Dissociation of the alphaIIbbeta3-integrin by EGTA stimulates the tyrosine kinase pp72(syk) without inducing platelet activation.
Incubation of human platelets with EGTA under conditions that dissociate the alphaIIbbeta3-integrin stimulated tyrosine phosphorylation of pp72(syk) (6.8-fold) and of proteins of 62 (2. 2-fold), 68 (2.5-fold) and 130 kDa (1.4-fold). Stimulation of tyrosine phosphorylation of pp72(syk) was associated with an increase of pp72(syk) kinase activity. In contrast to pp72(syk), tyrosine phosphorylation of the focal adhesion kinase pp125(FAK) was not stimulated by EGTA. Preincubation of platelets with the monoclonal antibody P2, which binds to the alphaIIbbeta3 complex and thus stabilizes it, strongly reduced the increase of tyrosine phosphorylation of pp72(syk), p62, and p68 induced by EGTA. The Y2/51 monoclonal antibody, which recognizes only the beta3 glycoprotein, did not inhibit the stimulation of protein tyrosine phosphorylation evoked by EGTA. Stimulation of tyrosine phosphorylation of pp72(syk), p62, p68, and p130 induced by EGTA was not observed in thrombasthenic platelets, which lack the alphaIIbbeta3-integrin. The results indicate that the dissociation of the alphaIIbbeta3 complex in intact platelets activates pp72(syk). The mechanism of activation was found to be insensitive to inhibition by cAMP and cGMP and only partially dependent on cytosolic Ca2+, suggesting a close functional coupling of alphaIIbbeta3-integrin and pp72(syk). Since platelets retain their discoid shape after EGTA treatment, we further conclude that pp72(syk) stimulation alone is not sufficient for platelet activation.
Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, b. d. Universität München, Pettenkoferstrasse 9, 80336 Munich, Germany.
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Negrescu
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Siess
D000911:Antibodies, Monoclonal / Q000276:immunology; D004533:Egtazic Acid; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D047908:Intracellular Signaling Peptides and Proteins; D010766:Phosphorylation; D015539:Platelet Activation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000276:immunology / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D000072377:Syk Kinase; D014443:Tyrosine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D014443:Tyrosine; D004533:Egtazic Acid; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase