Swing-out of the β3 hybrid domain is required for αIIbβ3 priming and normal cytoskeletal reorganization, but not adhesion to immobilized fibrinogen.

Abstract

Structural and functional analyses of integrin αIIbβ3 has implicated swing-out motion of the β3 hybrid domain in αIIbβ3 activation and ligand binding. Using data from targeted molecular dynamics (TMD) simulations, we engineered two disulfide-bonded mutant receptors designed to limit swing-out (XS-O). XS-O mutants cannot bind the high Mr ligand fibrinogen in the presence of an activating mAb or after introducing mutations into the αIIb subunit designed to simulate inside-out signaling. They also have reduced capacity to be "primed" to bind fibrinogen by pretreatment with eptifibatide. They can, however, bind the small RGD venom protein kistrin. Despite their inability to bind soluble fibrinogen, the XS-O mutants can support adhesion to immobilized fibrinogen, although such adhesion does not initiate outside-in signaling leading to normal cytoskeletal reorganization. Collectively, our data further define the biologic role of β3 hybrid domain swing-out in both soluble and immobilized high Mr ligand binding, as well as in priming and outside-in signaling. We also infer that swing-out is likely to be a downstream effect of receptor extension.