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

Abstract

Summary Background 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. 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 the stability. Methods Studies assessing factor VIII stability involved monitoring decay rates of factor VIII at 55°C or in guanidinium, decay of factor VIIIa following A2 subunit dissociation, and thrombin generation at low (0.3 nM) factor VIII 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 factor VIII reagents with improved stability parameters.

Combining mutations that modulate inter-subunit interactions and proteolytic inactivation enhance the stability of factor VIIIa

“…However, the 336(P4-P3')562/Asp519Val/Glu665Val and 336(P4-P3')562/Ala108Ile/Asp519Val/Glu665Val variants were the most stable FVIIIa forms, showing ~90% activity after a 40 min incubation and yielding decay rate values that were reduced ~25-fold compared with WT FVIII value. This effect resulted from the Asp519Val/Glu665Val mutation which showed ~80% activity after 40 min representing an ~14-fold reduced rate of FVIIIa decay, a value similar to that observed previously and attributed to increased retention of the A2 subunit in FVIIIa (8).…”

“…We previously reported that the Asp519Val/Glu665Val mutation yields a FVIII with prolonged activity following activation by strengthening the interaction of the A2 subunit in FVIIIa (8). Furthermore, the Ala108Ile mutation was shown to yield a FVIII variant with improved FVIII thermal and chemical stability by optimizing hydrophobic interactions in a pocket at the interface between the A1 and C2 domains (9).…”

“…WT FVIII and FVIII variants with mutation of 336(P4-P3')562 [where residues 333-339 (PQLRMKN) that flank the fast FXa-cleavage site at Arg336 are replaced with residues flanking the slow cleavage site at Arg562 (residues 559-565, VDQRGNQ)] combined with mutation of Ala108Ile, Asp519Val/Glu665Val, or Asp519Val/Glu665Val /Ala108Ile were constructed as B-domainless FVIII, lacking residues Gln744-Ser1637 in the B-domain as described before (8,9,19,21). Recombinant WT and variant FVIII forms were stably expressed in BHK cells and purified as described previously (22).…”

“…Mutations to increase buried hydrophobic area and/or reduce the buried hydrophilic area often result in enhanced protein stability (6). Earlier studies showed that acidic residues localized to hydrophobic pockets at the A1-A2 interface (Asp519) and A2-A3 interface (Glu665 and Glu1984) when mutated to hydrophobic residues (Ala or Val), yielded favorable effects on FVIII/FVIIIa stability and /or activity (7,8). In addition, replacing Ala108, that localized to a hydrophobic pocket at the A1-C2 domain interface, with the more bulky Ile also showed a marked increase in FVIII stability (9).…”

Mass Spectrometry-assisted Study Reveals That Lysine Residues 1967 and 1968 Have Opposite Contribution to Stability of Activated Factor VIII

“…For a number of hemophilia A variants carrying single amino acid substitutions, it has been suggested that a decreased stability of FVIIIa is the cause for the bleeding disorder (21,22). Employing molecular modeling studies on the available FVIII structures in combination with site-directed mutagenesis, Fay and co-workers have now successfully identified several amino acid residues that enhance or decrease the stability of FVIII (21,(23)(24)(25).…”