RhoA downstream of Gq and G12/13 Pathways regulates Protease- activated Receptor-mediated dense granule release in platelets

Platelet secretion is an important physiological event in hemostasis. The protease activated receptors, PAR 1 and PAR 4, and the thromboxane receptor activate the G12/13 pathways, in addition to the Gq pathways. Here, we investigated the contribution of G12/13 pathways to platelet dense granule release. 2MeSADP, which does not activate G12/13 pathways, does not cause dense granule release in aspirin-treated platelets. However, supplementing 2MeSADP with YFLLRNP (60 μM), as selective activator of G12/13 pathways, resulted in dense granule release. Similarly, supplementing PLC activation with G12/13 stimulation also leads to dense granule release. These results demonstrate that supplemental signaling from G12/13 is required for Gq-mediated dense granule release and that ADP fails to cause dense granule release because the platelet P2Y receptors, although activate PLC, do not activate G12/13 pathways. When RhoA, downstream signaling molecule in G12/13 pathways, is blocked, PAR-mediated dense granule release is inhibited. Furthermore, ADP activated Rho A downstream of Gq and upstream of PLC. Finally, Rho A regulated PKCδ T505 phosphorylation, suggesting that Rho A pathways contribute to platelet secretion through PKCδ activation. We conclude that G12/13 pathways, through RhoA, regulate dense granule release and fibrinogen receptor activation in platelets.