A Soluble Recombinant Factor VIII Fragment Containing the A2 Domain Binds to Some Human Anti-Factor VIII Antibodies that Are not Detected by Immunoblotting

Scandella, Dorothea; Timmons, Lisa; Mattingly, M; Trabold, Norma C.; Hoyer, Leon W.

doi:10.1055/s-0038-1648520

Cited by 58 publications

(52 citation statements)

References 14 publications

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“…Its activity was determined in the one-stage clotting assay (33) to be 3300 units/mg, which is similar to a previously reported value (31). The recombinant C2 domain was produced as a soluble, secreted protein in Spodoptera frugiperda Sf9 insect cells (34), and it was purified by gel filtration and ion-exchange chromatography (21). vWf was purified from cryoprecipitate (Cutter Biological) (21).…”

Section: Methodssupporting

confidence: 72%

The Acidic Region of the Factor VIII Light Chain and the C2 Domain Together Form the High Affinity Binding Site for von Willebrand Factor

Saenko

Scandella

1997

Journal of Biological Chemistry

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A binding site for von Willebrand factor (vWf) was previously localized to the carboxyl terminus of the C2 domain of the light chain (LCh) of factor VIII (fVIII). The acidic region of the LCh, residues 1649 -1689, also controls fVIII⅐vWf binding by an unknown mechanism. Although anti-acidic region monoclonal antibodies prevent formation of the fVIII⅐vWf complex, the direct involvement of the acidic region in this binding has not been demonstrated. By limited proteolysis of LCh with Staphylococcus aureus V8 protease, we prepared 14-and 63-kDa LCh fragments, which begin with fVIII residues 1672 and 1795, respectively. Using surface plasmon resonance to measure binding interactions, we demonstrated that the 14-kDa fragment binds to vWf, but its affinity for vWf (K d 72 nM) was 19-fold lower than that of LCh. This was not due to an altered conformation of the acidic region within the 14-kDa fragment, since its affinity for an anti-acidic region monoclonal antibody was similar to that of LCh. All LCh derivatives lacking the acidic region (thrombin-cleaved LCh, recombinant C2, and 63-kDa fragment) had also greatly reduced affinities for vWf (K d 564 -660 nM) compared with LCh (K d 3.8 nM). In addition, the similar affinities of these derivatives for vWf indicated that apart from its acidic region, the LCh contains no vWf binding site other than the one within C2. The reduced affinities of the LCh derivatives lacking the acidic region for monoclonal antibody NMC-VIII/5 (epitope, C2 residues 2170 -2327) indicated that removal of the acidic region leads to a conformational change within C2. This change is likely to affect the conformation of the vWf binding site in C2, which overlaps the epitope of NMC-VIII/5; therefore, the acidic region also appears to be required to maintain the optimal conformation of this vWf binding site. Our results demonstrate that the acidic region and the C2 domain are both directly involved in forming a high affinity binding site for vWf.

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Section: Methodssupporting

confidence: 72%

The Acidic Region of the Factor VIII Light Chain and the C2 Domain Together Form the High Affinity Binding Site for von Willebrand Factor

Saenko

Scandella

1997

Journal of Biological Chemistry

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182

187

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“…Recombinant A2 domain polypeptides 336-606 (20) and 373-740 (10) were able to neutralize the FVIII inhibitory activity of a number of anti-A2 antibodies. Competition immunoradiometric assays in which several inhibitor IgG preparations were evaluated for inhibition ofbinding ofa radiolabeled inhibitor IgG to recombinant A2 have indicated that, despite the large region that currently defines the A2 epitope, anti-A2 inhibitors appear to bind to the same or to closely spaced epitopes (7). The above results suggest that a common inhibitor epitope is located within residues 373-606 of the A2 domain.…”

mentioning

confidence: 95%

Inhibition of human factor VIIIa by anti-A2 subunit antibodies.

Lollar¹,

Parker²,

Curtis³

et al. 1994

J. Clin. Invest.

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“…Inhibitor IgG was prepared as described previously. 14 The inhibitors in HR, LK, AA, and RvR antibodies were specific for the C2 domain on antibody neutralization assays. 2 AJ was identified as C2 specific by using a panel of recombinant hybrid human/porcine fVIII molecules.…”

Section: Introductionmentioning

confidence: 99%

Antigenicity of putative phospholipid membrane-binding residues in factor VIII

Barrow

Healey

Jacquemin

et al. 2001

Blood

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Most inhibitory antibodies to human factor VIII (fVIII) bind to epitopes in the A2, ap-A3, or C2 domains. The anticoagulant action of antibodies to the C2 domain is due to inhibition of binding of fVIII to phospholipid. The x-ray structure of the human fVIII C2 domain shows a putative hydrophobic, 3-prong, phospholipid membrane-binding site consisting of Met2199/Phe2200, Val2223, and Leu2251/Leu2252. Additionally, Lys2227, near Val2223, is part of a ring of positively charged residues that may contribute to electrostatic interaction of fVIII with negatively charged phosphatidylserine. In this study, 8 active mutants of human fVIII (Met2199Ile, Leu2252Phe, Phe2200Leu, Val2223Ala, Lys2227Glu, Met2199Ile/Phe2200Leu, Val2223Ala/Lys2227Glu, and Met2199Ile/Phe2200Leu/Val2223Ala/Lys2227Glu), which were constructed on the basis of differences between human, porcine, murine, and canine fVIII at proposed phospholipid binding sites, were expressed. The antigenicity of the mutants toward 5 C2-specific polyclonal human antibodies was measured by using the Bethesda assay. A human monoclonal anti-C2 antibody, BO2C11, and a murine C2-specific monoclonal antibody, NMC VIII-5, were also included in the analysis. In comparison with wild-type, B-domainless fVIII, the Met2199Ile, Phe2200Leu, and Leu2252 single mutants had lower antigenicity toward most of the inhibitors. In contrast, the Val2223Ala and Lys2227Glu mutants usually showed increased antigenicity. These results suggest that C2 inhibitors frequently target the Met2199/Phe2200 and Leu2251/Leu2252 β-hairpins and are consistent with the hypothesis that these residues participate in binding to phospholipid membranes. In contrast, Val2223 and Lys2227 may oppose antibody binding sterically or through stabilization of a low-affinity membrane-binding conformation of the C2 domain.

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A Soluble Recombinant Factor VIII Fragment Containing the A2 Domain Binds to Some Human Anti-Factor VIII Antibodies that Are not Detected by Immunoblotting

Cited by 58 publications

References 14 publications

The Acidic Region of the Factor VIII Light Chain and the C2 Domain Together Form the High Affinity Binding Site for von Willebrand Factor

The Acidic Region of the Factor VIII Light Chain and the C2 Domain Together Form the High Affinity Binding Site for von Willebrand Factor

Inhibition of human factor VIIIa by anti-A2 subunit antibodies.

Antigenicity of putative phospholipid membrane-binding residues in factor VIII

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