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Biological Properties of Hybrid Plasminogen Activators

  • P. P. Hung
  • J. Wilhelm
  • N. K. Kalyan
  • S. M. Cheng
  • S. G. Lee
  • H. L. James
  • D. Nachowiak
  • C. J. Weinheimer
  • B. E. Sobel
  • S. R. Bergmann
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 281)

Summary

A number of hybrid plasminogen activator genes were constructed from the t-PA and u-PA cDNAs and expressed using a bovine papilloma virus vector and mouse C-127 cells. Hybrid A was constructed by replacing the finger (F) and EGF domains of t-PA with the EGF and Ku domains of u-PA, while hybrids B and C had an extra Ku inserted before or after the double kringle (K1-K2) region of t-PA respectively. While all the hybrids showed comparable enzymatic activities towards a small substrate (S-2288), they had different activities in binding to fibrin clots as well in the fibrin-dependent plasminogen activation, the order of activities being: t-PA ≥ hybrid B > hybrid C > hybrid A. Carbohydrate analysis showed that while hybrid C, like rt-PA, had at least one high-mannose type sugar chain (probably at residue 117 in K1), the other hybrids had only complex-type carbohydrates suggesting that domain interaction in t-PA might influence glycan processing. Pharmacokinetic studies in dog showed that hybrid B had a significantly longer plasma half-life than rt-PA. Thrombolytic efficacies of hybrid B and rt-PA were compared in dog model using an artificially induced coronary thrombus. Complete thrombolysis was achieved with 18 mg and 50 mg dosages for hybrid B and rt-PA respectively. These data show the superior pharmacokinetic and thrombolytic properties of hybrid B compared to rt-PA.

Keywords

Plasminogen Activator Fibrin Clot Sugar Chain Hybrid Band Kringle Domain 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. 1.
    D. Collen, On the regulation and control of fibrinolysis. Thromb. Haemost., 43:77–89 (1980).PubMedGoogle Scholar
  2. 2.
    A.-J. Tiefenbrunn, and B. E. Sobel, The impact of coronary thrombolysis of myocardial infarction. Fibrinolysis, 3:1–15 (1989).Google Scholar
  3. 3.
    N. U. Bang, Tissue-type plasminogen activator mutants. Theoretical and clinical considerations. Circulation, 79:1391–1392 (1989).CrossRefGoogle Scholar
  4. 4.
    H. R. Lijnen, and D. Collen, Tissue-type plasminogen activator. Ann. Biol. Clin., 45:198–201 (1987).Google Scholar
  5. 5.
    E. W. Davie, A. Inchinose, and S. P. Leytus, Structural features of the proteins participating in blood circulation and fibrinolysis. Cold Spring Harbor Symp. Quant. Biol., 51:509–514 (1986).PubMedCrossRefGoogle Scholar
  6. 6.
    L. Patty, Evolution of the proteases of blood coagulation and fibrinolysis by assembly for modules. Cell, 41:657–663 (1985).CrossRefGoogle Scholar
  7. 7.
    J. W. McLean, J. E. Tomlinson, W.-J. Kuang, D. L. Eaton, E. Y. Chen, G. M. Fless, A. M. Scanu, and R. M. Lawn, cDNA sequence of human apolipoprotein (a) is homologous to plasminogen. Nature, 330:132–137 (1987).PubMedCrossRefGoogle Scholar
  8. 8.
    H. Pannekoek, C. de Vries, and A.-J. van Zonneveld, Mutants of human tissue-type plasminogen activator (t-PA): Structural aspects and functional properties. Fibrinolysis, 2:123–132 (1988).Google Scholar
  9. 9.
    V. Gurewich, R. Pannell, S. Louie, P. Kelley, R. L. Suddith, and R. Greenlee, Effective and fibrin-specific clot lysis by a zymogen precursor form of urokinase (pro-urokinase): a study in vitro and in two animal species. J. Clin. Invest., 73:1731–1739 (1984).PubMedCrossRefGoogle Scholar
  10. 10.
    S. G. Lee, N. K. Kalyan, J. Wilhelm, W.-T. Hum, R. Rappaport, S.-M. Cheng, S. Dheer, C. Urbano, R. W. Hartzell, M. Ronchetti-Blume, M. Levner, and P. P. Hung, Construction and expression of hybrid plasminogen activators prepared from tissue-type plasminogen activator and urokinase-type plasminogen activator genes. J. Biol. Chem., 263:2917–2924 (1988).PubMedGoogle Scholar
  11. 11.
    M. Ranby, N. Bergsdorf, G. Pohl, and P. Wallen, Isolation of two variants of native one-chain tissue plasminogen activator. FEBS Letters, 146:289–292 (1982).PubMedCrossRefGoogle Scholar
  12. 12.
    N. K. Kalyan, S. G. Lee, J. Wilhelm, K. P. Fu, W.-T. Hum, R. Rappaport, R. W. Hartzell, C. Urbano, and P. P. Hung, Structure-function analysis with tissue-type plasminogen activator: effect of deletion of NH2-terminal domains on its biochemical and biological properties. J. Biol. Chem., 263:3971–3978 (1988).PubMedGoogle Scholar
  13. 13.
    G. Pohl, L. Kenne, B. Nilsson, and M. Einarsson, Isolation and characterization of three different carbohydrate chains from melanoma tissue plasminogen activator. Eur. J. Biochem., 170:69–75 (1987).PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1990

Authors and Affiliations

  • P. P. Hung
    • 1
  • J. Wilhelm
    • 1
  • N. K. Kalyan
    • 1
  • S. M. Cheng
    • 1
  • S. G. Lee
    • 1
  • H. L. James
    • 2
  • D. Nachowiak
    • 2
  • C. J. Weinheimer
    • 2
  • B. E. Sobel
    • 2
  • S. R. Bergmann
    • 2
  1. 1.Wyeth-Ayerst ResearchPhiladelphiaUSA
  2. 2.Washington UniversitySt. LouisUSA

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