Abstract
Factor VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), while the contiguous A1A2 domains are separate subunits in the cofactor, factor VIIIa. The intrinsic instability of the cofactor results from weak affinity interactions of the A2 subunit within factor VIIIa. 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, 2008, in press). These mutations did not appreciably affect factor VIII specific activity or thrombin generation parameters. Factor VIII structure studies suggest D519 is buried at the A1/A2 domain interface, while E665 and E1984 localize at the A2/A3 domain interface. In the current study we generated combination mutants at these three sites to examine any additive and/or synergistic effect of these mutations. Factor VIII variants generated included double mutants with a mutation at D519 and mutation at either E665 or E1984 (Group A), double mutants with a mutation at E665 and mutation at E1984 (Group B), and triple mutants with a mutation at D519 and mutations at both E665 and E1984 (Group C). Most of the mutants retained normal specific activity values compared to wild type (WT) with exceptions noted for E665A/E1984A, E665A/E1984V and D519V/E665V/E1984V which showed ~2-fold reductions in this parameter. Studies assessing factor VIII stability involved monitoring the rates of loss of factor VIII activity by factor Xa generation assay following incubation of factor VIII (4 nM) at 55ºC. The rate of decay of factor VIIIa was monitored over time at 23ºC using the factor Xa generation assay following activation of factor VIII (1.5 nM) with thrombin. Data were fitted to single exponential decay equations and rates of decay were compared. The Group A variants D519A/E665A, D519A/D665V, and D519V/E665V showed significant enhancement (up to ~1.3-fold for the D519A/D665V variant) in factor VIII thermal stability as compared with the best single mutation in that pairing, and representing actual decay rates that approached 45% the WT value. On the other hand, the relative factor VIII decay rates for three of the four of the Group B mutants were somewhat increased compared with the best single mutation in the pairing. No significant changes were observed for the Group C mutants. Evaluation of factor VIIIa stability revealed large enhancements of up to ~4-fold compared with the single mutants for all of the Group A variants. Group B variants yielded poorer results when compared with the better individual mutation in the pairing. The triple mutations (Group C) showed the largest factor VIIIa stability enhancements with maximal stability observed for D519V/E665V/E1984A, which showed a decay rate that was ~10% the WT value. A calibrated thrombin generation assay using a fluorogenic substrate was performed on selected mutants using a sub-physiologic factor VIII concentration (0.2 nM). Enhancements in selected parameter values were observed for the D519V/E665V variant (~2.3-fold increase in the peak height and ~1.5-fold increase in endogenous thrombin potential compared with WT), while the D519A/E665V, D519V/E1984A, and D519V/E665V/E1984A variants showed 1.2 to 1.7-fold increases in these parameter values. These observations may suggest a greater capacity for thrombin generation per unit concentration factor VIII for these variants. 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 factor VIII reagents with improved stability parameters.
Combining mutations that modulate inter-subunit interactions and proteolytic inactivation enhance the stability of factor VIIIa
