Identification of surface epitopes of human coagulation factor Va that are important for interaction with activated protein C and heparin.
Inactivation of factor Va (FVa) by activated protein C (APC) is a key reaction in the down-regulation of thrombin formation. FVa inactivation by APC is correlated with a loss of FXa cofactor activity as a result of three proteolytic cleavages in the FVa heavy chain at Arg306, Arg506, and Arg679. Recently, we have shown that heparin specifically inhibits the APC-mediated cleavage at Arg506 and stimulates cleavage at Arg306. Three-dimensional molecular models of APC docked at the Arg306 and Arg506 cleavage sites in FVa have identified several FVa amino acids that may be important for FVa inactivation by APC in the absence and presence of heparin. Mutagenesis of Lys320, Arg321, and Arg400 to Ala resulted in an increased inactivation rate by APC at Arg306, which indicates the importance of these residues in the FVa-APC interaction. No heparin-mediated stimulation of Arg306 cleavage was observed for these mutants, and stimulation by protein S was similar to that of wild type FVa. With this, we have now demonstrated that a cluster of basic residues in FVa comprising Lys320, Arg321, and Arg400 is required for the heparin-mediated stimulation of cleavage at Arg306 by APC. Furthermore, mutations that were introduced near the Arg506 cleavage site had a significant but modest effect on the rate of APC-catalyzed FVa inactivation, suggesting an extended interaction surface between the FVa Arg506 site and APC.
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, 6200MD, The Netherlands.
Kenneth
K
Segers
Björn
B
Dahlbäck
Jan
J
Rosing
Gerry A F
GA
Nicolaes
D019943:Amino Acid Substitution; D018076:DNA, Complementary / Q000235:genetics; D000939:Epitopes / Q000032:analysis*; D015943:Factor Va / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D005786:Gene Expression Regulation; D014644:Genetic Variation; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D011486:Protein C / Q000378:metabolism*; D017293:Protein S / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
D018076:DNA, Complementary; D000939:Epitopes; D011486:Protein C; D017293:Protein S; D011994:Recombinant Proteins; D015943:Factor Va; D006493:Heparin; D013917:Thrombin