Submitted February 27, 2008
Accepted July 8, 2008
Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface
Hironao Wakabayashi, Fatbardha Varfaj, Jennifer DeAngelis, and Philip J Fay*
Biochemistry and Biophysics, University of Rochester, Rochester, NY, United States
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, United States
* Corresponding author; email: philip_fay{at}urmc.rochester.edu.
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. The charged residues Glu272, Asp519, Glu665, and Glu1984 appear buried at the interface of the A2 domain with either the A1 or A3 domain, and thus may impact protein stability. To determine the effects of these residues on procofactor/cofactor stability, these residues were individually replaced with either Ala or Val and stable BHK cell lines expressing the B-domainless proteins were prepared. Specific activity and thrombin generation parameters for 7 of the 8 variants were >80% the wild type (WT) value. Factor VIII activity at 52-60°C and the decay of factor VIIIa activity following thrombin activation were monitored using factor Xa generation assays. Six of the 7 variants showing WT-like activity demonstrated enhanced stability, with the Glu1984Val variant showing a 2-fold increase in thermostability and an ~ 4-8-fold increase in stability of factor VIIIa. These results indicate that replacement of buried charged residues is an effective alternative to covalent modification in increasing factor VIII (VIIIa) stability.
