Variants of Tissue-type Plasminogen Activator That Display Extraordinary Resistance to Inhibition by the Serpin Plasminogen Activator Inhibitor Type 1

• Published in: The Journal of biological chemistry 1997-06, Vol.272 (23), p.14580-14585
• Main Authors: Tachias, K, Madison, E L
• Format: Article
• Language: English
• Subjects: Arginine; Base Sequence; Chromogenic Compounds; DNA Primers; Genetic Variation; Kinetics; Macromolecular Substances; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligopeptides; Plasminogen Activator Inhibitor 1 - pharmacology; Point Mutation; Recombinant Proteins - antagonists & inhibitors; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Tissue Plasminogen Activator - antagonists & inhibitors; Tissue Plasminogen Activator - biosynthesis; Tissue Plasminogen Activator - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.23.14580

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.