Comparison of the Release of microRNA and extracellular vescicles from platelets in response to different agonists

Abstract

On activation platelets release microRNA and extracellular vesicles (EV) into the circulation. The release of EV from platelets has been shown to be dependent on the agonist; here we investigated whether the microRNA profile or EV released from platelets was also agonist specific. Washed platelets from healthy subjects were maximally stimulated with agonists specific for the receptors for collagen (GPVI), thrombin (PAR1/PAR4), or ADP (P2Y1/P2Y12) with/without inhibiting secondary mediators, using aspirin to block COX-1 and apyrase to remove ADP. The released microRNA was profiled using TaqMan microRNA microarray cards. Platelet-derived EV (pdEV) were characterised by size (Nanoparticle Tracking Analysis; NTA), for procoagulant activity (Annexin-V binding and support of thrombin generation), and for the EV markers CD63 and HSP70. Platelet activation triggered the release of 57-79 different microRNA, dependent upon agonist, with a core of 46 microRNA observed with all agonists. There was a high level of correlation between agonists (r >0.98; p<0.0001 for all), and with the microRNA content of the parent platelets (r2 >0.98; p<0.0001). The 46 microRNA seen in all samples are predicted to have significant effects on the translation of proteins involved in endocytosis, cell cycle control and differentiation. MiR-223-3p was most abundant in all samples and has previously been implicated in myeloid lineage development and demonstrated to have antiinflammatory effects. Stimulation through GPVI produced a pdEV population with significantly more procoagulant activity than the other agonists. Apyrase significantly reduced microRNA and pdEV release, while aspirin had little effect. These data suggest that all tested agonists trigger the release of a similar microRNA profile whilst the procoagulant activity of the pdEV was agonist dependent. ADP was shown to play an important role in the release of both microRNA and pdEV