Association of MicroRNAs and YRNAs With Platelet Function

Abstract

M icroRNAs (miRNAs) are small noncoding RNAs with cell-type specific expression patterns that are released by cells into the circulation as part of membranous particles or protein complexes.1 Thus, miRNAs can be readily quantified by real-time polymerase chain reactions (qPCRs) in plasma and serum and have generated increasing interest as potential new biomarkers.2 Our group has previously identified plateletrelated miRNA signatures that are predictive of cardiovascular events. 3 In addition, we measured miRNAs in healthy volunteers and in patients with symptomatic atherosclerosis before and after initiation of dual antiplatelet therapy and demonstrated reduced plasma levels of platelet-related miRNAs on platelet inhibition. Kaudewitz et al Plasma MicroRNAs and Platelet Function 421Dual oral antiplatelet therapy (acetylsalicylic acid [ASA]+a P2Y 12 inhibitor) is commonly used for the management of non-ST-segment-elevation acute coronary syndromes (ACS) and ST-segment-elevation myocardial infarction.5 ASA irreversibly inhibits cyclooxygenase 1 in platelets, thereby repressing thromboxane A 2 (TxA 2 ) synthesis and, consequently, platelet activation. Clopidogrel, prasugrel, and ticagrelor target the P2Y 12 receptor for ADP. However, interindividual variability in the platelet response to clopidogrel has been reported. Prasugrel and ticagrelor exhibit a more consistent antiplatelet effect and have shown benefits over clopidogrel in patients with ACS but also increase the risk of bleeding. 6,7 It is currently unclear whether plasma levels of platelet-related miRNAs correlate with the residual platelet activity in patients with ACS and how different antiplatelet agents alter miRNAs.In this study, we used RNA sequencing to characterize small RNAs in plasma. Then, we compared the effect of different antiplatelet agents and explored the association of small RNAs (miRNAs and YRNAs) with platelet function tests in patients with ACS. Moreover, we correlated their plasma levels to platelet activation markers in the prospective, population-based Bruneck study 3 and investigated whether a single-nucleotide polymorphism (SNP) that facilitates miR-126 processing 8 alters circulating miR-126 levels and platelet reactivity. These epidemiological observations were complemented by preclinical studies, assessing platelet function in mice on treatment with antagomiRs directed against miR-126 and by mechanistic studies measuring miR-126 targets. MethodsAn expanded Methods section is available in the Online Data Supplement. Next-Generation SequencingSmall RNA libraries were generated from non-normalized RNA (ranging from 375 pg to 1 ng) extracted from equal volumes of platelet-poor plasma (PPP) and platelet-rich plasma (PRP) from healthy human volunteers. Before library preparation, RNA was spiked with equal amounts of C. elegans miR-39 star (cel-miR-39*) to assist in normalization. Libraries were prepared using the small RNA library preparation kit version 2.0 (Illumina Cambridge Ltd) according to manufacturer's protocol with limi...

microRNAs in cardiovascular disease – clinical application

“…Inhibition of miR-126-3p in mice attenuates platelet aggregation and levels of circulating miR-126-3p show positive correlations with platelet function tests in mice and men [62]. These findings interlink miR-126-3p with platelet-associated vascular and EC function and raise further questions as to what extend pharmacological platelet inhibition might interfere with accurate quantification measures in miRNA analyses in general (see below).…”

“…Therefore, recently, analyses were conducted revealing that circulating miR-92a levels in plasma of aspirin treated patients showed a decreasing slope according to increasing aspirin response and, intriguingly, miR-92a was able to identify aspirin responders from non-responders with high sensitivity and specificity [154]. Regarding the same mechanism, Kaudewitz et al have proven a correlation of miRNAs with established platelet function tests in patients with ACS and platelet activation markers in the general population and identified miR-126 to affect platelet reactivity [62]. Interestingly, the miR-126 pathway modulating platelet function impacts on P2Y12 expression [62] -the target of modern thrombocyte aggregation inhibitors such as clopidogrel, prasugrel, and ticagrelor, which are widely used in patients with CAD.…”

miRNA in Cancer Pathology

“…MiR-223 and miR-126, other copious platelet miRNAs and YRNAs exhibited significant positive correlations with the vasodilatorstimulated phosphoprotein phosphorylation assay. Kaudewitz et al (2016) also showed that YRNAs, miR-126 and miR-223 were also amongst the small RNAs showing the utmost reliance on platelets. In addition, a single nucleotide polymorphism that enables processing of pri-miR-126 to mature miR-126 accounted for a rise in circulating platelet stimulation markers.…”

“…Wang et al (2016) have recently emphasized the pathogenesis of chronic myeloid leukaemia (CML), functional significance of miRs and lncRNAs (long non-coding RNAs) in regulation of CML progress and complicated mechanisms fundamental to their action. Kaudewitz et al (2016) believed that it is uncertain whether plasma miRNA levels are related to platelet function. For this they aimed to link small RNAs to platelet reactivity.…”

“…Finally, inhibition of miR-126 in mice reduced platelet accumulation and it directly and indirectly affects ADAM9 and P2Y12 receptor expression. It is concluded that levels of plateletrelated plasma miRNAs and YRNAs correlate with platelet function tests in ACS patients and platelet stimulation markers in the general population and that changes in miR-126 affect platelet reactivity (Kaudewitz et al, 2016). An experimental-validated map of RAS-MAPK oncomiRs and tumour suppressor miRNAs from transmembrane receptor to downstream ERK proteins has been recently provided (Masliah-Planchon et al, 2016).According to which, MiRNAs could be further regarded as probable genetic biomarkers for diagnosis, prognosis, or therapeutic purpose.…”