Structure of the Human Factor VIII C2 Domain in Complex with the 3E6 Inhibitory Antibody.

Blood coagulation factor VIII is a glycoprotein cofactor that is essential for the intrinsic pathway of the blood coagulation cascade. Inhibitory antibodies arise either spontaneously or in response to therapeutic infusion of functional factor VIII into hemophilia A patients, many of which are specific to the factor VIII C2 domain. The immune response is largely parsed into "classical" and "non-classical" inhibitory antibodies, which bind to opposing faces cooperatively. In this study, the 2.61 Å resolution structure of the C2 domain in complex with the antigen-binding fragment of the 3E6 classical inhibitory antibody is reported. The binding interface is largely conserved when aligned with the previously determined structure of the C2 domain in complex with two antibodies simultaneously. Further inspection of the B factors for the C2 domain in various X-ray crystal structures indicates that 3E6 antibody binding decreases the thermal motion behavior of surface loops in the C2 domain on the opposing face, thereby suggesting that cooperative antibody binding is a dynamic effect. Understanding the structural nature of the immune response to factor VIII following hemophilia A treatment will help lead to the development of better therapeutic reagents.

Structure of the factor VIII C2 domain in a ternary complex with 2 inhibitor antibodies reveals classical and nonclassical epitopes.

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.