FVIII residues 2196, 2199, 2201, and 2204 contribute to DRB1*01:01 binding. The anchor residues contributing peptide-DRB1*01:01 affinity were determined by competition binding assays with reference peptide HA_306-318 using FVIII_2194-2205 peptides having single Arg (A) or Ala (B) substitutions at every possible position. The Arg substitution was chosen to map the anchor residues as this bulky, charged side chain is rarely accommodated by pockets within the MHCII peptide-binding groove. Ala substitutions are also informative, as they can perturb binding by removing interactions contributing to peptide-MHCII affinity. Results are expressed as relative affinities (IC₅₀ of FVIII_2194-2205/IC₅₀ of the substituted peptide) ± SE. The IC₅₀ of FVIII_2194-2205 was 0.26 ± 0.02 μM.