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 two classes of anti-C2 inhibitory antibodies that bind to factor VIII simultaneously has been investigated by small angle X-ray scattering and X-ray crystallography. The C2 domain/3E6 FAB/G99 FAB stable ternary complex, both in solution and in its crystalline state, illustrates that each antibody epitope resides on opposing faces of the factor VIII C2 domain. The 3E6 epitope is a classical antibody that forms direct contacts to the C2 domain at two loops consisting of Glu2181-Ala2188 and Thr2202-Arg2215, which inhibits the binding of the C2 domain to von Willebrand Factor and phospholipid surfaces. The G99 is a non-classical antibody that prevents proteolytic activation of factor VIII, and its 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 charges present on both C2 epitopes and complementary negative charges on each antibody. A new model of phospholipid 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. Furthermore, a 1.7 Å X-ray crystal structure of the porcine factor VIII C2 domain has also been determined, which supports the presented model for phospholipid binding. These results illustrate the complex nature of the polyclonal immune response against the factor VIII C2 domain, and further define the epitopes for both classical and non-classical inhibitory antibodies.
Disclosures:
No relevant conflicts of interest to declare.
Structure of the factor VIII C2 domain in a ternary complex with 2 inhibitor antibodies reveals classical and nonclassical epitopes
