Peptide-specific antibodies as probes of the topography of the Ca2+-binging motifs in alphaIIbbeta3.
Antibodies against synthetic peptides corresponding to four Ca2+-binding motifs of the alphaIIb subunit have been obtained and used as molecular probes to analyze the topography of the alphaIIbeta3 complex. The specificity of the antibodies has been characterized by ELISA and Western immunoblotting in terms of binding capacity and affinity to the isolated alphaIIbbeta3 and its alphaIIb subunit. Our data suggest that: (a) all four Ca2+-binding motifs of the alphaIIb are partially 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 complete inhibition of platelet aggregation. (b) The conformation of amino acid stretches which form the second Ca2+-binding motif of alphaIIb is particularly dependent upon the presence of cation, and this region undergoes significant conformational alterations upon Ca2+ expulsion.
Niewiarowska
J
J
Department of Biophysics, Medical University in Lodz, Poland.
Swiderska
M
M
Majewski
T
T
Cierniewski
C S
CS
D000595:Amino Acid Sequence; D000906:Antibodies / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D000918:Antibody Specificity; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology; D010974:Platelet Aggregation / Q000276:immunology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding
D000906:Antibodies; D002135:Calcium-Binding Proteins; D000939:Epitopes; D015335:Molecular Probes; D009842:Oligopeptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D002118:Calcium