analysis of CD9, CD32 and p67 signalling: use of degranulated platelets indicates direct involvement of CD9 and p67 in integrin activation

The use of agonist monoclonal antibodies (mAbs) to probe the signalling function of platelet membrane proteins is severely limited by the dependence of the mAb effect on Fc–FcγRII interaction. Furthermore, in addition to its anchoring role, the FcγRII receptor itself generates a stimulation signal resulting in granule secretion. Platelet stimulation by the released granule contents can then further obscure the original activation signal. Here we demonstrate that these problems are largely overcome by the use of platelets which had been degranulated with thrombin prior to stimulation with mAbs. We found that, like intact cells, degranulated platelets could also be activated and induced to aggregate by mAbs against a 67 kD membrane protein (known as PTA1) and CD9, and by crosslinked CD32 (FcγRII). However, the signal generated by crosslinked FcγRII was weak compared with that induced by the other monoclonal antibodies. Thus, by diminishing the FcγRII signal contribution, we have succeeded for the first time to clearly dissect the target antigen signal from that generated by FcγRII. 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 generated by CD9 and PTA1 antigens differed significantly in their sensitivity to PKC inhibition or ADP-ribosylation of the small GTP-binding protein rhoA. Despite these differences, the signals initiated by all three antigens converged to a common signalling pathway which included activation of tyrosine kinase(s). The pattern of protein phosphorylation strongly resembled that induced by gpIIb/IIIa-mediated platelet interaction with macromolecular ligands and by mutual cell contact. The multiple intercellular links formed by mAb would have a similar effect since the Fc-receptor anchorage required for antigen stimulation is already 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.