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 affini ty binding state in response to extracellular agonists such as thrombi n, ADP, or collagen. During this activation, the receptor undergoes a number of conformational changes. To characterize the different confor mations of alpha IIb beta 3, we expressed recombinant alpha IIb beta 3 in human embryonic kidney (HEK) 293 cells. Antigenic and peptide reco gnition specificities of the full-length recombinant receptor resemble d those of the native receptor in platelets. We used an array of pepti dic and nonpeptidic arginine-glycine-aspartic acid (RGD) mimics that s pecifically bind to human platelet alpha IIb beta 3 to determine the a ffinity state of the receptor. Some of these RGD mimics were previousl y shown to clearly discriminate between resting and activated alpha II b beta 3. Solution-phase binding of these RGD mimics to the recombinan t cells suggested that in HEK 293 cells the full-length alpha IIb beta 3 is expressed in a ''transitional'' activation state. This observati on was confirmed by the binding of the activation-specific, monoclonal anti-alpha IIb beta 3 antibody PAC1 to cells expressing the full-leng th recombinant alpha IIb beta 3. Deletion of the entire cytoplasmic do main 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-i ndependent aggregation of cells in the presence of fibrinogen. Thus, b y using RGD mimics, we have identified a functional transitional activ ation state of alpha IIb beta 3 that is capable of mediating fibrinoge n-dependent cell aggregation.