Human Inhibitor Antibodies Specific for the Factor VIII A2 Domain Disrupt the Interaction between the Subunit and Factor IXa

Philip J Fay,Dorothea Scandella

doi:10.1074/jbc.274.42.29826

Philip J Fay, Dorothea Scandella

Open Access

https://doi.org/10.1074/jbc.274.42.29826

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Abstract

Factor VIIIa, a heterotrimer of the A1, A2, and A3-C1-C2 subunits, increases the catalytic efficiency for factor IXa-catalyzed activation of factor X. A significant fraction of naturally occurring, anti-factor VIII inhibitor antibodies reacts with the A2 domain. Utilizing the capacity for isolated A2 subunit to stimulate factor IXa activity, we show that a panel of these inhibitors block this activity. Inhibition of activity parallels the antibody potency as measured in the Bethesda assay. These antibodies also block the A2-dependent increases in fluorescence anisotropy of fluorescein-Phe-Phe-Arg factor IXa. Similar to the IgG fractions, a peptide representing the sequence of the inhibitor epitope (A2 residues 484-509) blocked the A2-dependent stimulation of factor IXa. These results indicate that antibodies possessing this specificity directly inhibit the interaction of A2 subunit with factor IXa, thus abrogating the contribution of this subunit to cofactor activity. Furthermore, these results also suggest that factor VIII residues 484-509 contribute to a factor IXa-interactive site.

Highlights

Factor VIIIa, a heterotrimer of the A1, A2, and A3C1-C2 subunits, increases the catalytic efficiency for factor IXa-catalyzed activation of factor X
Differential Inhibition of the A2-dependent Stimulation of Factor IXa by Anti-A2 Monoclonal Antibodies—Two monoclonal antibodies specific for distinct regions in the A2 subunit were evaluated for their ability to inhibit the A2-dependent stimulation of factor IXa-catalyzed conversion of factor X. mAb R8B12, a high affinity antibody used for immunopurification of factor VIII or A2 subunit [4], recognizes a C-terminal region of A2 contained within residues 563–740 [19]
Inhibitor Antibodies Block the A2-dependent Stimulation of Factor IXa—Because the epitope recognized by mAb 413 cross-reacts with antibodies obtained from patients possessing factor VIII inhibitors, the above experiment suggested that a possible mechanism for these anti-A2 inhibitors is perturbation of the interaction of the A2 subunit of factor VIIIa with factor IXa

Summary

Introduction

Factor VIIIa, a heterotrimer of the A1, A2, and A3C1-C2 subunits, increases the catalytic efficiency for factor IXa-catalyzed activation of factor X. The activated form of factor VIII, factor VIIIa, functions as a protein cofactor for the serine protease factor IXa to form an anionic phospholipiddependent complex referred to as the intrinsic factor Xase. A significant fraction (ϳ20%) of individuals with hemophilia A who are treated with either purified plasma-derived or recombinant factor VIII develop an immune response to factor VIII [11]. These antibodies typically appear in situations where large deletions or inversions in the factor VIII gene are present [12], and some of them inhibit factor VIII activity. A significant fraction of anti-A2 inhibitors characterized to date show a shared epitope with mAb1 413, which recognizes a region comprised of residues 484 –508 [18]

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