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Thromb Haemost 1992; 68(03): 291-296
DOI: 10.1055/s-0038-1656367
Original Article
Schattauer GmbH Stuttgart

Molecular Cloning and Cell-Free Expression of Mouse Antithrombin III

John K Wu
1   The Canadian Red Cross Society Blood Transfusion Service, McMaster University, Hamilton, Ontario, Canada
,
William P Sheffield
1   The Canadian Red Cross Society Blood Transfusion Service, McMaster University, Hamilton, Ontario, Canada
,
Morris A Blajchman
1   The Canadian Red Cross Society Blood Transfusion Service, McMaster University, Hamilton, Ontario, Canada
2   The Department of Pathology, McMaster University, Hamilton, Ontario, Canada
› Author Affiliations
Further Information

Publication History

Received 05 December 1991

Accepted after revision 06 April 1992

Publication Date:
04 July 2018 (online)

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Summary

The homology between antithrombin III (AT-III) of mouse, of man, and that of other species was investigated. Preliminary experiments showed that mouse AT-III inhibited human α-thrombin efficiently (second order rate constant [K 2nd] 5.8 × 103 M–1 s–1) as compared to human AT-III (K 2nd 6.7 × 103 M–1), but was not recognized on immunoblots by antibodies that recognized both human and rabbit AT-III. In order to compare AT-III from different species at the molecular level, a cDNA clone for murine AT-III was isolated from a λZAP mouse liver cDNA library on the basis of hybridization to a rabbit AT-III cDNA probe. The 1509 bp murine AT-III cDNA consists of a 1398 bp open reading frame, preceded by a 15 bp 5’ untranslated region, followed by a 75 bp 3’ untranslated region. The deduced primary protein structure consists of a 32 amino acid signal sequence, with a mature portion of 433 residues. Mature murine AT-III is 89% identical to its human counterpart, 86% identical to bovine AT-III, and 82% identical to that of the rabbit. Constructs lacking the nucleotides encoding the signal sequence were engineered and expressed in a cell-free system. The resulting 47 kDa non-glycosylated translation product was capable of being cleaved by human α-thrombin, of forming SDS-stable complexes with the protease, and of binding to immobilized heparin. Isolation of the murine AT-III cDNA will make feasible molecularly defined experiments with murine AT-III in the mouse system.

 

  • References

  • 1 Rosenberg RD, Damus PS. The purification and mechanism of antithrombin-heparin cofactor. J Biol Chem 1973; 248: 6490-6505
    PubMedGoogle Scholar
  • 2 Buchanan MR, Boneu B, Ofosu F, Hirsh J. The relative importance of thrombin inhibition and factor Xa inhibition to the antithrombotic effects of heparin. Blood 1985; 65: 198-201
    PubMedGoogle Scholar
  • 3 Chandra T, Stackhouse R, Kidd V, Robson KJH, Woo SLC. Sequence homology between human α-1-chymotrypsin, α-1-antitrypsin and antithrombin III. Biochemistry 1983; 22: 5055-5060
    CrossrefPubMedGoogle Scholar
  • 4 Bjork I, Jackson CM, Jornvall H, Laurie KH, Nordling K, Salsgiver WJ. The active site of antithrombin. J Biol Chem 1982; 257: 2406-2411
    PubMedGoogle Scholar
  • 5 Owen WG. Evidence for the formation of an ester between thrombin and heparin cofactor. Biochim Biophys Acta 1975; 405: 380-387
    CrossrefPubMedGoogle Scholar
  • 6 Pizzo SV. Serpin Receptor 1: A hepatic receptor that mediates the clearance of antithrombin III protease complexes. Am J Med 1989; 87 (Suppl. 3B) 10S-14S
    PubMedGoogle Scholar
  • 7 Stern D, Nawroth P, Marcum J, Hendley D, Kisiel W, Rosenberg R, Stern K. Interaction of antithrombin III with bovine aortic segments. Role of heparin in binding and enhanced anticoagulant activity. J Clin Invest 1985; 75: 272-279
    PubMedGoogle Scholar
  • 8 Hatton MW, Moar SL, Richardson M. Evidence that rabbit [125]I-antithrombin binds to proteoheparin sulphate at the subendothelium of the rabbit aorta in vitro. Blood Vessels 1988; 25: 12-27
    PubMedGoogle Scholar
  • 9 Verstraete M. Pharmacotherapeutic aspects of unfractionated and low molecular weight heparins. Drugs 1990; 40: 498-530
    CrossrefPubMedGoogle Scholar
  • 10 Sas G, Peto I, Banhegyi D, Blasko G, Dornjan G. Heterogeneity of the “classical” antithrombin III deficiency. Thromb Haemostas 1980; 43: 133-136
    PubMedGoogle Scholar
  • 11 Rosenberg RD. Actions and interactions of antithrombin and heparin. N Engl J Med 1975; 292: 146-151
    CrossrefPubMedGoogle Scholar
  • 12 Abildgaard V. Antithrombins and related inhibitors of coagulation. In: Recent Advances in Blood Coagulation. Poller L. (ed) Churchill Livingstone Edinburgh: 1981. pp 151-173
    Google Scholar
  • 13 Petersen TE, Dudek-Wojciechowska G, Sottrup-Jensen L. Primary structure of antithrombin III (heparin cofactor). Partial homology between α-1-antitrypsin and antithrombin III. The Physiological Inhibitors of Coagulation and Fibrinolysis Collen D, Wiman B, Verstraete M. Elsevier North-Holland Biomedical Press, Amsterdam: 1979: 43-54
    Google Scholar
  • 14 Medjoub H, Leret M, Boulanger Y, Maman M, Choay J, Reinbolt J. The complete amino acid sequence of bovine antithrombin (ATIII). J Prot Chem 1991; 10: 205-212
    CrossrefPubMedGoogle Scholar
  • 15 Bock SC, Wion KL, Vehar GA, Lawn RM. Cloning and expression of the cDNA for human antithrombin III. Nucleic Acids Res 1982; 10: 8113-8125
    CrossrefPubMedGoogle Scholar
  • 16 Prochownik EV, Markham AF, Orkin SH. Isolation of a cDNA clone for human antithrombin III. J Biol Chem 1983; 258: 8389-8394
    PubMedGoogle Scholar
  • 17 Sheffield WP, Brothers AB, Hatton MWC, Clarke BJ, Blajchman MA. Molecular cloning and expression of rabbit antithrombin III. Blood, (in press) 1992
    PubMedGoogle Scholar
  • 18 Short JM, Fernandez JM, Sorge JA, Huse WD. λZAP: A bacteriophage λ expression vector with in vivo excision properties. Nucleic Acids Res 1988; 16: 7583-7600
    CrossrefPubMedGoogle Scholar
  • 19 Falcone D, Andrews DW. Both 5’ untranslated region and sequences surrounding the start site contribute to efficient initiation of translation in vitro. Mol Cell Biol 1991; 11: 2656-2664
    CrossrefPubMedGoogle Scholar
  • 20 Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. (eds) Current Protocols in Molecular Biology. Wiley-Interscience: New York: 1987
    Google Scholar
  • 21 Thomas PS. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci (USA) 1980; 77: 5201-5211
    CrossrefPubMedGoogle Scholar
  • 22 Saiki RK, Scharf S, Faloona F, Mully KB, Horn GT, Ehrlich HA, Arnheim N. Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985; 230: 1350-1354
    CrossrefPubMedGoogle Scholar
  • 23 Krieg PA, Melton DA. Functional messenger RNAs produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res 1984; 12: 7057
    CrossrefPubMedGoogle Scholar
  • 24 Austin RC, Rachubinski RA, Fernandez-Rachubinski F, Blajchman MA. Expression in a cell-free system of normal and variant forms of human antithrombin III. Ability to bind heparin and react with α-thrombin. Blood 1990; 76: 1521-1529
    PubMedGoogle Scholar
  • 25 Lamontagne L, Gauldie J. Ontogeny of antithrombin III in mouse and rat. Thromb Res 1980; 20: 417-424
    CrossrefPubMedGoogle Scholar
  • 26 Kovacs T, Kalapas M, Spolarius Z, Ganzo T, Antoni F, Machovich R. Interaction of thrombin, antithrombin III and their complex with hepatocytes: Comparison of molecular components of human and mouse origin. Thromb Res 1987; 46: 875-880
    CrossrefPubMedGoogle Scholar
  • 27 Jordan RE. Antithrombin III in vertebrate evolution. Arch Biochem Biophys 1893; 227: 587-595
    PubMedGoogle Scholar
  • 28 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254
    CrossrefPubMedGoogle Scholar
  • 29 Abildgaard U, Lie M, Odegard OR. Antithrombin (heparin cofactor) assay with “new” chromogenic substrates (S-2238) and chromozym TH. Thromb Res 1977; 22: 549-553
    PubMedGoogle Scholar
  • 30 Towbin H, Staehlin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4354
    CrossrefPubMedGoogle Scholar
  • 31 Kurachi K, Schmer G, Hermodson MA, Teller DC, Davie EW. Characterization of human, bovine, and horse antithrombin III. Biochemistry 1979; 15: 368-373
    PubMedGoogle Scholar
  • 32 Olson ST, Sheffer R, Stephens AW, Hirs CHW. Molecular basis of the reduced reactivity of Antithrombin-III-Denver with thrombin and factor Xa. Thromb Haemostas 1991; 65: 670a
    PubMedGoogle Scholar
  • 33 Austin RC, Rachubinski RA, Blajchman MA. Site-directed mutagenesis of alanine-382 of human antithrombin III. FEBS Lett 1991; 280: 254-258
    CrossrefPubMedGoogle Scholar