title: Substitution of asparagine for arginine 347 of recombinant factor Xa markedly reduces factor Va binding.
abstract: Herein we describe a recombinant factor X (fX) with a single substitution at position 347 (fXR347N). Activated fXR347N had a reduced affinity for factor Va (fVa), although the catalytic impact of fVa binding remained intact. The mutation was selective as demonstrated by normal activation and inhibition, except in the presence of subsaturating heparin where the rate of inhibition by antithrombin III (ATIII) was 15% of normal. The reactivity of fXaR347N toward prothrombin was equivalent to wild-type fXa (fXaWT) in the absence of fVa and phospholipid. Addition (without phospholipid) of fVa dramatically increased the catalytic efficiency of fXaWT toward prothrombin but had a negligible effect on fXaR347N. On addition of phosphatidylcholine:phosphatidylserine (PC:PS, 3:1) vesicles, fXaR347Ndisplayed an increased catalytic activity in response to fVa, but the apparent affinity for fVa on the phospholipid surface was 5-20-fold lower than that of fXaWT. On an activated platelet surface, however, fXaWT and fXaR347N activated prothrombin similarly. In a competitive binding assay that measures the displacement of radiolabeled fXa from fVa on a phospholipid surface, fXaR347N was approximately 10-fold less effective than fXaWT. Substitution of fXa at position 347 selectively attenuates the interaction between fXa and fVa without affecting its catalytic activity.
authors.affiliation: Departments of Pathology and Medicine, Division of Laboratory Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA. amy.e.rudolph@monsanto.com
authors.forename: A E
authors.initials: AE
authors.lastname: Rudolph
authors.forename: R
authors.initials: R
authors.lastname: Porche-Sorbet
authors.forename: J P
authors.initials: JP
authors.lastname: Miletich
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D000990:Antithrombin III / Q000494:pharmacology; D001120:Arginine / Q000235:genetics* / Q000378:metabolism; D001216:Asparagine / Q000235:genetics* / Q000378:metabolism; D001667:Binding, Competitive / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015943:Factor Va / Q000378:metabolism; D015951:Factor Xa / Q000235:genetics* / Q000378:metabolism*; D065427:Factor Xa Inhibitors; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D011516:Prothrombin / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D013499:Surface Properties
chemical_list: D004791:Enzyme Inhibitors; D065427:Factor Xa Inhibitors; D010743:Phospholipids; D011994:Recombinant Proteins; D015943:Factor Va; D001216:Asparagine; D000990:Antithrombin III; D011516:Prothrombin; D001120:Arginine; D015951:Factor Xa