title: Purification and characterization of recombinant antistasin: a leech-derived inhibitor of coagulation factor Xa.
abstract: Antistasin (ATS) is a selective, tight-binding inhibitor of blood coagulation Factor Xa originally isolated from the salivary glands of the Mexican leech Haementeria officinalis. In order to provide sufficient quantities of ATS to further investigate the role of Factor Xa in blood coagulation, a recombinant version of ATS has been produced in an insect baculovirus host-vector system. In this study, we describe the purification and in vitro and in vivo characterization of a single recombinant antistasin (rATS) isoform. The purified protein constitutes a minor isoform relative to the more abundant ATS isoforms present in leech salivary gland extracts. In vitro, rATS inhibits purified human Factor Xa stoichiometrically, prolongs plasma-based clotting assays at nanomolar concentrations, and like native ATS, is cleaved at a single position by Factor Xa during the course of inhibition. An initial evaluation of the in vivo efficacy of rATS was addressed utilizing a rhesus monkey model of mild disseminated intravascular coagulation. rATS was shown to fully suppress thromboplastin-induced fibrinopeptide A generation in a dose-dependent fashion. The availability of rATS should provide a valuable tool for the critical evaluation of the specific role played by Factor Xa in coagulation.
authors.affiliation: Department of Biological Chemistry, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
authors.forename: E M
authors.initials: EM
authors.lastname: Nutt
authors.forename: D
authors.initials: D
authors.lastname: Jain
authors.forename: A B
authors.initials: AB
authors.lastname: Lenny
authors.forename: L
authors.initials: L
authors.lastname: Schaffer
authors.forename: P K
authors.initials: PK
authors.lastname: Siegl
authors.forename: C T
authors.initials: CT
authors.lastname: Dunwiddie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001777:Blood Coagulation / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D065427:Factor Xa Inhibitors; D005344:Fibrinopeptide A / Q000378:metabolism; D007447:Invertebrate Hormones / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D007536:Isomerism; D007865:Leeches; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012469:Salivary Glands / Q000737:chemistry*; D013917:Thrombin / Q000378:metabolism; D013925:Thromboplastin / Q000494:pharmacology
chemical_list: D065427:Factor Xa Inhibitors; D007447:Invertebrate Hormones; D011994:Recombinant Proteins; C053155:antistasin; D005344:Fibrinopeptide A; D013925:Thromboplastin; D013917:Thrombin