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

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, cause s the aggregation of intact platelets while inhibiting the phosphorylation of platelet surface proteins. Photoaffinity labeling with 100 nM 8-azido-[a lpha(32)P] ATP identified this ecto-PKC as a single surface protein of 43 k Da sensitive to proteolysis by extracellular 0.0005% trypsin. Inhibition of the binding of 8-azido[alpha(32)P] ATP to the 43-kDa surface protein by MA b 1.9 identified this site as the active domain of ecto-PKC. Covalent bindi ng of the azido-ATP molecule to the 43-kDa surface protein inhibited the ph osphorylative activity of the platelet ecto-PKC. Furthermore, PKC pseudosub strate inhibitory peptides directly induced the aggregation of platelets an d inhibited azido-ATP binding to the 43-kDa protein. Platelet aggregation i nduced by MAb 1.9 and by PKC inhibitory peptides required the presence of f ibrinogen and resulted in an increase in the level of intracellular free ca lcium concentration. This increase in intracellular free calcium concentrat ion induced by MAb 1.9 was found to be dependent on the binding of fibrinog en to activated GPIIb/IIIa integrins, suggesting that MAb 1.9 causes Ca2+ f lux through the fibrinogen receptor complex. We conclude that a decrease in the state of phosphorylation of platelet surface proteins caused by inhibi tion of ecto-PKC results in membrane rearrangements that can induce the act ivation of latent fibrinogen receptors, leading to platelet aggregation. Ac cordingly, the maintenance of a physiological steady state of phosphorylati on of proteins on the platelet surface by ecto-PKC activity appears to be o ne of the homeostatic mechanisms that maintain fibrinogen receptors of circ ulating platelets in a latent state that cannot bind fibrinogen.