PEPTIDE-SPECIFIC ANTIBODIES AS PROBES OF THE TOPOGRAPHY OF THE CA2-BINDING MOTIFS IN ALPHA(IIB)BETA(3)()

Antibodies against synthetic peptides corresponding to four Ca2+-bindi ng motifs of the alpha(IIb) subunit have been obtained and used as mol ecular probes to analyze the topography of the alpha(IIb)beta(3) compl ex. The specificity of the antibodies has been characterized by ELISA and Western immunoblotting in terms of binding capacity and affinity t o the isolated alpha(IIb)beta(3) and its alpha(IIb) subunit. Our data suggest that: (a) all four Ca2+-binding motifs of the alpha(IIb) are p artially exposed on the surface of the intact molecule and accessible to antipeptide antibodies. However, they are not in close vicinity to the ligand recognition domain since the antibodies do not produce comp lete inhibition of platelet aggregation. ib) The conformation of amino acid stretches which form the second Ca2+-binding motif of alpha(IIb) is particularly dependent upon the presence of cation, and this regio n undergoes significant conformational alterations upon Ca2+ expulsion .