Serpin-resistant mutants of human tissue-type plasminogen activator

Edwin L. Madison, Elizabeth J. Goldsmith, Robert D. Gerard, Mary Jane H Gething, Joseph F. Sambrook

Research output: Contribution to journalArticle

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Abstract

TISSUE-type plasminogen activator (t-PA) converts the inactive zymogen, plasminogen, into the powerful protease, plasmin, which then degrades the fibrin meshwork of thrombi1-3. To prevent systemic activation of plasminogen, plasma contains several inhibitors of t-PA, the most important of which is plasminogen activator inhibitor-1 (PAI-1), a member of the serpin superfamily4-6. As the ability to produce serpin-resistant variants of t-PA could increase the potential of this enzyme as a thrombolytic agent, we have used the known three-dimensional structure of the complex between trypsin and bovine pancreatic trypsin inhibitor (BPTI) to model the interactions between the active site of human t-PA and PAI-1. On the basis of this model we then altered by site-directed mutagenesis those amino acids of t-PA predicted to make contact with PAI-1 but not with the substrate plasminogen. We report here that although the resulting mutants have enzymatic properties similar to those of wild-type t-PA, they display significant resistance to inhibition by PAI-1. For example, following incubation with an amount of the serpin that completely inhibits the wild-type enzyme, one variant retains 95% of its initial activity. This mutant is also resistant to inhibition by the complex mixture of serpins present in human plasma.

Original languageEnglish (US)
Pages (from-to)721-724
Number of pages4
JournalNature
Volume339
Issue number6227
StatePublished - 1989

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Serpins
Plasminogen Activators
Tissue Plasminogen Activator
Plasminogen Activator Inhibitor 1
Plasminogen
Plasminogen Inactivators
Enzyme Precursors
Aprotinin
Fibrinolytic Agents
Fibrinolysin
Enzymes
Site-Directed Mutagenesis
Fibrin
Complex Mixtures
Trypsin
Catalytic Domain
Peptide Hydrolases
Amino Acids

ASJC Scopus subject areas

  • General

Cite this

Madison, E. L., Goldsmith, E. J., Gerard, R. D., Gething, M. J. H., & Sambrook, J. F. (1989). Serpin-resistant mutants of human tissue-type plasminogen activator. Nature, 339(6227), 721-724.

Serpin-resistant mutants of human tissue-type plasminogen activator. / Madison, Edwin L.; Goldsmith, Elizabeth J.; Gerard, Robert D.; Gething, Mary Jane H; Sambrook, Joseph F.

In: Nature, Vol. 339, No. 6227, 1989, p. 721-724.

Research output: Contribution to journalArticle

Madison, EL, Goldsmith, EJ, Gerard, RD, Gething, MJH & Sambrook, JF 1989, 'Serpin-resistant mutants of human tissue-type plasminogen activator', Nature, vol. 339, no. 6227, pp. 721-724.
Madison EL, Goldsmith EJ, Gerard RD, Gething MJH, Sambrook JF. Serpin-resistant mutants of human tissue-type plasminogen activator. Nature. 1989;339(6227):721-724.
Madison, Edwin L. ; Goldsmith, Elizabeth J. ; Gerard, Robert D. ; Gething, Mary Jane H ; Sambrook, Joseph F. / Serpin-resistant mutants of human tissue-type plasminogen activator. In: Nature. 1989 ; Vol. 339, No. 6227. pp. 721-724.
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