Structural data of integrin ␣IIb␤3 have been interpreted as supporting a model in which: 1) the receptor exists primarily in a "bent," low affinity conformation on unactivated platelets and 2) activation induces an extended, high affinity conformation prior to, or following, ligand binding. Previous studies found that "clasping" the ␣IIb head domain to the ␤3 tail decreased fibrinogen binding. To study the role of ␣IIb extension about the genu, we introduced a disulfide "clamp" between the ␣IIb thigh and calf-1 domains. Clamped ␣IIb␤3 had markedly reduced ability to bind the large soluble ligands fibrinogen and PAC-1 when activated with monoclonal antibody (mAb) PT25-2 but not when activated by Mn 2؉ or by coexpressing the clamped ␣IIb with a ␤3 subunit containing the activating mutation N339S. The clamp had little effect on the binding of the snake venom kistrin (M r 7,500) or ␣IIb␤3-mediated adhesion to immobilized fibrinogen, but it did diminish the enhanced binding of mAb AP5 in the presence of kistrin. Collectively, our studies support a role for ␣IIb extension about the genu in the binding of ligands of 340,000 and 900,000 M r with mAb-induced activation but indicate that it is not an absolute requirement. Our data are consistent with ␣IIb extension resulting in increased access to the ligand-binding site and/or facilitating the conformational change(s) in ␤3 that affect the intrinsic affinity of the binding pocket for ligand.The platelet ␣IIb␤3 receptor plays an important role in both hemostasis and thrombosis (1). Ligand binding to ␣IIb␤3 is controlled by an activation process that affects the conformation of the receptor and ligand binding, in turn, can also affect the conformation of the receptor (2). Several different conformations of ␣IIb␤3 have been identified based on inferences from biochemical analyses (3), studies employing monoclonal antibodies (4 -7) and electron microscopy (8 -10), comparison of the crystal structures of the liganded ␣IIb␤3 headpiece (11) and the unliganded complete ectodomain (12), and analysis of the unliganded and liganded ectodomain of the related ␣V␤3 receptor (13,14). Receptor extension about the regions encompassing the thigh, genu, and calf-1 domains of ␣IIb and the plexin-semaphorin-integrin (PSI), 2 integrin epidermal growth factor-1 (IEGF-1), and IEGF-2 domains of ␤3 or comparable regions of other integrin receptors has been proposed to play an important role in receptor activation (12,(15)(16)(17)(18), but there is uncertainty about whether this conformational change occurs prior to or after ligand binding (19 -21). Thus, "cross-clasping" the ␣IIb headpiece ␤-propeller domain to the ␤3 IEGF-4 domain in the tail region via a newly engineered disulfide bond prevented the binding of fibrinogen induced by activating mAb in concert with the activating divalent cation Mn 2ϩ , and a similar effect was observed with cross-clasped ␣V␤3 (16). In both cases the loss of ligand binding could be rescued by reducing the cross-clasped receptors with dithiothreitol (DTT). In contr...

Structural data of integrin αIIbβ3 have been interpreted as supporting a model in which: the receptor exists primarily in a “bent,” low affinity conformation on unactivated platelets, and activation induces an extended, high affinity conformation prior to, or following, ligand binding. Previous studies found that “clasping” the αIIb head domain to the β3 tail decreased fibrinogen binding. To study the role of αIIb extension about the genu we created and analyzed an energy-optimized model of the complete extracellular domain of αIIbβ3 and then introduced a disulfide “clamp” between the αIIb thigh and calf-1 domains by mutating αIIb R597 and Y645 to cysteine residues. Increased mobility of the clamped mutant on SDS-PAGE under nonreducing, but not reducing conditions, relative to the WT receptor supported the successful introduction of a new disulfide bond. Clamped αIIbβ3 had markedly reduced ability to bind the large soluble ligands fibrinogen [induced by mAb AP5; net normalized fluorescence intensity (NNFI) of 17 ± 2 for normal αIIbβ3 and 0.4 ± 0.2 for clamped αIIbβ3; n=3; p<0.001] and PAC1 [induced by mAb PT25-2; NNFIs of 17 ± 7 and 2 ± 1, respectively (n=3; p<0.001)]. The defect in fibrinogen and PAC-1 binding could be partially restored (~40–50%) by treating the cells with the reducing agent DTT. In contrast, the clamp had little effect on the binding of the Mr 7,500 snake venom kistrin (non-significant ~30% reduction) or αIIbβ3-mediated adhesion to immobilized fibrinogen (non-significant ~20% reduction). Remarkably, the defect in binding the large soluble ligands was rescued by co-expressing clamped αIIb with the β3- activating mutation N339S. The clamped αIIbβ3 also displayed a defect in kistrin-induced (200 nM) exposure of the mAb AP5 epitope in the PSI domain of β3 (NNFI 50 ± 11 for normal αIIbβ3 vs. 24 ± 5 for clamped αIIbβ3; p<0.001; n=5), and this abnormality was also reversed by DTT. By analyzing the solvent accessible areas in our model of αIIbβ3 with and without extension, we determined that extension results in a 370 Å2 reduction in buried solvent-accessible area in the PSI domain as a result of losing some of its interactions with the IEGF-2 domain. We hypothesize that clamping αIIb may, therefore, partially preserve these PSI-IEGF-2 domain interactions. Collectively, our studies support a role for αIIb extension in the binding of large, but not small ligands, and suggest that αIIb extension is less important for interactions with immobilized than soluble fibrinogen. Our data are consistent with αIIb extension resulting in increased access to the ligand binding site and/or destabilizing β3 headpiece-tailpiece interactions and thus facilitating the conformational change(s) that affect the intrinsic affinity of the binding pocket for ligand. They may also provide an explanation for the paradoxical finding that platelet binding of soluble fibrinogen requires exogenous activation, whereas platelet adhesion to immobilized fibrinogen does not.