Enhanced factor VIIIa stability of A2 domain interface variants results from an increased apparent affinity for the A2 subunit

Factor (F)VIIIa, a heterotrimer comprised of A1, A2, and A3C1C2 subunits, is labile due to the tendency of the A2 subunit to dissociate from the A1/A3C1C2 dimer. As dissociation of the A2 subunit inactivates FVIIIa activity, retention of A2 defines FVIIIa stability and thus, FXase activity. Earlier results showed that replacing residues D519, E665, and E1984 at the A2 domain interface with Ala or Val reduced rates of FVIIIa decay, increasing FXa and thrombin generation. We now show the enhanced FVIIIa stability of these variants results from increases in inter-A2 subunit affinity. Using a FVIIIa reconstitution assay to monitor inter-subunit affinity by activity regeneration, the apparent K-d value for the interaction of wild-type (WT) A2 subunit with WT A1/A3C1C2 dimer (43 +/- 2 nM) was significantly higher than values observed for the A2 point mutants D519A/V, E665A/V, and E1984A/V which ranged from similar to 5 to similar to 19 nM. Val was determined to be the optimal hydrophobic residue at position 665 (apparent K-d = 5.1 +/- 0.7 nM) as substitutions with Ile or Leu at this position increased the apparent K-d value by similar to 3- and similar to 7-fold, respectively. Furthermore, the double mutant (D519V/E665V) showed an similar to 47-fold lower apparent K-d value (0.9 +/- 0.6 nM) than WT. Thus these hydrophobic mutations at the A2 subunit interfaces result in high binding affinities for the A2 subunit and correlate well with previously observed reductions in rates in FVIIIa decay.