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 Arg2209–Ser2216 and Leu2178–Asp2187 loops. In contrast, antibody G99 recognizes the C2 domain primarily through the Pro2221–Trp2229 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.
Journal of Biological Chemistry
Required Publisher's Statement
"This research was originally published in the Journal of Biological Chemistry. Werther RA, Walter JD, †Polozova MS, Pohlman J, Healey JF, Meeks SL, Lollar P, Spiegel PC. Journal of Biological Chemistry. 2013; 288:9905-9914. © the American Society for Biochemistry and Molecular Biology."
First Published on February 15, 2013, doi:10.1074/jbc.M112.424564
Spiegel, P. Clint; Walter, Justin D.; Werther, Rachel A.; Polozova, Maria S.; Pohlman, Julie; Healey, John F.; Meeks, Shannon L.; and Lollar, Pete, "Characterization and Solution Structure of the Factor VIII C2 Domain in a Ternary Complex with Classical and Non-classical Inhibitor Antibodies" (2013). Chemistry. Paper 9.