Arginine-glycine-aspartic acid mimics can identify a transitional activation state of recombinant alpha IIb beta 3 in human embryonic kidney 293 cells

The platelet-specific integrin alpha IIb beta 3 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 alpha IIb beta 3, we expressed recombinant alpha IIb beta 3 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 alpha IIb beta 3 to determine the affinity state of the receptor. Some of these RGD mimics were previously shown to clearly discriminate between resting and activated alpha IIb beta 3. Solution-phase binding of these RGD mimics to the recombinant cells suggested that in HEK 293 cells the full-length alpha IIb beta 3 is expressed in a ''transitional'' activation state. This observation was confirmed by the binding of the activation-specific, monoclonal anti-alpha IIb beta 3 antibody PAC1 to cells expressing the full-length recombinant alpha IIb beta 3. Deletion of the entire cytoplasmic domain of the beta 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 alpha IIb beta 3 that is capable of mediating fibrinogen-dependent cell aggregation.