Thrombin-activated factor VIII (FVIIIa) is a heterotrimer with the A2 subunit in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIIIa. A homology model (Blood 89:2413, 1997) of the triplicated A domains of factor VIII (FVIII) predicts a pseudo-threefold axis at the tightly packed hydrophobic core with several interdomain interactions. These lie at the interface of A1-A2, A2-A3 and A1-A3. We have previously demonstrated that hemophilia A mutations (R531H, A284E, S289L) within the predicted A1-A2 and A1-A3 interface disrupt potential intersubunit hydrogen bonds and have the molecular phenotype of increased rate of inactivation of FVIIIa due to increased rate of A2 subunit dissociation. Patients with these mutations exhibit a clinical phenotype where the FVIII activity by one-stage(1-st) assay is at least two-fold higher than by two-stage(2-st) assay. We have now also explored mutations within the predicted A2-A3 interface (N694I, R698W and R698L) that also have the phenotype of 1-st/2-st activity discrepancy. These mutations exhibit the same molecular mechanism of increased instability of FVIIIa as those mutations described along the A1-A2 and A1-A3 interfaces. This suggests that the entire tightly packed hydrophobic core within the predicted pseudo-threefold axis contributes to stabilization of FVIIIa.