Hemophilia A mutations associated with 1-stage/2-stage activity discrepancy disrupt protein-protein interactions within the triplicated A domains of thrombin-activated factor VIIIa

Abstract

AbstractThrombin-activated factor VIII (FVIIIa) is a heterotrimer with the A2 subunit (amino acid residues 373-740) in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIIIa. Patients with hemophilia A have been described whose plasmas display a discrepancy between their FVIII activities, where the 1-stage activity assay displays greater activity than the 2-stage activity assay. The molecular basis for one of these mutations, ARG531HIS, is an increased rate of A2 subunit dissociation. Examination of a homology model of the A domains of FVIII predicted ARG531 to lie at the interface of the A1 and A2 subunits and stabilize their interaction. Indeed, patients with mutations either directly contacting ARG531 (ALA284GLU, ALA284PRO) or closely adjacent to the A1-A2 interface in the tightly packed hydrophobic core (SER289LEU) have the same phenotype of 1-stage/2-stage discrepancy. TheALA284GLU andSER289LEU mutations in FVIII were produced by transfection of COS-1 monkey cells. Compared to FVIII wild-type both mutants had reduced specific activity by 1-stage clotting activity and at least a 2-fold lower activity by 2-stage analysis (COAMATIC), similar to the reported clinical data. Analysis of immunoaffinity purified ALA284GLU andSER289LEU proteins in an optical biosensor demonstrated that A2 dissociation was 3-fold faster for both FVIIIa mutants compared to FVIIIa wild-type. Therefore, these mutations within the A1 subunit of FVIIIa introduce a similar destabilization of the FVIIIa heterotrimer compared to the ARG531HISmutation within the A2 subunit and support that these residues stabilize the A domain interface of FVIIIa.