Download PDF
Thromb Haemost 1988; 60(01): 107-112
DOI: 10.1055/s-0038-1647645
Original Article
Schattauer GmbH Stuttgart

Investigation by HPLC of the Catabolism of Recombinant Tissue Plasminogen Activator in the Rat

Roy Harris
*   The National Institute for Biological Standards and Control, Madrid, Spain, South Mimms, Potters Bar, Herts UK
,
Louis Garcia Frade
**   The Ramon y Cajal Hospital, Madrid, Spain
,
Lesley J Creighton
*   The National Institute for Biological Standards and Control, Madrid, Spain, South Mimms, Potters Bar, Herts UK
,
Paul S Gascoine
*   The National Institute for Biological Standards and Control, Madrid, Spain, South Mimms, Potters Bar, Herts UK
,
Maher M Alexandroni
*   The National Institute for Biological Standards and Control, Madrid, Spain, South Mimms, Potters Bar, Herts UK
,
Stephen Poole
*   The National Institute for Biological Standards and Control, Madrid, Spain, South Mimms, Potters Bar, Herts UK
,
Patrick J Gaffney
*   The National Institute for Biological Standards and Control, Madrid, Spain, South Mimms, Potters Bar, Herts UK
› Author Affiliations
Further Information

Publication History

Received 26 February 1988

Accepted after revision 28 April 1988

Publication Date:
30 June 2018 (online)

Also available at
  PDF Download  Permissions and Reprints

Summary

The catabolism of recombinant tissue plasminogen activator (rt-PA) was investigated after injection of radiolabelled material into rats. Both Iodogen and Chloramine T iodination procedures yielded similar biological activity loss in the resultant labelled rt-PA and had half lives in the rat circulation of 1 and 3 min respectively. Complex formation of rt-PA was investigated by HPLC gel exclusion (TSK G3000 SW) fractionation of rat plasma samples taken 1-2 min after 125I-rt-PA injection. A series of radiolabelled complexes of varying molecular weights were found. However, 60% of the counts were associated with a single large molecular weight complex (350–500 kDa) which was undetectable by immunologically based assays (ELISA and BIA) and showed only low activity with a functional promoter-type t-PA assay. Two major activity peaks in the HPLC fractions were associated with Tree t-PA and a complex having a molecular weight of ̴ 180 kDa. HPLC fractionation to produce these three peaks at various timed intervals after injection of 125I-rt-PA showed each to have a similar initial rate half life in the rat circulation of 4-5 min. The function of these complexes as yet is unclear but since a high proportion of rt-PA is associated with a high molecular weight complex with a short half life in the rat, we suggest that the formation of this complex may be a mechanism by which t-PA activity is initially regulated and finally cleared from the rat circulation.

Key words

Recombinant tissue plasminogen activator - Rat - Catabolic behaviour - HPLC
 

  • References

  • 1 Hedner O, Nilsson IM. The role of fibrinolysis. Clin Haematol 1981; 10: 327-342
    PubMedGoogle Scholar
  • 2 Hoylaerts M, Rijken DC, Lijnen HR, Collen D. Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin J Biol Chem 1982; 257: 2912-2919
    PubMedGoogle Scholar
  • 3 Gaffney PJ. Fibrinolysis in “Thrombosis & Haemostasis”. Bloom AL, Thomas DP. (eds) Churchill-Livingstone; Edinburgh: 1987. pp 223-244
    Google Scholar
  • 4 Collen D. On the regulation and control of fibrinolysis. Thromb Haemostas 1980; 43: 77-89
    PubMedGoogle Scholar
  • 5 Verstraete M, Bournameaux M, De Cock F, Van der Werf F, Collen D. Pharmacokinetics and systemic fibrinogenolytic effects of recombinant tissue-type plasminogen activator in humans;. J Pharm Exp Ther 1985; 235: 506-512
    PubMedGoogle Scholar
  • 6 Korninger C, Stassen JM, Collen D. Turnover of human extrinsic (tissue-type) plasminogen activator in rabbits. Thromb Haemostas 1981; 46: 658-661
    PubMedGoogle Scholar
  • 7 Beebe DP, Aronson DL. Turnover of human tissue plasminogen activator in rabbits. Thromb Res 1986; 43: 663-674
    CrossrefPubMedGoogle Scholar
  • 8 Nilsson S, Einarsson M, Ekvam S, Haggroth L, Mattsson Ch. Turnover of tissue plasminogen activator in normal and hepatectomized rabbits. Thromb Res 1985; 39: 511-521
    CrossrefPubMedGoogle Scholar
  • 9 Fuchs HE, Berger H, Pizzo SV. Catabolism of human tissue plasminogen activator in mice. Blood 1985; 65: 539-544
    PubMedGoogle Scholar
  • 10 Rijken DC, Juhan-Vague I, Collen D. Complexes between tissue type plasminogen activator and proteinase inhibitors in human plasma, identified with an immunoradiometric assay. J Lab Clin Med 1983; 101: 285-294
    PubMedGoogle Scholar
  • 11 Thorsen S, Phillips M. Isolation of tissue type plasminogen activator-inhibitor complexes from human plasma. Evidence for a rapid plasminogen activator inhibitor Biochim Biophys Acta 1984; 802: 111-118
    CrossrefPubMedGoogle Scholar
  • 12 TIMI Study Group. The thrombolysis in myocardial infarction trial. N Engl J Med 1985; 312: 932-936
    PubMedGoogle Scholar
  • 13 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. Nature 1983; 301: 214-221
    CrossrefPubMedGoogle Scholar
  • 14 Garcia FradeL J, Poole S, Hanley S, Creighton LJ, Curtis AD, Gaffney PJ. Bioavailability in rats of human recombinant tissue plasminogen activator after subcutaneous and intramuscular injection. Thromb Haemostas 1986; 56: 299-301
    PubMedGoogle Scholar
  • 15 Campbell MN, Johnson G. Chloramine T-review. Chem Rev 1978; 78: 65
    CrossrefPubMedGoogle Scholar
  • 16 Fraker PJ, Speck JC. Protein and cell membrane iodinations with sparingly soluble chloramide. Biochem Biophys Res Commun 1978; 80: 849-857
    CrossrefPubMedGoogle Scholar
  • 17 Verheijen JH, Mullart E, Chang GT G, Kluft C, Wijngaards G. A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma. Thromb Haemostas 1982; 48: 266-269
    PubMedGoogle Scholar
  • 18 Mahmoud M, Gaffney PJ. Bioimmunoassay (BIA) of tissue plasminogen activator (t-PA) and its specific inhibitor (t-PA/INH). Thromb Haemostas 1985; 53: 356-359
    PubMedGoogle Scholar
  • 19 Rijken DC, Van Hinsberg VW M, Sens EM C. Quantitation of tissue type plasminogen activator in human endothelial cultures by use of an enzyme immunoassay. Thromb Res 1984; 33: 145-153
    CrossrefPubMedGoogle Scholar
  • 20 Gaffney PJ, Curtis AD. A collaborative study of a proposed international standard for tissue plasminogen activator (t-PA). Thromb Haemostas 1985; 53: 134-136
    PubMedGoogle Scholar
  • 21 Bergmann SR, Fox KA A, Ter-Pogossian MM, Sobel BE, Collen D. Clot-selective coronary thrombolysis with tissue type plasminogen activator. Science 1983; 220: 1181-1183
    CrossrefPubMedGoogle Scholar
  • 22 Collen D, Lijnen HR. New approaches in thrombolytic therapy. Arteriosclerosis 1984; 4: 579-585
    CrossrefPubMedGoogle Scholar
  • 23 Verstraete M, Bory M, Collen D, Erbel R, Lennane RJ. et al Randomised trial of intravenous recombinant tissue-type plasminogen activator versus intravenous streptokinase in acute myocardial infarction. Lancet 1985; 1: 842-847
    PubMedGoogle Scholar
  • 24 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
    CrossrefPubMedGoogle Scholar
  • 25 Emeis JJ, Van de Hoogen CM, Jense D. Hepatic clearance of tissue-type plasminogen activators in rats. Thromb Haemostas 1985; 54: 661-664
    PubMedGoogle Scholar
  • 26 Korninger C, Collen D. Neutralisation of human extrinsic (tissue type) plasminogen activator in human plasma: no evidence for a specific inhibitor. Thromb Haemostas 1981; 46: 662-665
    PubMedGoogle Scholar
  • 27 Kruithof EK O, Tran-Thang C, Ransijn A, Bachmann F. Demonstration of a fast acting inhibitor of plasminogen activator in human plasma. Blood 1984; 64: 907-913
    PubMedGoogle Scholar
  • 28 Verheijen JH, Chang GT G, Kluft C. Evidence for the occurrence of a fast acting inhibitor for tissue-type plasminogen activator in human plasma. Thromb Haemostas 1984; 51: 392-395
    PubMedGoogle Scholar
  • 29 Wiman B, Chmielewska J, Ranby M. Inactivation of tissue plasminogen activator in plasma. Demonstration of a complex with a new rapid inhibitor J Biol Chem 1984; 259: 3644-3647
    PubMedGoogle Scholar
  • 30 Nilsson IM, Tengborn L. A family with thrombosis associated with high level of tissue plasminogen activator inhibitor. Haemostasis 1984; 14: 24
    PubMedGoogle Scholar
  • 31 Juhan-Vague I, Moerman B, De Cock F, Aillaud MF, Collen D. Plasma levels of a specific inhibitor of tissue type plasminogen activator (and urokinase) in normal and pathological conditions. Thromb Res 1984; 33: 523-530
    CrossrefPubMedGoogle Scholar