Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface

Abstract

Factor VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), whereas 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 more than 80% the wild-type value. Factor VIII activity at 52 degrees C to 60 degrees C and the decay of factor VIIIa activity after thrombin activation were monitored. Six of the 7 variants showing wild-type-like activity demonstrated enhanced stability, with the Glu1984Val variant showing a 2-fold increase in thermostability and an approximately 4- to 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.

Figure 1

SDS-PAGE and Western blot analysis of factor VIII mutants and WT factor VIII. (A) Purified WT and mutant factor VIII proteins (0.77 μg) after SDS-PAGE on 8% polyacrylamide gels were visualized by GelCode. (B) Purified WT and mutant factor VIII proteins (0.34 μg) were electrophoresed on 8% polyacrylamide gels, transferred to PVDF membranes, and probed by biotinylated R8B12 antibody. Bands were visualized by chemifluorescence. WT (lane 1), Glu272Ala (lane 2), Glu272Val (lane 3), Asp519Ala (lane 4), Asp519Val (lane 5), Glu665Ala (lane 6), Glu665Val (lane 7), Glu1984Ala (lane 8), and Glu1984Val (lane 9). MW indicates molecular weight marker; sFVIII, single chain form factor VIII; HC, heavy chain; LC, light chain. An apparent stoichiometry ratio of single chain form to heterodimer of WT and mutant factor VIII forms were 0.96 (WT), 0.64 (Glu272Ala), 0.92 (Glu272Val), 0.74 (Asp519Ala), 0.8 (Asp519Val), 0.64 (Glu665Ala), 0.63 (Glu665Val), 0.91 (Glue1984Ala), and 0.5 (Glu1984Val).

Figure 2

Specific activity of factor VIII mutants relative to WT factor VIII and thrombin generation assays. (A) Specific Activity. Activity values were determined using a 1-stage clotting assay () and 2-stage chromogenic factor Xa generation assay (■). (B,C) Thrombogram of factor VIII proteins. WT (), Glu272Ala (□), Glu272Val (■), Asp519Ala (○), Asp519Val (●), Glu665Ala (△), Glu665Val (▲), Glu1984Ala (◇), and Glu1984Val (♦). (D) Parameter values obtained from thrombin generation assays. Thrombograms show the average values of triplicated samples. The parameter values were expressed as values (%) relative to WT. The actual values for WT were 7.5 plus or minus 0.5 minutes (lag time), 13.7 plus or minus 0.3 minutes (peak time), 157.3 plus or minus 14.7 nM (peak value), and 979.8 plus or minus 37.9 nM/min (ETP). Lag time (□), peak time (), peak value (■), and ETP (▧). Error bars represent SD values averaged from 3 separate determinations.

Figure 3

Activity decay of WT and mutant factor VIII. Factor VIII (4 nM) was incubated at various temperatures (52°C-60°C); and at the indicated times, aliquots were removed and assayed for activity by factor Xa generation assays. Data were fitted by nonlinear least squares regression, and decay rates were obtained. Each point represents the value averaged from 3 separate determinations. Results are shown for WT (, ×), Glu272Ala (□), Glu272Val (■), Asp519Ala (○), Asp519Val (●), Glu665Ala (△), Glu665Val (▲), Glu1984Ala (◇), Glu1984Val (♦), and full-length Kogenate factor VIII (). (A) Representative factor VIII decay curves after 55°C incubation. (B) Plots of factor VIII decay rate at various temperatures. (Inset) Magnified view of the decay results incubated at 52°C to 55°C.

Figure 4

Activity decay of factor VIII in plasma at 37°C. Factor VIII (1 nM) was incubated at 37°C in factor VIII–deficient plasma and at the indicated times aliquots were removed and assayed using the one-stage clotting assays. Results are shown for WT (, ×), Asp519Ala (○), Asp519Val (●), Glu665Ala (△), Glu665Val (▲), Glu1984Ala (◇), and Glu1984Val (♦). Data were fitted by nonlinear least squares regression, and each point represents the value averaged from 3 separate determinations.

Figure 5

Activity decay of WT and mutant factor VIIIa in the absence and presence of factor IXa. (A) Thrombin-activated factor VIIIa (4 nM) was incubated at 23°C, aliquots were taken at indicated time points, and activity was measured by factor Xa generation assay. (B) Activity decay of WT and mutant factor VIIIa in the presence of factor IXa. Factor VIII (4 nM) was activated with thrombin in the presence of 40 nM factor IXa, aliquots were taken at indicated time points, and activity was measured by factor Xa generation assay. Results are shown for WT (, ×), Glu272Ala (□), Glu272Val (■), Asp519Ala (○), Asp519Val (●), Glu665Ala (△), Glu665Val (▲), Glu1984Ala (◇), and Glu1984Val (♦). Data were fitted by nonlinear least squares regression, and each point represents the value averaged from 3 separate determinations.

Figure 6

Residues surrounding Asp519, Glu272, Glu1984, and Glu665. Factor VIII surface models of indicated regions based on the A domain homology model are drawn by Swiss PDB viewer; A1 domain (residues 1-336), A2 domain (residues 373-711), and A3 domain, (residues 1690-2332). Hydrogen, carbon, oxygen, sulfur, and nitrogen are colored cyan, white, red, yellow, and blue, respectively. There are no possible hydrogen acceptor or donor from the residues near the residues Asp519 (A), Glu272 (B), Glu1 984 (C), and Glu665 (D). (Inset) Factor VIII surface model of individual domains are drawn by Swiss PDB viewer and colored as yellow (A1), transparent blue (A2), red (A3), green (C1), and gray (C2). White dots indicate the location of side chain atoms of the indicated residues (Asp519, Glu272, Glu1984, and Glu665) as shown in the panels A, B, C, and D, respectively.