title: Serpin-resistant mutants of human tissue-type plasminogen activator.
abstract: Tissue-type plasminogen activator (t-PA) converts the inactive zymogen, plasminogen, into the powerful protease, plasmin, which then degrades the fibrin meshwork of thrombi. 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 superfamily. 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.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
authors.forename: E L
authors.initials: EL
authors.lastname: Madison
authors.forename: E J
authors.initials: EJ
authors.lastname: Goldsmith
authors.forename: R D
authors.initials: RD
authors.lastname: Gerard
authors.forename: M J
authors.initials: MJ
authors.lastname: Gething
authors.forename: J F
authors.initials: JF
authors.lastname: Sambrook
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D006023:Glycoproteins / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D015849:Plasminogen Inactivators; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D012689:Sequence Homology, Nucleic Acid; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors / Q000235:genetics*; D014357:Trypsin / Q000235:genetics; D014361:Trypsin Inhibitors / Q000494:pharmacology
chemical_list: D006023:Glycoproteins; D015849:Plasminogen Inactivators; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D014357:Trypsin; D010959:Tissue Plasminogen Activator