title: Ecotin is a potent anticoagulant and reversible tight-binding inhibitor of factor Xa.
abstract: Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, has been characterized as an extremely potent anticoagulant and reversible tight-binding inhibitor of human factor Xa (FXa). The ecotin gene was cloned by PCR, highly expressed in E. coli, and purified from the E. coli periplasm. The binding of ecotin to FXa was stoichiometric with an equilibrium dissociation constant Ki of 54 pM. The association rate constant was 1.35 x 10(6) M-1 s-1, and the dissociation rate constant, measured in the presence of human leukocyte elastase (HLE) to prevent reassociation of ecotin with FXa, was 6.5 x 10(-5) s-1. Ecotin prolonged clotting time ca. 10-fold at 0.3 microM and at 2 microM in activated partial thromboplastin time and prothrombin time assays, respectively. Ecotin did not effectively inhibit the human plasma proteases thrombin, tissue factor.factor VIIa, factor XIa, activated protein C, plasmin, or tissue plasminogen activator (t-PA); however, it did potently inhibit factor XIIa, plasma kallikrein, HLE, and bovine trypsin and chymotrypsin. Coincubation of ecotin and FXa at 10 microM each resulted in a (ecotin)2.(FXa)2 complex as determined by gel filtration. Dimerization of ecotin alone was measured by fluorescence titration which yielded a Kd of ca. 390 nM. FXa cleaved ecotin slowly at pH 4.0 between M84 and M85. Replacement of the P1 Met84 residue with Arg and Lys led to FXa inhibitors with Ki values of 11 and 21 pM, respectively. The P1 Arg and Lys mutants also significantly inhibited thrombin, factor XIa, activated protein C, plasmin, factor XIIa, kallikrein, and bovine trypsin and chymotrypsin but did not inhibit tissue factor.factor VIIa, t-PA, or HLE.
authors.affiliation: Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080.
authors.forename: J L
authors.initials: JL
authors.lastname: Seymour
authors.forename: R N
authors.initials: RN
authors.lastname: Lindquist
authors.forename: M S
authors.initials: MS
authors.lastname: Dennis
authors.forename: B
authors.initials: B
authors.lastname: Moffat
authors.forename: D
authors.initials: D
authors.lastname: Yansura
authors.forename: D
authors.initials: D
authors.lastname: Reilly
authors.forename: M E
authors.initials: ME
authors.lastname: Wessinger
authors.forename: R A
authors.initials: RA
authors.lastname: Lazarus
mesh_terms: D000925:Anticoagulants; D001426:Bacterial Proteins / Q000235:genetics / Q000494:pharmacology*; D001483:Base Sequence; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D065427:Factor Xa Inhibitors; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D033903:Periplasmic Proteins; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000925:Anticoagulants; D001426:Bacterial Proteins; D017931:DNA Primers; C039076:Eco protein, E coli; D029968:Escherichia coli Proteins; D065427:Factor Xa Inhibitors; D033903:Periplasmic Proteins