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Thromb Haemost 1988; 59(03): 523-528
DOI: 10.1055/s-0038-1647527
Original Article
Schattauer GmbH Stuttgart

Isolation, Identification and Pharmacokinetic Properties of Human Tissue-Type Plasminogen Activator Species: Possible Localisation of a Clearance Recognition Site

Ian Dodd
The Beecham Pharmaceuticals Research Division, Biosciences Research Centre, Great Burgh, Epsom, Surrey, UK
,
Barbara Nunn
The Beecham Pharmaceuticals Research Division, Biosciences Research Centre, Great Burgh, Epsom, Surrey, UK
,
Jeff H Robinson
The Beecham Pharmaceuticals Research Division, Biosciences Research Centre, Great Burgh, Epsom, Surrey, UK
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Publikationsverlauf

Received 10. November 1987

Accepted after revision 04. März 1988

Publikationsdatum:
29. Juni 2018 (online)

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Summary

Purified preparations of recombinant tissue-type plasminogen activator (t-PA) from the recombinant Bowes melanoma cell line TRBM6 were shown to contain multiple species of plasminogen activator. Using a combination of chromatography on Sephadex G25, Sephadex G75 and Heparin Sepharose CL6B we have isolated two fibrinolytically active species, which, under nonreduced SDS PAGE, have apparent Mr = 38,000 and 56,000. Double immunodiffusion studies indicated that both species were closely related to both the t-PA B chain and t-PA itself. N-terminal sequencing identified the Mr = 38,000 species as ala160-t-PA (essentially ΔFGKI t-PA) and the Mr = 56,000 species as ser1-tyr2-gln3-glyx-cys51 t-PA (ΔF t-PA), the latter probably produced by alternative splicing of the t-PA gene. The pharmacokinetic properties of N,N dirnethy1-4-aminobenzoyl (DAB) derivatives of these activators and native t-PA were determined in the guinea pig. Whereas DAB → ΔF t-PA showed a similar, rapid plasma disappearance profile to that of DAB → t-PA, DAB → ΔFGKI t-PA was cleared significantly slower. These results suggest that a rapid clearance recognition site resides on either the growth factor or kringle 1, or both, domains of t-PA.

Keywords

Tissue-type plasminogen activator - Fibrinoly - Pharmacokinetics
 

  • References

  • 1 Van de Werf F, Ludbrook PA, Bergmann SR, Tiefenbrunn AJ, Fox KA A, De Geest H, Verstraete M, Collen D, Sobel BE. Coronary thrombolysis with tissue-typ plasminogen activator in patients with evolving myocardial infarction. N Engl J Med 1984; 310: 609-613
    CrossrefPubMedSuche in Google Scholar
  • 2 Verstraete M, Bernard R, Bory M, Brower RW, Collen D, De Bono DP, Erbel R, Huhmann W, Lennane RJ, Lubsen J, Mathey D, Meyer J, Michels HR, Rutsch W, Schartl M, Schmidt W, Vebis R, Von Essen R. Randomized trial of intravenous recombinant tissue-type plasminogen activator versus intravenous streptokinase in acute myocardial infarction. Lancet 1985; 8433: 842-847
    PubMedSuche in Google Scholar
  • 3 Collen D, Topol EJ, Tiefenbrunn AJ, Gold HK, Weisfeldt ML, Sobel BE, Leinbach RC, Brinker JA, Ludbrook PA, Yasuda T, Bulkley BH, Robison AK, Hutter AM, Bell WR, Spadaro JJ, Khaw BA, Grossbard EB. Coronary thrombolysis with recombinant human tissue-type plasminogen activator: a prospective, randomized, placebo-controlled trial. Circulation 1984; 70: 1012-1017
    CrossrefPubMedSuche in Google Scholar
  • 4 Klausner A. Researchers probe second-generation t-PA. Bio/Technology 1986; 4: 706-711
    CrossrefPubMedSuche in Google Scholar
  • 5 Ezzell C. US patents in the balance. Nature 1987; 328: 189
    PubMedSuche in Google Scholar
  • 6 Nilsson S, Wallen P, Mellbring G. In vivo metabolism of human tissue-type plasminogen activator. Scand J Haematol 1984; 33: 49-53
    PubMedSuche in Google Scholar
  • 7 Rijken DC, Emeis JJ. Clearance of the heavy and light polypeptide chains of human tissue-type plasminogen activator in rats. Biochem J 1986; 238: 643-646
    CrossrefPubMedSuche in Google Scholar
  • 8 Rijken DC, Collen D. Purification and characterisation of the plasminogen activator secreted by human melanoma cells in culture. J Biol Chem 1981; 256: 7035-7041
    PubMedSuche in Google Scholar
  • 9 Pennica D, Holmes WE, Kohr WJ, Harkins RN, Vehar GA, Ward CA, Bennett WF, Yelverton E, Seeburg PH, Heyneker HL, Goeddel DV, Collen D. Cloning and expression of human tissue-type plasminogen activator cDNA in E.coli. Nature 1983; 301: 214-221
    CrossrefPubMedSuche in Google Scholar
  • 10 Pohl G, Kallstrom M, Bergsdorf N, Wallen P, Jornvall H. Tissue plasminogen activator: Peptide analyses confirm an indirectly derived amino acid sequence, identify the active site serine residue, establish glycosylation sites, and localize variant differences. Biochemistry 1984; 23: 3701-3707
    CrossrefPubMedSuche in Google Scholar
  • 11 Banyai L, Varadi A, Patthy L. Common evolutionary origin of the fibring-binding structures of fibronectin and tissue-type plasminogen activator. FEBS Lett 1983; 163: 37-41
    CrossrefPubMedSuche in Google Scholar
  • 12 Kagitani H, Tagawa M, Hatanaka K, Ikari T, Saito A, Bando H, Okada K, Matsuo O. Expression in E.coli of finger-domain lacking tissue-type plasminogen activator with high fibrin affinity. FEBS Lett 1985; 189: 145-149
    CrossrefPubMedSuche in Google Scholar
  • 13 Gilbert LC, Wachsmann JT. Characterisation and partial purification of the plasminogen activator from human neuroblastoma cell line, SK-N-SH. Biochim Biophys Acta 1982; 704: 450-460
    CrossrefPubMedSuche in Google Scholar
  • 14 Dodd I, Jalalpour S, Southwick W, Newsome P, Browne MJ, Robinson JH. Large scale, rapid purification of recombinant tissue-type plasminogen activator. FEBS Lett 1986; 209: 13-17
    CrossrefPubMedSuche in Google Scholar
  • 15 Browne MJ, Dodd I, Carey JE, Chapman CG, Robinson JH. Increased yield of human tissue-type plasminogen activator by means of recombinant DNA technology. Thromb Haemostas 1985; 54: 422-424
    PubMedSuche in Google Scholar
  • 16 Smith RAG. An active-site titrant for human tissue-type plasminogen activator. Biochem J 1986; 239: 477-479
    CrossrefPubMedSuche in Google Scholar
  • 17 Dodd I, Fears R, Robinson JH. Isolation and preliminary characterisation of active B-chain of recombinant tissue-type plasminogen activator. Thromb Haemostas 1986; 55: 94-97
    PubMedSuche in Google Scholar
  • 18 Petersen TE, Thogersen HC, Skorstengaard K, Vibe-Pedersen K, Sahl P, Sottrup-Jensen L, Magnusson S. Partial primary structure of bovine plasma fibronectin: Three types of internal homology. Proc Natl Acad Sci 1983; 50: 137-141
    PubMedSuche in Google Scholar
  • 19 Korninger C, Stassen JM, Collen D. Turnover of human extrinsic (tissue-type) plasminogen activator in rabbits. Thromb Haemostas 1981; 46: 658-661
    PubMedSuche in Google Scholar
  • 20 Smith RA G, Dupe RJ, English PD, Green J. Fibrinolysis with acyl enzymes: a new approach to thrombolytic therapy. Nature 1981; 290: 505-508
    CrossrefPubMedSuche in Google Scholar
  • 21 Ny T, Elgh F, Lund B. The structure of the human tissue-type plasminogen activator gene: Correlation of intron and exon structures to functional and structural domains. Proc Natl Acad Sci USA 1984; 81: 5355-5359
    CrossrefPubMedSuche in Google Scholar
  • 22 MacGregor IR, Micklem LR, James K, Pepper DS. Characterisation of epitopes on human tissue plasminogen activator recognized by a group of monoclonal antibodies. Thromb Haemostas 1985; 53: 45-50
    PubMedSuche in Google Scholar
  • 23 Verheijen JH, Caspers MP M, Chang GTG, de Munk GA, Pouwels PH, Enger-Valk BE. Involvement of finger domain and kringle 2 domain of tissue-type plasminogen activator in fibrin binding and stimulation of activity by fibrin. Embo J 1986; 5: 3525-3530
    PubMedSuche in Google Scholar
  • 24 Van Zonneveld A-J, Veerman H, Pannekoek H. Autonomous functions of structural domains on human, tissue-type plasminogen activator. Proc Natl Acad Sci USA 1986; 83: 4670-4674
    CrossrefPubMedSuche in Google Scholar