Epitope mapping of mAbs IAC-1 and 1D12 with human/mouse chimeras of the α₂ I-domain. (A) Sequence alignment of human (hu) VWF A1, α_M I, α₂ I, and mouse (mo) α₂ I-domain with ClustalW.¹⁹  Only murine residues that differ from the human sequence are shown. β-strands (open boxes) and α-helices (gray shaded boxes) were derived from the crystal structures.^7,20,21  Replacement of residues resulting in gain-of-function mutants are in bold. Underlined regions indicate the sequences substituted to give chimeras A to F. Dilution series of mAb IAC-1 (B) and 1D12 (C) were incubated with wells coated with wild-type α₂ I-domain (•) or chimeras A (♦), B (▪), C (○), D (▾), or F (▴), and mAb binding was detected with GAM-HRP. Insets: Western blots of the chimeric α₂ I-domains incubated with mAb IAC-1 (B) or 1D12 (C).