TAmiRNA GmbH, 1110 Vienna, Austria.
Department of Vascular Biology and Thrombosis Research, Center of Physiology and Pharmacology, Medical University of Vienna, Schwarzspanierstraße 17, 1090 Vienna, Austria.
Cells. 2022 Apr 7;11(8):1254. doi: 10.3390/cells11081254.
Dysregulation of platelet function is causally connected to thrombus formation and cardiovascular diseases. Therefore, assessing platelet reactivity is crucial. However, current platelet function tests come with pitfalls, limiting clinical use. Plasma miRNA signatures have been suggested as novel biomarkers for predicting/diagnosing cardiovascular diseases and monitoring antiplatelet therapy. Here, we provide results from a comprehensive study on the feasibility of using circulatory platelet miRNAs as surrogate markers of platelet activation. We performed small RNA-Seq on different blood cell types to confirm known and identify novel platelet-enriched miRNAs and validated a panel of 16 miRNAs using RT-qPCR. To identify the main carrier of these blood-based platelet miRNAs, we enriched and analyzed distinct microvesicle populations. Platelets were stimulated with GPVI and P2Y12 agonists in vitro to monitor the release of the selected miRNAs following activation. Finally, the miRNA panel was also measured in plasma from mice undergoing the Folts intervention (recurrent thrombus formation in the carotid artery). Applying an unbiased bioinformatics-supported workflow to our NGS data, we were able to confirm a panel of previously established miRNA biomarker candidates and identify three new candidates (i.e., miR-199a-3p, miR-151a-5p, and miR-148b-3p). Basal levels of platelet-derived miRNAs in plasma were mainly complexed with proteins, not extracellular vesicles. We show that changes in miRNA levels due to platelet activation are detectable using RT-qPCR. In addition, we highlight limitations of studying the in vitro release of miRNAs from platelets. In vivo thrombosis resulted in significant elevations of platelet-derived miRNA levels in mice. In conclusion, we provide in-depth evidence that activated platelets release miRNAs, resulting in measurable changes in circulatory miRNA levels, rendering them promising biomarker candidates.
血小板功能失调与血栓形成和心血管疾病有因果关系。因此，评估血小板反应性至关重要。然而，目前的血小板功能测试存在缺陷，限制了其临床应用。血浆 miRNA 特征已被提议作为预测/诊断心血管疾病和监测抗血小板治疗的新型生物标志物。在这里，我们提供了一项关于使用循环血小板 miRNA 作为血小板激活替代标志物的可行性的综合研究结果。我们对不同的血细胞类型进行了 small RNA-Seq，以确认已知和鉴定新的血小板丰富的 miRNA，并使用 RT-qPCR 验证了一组 16 个 miRNA。为了确定这些基于血液的血小板 miRNA 的主要载体，我们富集并分析了不同的微泡群体。在体外，用 GPVI 和 P2Y12 激动剂刺激血小板，以监测激活后选定 miRNA 的释放。最后，还在接受 Folts 干预（颈动脉内反复血栓形成）的小鼠的血浆中测量了 miRNA 谱。应用无偏倚的基于生物信息学的工作流程对我们的 NGS 数据进行分析，我们能够确认一组先前建立的 miRNA 生物标志物候选物，并鉴定出三个新的候选物（即 miR-199a-3p、miR-151a-5p 和 miR-148b-3p）。血浆中血小板衍生 miRNA 的基础水平主要与蛋白质结合，而不是与细胞外囊泡结合。我们表明，使用 RT-qPCR 可以检测到由于血小板激活导致的 miRNA 水平的变化。此外，我们还强调了研究血小板释放 miRNA 的体外释放的局限性。体内血栓形成导致小鼠血小板衍生 miRNA 水平显著升高。总之，我们提供了深入的证据表明，激活的血小板释放 miRNA，导致循环 miRNA 水平发生可测量的变化，使其成为有前途的生物标志物候选物。
