A CD9, αIIbβ3, integrin‐associated protein, and GPIb/V/IX complex on the surface of human platelets is influenced by αIIbβ3 conformational states

Open Access
Publisher Website
Google Scholar
Abstract
A noncovalently associated complex comprising of CD9, the fibrinogen (Fg) receptor αIIbβ3, integrin‐associated protein (IAP), and glycoprotein (GP) Ib/V/IX complex was isolated from Chaps‐solubilized human platelets. The CD9 complex was immunoprecipitated by mAbs specific for CD9 (mAb7), IAP (BRIC126), GPIb (SZ1), GPIX (GR‐P), β3 (AP3) and αIIb (C3). Additionally, the association between CD9 and αIIbβ3 was demonstrated by ELISA. In this system, CD9 did not bind to vitronectin receptor (αvβ3) suggesting that CD9/αIIbβ3 association was αIIb‐subunit or αIIbβ3‐complex dependent. D3, an αIIbβ3‐activating mAb that is also an anti‐LIBS (ligand‐induced binding site), immunoprecipitated primarily αIIbβ3 with GPIb and IAP. CD9 was not detected in D3 immunoprecipitates. D3 binding induced platelet aggregation via direct αIIbβ3 activation and was upregulated by the αIIbβ3 antagonist eptifibatide. In contrast, AP3 and C3 exhibited neither effect. In addition, D3 also inhibited whole blood clot retraction, in contrast to AP3 and C3, suggesting that conformational constraints on αIIbβ3 by D3 binding not only influenced the CD9 complex but also affected αIIbβ3 post receptor occupancy events. The CD9 complex was immunoprecipitated in the presence of eptifibatide, demonstrating that αIIbβ3 receptor 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 α‐thrombin in the presence of CaCl2 compared with that present in EDTA. Stirred platelets showed fibrinogen‐mediated aggregation by α‐thrombin in the presence of CaCl2 but not with EDTA, suggesting that fibrinogen crosslinking of CD9 complexes via αIIbβ3 could be partially responsible for this increase. These findings imply that the platelet CD9 complex is independent of platelet activation although it is dependent upon the conformation state of αIIbβ3.

This publication has 32 references indexed in Scilit: