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Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2552-2558
Cleavage of Factor V at Arg 506 by Activated Protein C and the
Expression of Anticoagulant Activity of Factor V
Elisabeth Thorelli,
Randal J. Kaufman, and
Björn Dahlbäck
From the Department of Clinical Chemistry, Lund University,
University Hospital Malmö, Sweden and the Howard Hughes Medical
Institute, Department of Biological Chemistry, University of Michigan
Medical Center, Ann Arbor, MI.
Activated protein C (APC) inhibits coagulation by cleaving and
inactivating procoagulant factor Va (FVa) and factor VIIIa (FVIIIa).
FV, in addition to being the precursor of FVa, has anticoagulant properties; functioning in synergy with protein S as a cofactor of APC
in the inhibition of the FVIIIa-factor IXa (FIXa) complex. FV:Q506
isolated from an individual homozygous for APC-resistance is less
efficient as an APC-cofactor than normal FV (FV:R506). To investigate
the importance of the three APC cleavage sites in FV (Arg-306, Arg-506,
and Arg-679) for expression of its APC-cofactor activity, four
recombinant FV mutants (FV:Q306, FV:Q306/Q506, FV:Q506, and FV:Q679)
were tested. FV mutants with Gln (Q) at position 506 instead of Arg (R)
were found to be poor APC-cofactors, whereas Arg to Gln mutations at
positions 306 or 679 had no negative effect on the APC-cofactor
activity of FV. The loss of APC-cofactor activity as a result of the
Arg-506 to Gln mutation suggested that APC-cleavage at Arg-506 in FV is
important for the ability of FV to function as an APC-cofactor. Using
Western blotting, it was shown that both wild-type FV and mutant FV was
cleaved by APC during the FVIIIa inhibition. At optimum concentrations of wild-type FV (11 nmol/L) and protein S (100 nmol/L), FVIIIa was
found to be highly sensitive to APC with maximum inhibition occurring
at less than 1 nmol/L APC. FV:Q506 was inactive as an APC-cofactor at
APC-concentrations  1 nmol/L and only partially active at higher APC
concentrations. Our results show that increased expression of FV
anticoagulant activity correlates with APC-mediated cleavage at Arg-506
in FV, but not with cleavage at Arg-306 nor at Arg-679.
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