and miR-223, were also increased in serum compared with platelet-poor plasma. In contrast, antiplatelet therapy significantly reduced miRNA levels. Using custom-made quantitative real-time polymerase chain reaction plates, 92 miRNAs were assessed in a dose-escalation study in healthy volunteers at 4 different time points: at baseline without therapy, at 1 week with 10 mg prasugrel, at 2 weeks with 10 mg prasugrel plus 75 mg aspirin, and at 3 weeks with 10 mg prasugrel plus 300 mg aspirin. Findings in healthy volunteers were confirmed by individual TaqMan quantitative real-time polymerase chain reaction assays (n=9). Validation was performed in an independent cohort of patients with symptomatic atherosclerosis (n=33), who received low-dose aspirin at baseline. Plasma levels of platelet miRNAs, such as miR-223, miR-191, and others, that is, miR-126 and miR-150, decreased on further platelet inhibition. Conclusions: Our study demonstrated a substantial platelet contribution to the circulating miRNA pool and identified miRNAs responsive to antiplatelet therapy. It also highlights that antiplatelet therapy and preparation of blood samples could be confounding factors in case-control studies relating plasma miRNAs to cardiovascular disease.