Summary. Background: Thromboxane A 2 (TXA 2 ) is a positive feedback lipid mediator that is generated upon stimulation of platelets with various agonists. Aspirin works as an antithrombotic drug by blocking the generation of TXA 2 . The aim of this study was to evaluate the role of the purinergic P2Y receptors in thrombin-induced TXA 2 generation. Results: PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin, induced the activation of cytosolic phospholipase A 2 (cPLA 2 ), release of arachidonic acid (AA) from membranebound phospholipids, and subsequent TXA 2 generation in human platelets. The actions of these agonists were significantly inhibited in the presence of the P2Y 12 receptor antagonist, AR-C69931MX, but not the P2Y 1 receptor antagonist, MRS2179. In addition, AYPGKF-and thrombin-induced TXA 2 generation was significantly reduced in platelets from mice dosed with clopidogrel, confirming the results obtained with the human platelets. Also, Pearl mouse platelets that lack releasable nucleotides generated significantly less TXA 2 when compared with the wild-type littermates in response to PAR stimulation. Inhibition of extracellular signal-regulated protein kinase 1/2 (Erk 1/2) activation using U0126, an inhibitor of MAP kinase kinase (MEK), suppressed PAR-mediated cPLA 2 phosphorylation and TXA 2 generation. Further, platelets that were pretreated with AR-C69931MX, as well as Pearl mouse platelets, displayed the reduced levels of Erk1/2 phosphorylation upon stimulation with the PAR agonists. Conclusions: Based on these findings, we conclude that thrombin-induced Erk1/2 activation is essential for PAR-mediated TXA 2 generation, which is potentiated by the P2Y 12 receptor-mediated signaling pathway but not the P2Y 1 receptor-mediated signaling pathway. Finally, using selective inhibitors of Src kinases, we show that PAR-mediated Src activation precedes Erk1/2 activation.

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...

Platelet activation is associated with platelet shape change, fibrinogen receptor activation, platelet aggregation, secretion of granule contents and phospholipase A2 (PLA2) activation. Agonist-induced PLA2 activation is important for liberation of arachidonic acid from membrane phospholipids, which is then subsequently converted to thromboxane A2 (TXA2) via the sequential effects of cyclo-oxygenase and thromboxane synthase respectively. Thus the generated TXA2 plays a very predominant role in potentiating the effects of other physiological platelet agonists like collagen, thrombin and ADP. ADP-induced TXA2 generation requires co-stimulation of the P2Y1 and P2Y12 receptors, fibrinogen receptor activation and subsequent outside-in signaling. We recently demonstrated that G-protein gated inwardly rectifying potassium channels (GIRKs) are important for P2Y12 receptor-mediated platelet functional responses, namely platelet aggregation, dense granule secretion and Akt phosphorylation. In this study, we evaluated the role of GIRK channels in ADP- mediated TXA2 production in human platelets. ADP-mediated TXA2 generation was inhibited in the presence of SCH23390 and U50488H in a concentration-dependent manner, with maximal inhibition occurring at 10 μM. At these concentrations of GIRK blockers, none of the other P2Y12 receptor-dependent platelet functional responses including aggregation, Akt phosphorylations were affected. We confirmed our findings by performing similar experiments with 2-MeSADP, a potent and selective P2Y1 and P2Y12 agonist. We observed that GIRK channel blockers inhibit 2-MeSADP-mediated TXA2 generation. These results suggest the existence of two different populations of GIRK channels;-one that plays an important role in TXA2 formation (sensitive to low concentrations of the GIRK blockers) and the other population that plays an important role in platelet aggregation (sensitive to higher concentrations of GIRK blocker). Experiments done in the presence of SKF38393, a structurally similar but inactive analog of SCH23390 did not have any effect on ADP or 2-MeSADP-induced TXA2 production in the non-aspirinated platelets. Finally, we also observed that arachidonic acid-induced TXA2 production is not affected by 10 μM concentrations of SCH23390 and U50488H. These data suggest that GIRK channel blockers inhibit TXA2 formation by interfering with agonist-induced PLA2 activation. Based on these findings we conclude that there exist different populations of GIRK channels, one that contributes to ADP- and 2-MeSADP-induced TXA2 and the other that contributes to P2Y12 receptor-dependent platelet functional responses. Also, each of the two populations of GIRK channels has varying susceptibilities to the GIRK channel blockers.