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

Abstract

ADP-induced TXA 2 generation requires the costimulation of P2Y 1 , P2Y 12 , and the GPIIb/IIIa receptors. Signaling events downstream of the P2Y receptors that contribute to ADP-induced TXA 2 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 P2Y 12 receptor, in the regulation of ADP-induced TXA 2 generation. At 10-M concentrations, the 2 structurally distinct GIRK channel blockers, SCH23390 and U50488H, caused complete inhibition of ADP-induced cPLA 2 phosphorylation and TXA 2 generation, without affecting the conversion of AA to TXA 2 or ADP-induced primary platelet aggregation in aspirintreated platelets. In addition, Src family kinase selective inhibitors abolished 2Me-SADP-mediated cPLA 2 phosphorylation and TXA 2 generation. Furthermore, these GIRK channel blockers completely blocked G i -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 TXA 2 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 TXA 2 generation, via the regulation of the Src and IntroductionPlatelets are involved in the maintenance of hemostasis, and abnormal platelet activation could initiate acute thrombotic events. Platelet activation is associated with platelet shape change, granule secretion, aggregation, and the generation of positive feedback mediators such as adenosine diphosphate (ADP) (secreted), thrombin, and thromboxane A 2 (TXA 2 ) (generated). ADP, a very important platelet agonist and a key mediator of hemostasis and thrombosis, 1,2 is secreted from the platelet-dense granules following their stimulation by physiological platelet agonists such as TXA 2 , collagen, and thrombin. Released ADP consolidates and stabilizes the growing thrombus by acting via the G-proteincoupled P2Y 1 and P2Y 12 receptors. 3-5 G q -coupled P2Y 1 receptor stimulation results in phospholipase C ␤ (PLC ␤)-mediated platelet shape change, whereas the P2Y 12 receptor stimulation results in G␣ i -mediated inhibition of adenylyl cyclase and G␤␥-mediated activation of PI3-K␥, 6,7 Rap1B,8,9 and GIRKs. 10 Marked inhibition of platelet aggregation and prolonged bleeding times in patients with mutations in the P2Y 12 receptor, 11 in P2Y 12 -or G␣ i2 -null mice, 12,13 or in the presence of P2Y 12 receptor antagonists such as clopidogrel 14 confirm their critical role in the maintenance of hemostasis.TXA 2 is a potent platelet agonist and an arachidonic acid (AA) metabolite, produced via the cyclooxygenase pathway. 15 Activation of cytosolic phospholipase A 2 (cPLA 2 ) has been shown to be important for the release of AA from the sn-2 position of membrane-bound phospholipids. 16 Ph...

An Important Role of the Src Family Kinase Lyn in Stimulating Platelet Granule Secretion

“…However, Lyn knock-out platelets have also been shown to potentiate platelet aggregation and secretion induced by GPVI-selective agonists (37), suggesting the dual roles of Lyn in GPVI-mediated platelet activation. Similarly controversial, although one study suggests that SFKs are not required in thrombin-induced platelet aggregation (38), other studies indicate that SFK stimulates platelet activation induced by thrombin (39) and ADP (40,41). The controversies about the roles of Lyn in platelet activation are in a way similar to the controversies about the role of the NOcGMP pathway in platelet activation in that both stimulatory and inhibitory roles of this enzyme have been reported.…”

“…These results suggest that an important role of Lyn in platelet activation induced by collagen is to mediate ADP secretion. Previously, it has been shown that SFKs are important in stimulating TXA 2 synthesis (40,46), which is important in collagen-induced platelet aggregation. We found that collagen-induced TXA 2 production was totally abolished by PP2 in human platelets, which was not corrected by ADP supplementation (data not shown).…”

The Src Family Kinases and Protein Kinase C Synergize to Mediate Gq-dependent Platelet Activation

“…However, a very recent study reported that the SFK Fyn activated downstream of G 12/13 inhibits G q -dependent intracellular calcium mobilization, PKC activation, and thrombin-induced platelet aggregation (22). SFKs also play a role in G i -dependent platelet activation and TXA 2 synthesis (23,24).…”

Differential signaling mechanisms of HNP‐induced IL‐8 production in human lung epithelial cells and monocytes

“…The present study was to examine the downstream signaling mechanisms. Since Src tyrosine kinases is known to be a downstream effector of P2Y 2 signaling (Santiago-Perez et al, 2001;Katz et al, 2006;Shankar et al, 2006), thus we thought the HNP induced-IL-8 expression is independent of Src activation because of a lack of SH3 binding motif to Src kinase in the P2Y 6 (NCBI, April 2007). Interestingly, we demonstrate that stimulation with HNP resulted in Src kinase phosphorylation that is required for IL-8 production in both cell types studied but the specific phenotype(s) of the Src family kinases is yet to be identified.…”