ANTI-CD9 MONOCLONAL-ANTIBODY ACTIVATES P72(SYK) IN HUMAN PLATELETS

NNKY 1-19, anti-CD9 monoclonal antibody (MoAb), induced protein tyrosi ne phosphorylation of 125-, 97-, 75-, 64-, and 40-kDa proteins in huma n platelets, whereas F(ab')(2) fragments of NNKY 1-19 did not, suggest ing that the stimulation of Fc gamma II receptors is required for the induction of protein tyrosine phosphorylation. Tyrosine-phosphorylated proteins of 97 and 125 kDa were associated with aggregation, while NN KY 1-19-induced protein tyrosine phosphorylation was completely inhibi ted by prostaglandin I-2 (PGI(2)). The activity of p72(syk) was assess ed in immunoprecipitation kinase assays to determine at which step the signal transduction pathway leading to protein tyrosine phosphorylati on was suspended. NNKY 1-19 induced a rapid and transient increase in the p72(syk)-associated tyrosine kinase activity that peaked at 10 s a nd subsided to the original level a min after stimulation. Coinciding with this time course, p60(c-src) transiently associated with p72(syk) . In platelets preexposed to GRGDS peptides or PGI(2), NNKY 1-19 also increased the p72(syk)-associated tyrosine kinase activity and led to the association of p60(c-src) with p72(syk), However, in contrast to t he control without any inhibitor, the elevated tyrosine kinase activit y and the associated state of the two tyrosine kinases persisted as lo ng as 5 min after stimulation, F(ab')(2) fragments of NNKY 1-19 induce d changes similar to those observed with the effects of GRGDS peptides or PGI(2) treatment on intact IgG NNKY 1-19 stimulation. F(ab')(2) fr agments of another CD9 MoAb, PMA2, had effects on p72(syk) essentially similar to those of NNKY 1-19. These findings suggest that the bindin g of anti-CDS MoAb to CD9 on the platelet membrane per se induces an i ncrease in the p72(syk)-associated tyrosine kinase activity but that F c gamma II receptor-mediated signal(s) is required for the full activa tion of platelets and the appearance of tyrosine-phosphorylated protei ns. The elevated intracellular cAMP level induced by PGI(2) acts at a step distal to the activation of p72(syk) and inhibited the signal tra nsduction pathway leading to protein tyrosine phosphorylation and aggr egation. p72(syk) activation occurs in the absence of aggregation, but aggregation appears to reduce the elevated p72(syk) activity induced by anti-CDS MoAb.