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 RhoA downstream of Gq and upstream of PLC. Finally, RhoA regulated PKCδ T505 phosphorylation, suggesting that RhoA 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.