Combining mutations of charged residues at the A2 domain interface enhances factor VIII stability over single point mutations

Background: Factor (F) VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), while the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. Recently we reported that procofactor stability at elevated temperature and cofactor stability over an extended time course were increased following replacement of individual charged residues (Asp(D)519, Glu(E)665 or Glu(E)1984) with either Ala (A) or Val (V) (Wakabayashi et al., Blood, 112, 2761, 2008). Objectives: In the current study we generated combination mutants at these three sites to examine any additive and/or synergistic effects of these mutations on stability. Methods: Studies assessing FVIII stability involved monitoring decay rates of FVIII at 55 degrees C or in guanidinium, decay of FVIIIa following A2 subunit dissociation, and thrombin generation at low (0.3 nmol L-1) FVIII concentration. Results and conclusions: Similar tendencies were observed within each group of variants. Variants with mutations at D519 and either E665 or E1984 (Group A) generally showed significantly better stability as compared with single mutants. Most variants with mutations at E665 and at E1984 (Group B) did not show significant improvement. Triple mutants with mutations at D519, E665 and E1984 (Group C) showed improvement to a similar degree as the Group A double mutants. Overall, these results indicate that selected combinations of mutations to reduce charge and/or increase hydrophobicity at the A2/A1 and A2/A3 domain interfaces yield FVIII reagents with improved stability parameters.