Analysis of the tetraspanin CD9-integrin alpha(IIb)beta(3) (GPIIb-IIIa) complex in platelet membranes and transfected cells

The platelet integrin, alpha(IIb)beta(3) (GPIIb-IIIa), and the tetraspanin, CD9, are integral membrane proteins that are abundant in platelet membranes. We have identified several proteins, including CD9, which were co-precipitated by anti-alpha(IIb)beta(3) antibody from untreated, resting platelets that were solubilized with the poly(oxyethylene) non-ionic detergent, Brij-35. Immunoblot and quantitative immunoprecipitation showed that the association of alpha(IIb)beta(3) with CD9 is specific and stoichiometric. The interaction between CD9 and alpha(IIb)beta(3) is probably hydrophobic, as Triton X-100 and hydrophobic detergents of the Brij series completely dissociated the CD9-alpha(IIb)beta(3) complex. Recombinant CD9 and alpha(IIb)beta(3) can associate after transfection into Chinese hamster ovary cells, as seen by co-immunoprecipitation and co-localization in the periphery of spreading cells and in the lamellipodia of cells plated on fibrinogen. This co-localization is absent from focal adhesions. Furthermore, anti-CD9-coated latex beads co-clustered alpha(IIb)beta(3) with CD9. This work indicates that the tetraspanin, CD9, is associated with beta(3) integrins in resting platelets and transfected cells.