Mapping of the D′D3 domain-binding site on FVIII by HDX-MS. (A) HDX difference plots illustrating localized differential deuterium uptake by the FVIII component of rFVIIIFc in the presence and absence of the monomeric D′D3 construct for the FVIII heavy chain (upper panel) and light chain (lower panel). The x-axis indicates the 463 peptides derived from the B domain–deleted FVIII sequence ordered from the N to C terminus based on their respective midpoints, and the y-axis indicates the calculated mass difference caused by differential deuterium uptake for peptides derived from FVIII in the D′D3-bound and D′D3-free states. FVIII domain boundaries are indicated with arrows. Individually colored curves correspond to the average mass difference values calculated for data acquired at the time points indicated. Vertical gray bars represent the sum of differences across all time points for a given peptide. Positive values for x indicate peptides for which deuterium uptake is reduced in the presence of the D′D3 domain. Peptides and overlapping clusters of peptides exhibiting statistically significant perturbations in deuterium uptake according to criteria defined by Houde et al¹⁸  are numbered and indicated with colored horizontal bars or dots, with green indicating those for which noncontradictory peptide overlap patterns could be derived and red indicating those for which this was not possible within the given constraints for statistical significance. (B) Surface representations of the FVIII structure (PDB ID: 2R7E¹⁹ ) with individual domains colored as indicated. Numbered areas correspond to sites of differential deuterium uptake identified in (A). (C) Ribbon representations of the FVIII C1 and C2 domains with areas corresponding to differential deuterium uptake numbered as in (B).