Integrin alpha IIb beta 3 reconstituted into lipid bilayers is nonclustered in its activated state but clusters after fibrinogen binding

Integrin activation, ligand binding, and integrin clustering were analyzed using alpha IIb beta 3 reconstituted into phospholipid vesicles and into supported planar lipid bilayers, Strong and specific binding of fibrinogen and the gamma-chain dodecapeptide of fibrinogen to alpha IIb beta 3 indicated that the integrin is in an activated state after membrane reconstitution. Cryoelectron and fluorescence microscopy suggested a nonclustered state of the protein in the vesicle membrane. Supported planar lipid membranes were generated by fusion of vesicles in which approximately equal fractions of integrins were pointing inside-out and outside-in. This distribution led to an immobilization of about 40% of the integrin in supported bilayers due to attachment of the large extracellular domains to the quartz support. Fluorescence recovery after photobleaching indicated a diffusion coefficient of D = (0.70 +/- 0.06) x 10(-8) cm(2)/s, consistent with a nonclustered state of the mobile integrin, Upon fibrinogen binding, the integrins became immobile, and fluorescence micrographs showed a patchy distribution of fibrinogen-integrin complexes consisting of approximately 250 molecules. In addition to the expected dimer formation by bivalent fibrinogen, additionally induced fibrinogen clustering may account for the large size of the complexes. In contrast, binding of monovalent GRGDS pentapeptide or the gamma-chain dodecapeptide of fibrinogen altered neither the mobile fraction nor the association state of alpha IIb beta 3. Our data indicate that integrin alpha IIb beta 3 is activated while monodisperse, and became clustered upon fibrinogen binding, leading to an irreversibly bound state.