PMID: 9531040Apr 8, 1998Paper

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

Thrombosis and Haemostasis
K AmanoR J Kaufman

Abstract

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 site-directed mutagenesis. Analysis of these FVIII mutants expressed in COS-1 monkey cells demonstrated the thrombin-cleaved mutant R562K was resistant to APC cleavage at residue 562 but not at Arg336 and the thrombin cleaved mutant R3361 was mostly resistant to APC cleavage at residue 336, but was sensitive to APC cleavage at Arg562. The double mutant R336I/R562K was mostly resistant to cleavage at residue 336 and completely resistant to cleavage at residue 562. Thus, APC cleavage of FVIII does not require a specific order of cleavage at either residue. The functional inactivation by APC was studied using partially purified preparations of FVIII expressed in Chinese hamster ovary cells. Both single mutants were inactivated at similar rates but slower than wild-type FVIII, whereas the double mutant R336I/R562K was resistant to inactivation. The ability of a commercially available APC-resistance assay kit to detect APC resistant FVIII was tested by reconsti...Continue Reading

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