FVIII with alanine substitutions in positions 2090, 2092, and 2093 shows diminished binding to KM33. (A-B) KM33 was immobilized on microtiter plates and incubated with increasing concentrations of FVIII WT (●, solid line) or mutants FVIII-R2090A (▴, dashed-dotted line), FVIII-K2092A/F2093A (■, dashed line), or FVIII-R2090A/K2092A/F2093A (♦, dotted line). Bound FVIII was detected either with C2 domain–targeting Ab CLB-CAg117 (A) or A2 domain–targeting Ab CLB-CAg9 (B). Data show mean ± SD of 3 experiments and are expressed as percentage of maximum binding, whereby 100% corresponds to signal obtained with 1 U/mL FVIII WT. (C) Surface plasmon resonance analysis of FVIII-KM33 interaction. KM33 IgG1 (25nM) was passed over FVIII WT (solid line), FVIII-K2092A/F2093A (dashed line), or FVIII-R2090A/K2092A/F2093A (dotted line) immobilized on a sensor chip via C2 domain–targeting mAb EL-14. Dissociation was initiated on replacement of ligand solution by buffer. Data are representative of 3 independent experiments. WT indicates wild-type FVIII; 2090, FVIII-R2090A; 2092/93, FVIII-K2092A/F2093A; and 2090/92/93, FVIII-R2090A/K2092A/F2093A.