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

Abstract

Summary. 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-FcgRII interaction. Furthermore, in addition to its anchoring role, the FcgRII 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 (FcgRII). However, the signal generated by crosslinked FcgRII was weak compared with that induced by the other monoclonal antibodies. Thus, by diminishing the FcgRII signal contribution, we have succeeded for the first time to clearly dissect the target antigen signal from that generated by FcgRII. In addition to differences in the degree of aggregation, analysis of the signals generated by each mAb showed differences in Ca 2+ 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.

CD9 is expressed on the cell surface of human granulosa cells and associated with integrin alpha61

“…Other studies showed that anti-CD9 mAbs caused a rise in intracellular calcium and enhanced tyrosine phosphorylation in platelets and Schwann cells (Favier et al, 1989;Yatomi et al, 1993;Hadjiargyrou and Patterson et al, 1995). In platelets, intracytoplasmic signalling pathways mimic those caused by integrin β 3 (gpIIb/IIIa), suggesting that CD9 has a role in integrin-associated cell signalling (Slupsky et al, 1989(Slupsky et al, , 1997. We also observed that anti-CD9 mAb (ALB6) enhanced migration of a human choriocarcinoma-derived cell line, BeWo cells, and that integrin α 5 β 1 is involved in the effect of the anti-CD9 mAb on BeWo cell migration (Hirano et al, 1999).…”

CD36 associates with CD9 and integrins on human blood platelets

“…14 Our data raise the possibility that signal transduction by CD36 may involve CD9 and protein kinase C. Platelets exhibit similar responses to anti-CD36 and anti-CD9 antibodies. 23,41,42 Both antibodies activate ␣IIb␤3 to bind fibrinogen and cause platelet aggregation. Platelet aggregation that is induced by an antibody to either antigen is inhibited by protein kinase C inhibitors.…”

CD9 Participates in Endothelial Cell Migration During In Vitro Wound Repair

“…15,62 Expression of CD9 in a human B-cell line increases the tyrosine phosphorylation of 69-and 130-kDa bands in cells adhering to FN, 9 whereas in Schwann cells, anti-CD9 mAbs enhance the tyrosine phosphorylation of 8-to 220-kDa proteins. 5 In the experimental conditions under which we observed inhibition of lesion repair, ALMA.1 did not modify the tyrosine phosphorylation profile of ECs over a period of 48 hours, even in the presence of a phosphatase inhibitor, to reveal low phosphorylation levels, nor did clustering of CD9 modify this profile (data not shown).…”