Integrin beta3 regions controlling binding of murine mAb 7E3: implications for the mechanism of integrin alphaIIbbeta3 activation.
Abciximab, a derivative of the murine mAb 7E3, protects against ischemic complications of percutaneous coronary interventions by inhibiting ligand binding to the alphaIIbbeta3 receptor. In this study we identified regions on integrin beta3 that control 7E3 binding. Murine/human amino acid substitutions were created in two regions of the betaA domain that previous studies found to influence 7E3 binding: the C177-C184 loop and K125-N133. The T182N substitution and a K125Q mutation reduced 7E3 binding to human beta3 in complex with alphaIIb. The introduction of both the human C177-C184 region and human W129 into murine beta3 was necessary and sufficient to permit 7E3 binding to the human alphaIIb/murine beta3 complex. Although we cannot exclude allosteric effects, we propose that 7E3 binds between C177-C184 and W129, which are within 15 A of each other in the crystal structure and close to the beta3 metal ion-dependent adhesion site. We previously demonstrated that 7E3 binds more rapidly to activated than unactivated platelets. Because it has been proposed that alphaIIbbeta3 changes from a bent to an extended conformation upon activation, we hypothesized that 7E3 binds less well to the bent than the extended conformation. In support of this hypothesis we found that 7E3 bound less well to an alphaIIbbeta3 construct locked in a bent conformation, and unlocking the conformation restored 7E3 binding. Thus, our data are consistent with alphaIIbbeta3 existing in variably bent conformations in equilibrium with each other on unactivated platelets, and activation resulting in alphaIIbbeta3 adopting a more extended conformation.
Laboratory of Blood and Vascular Biology, The Rockefeller University, New York, NY 10021, USA.
Andrea
A
Artoni
JiHong
J
Li
Beau
B
Mitchell
Jian
J
Ruan
Junichi
J
Takagi
Timothy A
TA
Springer
Deborah L
DL
French
Barry S
BS
Coller
D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D004220:Disulfides; D000939:Epitopes; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D039661:Integrin beta3 / Q000378:metabolism*; D051379:Mice; D010446:Peptide Fragments / Q000378:metabolism; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D004220:Disulfides; D000939:Epitopes; D007074:Immunoglobulin G; D039661:Integrin beta3; D010446:Peptide Fragments; D019039:Platelet Glycoprotein GPIIb-IIIa Complex