Signaling through Gi Family Members in Platelets

Abstract

Platelet responses at sites of vascular injury are regulated by intracellular cAMP levels, which rise rapidly when prostacyclin (PGI₂) is released from endothelial cells. Platelet agonists such as ADP and epinephrine suppress PGI₂-stimulated cAMP formation by activating receptors coupled to G_i family members, four of which are present in platelets. To address questions about the specificity of receptor:G protein coupling, the regulation of cAMP formation in vivo and the contribution of G_i-mediated pathways that do not involve adenylyl cyclase, we studied platelets from mice that lacked the α subunits of one or more of the three most abundantly expressed G_i family members and compared the results with platelets from mice that lacked the PGI₂receptor, IP. As reported previously, loss of G_{i2 α} or G_{z α}inhibited aggregation in response to ADP and epinephrine, respectively, producing defects that could not be reversed by adding an adenylyl cyclase inhibitor. Platelets that lacked both G_{i2 α} and G_{z α} showed impaired responses to both agonists, but the impairment was no greater than in the individual knockouts. Loss of G_{i3 α} had no effect either alone or in combination with G_{z α}. Loss of either G_{z α} or G_{i2 α}impaired the ability of ADP and epinephrine to inhibit PGI₂-stimulated adenylyl cyclase activity and caused a 40%-50% rise in basal cAMP levels, whereas loss of G_{i3 α} did not. Conversely, deletion of IP abolished responses to PGI₂ and caused cAMP levels to fall by 30%, effects that did not translate into enhanced responsiveness to agonists ex vivo. From these results we conclude that 1) cAMP levels in circulating platelets reflect ongoing signaling through G_i2, G_z, and IP, but not G_i3; 2) platelet epinephrine (α_2A-adrenergic) and ADP (P2Y12) receptors display strong preferences among G_i family members with little evidence of redundancy; and 3) these receptor preferences do not extend to G_i3. Finally, the failure of ADP and epinephrine to inhibit basal, as opposed to PGI₂-stimulated, cAMP formation highlights the need during platelet activation for G_i signaling pathways that involve effectors other than adenylyl cyclase.