Activation of the mitogen-activated protein (MAP) kinase pathway in nucleated cells is dependent on both growth factor receptors and integrins engaged in cell adhesion. Human platelets are an interesting model for studying cell adhesion and the involvement of integrin engagement on extracellular signal-regulated kinase (ERK) activation, independently from the nuclear-DNA signal pathway. Maximal phosphorylation and activity of ERK2 occurred late during thrombin-induced platelet aggregation (90 s and later), an ␣ IIb ␤ 3 integrin-dependent event. Surprisingly, ␣ IIb ␤ 3 inhibition by the RGDS ligand peptide, or (Fab) 2 fragments of the AP-2 monoclonal antibody, resulted in a 2-fold enhancement in ERK2 phosphorylation and activity. A similar 2-fold enhancement of ERK2 activation was observed in thrombasthenic platelets which are defective in ␣ IIb ␤ 3 and do not aggregate. This suggests that ERK2 activation in thrombin-induced platelet aggregation is dependent on thrombin rather than on ␣ IIb ␤ 3 and is down-regulated by ␣ IIb ␤ 3 engaged in ligand (fibrinogen) binding and/or aggregation. Finally, in the absence of stirring which allows fibrinogen binding to ␣ IIb ␤ 3 but prevents aggregation, ERK2 was again overactivated. This overactivation appears to be consecutive to inhibition of aggregation itself and to ␣ IIb ␤ 3 ligand binding. We conclude that in platelets, ␣ IIb ␤ 3 engaged in aggregation down-regulates thrombin-induced ERK2 activation. To our knowledge, this is the first report of a down-regulation of the MAP kinase pathway by integrin engagement.

and pleckstrin @7 b,Da) (12). The hydrolysis of Pi Pz results also from the stimulation of Pl-Cywhich is independent of G protein and requires tyrosine kinase activity. 2) Thrombin Eeatment causes also a dramatic increase in the level of phosphotyrosines on multiple proteins which occur in three temporal waves (3,4), the third one being dependent on aIIbBr integrin engagement and plateletag gregation, since agents that block fibrinogen binding inhibit the tyrosine phosphorylation of the last wave. The increase in tyrosine phosphorylation is associated with the activation and subcellular relocation of a number of non receptor protein tyrosine kinases (PTKs) such as Src family kinases (60 kDa), Syk (72 lDa) and FAI((125 kDa). In addition to their catalytic domain shared by all PTK, Syk contains two additional src homology (SH2) and one SH3 domains. SH2 and SH3 domains play critical roles in regulating infra and intermolecular proteinprotein interactions (5,6). 3) Following agonist occupation of a diverse range of cell surface rcceptors, a phosphoinositide 3-kinase (Pi3 kinase) cataly zes the pho sphorylati on of ino sitolphosph olipids on the 3 position of the inositol ring (7). In platelets Pi3 kinase activation gives rise to phophatidylinositol 3, 4 Pz (Pr 3APz) andphophatidylinositol 3,4,5 P3 (Pi3,4,5P3) which are considered as second messengers (8). 4) The Mitogen Activated Protein Kinase (MAPK) is a conseryed eukaryotic signaling molecule that converts receptor signals into a variety of outputs. MAPK patways are extensively used for transcytoplasmic signaling in the nucleus where transcription of specific genes is induced through phosphorylation and activation of transcription factors (9). Platelet stimulation also leads to stimulation of the MAP kinase pathway (10, 11) in which low molecular weight GTP binding proteins such as 'tas" could participate (12). Once platelets are activated, unidentified intracellular events (PKC ?) acton oIIbFr to induce binding of fibrinogen to the exEacellular domain of the receptor (13). As a result, this integrin transmits signals. The outside-in integrin signaling through oIIbFr is critical for platelet function. It causes increased tyrosine phosphorylation of signaling proteins such as Syk and EAK, increased Ca2* mobilization, calpain activation, and increased cytoskeletal organization.

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