Effects of Limiting Extension at the αIIb Genu on Ligand Binding to Integrin αIIbβ3

Structural data of integrin alpha IIb beta 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 alpha IIb head domain to the beta 3 tail decreased fibrinogen binding. To study the role of alpha IIb extension about the genu, we introduced a disulfide "clamp" between the alpha IIb thigh and calf-1 domains. Clamped alpha IIb beta 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 Mn2+ or by coexpressing the clamped alpha IIb with a beta 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 alpha IIb beta 3-mediated adhesion to immobilized fibrinogen, but it did diminish the enhanced binding of mAbAP5 in the presence of kistrin. Collectively, our studies support a role for alpha 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 alpha IIb extension resulting in increased access to the ligand-binding site and/or facilitating the conformational change(s) in beta 3 that affect the intrinsic affinity of the binding pocket for ligand.