Functional implications of tyrosine protein phosphorylation in platelets. Simultaneous studies with different agonists and inhibitors.
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.
Bachelot
C
C
U150 INSERM, Hôp Lariboisière, Paris, France.
Cano
E
E
Grelac
F
F
Saleun
S
S
Druker
B J
BJ
Levy-Toledano
S
S
Fischer
S
S
Rendu
F
F
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003094:Collagen / Q000494:pharmacology; D004220:Disulfides / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008954:Models, Biological; D009842:Oligopeptides / Q000494:pharmacology; D010750:Phosphoproteins / Q000097:blood* / Q000302:isolation & purification; D010766:Phosphorylation; D010936:Plant Extracts / Q000494:pharmacology; D015539:Platelet Activation; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000097:blood; D013454:Sulfoxides; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D014443:Tyrosine
D001798:Blood Proteins; D004220:Disulfides; D009842:Oligopeptides; D010750:Phosphoproteins; D010936:Plant Extracts; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D013454:Sulfoxides; D014443:Tyrosine; D003094:Collagen; C048980:ajoene; C046617:arginyl-glycyl-aspartyl-serine; D011493:Protein Kinase C; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate