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1.
Blood ; 122(26): 4270-8, 2013 Dec 19.
Article in English | MEDLINE | ID: mdl-24085769

ABSTRACT

The factor VIII C2 domain is a highly immunogenic domain, whereby inhibitory antibodies develop following factor VIII replacement therapy for congenital hemophilia A patients. Inhibitory antibodies also arise spontaneously in cases of acquired hemophilia A. The structural basis for molecular recognition by 2 classes of anti-C2 inhibitory antibodies that bind to factor VIII simultaneously was investigated by x-ray crystallography. The C2 domain/3E6 FAB/G99 FAB ternary complex illustrates that each antibody recognizes epitopes on opposing faces of the factor VIII C2 domain. The 3E6 epitope forms direct contacts to the C2 domain at 2 loops consisting of Glu2181-Ala2188 and Thr2202-Arg2215, whereas the G99 epitope centers on Lys2227 and also makes direct contacts with loops Gln2222-Trp2229, Leu2261-Ser2263, His2269-Val2282, and Arg2307-Gln2311. Each binding interface is highly electrostatic, with positive charge present on both C2 epitopes and complementary negative charge on each antibody. A new model of membrane association is also presented, where the 3E6 epitope faces the negatively charged membrane surface and Arg2320 is poised at the center of the binding interface. These results illustrate the potential complexities of the polyclonal anti-factor VIII immune response and further define the "classical" and "nonclassical" types of antibody inhibitors against the factor VIII C2 domain.


Subject(s)
Antibodies/chemistry , Epitopes/chemistry , Factor VIII/chemistry , Hemophilia A/blood , Ternary Complex Factors/chemistry , Antibodies/immunology , Crystallography, X-Ray , Electrochemistry , Epitopes/immunology , Factor VIII/immunology , Hemophilia A/immunology , Humans , Protein Structure, Quaternary , Protein Structure, Tertiary , Ternary Complex Factors/immunology
2.
J Biol Chem ; 288(14): 9905-9914, 2013 Apr 05.
Article in English | MEDLINE | ID: mdl-23417672

ABSTRACT

The most significant complication for patients with severe cases of congenital or acquired hemophilia A is the development of inhibitor antibodies against coagulation factor VIII (fVIII). The C2 domain of fVIII is a significant antigenic target of anti-fVIII antibodies. Here, we have utilized small angle x-ray scattering (SAXS) and biochemical techniques to characterize interactions between two different classes of anti-C2 domain inhibitor antibodies and the isolated C2 domain. Multiple assays indicated that antibodies 3E6 and G99 bind independently to the fVIII C2 domain and can form a stable ternary complex. SAXS-derived numerical estimates of dimensional parameters for all studied complexes agree with the proportions of the constituent proteins. Ab initio modeling of the SAXS data results in a long kinked structure of the ternary complex, showing an angle centered at the C2 domain of ∼130°. Guided by biochemical data, rigid body modeling of subunits into the molecular envelope of the ternary complex suggests that antibody 3E6 recognizes a C2 domain epitope consisting of the Arg(2209)-Ser(2216) and Leu(2178)-Asp(2187) loops. In contrast, antibody G99 recognizes the C2 domain primarily through the Pro(2221)-Trp(2229) loop. These two epitopes are on opposing sides of the fVIII C2 domain, are consistent with the solvent accessibility in the context of the entire fVIII molecule, and provide further structural detail regarding the pathogenic immune response to fVIII.


Subject(s)
Factor VIII/chemistry , Blood Coagulation Factors/chemistry , Chromatography/methods , Enzyme-Linked Immunosorbent Assay/methods , Epitopes/chemistry , Factor VIII/metabolism , Hemophilia A/immunology , Humans , Immunoglobulin Fab Fragments/chemistry , Mutation , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Scattering, Small Angle , Solvents/chemistry
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