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.
Disclosures: Coller:Centocor, Inc.: Patents & Royalties.