Abstract

The platelet-specific integrin αIIbβ3 achieves a high affinity binding state in response to extracellular agonists such as thrombin, ADP, or collagen. During this activation, the receptor undergoes a number of conformational changes. To characterize the different conformations of αIIbβ3, we expressed recombinant αIIbβ3 in human embryonic kidney (HEK) 293 cells. Antigenic and peptide recognition specificities of the full-length recombinant receptor resembled those of the native receptor in platelets. We used an array of peptidic and nonpeptidic arginine-glycine-aspartic acid (RGD) mimics that specifically bind to human platelet αIIbβ3 to determine the affinity state of the receptor. Some of these RGD mimics were previously shown to clearly discriminate between resting and activated αIIbβ3. Solution-phase binding of these RGD mimics to the recombinant cells suggested that in HEK 293 cells the full-length αIIbβ3 is expressed in a “transitional” activation state. This observation was confirmed by the binding of the activation-specific, monoclonal anti-αIIbβ3 antibody PAC1 to cells expressing the full-length recombinant αIIbβ3. Deletion of the entire cytoplasmic domain of the β subunit was sufficient to convert the receptor in HEK 293 cells to a fully active form, as found in activated platelets. In addition, the full-length receptor was capable of mediating agonist-independent aggregation of cells in the presence of fibrinogen. Thus, by using RGD mimics, we have identified a functional transitional activation state of αIIbβ3 that is capable of mediating fibrinogen-dependent cell aggregation.