Integrin beta3 regions controlling binding of murine mAb 7E3: implications for the mechanism of integrin alphaIIbbeta3 activation

Abstract

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.

Fig. 1.

Effects of swapping of murine sequence in β3Hu on 7E3 binding. (A) Adhesion to immobilized fibrinogen of CHO cells transiently expressing β3Hu or β3Hu–M177-184 in association with human αIIb. Cells were preincubated with 2 mM Ca²⁺ and different antibodies. Adhesion is expressed as OD₅₅₀ of solubilized crystal violet-stained cells. Data presented are the mean ± standard deviation of three experiments. (B) Radiograph of immunoprecipitates under nonreduced and reduced conditions of ³⁵S-labeled cells using 10E5 or 7E3. Cells were transiently transfected with β3Hu or β3Hu–M177-184 in association with human αIIb.

Fig. 2.

Effects of introducing single murine substitutions in β3Hu C177–C184. (A) Radioautographs of ³⁵S-labeled cells immunoprecipitated with 10E5 or 7E3 and electrophoresed under nonreduced conditions in SDS/PAGE. (B) Immunoblot analysis using mAb AP3 (anti-β3) of 7E3 or 10E5 immunoprecipitates of lysates of cells transfected with β3Hu or T182N. T182N was immunoprecipitated by 10E5 and migrated as a closely spaced doublet. Only the minor band migrating with the same M_r as β3Hu was immunoprecipitated by 7E3. (C) Adhesion to immobilized fibrinogen of 293T cells expressing native and mutated β3. Cells were preincubated with 2 mM Ca²⁺. Data are the mean of two experiments and are normalized for adhesion of cells in the absence of antibodies.

Fig. 3.

Effect of substituting alanines for C177 and C184 on 10E5 and 7E3 binding. Lysates of constructs containing β3Hu or β3Hu–M178-183 and/or C177A+C184A residues were immunoprecipitated by 10E5 (Left) or 7E3 (Right), electrophoresed under nonreduced conditions in SDS/PAGE, and immunoblotted with anti-β3 mAb AP-3.

Fig. 4.

Engineering of 7E3 epitope into β3M. (A) Adhesion to immobilized fibrinogen of cells stably expressing native and mutated β3. Cells were preincubated with 2 mM Ca²⁺ and purified murine IgG, 10E5, or 7E3. Data are the mean of three experiments ± standard deviation and are normalized for adhesion of cells in the presence of purified murine mAb. (B) Radioautographs of immunoprecipitates using 10E5 or 7E3 of lysates of ³⁵S-labeled cells transfected with β3Hu, β3M, or chimeric β3 and electrophoresed under nonreduced conditions in SDS/PAGE.

Fig. 5.

The role of K125 in 7E3 binding. (A) Crystal structure of selected regions of the external domain of integrin αVβ3 in complex with the peptide RGDW as per Xiong et al. (31). The αV propeller is in blue, and the β3–βA domain is in red. The peptide is in green, and Mn²⁺ ions are depicted as yellow spheres. (B) Higher-power view of the top surface of the β3–βA domain in association with the peptide. The three yellow spheres represent the three Mn²⁺ ions in the ligand-associated metal binding site, MIDAS, and adjacent to MIDAS positions, respectively. The amino acids depicted in orange are the ones described in the text as important in 7E3 binding. (C) Effect of K125 substitutions on 10E5 and 7E3 binding. Data are the mean ± standard deviation of three independent experiments. (D and E) Molecular modeling of the effect of the Q125 substitution on C177–C184 and α-helix 1. Models are rotated 180° in comparison with A and B. Residues at positions 122, 125, and 182 are shown for β3Hu (D) and K125Q (E). Selected hydrogen bonds are depicted in gray and the new hydrogen bond between Y122 and K125Q is identified by a red arrow. Notice the reorientation of K125 as a result of the additional hydrogen bond, along with the loss of a portion of the α-helical structure of α-helix 1 (red arrowhead).

Fig. 6.

7E3 binding to αIIbβ3 and αIIb-R320C β3-R563C. (A) Immunoprecipitation with 10E5 of lysates of surface biotin-labeled 293 cells expressing αIIbβ3 or αIIb-R320C β3-R563C. Samples were electrophoresed under nonreduced and reduced conditions. (B) Fluorescent intensity of cells expressing αIIbβ3 or αIIb-R320C β3-R563C after incubation with Alexa⁶⁴⁷-7E3 IgG or Alexa⁶⁴⁷-10E5, with or without treatment with 2 mM DTT.

Fig. 7.

Crystal structure of entire extracellular domain of αVβ3 with amino acids Y122, K125, W129, and K181, which are implicated in the 7E3 epitope, shown in orange. The RGD peptide is shown in green. Figure was prepared with mol mol (32).