title: Enhanced rate of cleavage at Arg-306 and Arg-506 in coagulation factor Va by Gla domain-mutated human-activated protein C.
abstract: A Gla domain-mutated protein C variant, QGNSEDY, modified at positions 10-12, 23, 32-33, and 44, having enhanced affinity for negatively charged phospholipid and increased anticoagulant potential, was used to elucidate the importance of the interaction between the Gla domain and the phospholipid for the ability of activated protein C (APC) to inactivate factor Va (FVa). FVa degradation by wild type (WT)-APC and QGNSEDY-APC yielded similar fragments on Western blotting; QGNSEDY-APC was, however, considerably more efficient. The kinetic parameters for individual APC-mediated cleavages in FVa, i.e. at Arg-306 and Arg-506, were investigated at high and low phospholipid concentrations in the presence and absence of protein S. FVa variants 306Q679Q and 506Q679Q, which can only be cleaved at Arg-506 and Arg-306, respectively, were used. In the absence of protein S, QGNSEDY-APC was 17.8- and 4-fold more efficient than WT-APC in cleaving at Arg-306 and Arg-506, respectively, at high phospholipid. Similar values were obtained at low phospholipid. In the presence of protein S, QGNSEDYAPC was 6.8- and 3.2-fold more active than WT-APC in cleaving at Arg-306 and Arg-506, respectively, at high phospholipid. At low phospholipid, the corresponding values were 14- and 6.5-fold. In conclusion, the modification of the Gla domain in QGNSEDY-APC yielded increased rates of cleavage at both sites in FVa, the increase being particularly pronounced for the Arg-306 site in the absence of protein S. The results obtained with QGNSEDY-APC provide insights into the importance of the APC-phospholipid interaction for the APC-mediated cleavages at Arg-306 and Arg-506 in FVa.
authors.affiliation: Department of Laboratory Medicine, Division of Clinical Chemistry, Lund University, University Hospital, Malmö, S-20502 Malmö, Sweden.
authors.forename: Yong-Hui
authors.initials: YH
authors.lastname: Sun
authors.forename: Sinh
authors.initials: S
authors.lastname: Tran
authors.forename: Eva A
authors.initials: EA
authors.lastname: Norstrøm
authors.forename: Björn
authors.initials: B
authors.lastname: Dahlbäck
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism*; D004789:Enzyme Activation; D015943:Factor Va / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D010743:Phospholipids / Q000378:metabolism; D011486:Protein C / Q000235:genetics / Q000378:metabolism*; D017293:Protein S / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000925:Anticoagulants; D010743:Phospholipids; D011486:Protein C; D017293:Protein S; D021122:Protein Subunits; D011994:Recombinant Proteins; D015943:Factor Va; D001120:Arginine