PB1357 - Platelet PAR4 activation leads to EV release that has anti-inflammatory properties

Background: Extracellular vesicles (EVs) are small membrane vesicles that are released from cells after cellular activation. During infections thrombin mediated PAR4 activation of platelets lead to EV release that contain miRNAs. The EV-miRNAs can reduce pathologic activation of the inflammatory response during infections.

Aims: To elucidate if PAR4 activation is critical for release of platelet EVs and limiting pathological inflammatory overactivation

Methods: Platelets of WT and ΔPAR4 mice were stimulated with thrombin (1U/mL) to induce vesiculation. Large EVs and small EVs were isolated by ultracentrifugation and were analyzed by using nano particle counting by zetaview. MΦ were stimulated with poly IC (10μg/mL) or LPS (0.1μg/mL in 1%FBS) with or without resuspended LEVs, SEVs or EV-free Plt-R (platelet releasate) (1:10 diluted) for up to 24hrs.

Results: Our preliminary data confirmed that the thrombin receptor PAR4 is important for the generation of circulating EVs as ΔPAR4 mice have 50% less EVs as compared to WT mice. Our data show that global lack of PAR4 in mice results in dysregulated EV release. Releasate from thrombin stimulated platelets (Plt-R) modulated the inflammatory responses of MΦ, as it was able to reduce both the poly IC- and LPS-induced NFκB pathway activation in murine RawDualTM cells (Invivogen). Our observed reduction in NFκB pathway activation was associated with reduced TNFα production in poly IC or LPS stimulated MΦs . In addition, we found that the anti-inflammatory effect of Plt-R was mediated by the EVs portion of Plt-R which transfers anti-inflammatory miRNAs to the inflamed target cells.

Conclusion(s): Our data demonstrates that PAR4 on platelets is important for the release of circulating EVs. Platelets EVs carry miRNAs that act as anti-inflammatory factors reducing MΦ activation and maintain overall homeostasis.