Comprehensive Characterization of Platelet-Enriched MicroRNAs as Biomarkers of Platelet Activation

Abstract

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.

The Impact of the Anticoagulant Type in Blood Collection Tubes on Circulating Extracellular Plasma MicroRNA Profiles Revealed by Small RNA Sequencing

“…Among them, we selected the top ten species based on their abundance in plasma to form the list of potential RBC-derived circulating extracellular miRNAs. Similarly, we selected the top 10 potential platelet-derived miRNA species based on previous studies revealing the presence of platelet-specific miRNAs in extracellular fraction or using small RNA sequencing or microarray screening to analyze platelet miRNome [ 31 , 32 , 33 , 38 ]. Platelet-derived circulating miRNA levels are an important marker of in vivo or in vitro platelet activation resulting in the release of PMVs which contain specific miRNA signatures [ 31 , 33 , 34 , 38 ].…”

“…Plasma preparation conditions may also cause platelet activation and release of platelet microvesicles (PMVs) which contain specific miRNA signatures [ 31 , 32 , 33 , 34 , 35 ]. PMVs constitute the main fraction of all microvesicles in plasma [ 36 , 37 , 38 ].…”

Plasma levels of platelet-enriched microRNAs change during antiplatelet therapy in healthy subjects

“…Active or passive release from other cell types likely contributes to basal levels. MiR-223-3p and miR-150-5p are also expressed in leukocytes ( de Rie et al, 2017 ; Krammer et al, 2022 ). MiR-126-3p is abundant in endothelial cells, while miR-24-3p shows broad expression among different tissues ( de Rie et al, 2017 ).…”

“…The main carrier of extracellular miRNAs remains a matter of controversy and likely differs between miRNAs. We previously reported that basal levels of platelet-related miRNAs are mainly protein-bound ( Krammer et al, 2022 ). Activated platelets have been shown to secrete EVs with miRNAs as cargo ( Diehl et al, 2012 ; Gidlöf et al, 2013 ; Laffont et al, 2013 ).…”

“…The “simply RNA tissue” program was used and RNA was eluted in 50 μl nuclease-free water and stored at −80°C until further processing. Reverse transcription and quantitative PCR (qPCR) analysis were performed as previously described ( Krammer et al, 2022 ). In short, for cDNA synthesis, the miRCURY RT kit (Qiagen, Venlo, Netherlands) was used with 2 μl total RNA input.…”