Visualization of FVIII-C2 epitopes in the B-domain-deleted FVIII crystal structure. With the exception of type A inhibitors, the neutralizing mAbs analyzed here bound to an outside-facing surface of FVIII, where they would not be expected to interfere with the packing or orientation of FVIII domains. (A) The type BC and C epitopes recognized by nonclassical inhibitors are shown as space-filling purple spheres in the FVIII structure.²¹  The protein is oriented with the membrane-binding residues M2199, F2200, L2251, L2252, K2092, and F2093 pointing down. Also indicated by space-filling spheres are residues known to be at the interface between FVIIIa and activated factor IX in the intrinsic tenase complex: region i (FVIII residues 558-565) is colored light blue; region ii consists of residues near residue 712, which is colored gray; region iii (residues 1811-1818) is colored salmon. Note that the type BC epitope, which corresponds to a docking site for activated thrombin, is on the opposite side of FVIII to the FVIIIa-FIXa interface. (B) All of the residues identified as contributing to each of the 5 types of epitopes are shown as space-filling spheres in the FVIII crystal structure.²¹  (C) Venn diagram depictions of specific amino acid residues localized to the B-cell epitopes A, AB, B, BC, and C. Amino acid side chains that SPR assays, followed by visual inspection of the FVIII-C2 crystal structure, indicated contribute to functional B-cell epitopes in human FVIII. Amino acid residues in porcine FVIII that differ from the human FVIII sequence at positions corresponding to functional B-cell epitopes identified in this study are indicated in the second Venn diagram.