Identification of ecto-PKC on surface of human platelets: role in maintenance of latent fibrinogen receptors

• Published in: American journal of physiology. Heart and circulatory physiology 2000-06, Vol.278 (6), p.H2008-H2019
• Main Authors: Babinska, Anna, Hogan, Michael V, Sobocki, Tomasz, Sobocka, Malgorzata B, Ehrlich, Yigal H, Kornecki, Elizabeth
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - metabolism; Adult; Azides - metabolism; Blood Platelets - enzymology; Calcium - metabolism; Female; Homeostasis; Humans; Intracellular Membranes - metabolism; Magnesium - physiology; Male; Membrane Proteins - metabolism; Middle Aged; Phosphorylation; Platelet Aggregation - physiology; Platelet Glycoprotein GPIIb-IIIa Complex - physiology; Protein Kinase C - blood; Protein Kinases - blood
• ISSN: 0363-6135; 1522-1539
• DOI: 10.1152/ajpheart.2000.278.6.h2008

1  Department of Anatomy and Cell Biology, State University of New York, Health Science Center at Brooklyn, Brooklyn 11203; and the 2  College of Staten Island/Institute for Basic Research Center for Developmental Neuroscience, City University of New York, College of Staten Island, Staten Island, New York 11314 Human platelets express a protein phosphorylation system on their surface. A specific protein kinase C (PKC) antibody, monoclonal antibody (MAb) 1.9, which binds to the catalytic domain of PKC and inhibits its activity, causes the aggregation of intact platelets while inhibiting the phosphorylation of platelet surface proteins. Photoaffinity labeling with 100 nM 8-azido-[ 32 P]ATP identified this ecto-PKC as a single surface protein of 43 kDa sensitive to proteolysis by extracellular 0.0005% trypsin. Inhibition of the binding of 8-azido-[ 32 P]ATP to the 43-kDa surface protein by MAb 1.9 identified this site as the active domain of ecto-PKC. Covalent binding of the azido-ATP molecule to the 43-kDa surface protein inhibited the phosphorylative activity of the platelet ecto-PKC. Furthermore, PKC pseudosubstrate inhibitory peptides directly induced the aggregation of platelets and inhibited azido-ATP binding to the 43-kDa protein. Platelet aggregation induced by MAb 1.9 and by PKC inhibitory peptides required the presence of fibrinogen and resulted in an increase in the level of intracellular free calcium concentration. This increase in intracellular free calcium concentration induced by MAb 1.9 was found to be dependent on the binding of fibrinogen to activated GPIIb/IIIa integrins, suggesting that MAb 1.9 causes Ca 2+ flux through the fibrinogen receptor complex. We conclude that a decrease in the state of phosphorylation of platelet surface proteins caused by inhibition of ecto-PKC results in membrane rearrangements that can induce the activation of latent fibrinogen receptors, leading to platelet aggregation. Accordingly, the maintenance of a physiological steady state of phosphorylation of proteins on the platelet surface by ecto-PKC activity appears to be one of the homeostatic mechanisms that maintain fibrinogen receptors of circulating platelets in a latent state that cannot bind fibrinogen. ecto-protein kinase; extracellular adenosine 5'-triphosphate; glycoprotein IIb/IIIa; azido adenosine 5'-triphosphate binding; surface protein phosphorylation; intracellular Ca 2+ *  M. V. Hogan and T. Sobocki contributed equally to this work and their names