Multiple discontinuous ligand-mimetic antibody binding sites define a ligand binding pocket in integrin alpha(IIb)beta(3)

Integrin alpha(IIb)beta(3), a platelet fibrinogen receptor, is critically i nvolved in thrombosis and hemostasis. However, how ligands interact with al pha(IIb)beta(3) has been controversial, Ligand-mimetic anti-alpha(IIb)beta( 3) antibodies (PAC-1, LJ-CP3, and OP-G2) contain the RGD-like RYD sequence in their CDR3 in the heavy chain and have structural and functional similar ities to native ligands. We have located binding sites for ligand-mimetic a ntibodies in alpha(IIb) and beta(3) using human-to-mouse chimeras, which we expect to maintain functional integrity of alpha(IIb)beta(3). Here we repo rt that these antibodies recognize several discontinuous binding sites;in b oth the alpha(IIb) and beta(3) subunits; these binding sites are located in residues 156-162 and 229-230 of alpha(IIb) and residues 179-183 of beta(3) . In contrast, several nonligand-mimetic antibodies (e.g. 7E3) recognize si ngle epitopes in either subunit, Thus, binding to several discontinuous sit es in both subunits is unique to ligand-mimetic antibodies. Interestingly, these binding sites overlap with several (but not all) of the sequences tha t have been reported to be critical for fibrinogen binding (e.g. N-terminal repeats 2-3 but not repeats 4-7, of alpha(IIb)). These results suggest tha t ligand-mimetic antibodies and probably native ligands may make direct con tact with these discontinuous binding sites in both subunits, which may con stitute a ligand-binding pocket.