STIMULATORY ANTIBODY-INDUCED ACTIVATION AND SELECTIVE TRANSLOCATION OF PROTEIN-KINASE-C ISOENZYMES IN HUMAN PLATELETS

A novel stimulatory monoclonal antibody (Mab) termed Mab.F11 induces g ranular secretion and subsequent aggregation of human platelets. Mab.F 11 recognizes a unique 32 and 35 kDa protein duplex on the platelet me mbrane surface, called the F11 receptor; binding of Mab.F11 to its rec eptor results in increased intracellular phosphorylation of P47, the k nown protein kinase C (PKC) substrate pleckstrin. In order to determin e whether the mechanism of action of Mab.F11 involves direct activatio n of PKC, two types of functional assays for measuring PKC activity we re performed. Measurement of PKC activity in digitonin-permeabilized p latelets revealed that Mab.F11 produced a rapid, 2-3-fold increase in the control value in the phosphorylation of the PKC peptide substrate, PKC(19-31) Ser(25). The increase in PKC activity induced by Mab.F11 w as found to be associated with the platelet membrane; a 1.6-fold contr ol value increase in membrane PKC activity occurred rapidly, within 10 s of the addition of Mab.F11. The translocation from the cytoplasm to the membrane induced by Mab.F11 in PKC isoenzymes alpha and zeta was reversible, whereas translocation of the PKC isoenzymes delta, beta, e ta' and theta was irreversible, with PKC levels remaining elevated in the membrane for at least 15 min. Taken together, our results demonstr ate that in the initial stages of platelet activation by this stimulat ory antibody, the enhanced membrane PKC activity reflects the presence of all six isoenzymes. At later stages, PKC activity is reflective of four isoenzymes. These results demonstrate that separate groups of PK C isoenzymes must be involved in different aspects of platelet activat ion. The long lag period and prolonged activation time of platelets by Mab.F11 renders this agonist most suitable for identifying the isoenz ymes and their specific endogenous protein substrates involved in plat elet secretion and aggregation induced by platelet membrane protein an tibodies.