A CD9, alpha(IIb)beta 3, integrin-associated protein, and GPIb/V/IX complex on the surface of human platelets is influenced by alpha(IIb)beta 3 conformational states

A noncovalently associated complex comprising of CD9, the fibrinogen (Fg) r eceptor alpha(IIb)beta 3, integrin-associated protein (IAP), and glycoprote in (GP) Ib/V/IX complex was isolated from Chaps-solubilized human platelets . The CD9 complex was immunoprecipitated by mAbs specific for CD9 (mAb7), I AP (BRIC126), GPIb (SZ1), GPIX (GR-P), beta 3 (AP3) and alpha(IIb) (C3). Ad ditionally, the association between CD9 and alpha(IIb)beta 3 was demonstrat ed by ELISA. In this system, CD9 did not bind to vitronectin receptor (alph a(nu)beta 3) suggesting that CD9/alpha(IIb)beta 3 association was alpha(IIb )-subunit or alpha(IIb)beta 3-complex dependent. D3, an alpha(IIb)beta 3-ac tivating mAb that is also an anti-LIES (ligand-induced binding site), immun oprecipitated primarily alpha(IIb)beta 3 With GPIb, and IAP. CD9 was not de tected in D3 immunoprecipitates. D3 binding induced platelet aggregation vi a direct cu,pa activation and was upregulated by the alpha(IIb)beta 3 antag onist eptifibatide. In contrast, AP3 and C3 exhibited neither effect. In ad dition, D3 also inhibited whole blood clot retraction, in contrast to AP3 a nd C3, suggesting that conformational constraints on alpha(IIb)beta 3 by D3 binding not only influenced the CD9 complex but also affected alpha(IIb)be ta 3 post receptor occupancy events. The CD9 complex was immunoprecipitated in the presence of eptifibatide, demonstrating that alpha(IIb)beta-3 recep tor occupancy was not sufficient to cause complex dissociation. CD9 complex isolation was also independent of platelet activation, although a twofold increase in the quantity of CD9 complex was seen after platelet activation by alpha-thrombin in the presence of CaCl2 compared with that present in ED TA. Stirred platelets showed fibrinogen-mediated aggregation by alpha-throm bin in the presence of CaCl2 but not with EDTA, suggesting that fibrinogen crosslinking of CD9 complexes via alpha(IIb)beta 3 could be partially respo nsible for this increase. These findings imply that the platelet CD9 comple x is independent of platelet activation although it is dependent upon the c onformation state of alpha(IIb)beta 3.