title: Variants of tissue-type plasminogen activator that display extraordinary resistance to inhibition by the serpin plasminogen activator inhibitor type 1.
abstract: Fibrinolysis is regulated in part by the interaction of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1). Previous investigations suggest that three specific arginine residues, Arg-298, Arg-299, and Arg-304 of t-PA, play a critical role in this important regulatory interaction. Our earlier studies have demonstrated that conversion of any of these three residues to a glutamic acid residue reduced the rate of inhibition of t-PA by PAI-1 by factors varying from 58-64. In addition, we have reported that the second order rate constant for inhibition by PAI-1 of the variant t-PA/K296E,R298E,R299E is reduced by a factor of approximately 2800 compared with that of wild type t-PA. In this study, we have significantly extended our earlier observations by identifying t-PA variants that are substantially more resistant to inhibition by PAI-1 than any previously reported variants of t-PA or urokinase-type plasminogen activator. Single-chain t-PA/R275E,R298E, R299E,R304E, for example, is inhibited by PAI-1 approximately 120, 000 times less rapidly than single-chain, wild type t-PA. We also report the first direct comparison of the effects of charge reversal mutations of Arg-298, Arg-299, and/or Arg-304 on the properties of the single- and two-chain forms of t-PA. While these mutations confer extraordinary resistance to inhibition by PAI-1 to both forms of the enzyme, our observations reveal that the single-chain enzyme is affected to a greater extent than the two-chain enzyme. Two-chain, wild type t-PA is inhibited by PAI-1 approximately 1.4 times more rapidly than single-chain t-PA. The corresponding ratio increases to 7.6 or 6.7, respectively, for variants of t-PA containing the R298E, R299E or R298E,R299E,R304E mutations.
authors.affiliation: The Scripps Research Institute, Department of Vascular Biology, La Jolla, California 92037, USA.
authors.forename: K
authors.initials: K
authors.lastname: Tachias
authors.forename: E L
authors.initials: EL
authors.lastname: Madison
mesh_terms: D001120:Arginine; D001483:Base Sequence; D002863:Chromogenic Compounds; D017931:DNA Primers; D014644:Genetic Variation; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D017395:Plasminogen Activator Inhibitor 1 / Q000494:pharmacology*; D017354:Point Mutation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000737:chemistry
chemical_list: D002863:Chromogenic Compounds; D017931:DNA Primers; D046911:Macromolecular Substances; D009842:Oligopeptides; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D001120:Arginine; C049024:H-hexahydrotyrosyl-alanyl-arginine-4-nitroanilide; D010959:Tissue Plasminogen Activator