The platelet integrin alphaIIb-beta3 is one of seven integrins that recognize the RGD motif. When platelets are activated, alphaIIb-beta3 undergoes a change in conformation that renders this integrin more accessible to RGD ligands. The applicant has generated a monoclonal antibody AP7 that contains the RGD sequence in the third CDR of its heavy chain (H3). This antibody binds to both quiescent and reconformed states of the integrin, albeit with five-fold higher affinity to the latter. AP7.3, in which the AP7 H3 loop has been replaced by the corresponding sequence from PAC1 (another antibody that binds only to the reconformed integrin), exhibits an increased selectivity for reconformed integrin and a decrease in overall affinity that is identical in all respects to the behavior of recombinant PAC1 Fab fragments themselves. Both AP7.3 and PAC1 bind only a subset of integrin on the surface of activated platelets. The goal of the proposed research is to better understand the molecular basis of reconformation-sensitive RGD recognition by the integrin and to establish the biological significance of subpopulations of integrin. To address these objectives, the applicant will use engineered derivatives of AP7 as RGD ligands and recombinant, soluble, functional derivatives of human alphaIIb- beta3 heterodimers as receptor.
Four specific aims will be pursued: 1) to define features in the H3 loop of AP7.3 that allow it to selectively bind to the activated form of alphaIIb-beta3; 2) to determine the sites in alphaIIb- beta3 that bind AP7.3; 3) to determine why only a subpopulation of alphaIIb- beta3 binds AP7.3 and other activation-dependent antibodies; and 4) to determine the intermediate resolution structure of AP7/alphaIIb-beta3 complexes by two- dimensional electron crystallography. The applicant suggests that molecular features in alphaIIb-beta3 and the Fab fragments of the AP7 series of antibodies that are involved in the reconformation-sensitive interaction will be applicable to all RGD-containing ligands, including peptidomimetics.
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