No abstract

Extracellular signal-regulated kinase (ERK) activation pathways have been well characterized in a number of cell types but very few data are available for platelets. The thrombin-induced signaling pathway leading to ERK2 activation in platelets is largely uncharacterized. In this study, we investigated the kinases involved in thrombin-induced ERK2 activation in conditions of maximal ERK2 activation. We found that thrombin-induced mitogen-activated protein kinase/ERK kinase (MEK)1/2 activation was necessary for ERK2 phosphorylation. We obtained strong evidence that conventional protein kinase Cs (PKCs) and calcium are involved in thrombin-induced ERK2 activation. First, ERK2 and MEK1/2 phosphorylation was totally inhibited by low concentrations (1 W WM) of RO318425, a speci¢c inhibitor of conventional PKCs. Second, Ca 2+ , from either intracellular pools or the extracellular medium, was necessary for ERK2 activation and conventional PKC activation, excluding the involvement of a new class of calcium-insensitive PKCs. Third, LY294002 and wortmannin had no signi¢cant e¡ect on ERK2 activation, even at concentrations that inhibit phosphatidylinositol (PI)3-kinase (5 W WM to 25 W WM and 50 nM, respectively). This suggests that PI3-kinase was not necessary for ERK2 activation and therefore, that PI3-kinase-dependent atypical PKCs were not involved. Surprisingly, in contrast to proliferative cells, we found that the serine/threonine kinases Raf-1 and B-Raf were not an intermediate kinase between conventional PKCs and MEK1/2. After immunoprecipitation of Raf-1 and B-Raf, the basal glutathione S-transferase^MEK1 phosphorylation observed in resting platelets was not upregulated by thrombin and was still observed in the absence of anti-Raf-1 or anti-B-Raf antibodies. In these conditions, the in vitro cascade kinase assay did not detect any MEK activity. Thus in platelets, thrombin-induced ERK2 activation is activated by conventional PKCs independently of Raf-1 and B-Raf activation. ß

The stimulation of platelets by low doses of collagen induces extracellular signal-regulated kinase 2 (ERK2) activation. In this report, we demonstrate that collagen-induced ERK2 activation depends on thromboxane A 2 (TXA 2 ) formation and ADP release. The collagen-induced ERK2 activation was inhibited by indomethacin (88%) and by AR-C69931MX (70%), a speci¢c antagonist of P2Y12, a Gi-coupled ADP receptor. AR-C69931MX (10 W WM) inhibition was overcome by epinephrine (1 W WM), an agonist of the Gi-coupled K K 2A -adrenergic receptor, suggesting that the Gi-coupled receptor was necessary for ERK2 activation by collagen. By contrast, MRS 2179 (10 W WM), a speci¢c antagonist of P2Y1, a Gq-coupled ADP receptor, did not a¡ect collagen-induced ERK2 activation. Little or no ERK2 activation was observed with ADP alone (10 W WM). By contrast, U46619 (10 W WM), a stable analog of TXA 2 , induced ERK2 activation in an ADP-dependent manner, via the P2Y12 receptor. These results suggest that the Gi-dependent signaling pathway, stimulated by ADP or epinephrine, was not the only pathway required for ERK2 activation by collagen. Costimulation of the speci¢c G 12=13 -coupled TXA 2 receptor with a low dose of U46619 (10 nM) and of Gi-and Gq-coupled ADP receptor (10 W WM) induced very low levels of ERK2 activation, similar to those observed with ADP alone, suggesting that G 12=13 is not involved or not su⁄cient to induce the additional pathway necessary for ERK2 activation. The Gq-coupled TXA 2 receptor was required for ERK2 activation by U46619 (10 W WM) and low doses of collagen, clearly showing that a coordinated pathway through both Gq from TXA 2 and Gi from ADP was necessary for ERK2 activation. Finally, we demonstrate that ERK2 activation is involved in collagen-induced aggregation and secretion.