VWF-D′ solution structure. FVIII and VWF-D′ domain structures. Residues where substitutions affected or would be expected to affect binding affinity are shown in spherical representation (blue, positively charged; red, negatively charged; brown, hydrophobic; turquoise, neutral). (A) Hydrophobic FVIII-C2 residues M2199, F2200, L2251, and L2252 interact with VWF, whereas flanking surface-exposed residues R2215, R2220, and K2249 make this FVIII-C2 region positively charged. Five noncysteine hemophilic FVIII-C1 domain amino acid substitutions that affected FVIII-VWF binding (http://www.factorviii-db.org/) are indicated. The cleavage site for the FVIII light-chain (LC) acidic peptide is also indicated. VWF-D′ is oriented with its flexible TIL′ domain approaching FVIII. (B) VWF-D′ is oriented here with its rigid E domain at the top. Noncysteine amino acids whose substitutions are associated with type 2N VWD are shown in spherical representation, and those affecting charged side chains are labeled. Disulfide bonds are shown in orange stick representation.