Increasing Hydrophobicity and Disulfide Bridging Between A1 and C2 Subunits Improves Factor VIII Stability.
Authors: Wakabayashi Hironao , Griffiths Amy E , Fay Philip 
Published: October 2019

Abstract 3176

Poster Board III-100

Factor VIII (FVIII) consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), while the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. Previously we have generated FVIII mutants with enhanced stability by mutating residues located at A1-A2 or A2-A3 interfaces (Wakabayashi et al, Blood, 112, 2761-9, 2008, Wakabayashi et al, J. Thromb. Haemost. 7, 438-44, 2009). FVIII X-ray structures show close contacts between the Ca2+ binding site contained within the A1 domain and the C2 domain of LC. In this study we mutated residues located at this interface to examine the effects on FVIII(a) stability. Studies assessing FVIII thermal and chemical stability involved monitoring the rates of loss of FVIII activity by FXa generation assay following incubation of FVIII (4 nM) at 57°C or in various concentrations of guanidinium (0-1.2 M). The rate of decay of FVIIIa was monitored over time at 23°C using FXa generation assays following activation of FVIII (1.5 nM) with thrombin. Data were fitted to single exponential decay equations and rates of decay were compared. In one variant, a disulfide bond was introduced between the two domains by a double mutation at Arg121 in A1 and Leu2302 in the C2 domain to Cys (R121C/L2302C). In addition, based on the finding that there is a gap between the methyl groups of Ala108 (A1 domain) and Ala2328 (C2 domain) we mutated Ala108 to Val, Ile, or Leu to examine whether these mutants increase the stability of FVIII by an improved hydrophobic interaction at this site. Significant increases in FVIII thermal stability, up to 4-fold compared with WT, were observed in R121C/L2302C, Ala108Ile, and Ala108Leu. R121C/L2302C and Ala108Ile retained ∼80% FVIII activity as measured by FXa generation assay compared to WT value, however, that of Ala108Leu was ∼25% the WT value. Only Ala108Ile showed an improvement in chemical stability (10% increase in IC50 value as compared with WT FVIII) and FVIIIa decay due to A2 subunit dissociation was similar to WT FVIII (20-40% reduction in FVIIIa decay rate compared to WT). Ca2+ is necessary for FVIII function and EGTA (2 mM) reduced WT FVIII activity by ∼70%. However, EGTA-treated R121C/L2302C FVIII retained ∼100% activity, suggesting that the Ca2+ requirement for FVIII function may be substituted by covalent bonding between the Ca2+ binding region in A1 and C2 subunit. Furthermore, the Ala108Ile variant showed ∼60% activity remaining after EGTA treatment suggesting partial relief of this Ca2+ dependency for stability of the A1-C2 interaction. Next, we tested whether the mutations at the A1-C2 interface can be combined with mutations at A1-A2 or A2-A3 interfaces to generate a FVIII with further improved stability. Previously characterized FVIII variants, designated A domain mutants, showing up to 2-fold increases in thermal stability compared with WT FVIII included Asp519Ala, Asp519Val, Glu665Ala, Glu665Val, Glu1984Ala, and Glu1984Val. In combining those mutations with either R121C/L2302C or Ala108Ile, we obtained variants with >5-fold increases in thermal stability (9/12 mutants), with the Ala108Ile/Glu665Val variant showing the greatest increase (∼10-fold). Most of the mutants (9/12) showed normal FVIII activity values by FXa generation assay (>60%) and 15-30% increases in IC50 values for chemical stability as compared with WT. In addition, the high FVIIIa stability of the A domain mutants was largely preserved in the combined mutations. Collectively, these results suggest that alterations at this A1-C2 contact region by covalent modification or increasing hydrophobic interaction yields improved FVIII stability that can be combined with other high stability mutations to produce additive effects.

Disclosures

No relevant conflicts of interest to declare.