Anti-C1 domain antibodies epitope mapping by HDX MS. (A) Heat map demonstrating percentage of H/D exchange protection of HSA-C1 fVIII protein in the presence of the anti-C1 mAbs. The color scale represents the difference in percentage of deuterium exchange per residue between antigen alone and antigen/mAb mixtures at 120 seconds (antigen/mAb subtracted from antigen) in which the darker shades represent decreased deuterium exchange in the presence of anti-C1 mAbs. The red and purple shaded regions on the C1 domain corresponding to the red and purple bars below the heat map adapted from the crystal structure of fVIII (Protein Data Bank ID code 4BDV) represents regions of reduced deuterium exchange rate by group A mAbs. The red shaded region and bar corresponds to the proposed primary binding epitope for group A mAbs. The green bars below the heat map and green shaded region on the fVIII C1 domain crystal structure corresponds to the proposed phospholipid binding site for group AB mAb KM33 and group B mAb B136. (B) Binding regions for each anti-C1 mAb imposed on the C1 domain. Shaded regions represent sequences with decreased deuterium exchange in the presence of the anti-C1 mAb. Regions shaded red correspond to sequences with increased H/D exchange protection and orange shaded regions correspond to sequences with a lesser degree of protection in HDX-MS.