Mild hemophilia A caused by increased rate of factor VIII A2 subunit dissociation: Evidence for nonproteolytic inactivation of factor VIIIa in vivo

Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional factor VIII (FVIII) protein and are termed cross-reacting material (CRM)-positive. FVIII is a heterodimer (domain structure A1-A2-B/A3-C1-C2) that requires thrombin cleavage to elicit procoagulant activity, Thrombin-activated FVIII is a heterotrimer with the A2 subunit (amino acid residues 373 to 740) in a weak ionic interaction with the Al and A3-C1-C2 subunits, Dissociation of the A2 subunit correlates with inactivation of FVIII. Recently, a phenotype of CRM-positive hemophilia A patients has been characterized whose plasma displays a discrepancy between their FVIII activities, where the one-stage clotting assay displays greater activity than the two-stage clotting assay. One example is a missense mutation where (ARG)531 has been substituted by (HIS)531, A, FVIII cDNA construct was prepared containing the (ARG)531(HIS) mutation and the protein was expressed in COS-1 monkey cells by transient DNA transfection. Metabolic labeling with [S-35]. methionine demonstrated that (ARG)531(HIS) was synthesized at an equal rate compared with FVIII wild-type (WT) but had slightly reduced antigen in the conditioned medium, suggesting a modest secretion defect. A time course of structural cleavage of (ARG)531(HIS) demonstrated identical thrombin cleavage sites and rates of proteolysis as FVIII WT. Similar to the patient phenotypes, (ARG)531(HIS) had discrepant activity as measured by a one-stage activated partial thromboplastin time (aPTT) clotting assay (36% +/- 9.6% of FVIII WT) and a variation of the two-stage assay using a chromogenic substrate (COAMATIC; 19% +/- 6.9% of FVIII WT). Partially purified FVIII WT and (ARG)531(HIS) proteins were subjected to functional activation by incubation with thrombin. (ARG)531(HIS) demonstrated significantly reduced peak activity and was completely inactivated after 30 seconds, whereas FVIII WT retained activity until 2.5 minutes after activation. Because the (ARG)531(HIS) missense mutation predicts a charge change to the A2 subunit, we hypothesized that the (ARG)531(HIS) A2 subunit could be subject to more rapid dissociation from the heterotrimer. The rate of A2 dissociation, using an optical biosensor, was determined to be fourfold faster for (ARG)531(HIS) compared with FVIII Wi. Because the two-stage assay involves a preincubation phase before assay measurement, an increased rate of A2 dissociation would result in an increased rate of inactivation and reduced specific activity. (C) 1999 by The American Society of Hematology.