Summary
FVIIIa is labile due to the dissociation of A2 subunit. Previously, we introduced
hydrophobic mutations at select A1/A2/A3 subunit interfaces yielding more stable FVIII(a)
variants. Separately we showed that altering the sequence flanking the primary FXa
cleavage site in FVIIIa (Arg336) yielded reduced rates of proteolytic inactivation
of FVIIIa. In this study we prepared the FXa-cleavage resistant mutant (336(P4-P3’)562)
combined with mutations of Ala108Ile, Asp519Val/ Glu665Val or Ala108Ile/Asp519Val/Glu665Val
and examined the effects of these combinations relative to FVIII thermal stability,
rates of FVIIIa decay and proteolytic inactivation of FVIIIa by FXa. Thermal decay
rates for 336(P4-P3’)562/Ala108Ile, 336(P4-P3’)562/Asp519Val/ Glu665Val, and 336(P4-P3’)562/Ala108Ile/Asp519Val/Glu665Val
variants were reduced by ∼2– to 5-fold as compared with wild-type (WT) primarily reflecting
the effects of the A domain interface mutations. FVIIIa decay rates for 336(P4-P3’)562/Asp519Val/Glu665Val
and 336(P4-P3’)562/Ala108Ile/Asp519Val/Glu665Val variants were reduced by ∼25 fold,
indicating greater stability than the control Asp519Val/Glu665Val variant (∼14-fold).
Interestingly, 336(P4-P3’)562/Asp519Val/Glu665Val and 336(P4-P3’)562/Ala108Ile/ Asp519Val/Glu665Val
variants showed reduced FXa-inactivation rates compared with the 336(P4-P3’)562 control
(∼4-fold), suggesting A2 subunit destabilisation is a component of proteolytic inactivation.
Thrombin generation assays using the combination variants were similar to the Asp519Val/Glu665Val
control. These results indicate that combining multiple gain-of-function FVIII mutations
yields FVIII variants with increased stability relative to a single type of mutation.
Keywords
Factor VIII - factor VIIIa - factor Xa - protein stability - thrombin generation assay