Cleavage of factor V at Arg 506 by activated protein C and the expression of anticoagulant activity of factor V.
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.
Thorelli
E
E
Department of Clinical Chemistry, Lund University, University Hospital Malmö, Sweden.
Kaufman
R J
RJ
Dahlbäck
B
B
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine; D002417:Cattle; D017931:DNA Primers; D015949:Factor IXa / Q000378:metabolism; D005165:Factor V / Q000737:chemistry* / Q000378:metabolism*; D015944:Factor VIIIa / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011486:Protein C / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D011486:Protein C; D011994:Recombinant Proteins; D015944:Factor VIIIa; D005165:Factor V; D001120:Arginine; D015949:Factor IXa