Activation of G_12/13 Pathway Is Essential for the Dense Granule Release in Platelets.

Platelet secretion is an important physiological event in hemostasis. A number of agonists such as thrombin and thromboxane A2 induce platelet secretion. ADP does not cause dense granule release in aspirin-treated platelets, although ADP induce platelet shape change, aggregation and alpha granule release through G_q and G_i pathways. The protease activated receptors 1 and 4, and the thromboxane receptor activate the G_12/13 pathways in addition to the G_q pathways. We postulated that the platelet dense granule release reaction depends on both G_q and G_12/13, and in the absence of signaling through either G protein abolishes secretion. In other words, co-stimulation of G_q and G_12/13 pathways is a major requirement for dense granule release in platelets. We rationalize that because U46619 and thrombin can activate both G_q and G_12/13, they cause platelet dense granule release, whereas ADP fails to cause platelet secretion from dense granules because it does not activate G_12/13. As a first step towards testing this hypothesis, we supplemented ADP signaling in aspirin-treated platelets with selective activation of G_12/13 pathways using YFLLRNP, a partial agonist of PAR-1. YFLLRNP selectively active G_12/13 signaling pathway without activating G_q or G_i pathways at low concentrations. YFLLRNP (60 μM) or 2MeSADP (100 nM) failed to cause dense granule release. However, addition of YFLLRNP (60 μM) and 2MeSADP (100 nM) together caused dense granule release. We proceeded to confirm these results in G_q null mice by selectively activating phospholipase C (PLC), a downstream signaling molecule from G_q. In aspirin-treated Gα_q knockout mouse platelets 80 μM m-3M3FBS, a direct PLC activator, causes calcium mobilization from intracellular stores and platelet shape change, but does not cause dense granule secretion. We have previously shown that G_q/PLC pathways downstream of ADP are not sufficient for aggregation and require concomitant signaling from G_i for platelet aggregation. Thus, lack of aggregation by PLC activation alone in G?_q knockout mouse platelets is consistent with our previous observations. In G?_q null mouse platelets, m-3M3FBS (80 μM) or AYPGKF (500 μM) alone failed to cause dense granule release. However, addition of m-3M3FBS (80 μM) and AYPGKF (500 μM) together caused dense granule release. In addition, consistent with our previous findings, co-stimulation of G_12/13 pathways and G_i (AYPGKF + 2MeSADP) in G?_q knockout mouse platelets caused aggregation, but failed to cause dense granule release. We conclude 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.