Integrin β3 regions controlling binding of murine mAb 7E3: Implications for the mechanism of integrin αIIbβ3 activation

Abstract

Abciximab, a derivative of the murine mAb 7E3, protects against ischemic complications of percutaneous coronary interventions by inhibiting ligand binding to the ␣IIb␤3 receptor. In this study we identified regions on integrin ␤3 that control 7E3 binding. Murine͞ human amino acid substitutions were created in two regions of the ␤A 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 ␤3 in complex with ␣IIb. The introduction of both the human C177-C184 region and human W129 into murine ␤3 was necessary and sufficient to permit 7E3 binding to the human ␣IIb͞murine ␤3 complex. Although we cannot exclude allosteric effects, we propose that 7E3 binds between C177-C184 and W129, which are within 15 Å of each other in the crystal structure and close to the ␤3 metal ion-dependent adhesion site. We previously demonstrated that 7E3 binds more rapidly to activated than unactivated platelets. Because it has been proposed that ␣IIb␤3 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 ␣IIb␤3 construct locked in a bent conformation, and unlocking the conformation restored 7E3 binding. Thus, our data are consistent with ␣IIb␤3 existing in variably bent conformations in equilibrium with each other on unactivated platelets, and activation resulting in ␣IIb␤3 adopting a more extended conformation.T he integrin ␣IIb␤3 and ␣V␤3 receptors are important in a number of physiologic and pathologic phenomena, including hemostasis, thrombosis, tumor angiogenesis, and bone resorption (1, 2). The murine mAb 7E3 (3) blocks ligand binding to both ␣IIb␤3 and ␣V␤3 receptors (4, 5). Abciximab is a mouse͞ human chimeric Fab fragment of the mAb 7E3 that inhibits ␣IIb␤3-mediated platelet aggregation and is approved for human use to prevent the ischemic complications associated with percutaneous coronary interventions (6).Previous studies of 7E3 binding to cells expressing recombinant ␣IIb␤3 receptors demonstrated that: (i) swapping select murine for human ␣IIb sequences does not decrease 7E3 binding (7), (ii) removing the specificity determining loop (SDL) (K156-G189 sequence) from ␤3 results in loss of 7E3 binding (8), (iii) swapping the murine for human C177-C184 sequence within the SDL region in ␤3 results in loss of 7E3 binding (7), and (iv) swapping the murine S129-T133 sequence for the human W129-N133 sequence results in partial loss of 7E3 binding (7).The above human͞mouse swapping studies identified regions within ␤3 that affect 7E3 binding, but the biochemical and functional properties of these chimeric receptors were not characterized. In addition, the W129-N133 region contains two amino acid differences between human ␤3 (␤3Hu) and mouse ␤3 (␤3M) and the C177-C184 region contains three amino acid differences (Table 1). To define further the regions on ␣IIb␤3 th...

Mapping early conformational changes in αIIb and β3 during biogenesis reveals a potential mechanism for αIIbβ3 adopting its bent conformation

“…43 10E5 interacts exclusively with the "Cap" subdomain of the ␣IIb ␤-propeller, 15 whereas the 7E3 epitope reacts with a region adjacent to the MIDAS in the ␤A (I-like) domain of ␤3. 30 Their patterns of precipitation of radiolabeled ␣IIb and ␤3 were nearly identical ( Figure 1). At the 1-hour time point they precipitated pro-␣IIb, mature ␣IIb, and ␤3; at 2 and 4 hours the intensity of the pro-␣IIb band progressively decreased, and the intensities of the mature ␣IIb and ␤3 bands increased, consistent with ongoing ␣IIb␤3 biogenesis during this time period.…”

“…30 Transfections were performed using Lipofectamine 2000 (Gibco-BRL, Carlsbad, CA) according to the manufacturer's instructions, followed by selection in media containing 800 g/mL G418 for 2 to 4 weeks. To obtain a population of cells uniformly expressing high levels of ␣IIb␤3, cells were labeled with the mAb 10E5 (anti-␣IIb␤3) and sorted using a FACSVantage SE cell sorter (BectonDickinson, Rutherford, NJ).…”

αIIbβ3 binding to a fibrinogen fragment lacking the γ-chain dodecapeptide is activation dependent and EDTA inducible

“…Because our fragment resembles to a great extent the D98 fragment described by Lishko et al 48 (which was the fragment used in the study by Podolnikova et al 24 ), most importantly for the current study in not having an intact g-404-411 peptide, but may differ in minor aspects, we have chosen to name our fragment 'D98.' The mAb's 10E5 (anti-aIIbb3), 11,27 7E3 (anti-aIIbb3 1 aVb3, which binds in the region between the specificity loop and a1 helix of b3), 49,50 and 7E9 (anti-C-terminal g-12 peptide) 12 were described previously. The anti-LIBS mAb AP5 51 and the anti-aVb3 mAb LM609 52,53 were generously provided by Peter Newman and David Cheresh, respectively.…”

Mutation of a Conserved Asparagine in the I-like Domain Promotes Constitutively Active Integrins αLβ2 and αIIbβ3

“…Human fibrinogen was obtained from Sigma. These mAbs were kind gifts from different sources: MHM24 (␣ L ␤ 2 -specific and function-blocking) (25) and MHM23 (␤ 2 integrin heterodimer-specific) (26) were from A. J. McMichael (Institute of Molecular Medicine, Oxford, UK); KIM185 (␤ 2 integrin-activating mAb) (27) and KIM127 (␤ 2 integrin-specific and activation reporter mAb) (28) were from M. K. Robinson (CellTech, Slough, UK); and 10E5 (integrin ␣ IIb -specific and function-blocking) (29,30) and 7E3 (␤ 3 integrin recognizing mAb) (31) were from B. S. Coller (The Rockefeller University, New York, NY). The mAb MEM148 (␤ 2 integrin-specific and hybrid domain displacement reporter mAb) (22) was purchased from Serotec, Oxford, UK.…”