G-protein–gated inwardly rectifying potassium channels regulate ADP-induced cPLA2 activity in platelets through Src family kinases

ADP-induced TXA2 generation requires the costimulation of P2Y1, P2Y12, and the GPIIb/IIIa receptors. Signaling events downstream of the P2Y receptors that contribute to ADP-induced TXA2 generation have not been clearly delineated. In this study, we have investigated the role of G-protein-gated inwardly rectifying potassium channels (GIRKs), a recently identified functional effector for the P2Y12 receptor, in the regulation of ADP-induced TXA2 generation. At 10-microM concentrations, the 2 structurally distinct GIRK channel blockers, SCH23390 and U50488H, caused complete inhibition of ADP-induced cPLA2 phosphorylation and TXA2 generation, without affecting the conversion of AA to TXA2 or ADP-induced primary platelet aggregation in aspirin-treated platelets. In addition, Src family kinase selective inhibitors abolished 2MeSADP-mediated cPLA2 phosphorylation and TXA2 generation. Furthermore, these GIRK channel blockers completely blocked Gi-mediated Src kinase activation, suggesting that GIRK channels are upstream of Src family tyrosine kinase activation. In weaver mouse platelets, which have dysfunctional GIRK2 subunits, ADP-induced TXA2 generation was impaired. However, we did not observe any defect in 2MeSADP-induced platelet functional responses in GIRK2-null mouse platelets, suggesting that functional channels composed of other GIRK subunits contribute to ADP-induced TXA2 generation, via the regulation of the Src and cPLA2 activity.