ANALYSIS OF CD9, CD32 AND P67 SIGNALING - USE OF DEGRANULATED PLATELETS INDICATES DIRECT INVOLVEMENT OF CD9 AND P67 IN INTEGRIN ACTIVATION

The use of agonist monoclonal antibodies (mAbs) to probe the signallin g function of platelet membrane proteins is severely limited by the de pendence of the mAb effect on Fc-Fc gamma RII interaction. Furthermore , in addition to its anchoring role, the Fc gamma RII receptor itself generates a stimulation signal resulting in granule secretion. Platele t stimulation by the released granule contents can then further obscur e the original activation signal. Here we demonstrate that these probl ems are largely overcome by the use of platelets which had been degran ulated with thrombin prior to stimulation with mAbs. We found that, li ke intact cells, degranulated platelets could also be activated and in duced to aggregate by mAbs against a 67 kD membrane protein (known as PTA1) and CD9, and by crosslinked CD32 (Fc gamma RII). However, the si gnal generated by crosslinked Fc gamma RII was weak com pared with tha t induced by the other monoclonal antibodies. Thus, by diminishing the Fc gamma RII si,anal contribution, we have succeeded for the first ti me to clearly dissect the target antigen signal from that generated by Fc gamma RII. In addition to differences in the degree of aggregation , analysis of the signals generated by each mAb showed differences in Ca2+ fluxes and protein phosphorylation. Moreover, the signals generat ed by CD9 and PTA1 antigens differed significantly in their sensitivit y to PKC inhibition or ADP-ribosylation of the small GTP-binding prote in rhoA. Despite these differences, the signals initiated by all three antigens converged to a common signalling pathway which included acti vation of tyrosine kinase(s). The pattern of protein phosphorylation s trongly resembled that induced by gpIIb/IIIa-mediated platelet interac tion with macromolecular ligands and by mutual cell contact, The multi ple intercellular links formed by mAb would have a similar effect sinc e the Fc-receptor anchorage required for antigen stimulation is alread y known to be provided by adjacent cells. The present findings suggest that the function of both CD9 and PTA1 antigens is closely associated with gpIIb/IIIa activation.