The interplay of G protein-coupled signaling pathways in platelet activation

The platelet contains many receptors and intracellular signaling pathways that regulate platelet aggregation and procoagulant responses. We have characterized the receptor and signaling requirements for these platelet responses. PAR1 or PAR4-stimulation alone did not cause marked platelet prothrombinase activity, nor could they potentiate collagen-induced prothrombinase activity to a similar level as thrombin. Either receptor alone could not cause increases of phosphatidylserine exposure. Combined stimulation of both PAR receptors caused comparable levels of prothrombinase activity when compared with thrombin, though phosphatidylserine exposure remained less than levels caused by thrombin. Antagonism of the P2Y12, but not the P2Y1 receptor, caused a decrease in collagen- or thrombin-induced prothrombinase activity. Thus, platelet procoagulant responses are mediated by the stimulation of multiple receptors.; Activation of GPIIb/IIIa requires agonist-induced inside-out signaling through Gq, Gi, and Gz. We used selective stimulation of G protein pathways to investigate the contribution of G 12/13 activation to platelet fibrinogen receptor activation. YFLLRNP-mediated G12/13 signaling caused platelet aggregation and enhanced PAC-1 binding when combined with Gi signaling via P2Y12 or alpha 2A adrenergic receptor stimulation. Co-activation of both G12/13 and Gi resulted in an increase in intracellular calcium, and chelation of intracellular calcium decreased G12/13 and Gi-mediated platelet aggregation. Given that either G q or G12/13 pathways can synergize with Gi to cause platelet aggregation, we tested if the signaling downstream of the alpha 2A adrenergic receptor can also cause platelet fibrinogen receptor activation in the presence of Gi signaling. While neither P2Y12 activation nor alpha2A adrenergic receptor stimulation caused platelet aggregation, a combination of signaling through both of these receptors caused partial platelet aggregation without shape change. When P2Y12 receptor stimulation is combined with alpha2A adrenergic receptor stimulation, there is a small mobilization of intracellular calcium. Thus, the P2Y12 receptor is capable of causing aggregation if it is combined with either the G 12/13 pathway or alpha2A adrenergic receptor stimulation. Our results suggest that these two mechanisms of platelet aggregation are likely to involve distinct signaling that converge on GPIIb/IIIa activation.