Activation of human platelets by inhibition of surface protein phosphorylation: role for ecto-protein kinase in platelet homeostasis+. Babinska, Anna, Yigal H. Ehrlich, and Elizabeth Kornecki. State University of New York Health Science Center at Brooklyn, New York and the CSI/IBR Center for Developmental Neuroscience, CUNY at CSI, Staten Island, New York
APStracts 3:0230H, 1996.
We have determined that a monoclonal antibody which inhibits the activity of protein kinase C (PKC) by specifically interacting with an epitope in its catalytic domain directly activates human platelets by causing aggregation, followed by granular secretion. This antibody, termed M.Ab.1.9, binds directly to the surface of intact platelets, as determined by flow cytometry and immunofluorescence microscopy. The performance of assays using extracellular [[gamma]32P]ATP (50-100nM) indicates that M.Ab.1.9 inhibits the phosphorylation of five proteins on the platelet surface by an ecto -protein kinase. In contrast, monoclonal antibodies directed against the regulatory domain of PKC do not inhibit ecto-protein kinase activity and are completely ineffective in causing platelet activation. On the other hand, PKC pseudosubstrate inhibitory peptides that inhibit the activity of PKC cause platelet activation similar to that produced by M.Ab.1.9. The activation of platelets by protein kinase C inhibitors is blocked by the addition of the membrane impermeable phosphatase inhibitor, microcystin. Thus, the inhibition of surface protein phosphorylation together with continuous dephosphorylation, namely, a decrease in the phosphorylation state of surface proteins, is the cause of the activation of platelets by M.Ab.1.9 and PKC pseudosubstrate peptides. Based on these findings we conclude that the platelet ecto- protein kinase has characteristics of a constitutively-active protein kinase C (now termed platelet ecto-PKC), and that phosphorylation of surface proteins by platelet ecto-PKC protects platelets from spontaneous aggregation, and thus can play an important role in homeostatic mechanisms that maintain circulating platelets in a resting state. 

Received 5 December 1995; accepted in final form 21 May 1996.
APS Manuscript Number H1130-5.
Article publication pending Am. J. Physiol. (Heart Circ. Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 5 June 96