RhoA downstream of G q and G 12/13 pathways regulates protease-activated receptor-mediated dense granule release in platelets

Abstract

Platelet secretion is an important physiological event in hemostasis. The protease-activated receptors, PAR 1 and PAR 4, and the thromboxane receptor activate the G 12/13 pathways, in addition to the G q pathways. Here, we investigated the contribution of G 12/13 pathways to platelet dense granule release. 2MeSADP, which does not activate G 12/13 pathways, does not cause dense granule release in aspirin-treated platelets. However, supplementing 2MeSADP with YFLLRNP (60 μM), as selective activator of G 12/13 pathways, resulted in dense granule release. Similarly, supplementing PLC activation with G 12/13 stimulation also leads to dense granule release. These results demonstrate that supplemental signaling from G 12/13 is required for G q-mediated dense granule release and that ADP fails to cause dense granule release because the platelet P2Y receptors, although activate PLC, do not activate G 12/13 pathways. When RhoA, downstream signaling molecule in G 12/13 pathways, is blocked, PAR-mediated dense granule release is inhibited. Furthermore, ADP activated RhoA downstream of G q and upstream of PLC. Finally, RhoA regulated PKCδ T505 phosphorylation, suggesting that RhoA pathways contribute to platelet secretion through PKCδ activation. We conclude that G 12/13 pathways, through RhoA, regulate dense granule release and fibrinogen receptor activation in platelets.