title: Role of basic residues of the autolysis loop in the catalytic function of factor Xa.
abstract: The autolysis loop of factor Xa (fXa) has four basic residues (Arg(143), Lys(147), Arg(150), and Arg(154)) whose contribution to protease specificity of fXa has not been examined. Here, we substituted these basic residues individually with Ala in the fX cDNA and expressed them in mammalian cells using a novel expression/purification vector system. Following purification to homogeneity and activation by the factor X activator from Russell viper venom, the mutants were characterized with respect to their ability to assemble into the prothrombinase complex to activate prothrombin and interact with target plasma fXa inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin. We show that all mutants interacted with factor Va with normal affinities and exhibited wild-type-like prothrombinase activities toward prothrombin. Lys(147) and Arg(154) mutants were inhibited by TFPI approximately 2-fold slower than wild type; however, both Arg(143) and Arg(150) mutants were inhibited normally by the inhibitor. The reactivities of Arg(143) and Lys(147) mutants were improved approximately 2-fold with antithrombin in the absence but not in the presence of heparin cofactors. On the other hand, the pentasaccharide-catalyzed reactivity of antithrombin with the Arg(150) mutant was impaired by an order of magnitude. These results suggest that Arg(150) of the autolysis loop may specifically interact with the activated conformation of antithrombin.
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, 1402 S. Grand Boulevard, St Louis, MO 63104, USA.
authors.forename: Chandrashekhara
authors.initials: C
authors.lastname: Manithody
authors.forename: Likui
authors.initials: L
authors.lastname: Yang
authors.forename: Alireza R
authors.initials: AR
authors.lastname: Rezaie
mesh_terms: D000991:Antithrombins / Q000378:metabolism; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D015951:Factor Xa / Q000235:genetics / Q000378:metabolism*; D065427:Factor Xa Inhibitors; D006801:Humans; D007700:Kinetics; D008074:Lipoproteins / Q000494:pharmacology*; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis / Q000502:physiology; D009842:Oligopeptides / Q000032:analysis / Q000378:metabolism; D011516:Prothrombin / Q000378:metabolism*; D013925:Thromboplastin / Q000378:metabolism*
chemical_list: D000991:Antithrombins; D065427:Factor Xa Inhibitors; D008074:Lipoproteins; D009842:Oligopeptides; C051928:lipoprotein-associated coagulation inhibitor; D011516:Prothrombin; D013925:Thromboplastin; D001120:Arginine; C049024:H-hexahydrotyrosyl-alanyl-arginine-4-nitroanilide; D015951:Factor Xa; D008239:Lysine