Stabilizing Factor VIIIa By Combining Mutations That Modulate Inter-Subunit Interactions and Proteolytic Inactivation

Factor (F) VIIIa serves as a cofactor for FIXa forming intrinsic FXase complex. The activity of FVIIIa is labile due to the tendency for A2 subunit of the cofactor to dissociate, thereby inactivating FVIIIa and dampening FXase activity. Recently we have shown that a combination of hydrophobic point mutations generated FVIII(a) variants with improved FVIII thermal stability and enhanced A2 subunit retention in FVIIIa (Wakabayashi et al. J. Thromb. Haemost. 2012, 10: 492-495). Furthermore, FVIIIa is a target for proteolytic inactivation catalyzed by FXa, the product of FXase complex. We have shown this proteolysis further contributes to the down-regulation of FXase and that altering the sequence flanking the primary FXa cleavage site in FVIIIa (at Arg336) yields marked reductions in the rates of proteolytic inactivation of FVIIIa catalyzed by FXa (DeAngelis et al. J. Biol. Chem. 2012, 287: 15409-15417). In this study we prepared various combinations of the above mutations to obtain a panel of novel FVIII molecules and examined the attributes of these reagents in assays monitoring FVIII thermal stability, FVIIIa spontaneous decay rates, rates of proteolytic inactivation by FXa, and thrombin generation potential.