Asparaginyl-arginine

Platelets contain high levels of Src family kinases (SFKs), but their functional role downstream of G protein pathways has not been completely understood. We found that platelet shape change induced by selective G(12/13) stimulation was potentiated by SFK inhibitors, which was abolished by intracellular calcium chelation. Platelet aggregation, secretion, and intracellular Ca(2+) mobilization mediated by low concentrations of SFLLRN or YFLLRNP were potentiated by SFK inhibitors. However, 2-methylthio-ADP-induced intracellular Ca(2+) mobilization and platelet aggregation were not affected by PP2, suggesting the contribution of SFKs downstream of G(12/13), but not G(q)/G(i), as a negative regulator to platelet activation. Moreover, PP2 potentiated YFLLRNP- and AYPGKF-induced PKC activation, indicating that SFKs downstream of G(12/13) regulate platelet responses through the negative regulation of PKC activation as well as calcium response. SFK inhibitors failed to potentiate platelet responses in the presence of G(q)-selective inhibitor YM254890 or in G(q)-deficient platelets, indicating that SFKs negatively regulate platelet responses through modulation of G(q) pathways. Importantly, AYPGKF-induced platelet aggregation and PKC activation were potentiated in Fyn-deficient but not in Lyn-deficient mice compared with wild-type littermates. We conclude that SFKs, especially Fyn, activated downstream of G(12/13) negatively regulate platelet responses by inhibiting intracellular calcium mobilization and PKC activation through G(q) pathways.

Platelet secretion is an important physiological event in hemostasis. The protease-activated receptors, PAR 1 and PAR 4, and the thromboxane receptor activate the G(12/13) pathways, in addition to the G(q) pathways. Here, we investigated the contribution of G(12/13) pathways to platelet dense granule release. 2MeSADP, which does not activate G(12/13) pathways, does not cause dense granule release in aspirin-treated platelets. However, supplementing 2MeSADP with YFLLRNP (60muM), as selective activator of G(12/13) pathways, resulted in dense granule release. Similarly, supplementing PLC activation with G(12/13) stimulation also leads to dense granule release. These results demonstrate that supplemental signaling from G(12/13) is required for G(q)-mediated dense granule release and that ADP fails to cause dense granule release because the platelet P2Y receptors, although activate PLC, do not activate G(12/13) pathways. When RhoA, downstream signaling molecule in G(12/13) pathways, is blocked, PAR-mediated dense granule release is inhibited. Furthermore, ADP activated RhoA downstream of G(q) and upstream of PLC. Finally, RhoA regulated PKCdelta T505 phosphorylation, suggesting that RhoA pathways contribute to platelet secretion through PKCdelta activation. We conclude that G(12/13) pathways, through RhoA, regulate dense granule release and fibrinogen receptor activation in platelets.

Rho/Rho-kinase downstream of G12/13 plays an important role in the regulation of calcium-independent platelet shape change. We have previously shown that proline-rich tyrosine kinase 2 (Pyk2) is activated downstream of G12/13 pathways. In this study, we evaluated the role of Pyk2 in G12/13-induced platelet shape change. We used low concentrations of YFLLRNP, a heptapeptide binding to protease-activated receptor 1 (PAR1), or PAR4-activating peptide AYPGKF in the presence of Gαq inhibitor YM254890 to selectively stimulate G12/13 pathways. We found that G12/13-induced platelet shape change was completely inhibited in the presence of Pyk2 inhibitors AG17 and TAT-Pyk2-CT, suggesting an important role of Pyk2 in platelet shape change. In addition, AYPGKF-induced shape change in Gq -/- platelets was completely inhibited in the presence of AG17 or RhoA/p160ROCK inhibitor Y27632, confirming the role of Pyk2 in RhoA-dependent shape change. Furthermore, AYPGKF-induced platelet aggregation and dense granule secretion were inhibited by blocking Pyk2 or RhoA. Finally, G12/13-induced myosin phosphatase target subunit 1 (MYPT1) phosphorylation was inhibited by AG17, confirming that Pyk2 regulates RhoA/p160ROCK activation in platelets. These results demonstrate that Pyk2 downstream of G12/13 pathways regulates platelet shape change as well as platelet aggregation and dense granule secretion through the regulation of RhoA/p160ROCK.