MUTATION AT EITHER ARG336 OR ARG562 IN FACTOR-VIII IS INSUFFICIENT FOR COMPLETE RESISTANCE TO ACTIVATED PROTEIN-C (APC)-MEDIATED INACTIVATION - IMPLICATIONS FOR THE APC RESISTANCE TEST

Activated protein C (APC)-mediated inactivation of factor VIII (FVIII) correlates with cleavage at either Arg336 and/or Arg562. To elucidate the APC cleavage requirements for inactivation of FVIII, APC cleavage site mutants in FVIII (R336I, R562K and R336I/R562K) were made by sit e-directed mutagenesis. Analysis of these FVIII mutants expressed in C OS-1 monkey cells demonstrated the thrombin-cleaved mutant R562K was r esistant to APC cleavage at residue 562 but not at Arg336 and the thro mbin cleaved mutant R336I was mostly resistant to APC cleavage at resi due 336, but was sensitive to APC cleavage at Arg562. The double mutan t R336I/R562K was mostly resistant to cleavage at residue 336 and comp letely resistant to cleavage al residue 562. Thus, APC cleavage of FVI II does not require a specific order of cleavage al either residue. Th e functional inactivation by APC was studied using partially purified preparations of FVIII expressed in Chinese hamster ovary cells. Both s ingle mutants were inactivated at similar rates but slower than wild-t ype FVIII, whereas the double mutant R336I/R562K was resistant to inac tivation. The ability of a commercially available APC-resistance assay kit to detect APC resistant FVIII was tested by reconstituting FVIII deficient plasma with the APC resistant mutants. Only the R336I/R562K demonstrated a reduced APC-resistance ratio, indicating that this assa y can not detect the single APC cleavage site mutant of FVIII. These r esults suggest that APC-mediated cleavage at either Arg336 or Arg562 p artially inactivate FVIII.