Functional implications of tyrosine protein phosphorylation in platelets : simultaneous studies with different agonists and inhibitors

• Published in: Biochemical journal 1992-06, Vol.284 (3), p.923-928
• Main Authors: BACHELOT, C, CANO, E, GRELAC, F, SALEUN, S, DRUKER, B. J, LEVY-TOLEDANO, S, FISCHER, S, RENDU, F
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Blood coagulation. Blood cells; Blood Platelets - drug effects; Blood Platelets - metabolism; Blood Proteins - isolation & purification; Blood Proteins - metabolism; Collagen - pharmacology; Disulfides - pharmacology; Fundamental and applied biological sciences. Psychology; Humans; In Vitro Techniques; Models, Biological; Molecular and cellular biology; Oligopeptides - pharmacology; Phosphoproteins - blood; Phosphoproteins - isolation & purification; Phosphorylation; Plant Extracts - pharmacology; Platelet; Platelet Activation; Platelet Aggregation - drug effects; Platelet Aggregation Inhibitors - pharmacology; Platelet Membrane Glycoproteins - metabolism; Protein Kinase C - antagonists & inhibitors; Protein Kinase C - blood; Tetradecanoylphorbol Acetate - pharmacology; Thrombin - pharmacology; Tyrosine
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2840923

During activation of platelets by agonists, a number of proteins become phosphorylated at tyrosine residues. Using immunoblotting with a monoclonal anti-phosphotyrosine antibody, we have compared the different phosphotyrosine-protein (PTP) profiles of platelets stimulated with thrombin, collagen, ADP, arachidonic acid, phorbol myristate acetate and P256, an anti-glycoprotein-IIb-IIIa (GPIIb-IIIa) monoclonal antibody (mAb). Only a few PTPs were observed in resting platelets, of molecular masses 130, 64, 56-60 and 36 kDa. After stimulation by different agonists these proteins were more intensely phosphorylated and additional PTPs appeared with molecular masses of 170, 150, 140, 120, 105/97 (doublet), 85, 80, 75 and 45 kDa. The kinetics of phosphorylation differed from one agonist to another, but no significant differences in the overall patterns were detected, except in presence of ADP and P256-F(ab')2, which induced only the additional tyrosine phosphorylation of the 64 kDa protein and to a lesser extent that of a 75 kDa protein. The use of various agonists and the inhibitors (staurosporine, ajoene and RGDS) permitted a better characterization of the relationship between the different steps of activation and phosphorylation on tyrosine residues. The studies suggest the following conclusions: (i) stimulation of tyrosine phosphorylation occurs after activation of protein kinase C; (ii) there is a relationship between ligand binding to GPIIb-IIIa and the tyrosine phosphorylation of the 64 kDa protein; and (iii) there is a close relationship between PTP formation and the intensity of platelet activation and aggregation.