C1 domain mutations alter FVIII endocytosis by APCs. (A-B) Final concentration of 15nM FVIII WT or mutants FVIII-R2090A, FVIII-K2092A/F2093A, or FVIII-R2090A/K2092A/F2093A was added to MDDCs (white bars), MDMΦs (gray bars), or BMDCs (black bars) for 30 minutes at 37°C. Internalized FVIII was detected by adding CLB-CAg117-FITC in the presence of saponin. Representative histograms (gray-filled histograms show untreated cells, open histograms represent cells treated with FVIII; A) and quantification from ≥ 3 independent experiments (B) are shown (mean ± SD). Results are expressed as the percentage of mean fluorescence intensity (MFI), whereby 100% corresponds to MFI obtained with FVIII WT. (C) Increasing concentrations (0-40nM) FVIII WT (●, solid line), FVIII-K2092A/F2093A (■, dashed line), or FVIII-R2090A/K2092A/F2093A (♦, dotted line) were incubated with MDDCs at 37°C. 100% MFI refers to MFI measured for 40nM FVIII WT. Data show mean ± SD from 3 independent experiments. WT indicates wild-type FVIII; 2090, FVIII-R2090A; 2092/93, FVIII-K2092A/F2093A; and 2090/92/93, FVIII-R2090A/K2092A/F2093A.