Phosphorylation of human platelet glycoprotein IIIa (GPIIIa). Dissociation from fibrinogen receptor activation and phosphorylation of GPIIIa in vitro.
Glycoprotein IIb-IIIa (GPIIb-IIIa) is the fibrinogen receptor on activated platelets. GPIIIa is phosphorylated in resting platelets and the incorporation of 32Pi increases with platelet activation. To address the functional significance of this modification, the stoichiometry of GPIIIa phosphorylation was determined in resting and activated platelets by estimating the specific activity of metabolic [gamma-32P]ATP from the specific activity of phosphatidic acid. Approximately 0.01 mol of P/mol of GPIIIa was phosphorylated in resting platelets and 0.03 mol of P/mol of GPIIIa was phosphorylated in thrombin-, phorbol ester-, or U46619-treated platelets. Myosin light chain (MLC) phosphorylation served as a positive control for this method (1.2 mol of P/mol of MLC). Phosphorylation of purified GPIIb-IIIa by human platelet protein kinase C (PKC) resulted in levels of GPIIIa phosphorylation similar to that in platelets (0.05 mol of P/mol of GPIIIa). However, while GPIIIa in platelets was phosphorylated primarily on threonine, purified GPIIIa treated with PKC was phosphorylated primarily on serine. These results suggest that PKC may not directly phosphorylate GPIIIa in intact platelets. Ca2+/calmodulin-dependent kinase II phosphorylated purified GPIIIa to higher levels (0.5 mol of P/mol of GPIIIa) with phosphorylation on both threonine and serine. The limited phosphorylation of GPIIIa in intact platelets suggests that this event is unlikely to affect functions involving large populations of GPIIb-IIIa, such as its conversion to a fibrinogen receptor. However, these results may suggest the existence of a more readily phosphorylated subpopulation of GPIIb-IIIa with potentially distinct structural or functional properties.
Hillery
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CA
Department of Pediatrics, School of Medicine, University of North Carolina, Chapel Hill 27599.
Smyth
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SS
Parise
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D000596:Amino Acids / Q000032:analysis; D001345:Autoradiography; D001792:Blood Platelets / Q000201:enzymology / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation; D015539:Platelet Activation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011233:Precipitin Tests; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
D000596:Amino Acids; D010703:Phorbol Esters; D010980:Platelet Membrane Glycoproteins; D011450:Prostaglandin Endoperoxides, Synthetic; D011494:Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D013917:Thrombin