We investigated the role of two MAP kinases, ERK2 and p38, in platelet adhesion and spreading over collagen matrix in static and blood flow conditions. P38 was involved in collagen-induced platelet adhesion and spreading in static adhesion conditions, whereas ERK2 was not. In blood flow conditions, with shear rates of 300 or 1500 s ؊1 , ERK2 and p38 displayed differential involvement in platelet adhesion, depending on the presence or absence of the von Willebrand factor (vWF). Low collagen coverage densities (0.04 g/cm 2 ) did not support vWF binding. During perfusions over this surface, platelet adhesion was not affected by the inhibition of ERK2 phosphorylation by PD 98059. However, abolishing p38 activation by SB 203580 treatment reduced platelet adhesion by 67 ؎ 9% at 300 s ؊1 and 56 ؎ 2% at 1500 s ؊1 . In these conditions, the p38 activity required for platelet adhesion depends on the ␣2␤1 collagen receptor. At higher collagen coverage densities (0.8 g/cm 2 ) supporting vWF binding, the inhibition of ERK2 activity by PD 98059 decreased adhesion by 47 ؎ 6% at 300 s ؊1 and 72 ؎ 3% at 1500 s ؊1 , whereas p38 inhibition had only a small effect. The ERK2 activity required for platelet adhesion was dependent on the interaction of vWF with GPIb. In conclusion, ERK2 and p38 have complementary effects in the control of platelet adhesion to collagen in a shear stress-dependent manner.The adhesion and aggregation of platelets in response to vascular injury plays a key role in hemostasis and thrombosis. Fibrillar collagen is the most abundant thrombogenic molecule among the macromolecular constituents of the subendothelial layer. Following exposure to collagen, platelets rapidly adhere, spread, become active, and then aggregate (1, 2). This interaction of collagen with platelets is both direct and indirect. Under the high shear stress conditions found in small arteries, the von Willebrand factor (vWF), 1 which binds to newly exposed collagen fibers, is required to capture flowing platelets (3) via the GPIb-IX-V complex on the platelet surface. This interaction (vWF-GPIb) cannot support the assembly of a stable platelet thrombus. Subsequent firm platelet adhesion and activation are mediated by two major surface receptors for collagen, the integrin ␣2␤1 and glycoprotein VI (4 -6). The relative contributions of these two receptors to collagen-mediated adhesion are unclear, and various models have been proposed (7). According to the two-site, two-step model, the major candidate for involvement in platelet deposition and stabilization on collagen is ␣2␤1, with GPVI mediating activation and initiating the signaling pathway involved in thrombus formation (8). Platelet activation leads to an increase in the affinity of ␣2␤1, which in turn leads to an increase in adhesion stability. Another model has been proposed in which the absence of functional GPVI impairs adhesion under static and shear conditions (9, 10). In this model, GPVI initiates signaling pathways, leading to the activation of the ␣2␤1 integrin and of other integri...

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.

Although the involvement of protease-activating receptor PAR1 and PAR4 is well established in platelet aggregation, their role in platelet adhesion and spreading has yet to be characterized. We investigated platelet adhesion and spreading on a fibrinogen matrix after PAR1 and PAR4 stimulation in correlation with the activation of two MAPKs, ERK2 and p38. Of the two PAR-activating peptides (PAR-APs), PAR1-AP and PAR4-AP, which both induce adhesion, only PAR4-AP induced full platelet spreading. Although both PAR1-AP and PAR4-AP induced ADP secretion, which is required for platelet spreading, only PAR4-AP induced sustained Ca(2+) mobilization. In these conditions of PAR4 induction, ERK2 and p38 activation were involved in platelet spreading but not in platelet adhesion. p38 phosphorylation was dependent on ADP signaling through P2Y12, its receptor. ERK2 phosphorylation was triggered through integrin alphaIIbbeta3 outside-in signaling and was dependent on the Rho pathway. ERK2 and p38 activation induced phosphorylation of the myosin light chain and actin polymerization, respectively, necessary for cytoskeleton reorganization. These findings provide the first evidence that thrombin requires PAR4 for the full spreading response. ERK2 and p38 and sustained Ca(2+) mobilization, involved in PAR4-induced platelet spreading, contribute to the stabilization of platelet thrombi at sites of high thrombin production.