title: Conformational conversion of antithrombin to a fully activated substrate of factor Xa without need for heparin.
abstract: Regulation of the inhibitory activity of antithrombin, the principal inhibitor of the blood-clotting proteinases factor Xa and thrombin, is accomplished by binding to heparin. We report here an antithrombin variant in which serine at position 380, 14 residues N-terminal from the reactive bond and at a hinge point in the structure, was replaced by cysteine to test a proposed mechanism of heparin activation of antithrombin as an inhibitor of factor Xa. By derivatization of this cysteine with a bulky group, fluorescein, the antithrombin became permanently and fully activated toward reaction with factor Xa in a manner analogous to heparin activation, albeit as a substrate. These findings establish a structural basis for the mechanism of heparin activation of antithrombin against factor Xa in agreement with that proposed from an X-ray structure of antithrombin.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Illinois at Chicago 60612-4316, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Huntington
authors.forename: P G
authors.initials: PG
authors.lastname: Gettins
mesh_terms: D000818:Animals; D000990:Antithrombin III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D015951:Factor Xa / Q000378:metabolism*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D014644:Genetic Variation; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; C015296:5-iodoacetamidofluorescein; D000990:Antithrombin III; D006493:Heparin; D015951:Factor Xa