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 dysfunct ional factor VIII (FVIII) protein and are termed cross-reacting material (C RM)-positive. FVIII is a heterodimer (domain structure A1-A2-B/A3-C1-C2) th at requires thrombin cleavage to elicit procoagulant activity, Thrombin-act ivated 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, Dis sociation 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 t he one-stage clotting assay displays greater activity than the two-stage cl otting assay. One example is a missense mutation where (ARG)531 has been su bstituted 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 d emonstrated that (ARG)531(HIS) was synthesized at an equal rate compared wi th 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 an d rates of proteolysis as FVIII WT. Similar to the patient phenotypes, (ARG )531(HIS) had discrepant activity as measured by a one-stage activated part ial thromboplastin time (aPTT) clotting assay (36% +/- 9.6% of FVIII WT) an d a variation of the two-stage assay using a chromogenic substrate (COAMATI C; 19% +/- 6.9% of FVIII WT). Partially purified FVIII WT and (ARG)531(HIS) proteins were subjected to functional activation by incubation with thromb in. (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 mut ation 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 t he heterotrimer. The rate of A2 dissociation, using an optical biosensor, w as determined to be fourfold faster for (ARG)531(HIS) compared with FVIII W i. Because the two-stage assay involves a preincubation phase before assay measurement, an increased rate of A2 dissociation would result in an increa sed rate of inactivation and reduced specific activity. (C) 1999 by The Ame rican Society of Hematology.