Investigation of the specific effects of different mAb known to stimulate platelets (agonist mAb) is complicated by interaction of the Fc portion of these mAb with the platelet FcyRII. This has led to the conclusion that nearly all agonist-mAb-induced activation of platelets is mediated by this receptor. However, the target antigen-mediated signal can be analysed provided that the effects of FcyRII engagement can either be reduced or eliminated. We have therefore blocked platelet FcyRll with IV.3 Fab fragments (an anti-FcyRII mAb), and stimulated the platelets by cross-linking intact agonist mAb with F(ab')> fragments of an Fc-specific anti-mouse antibody. By analysing functional platelet responses and proteintyrosine phosphorylation, we found that such non-FcyRII-mediated cross-linking of CD9, CD42 and glycoprotein (gp) IIb/IIIa generates closely similar signals. Since this may indicate molecular associations, we analyzed the surface topography of platelets using the chemical cross-linking agent dithiobis(su1fosuc-cinimidyl propionate). We found that a proportion of CDY, gpIIb/IIIa and CD42 molecules associate with each other on the platelet surface membrane. Thus, our results suggest that these antigens are able to form a larger molecular complex and induce similar signals. Furthermore, cross-linking of CD9 and CD42 stimulated thrombasthenic platelets completely lacking gpIIb/IIIa. These data therefore indicate that CDY and CD42 can signal independently of gpIIb/IIIa, and that signals generated by all these molecules may converge on a common pathway.Keywords: glycoprotein Tb ; integrin ; CD9 ; transmembrane-4 superfamily ; signal transduction.Many cell receptors are now known to be multicomponent complexes involving ligand-binding and signalling proteins. Members of the transmembrane-4 superfamily (or tetraspan superfamily) of membrane proteins, which includes CD9 , are frequently found within such complexes, where they are thought to participate in receptor signalling (reviewed by Wright and Tomlinson, 1994). The B-cell and T-cell antigen receptors are particularly good examples of such multicomponent structures (Matsumoto et al., 1993 ;Imai et al., 1995).Integrin heterodimers can form complexes with other membrane proteins, including a transmembrane-4 protein, CD9 (Slupsky et al., 1989;Rubinstein et al., 1994;Nakamura et al., 1995). Thus, in stimulated platelets, the principal integrin, glycoprotein (gp)IIb/IIIa (also known as a , r h /~3 , CD41/CD61), has been shown by chemical cross-linking to be associated with CD9 (Slupsky et al., 1989). In addition, there is evidence that gpIIb/IIIa and gpIb/TX [CD42b/CD42a; a receptor for von Willebrand factor and thrombin (Roth, 1991 ;Gralnick et al., 1994)] may also be associated (Fox, 1985;Davies and Palek, 1982;Jung and Moroi, 1983 These observations raise the possibility that gpIIb/IIIa, gpIbl IX and CD9 can all associate into multicomponent adhesion/ signalling receptor complexes. In the present paper, we provide evidence from mAb-probing and chemical-cross-linki...