Platelets and inflammation - insights from platelet non-coding RNA content and release in the Bruneck Study and the PACMAN-AMI trial

Aims Platelets contain non-coding RNAs (ncRNAs), and their measurement may complement platelet aggregometry. Methods and results In the community-based Bruneck Study (n = 338), we generated platelet-rich plasma (PRP), platelet-poor plasma (PPP) and platelets. PRP was subjected to aggregometry using various agonists and processed to platelet releasates thereafter. Releasates, PPP and platelets underwent real-time polymerase chain reactions to measure ncRNAs. Platelet ncRNA release appeared agonist-specific, dose-dependent, and inhibited by aspirin. Collagen triggered the strongest release for most ncRNAs, whereas miR-150 was hyperresponsive to ADP, and miR-21 was hyperresponsive to arachidonic acid. Comparing the dynamic range of ncRNA release to aggregation, aggregation reached a maximum at high agonist concentrations, while ncRNAs continued to rise. Cohort-wide associations showed that inflammation parameters like neutrophil counts and C-reactive protein correlated inversely with platelet aggregation and ncRNA release. Similarly, a high leukocyte-derived RNA content in isolated platelets correlated inversely with aggregation. Inverse correlations were absent in aspirin users. Through experiments on plasma-free platelet releasates and platelets, including size-exclusion chromatography, ultracentrifugation and degradation assays, we discovered that microRNAs and YRNAs are carried by proteins and readily released, while circular-, long non-coding- and messenger RNAs are carried by vesicles and preferentially retained. Finally, we assessed ncRNA responses to short- and long-term dual antiplatelet therapy (DAPT) in plasma from 265 patients with acute myocardial infarction (AMI) of the PACMAN-AMI trial. Most of the DAPT effect was already achieved by 4 weeks, with a further reduction at 52 weeks, revealing a short- and long-term DAPT effect not captured by aggregometry. Conclusions Inflammation and leukocyte-derived RNAs in isolated platelets are associated with reduced platelet responses ex vivo, potentially reflecting exhaustion through pre-activation in vivo. We show that protein-bound ncRNAs are readily released from platelets, whereas vesicle-bound ncRNAs are preferentially retained. We highlight the potential of ncRNAs as biomarkers complementing aggregometry.