Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Engineering plasminogen activator inhibitor 1 mutants with increased functional stability

D A Lawrence1, S T Olson, S Palaniappan

  • 1Department of Internal Medicine, University of Michigan, Ann Arbor 48109-0650.

Biochemistry
|March 29, 1994
PubMed
Summary

Researchers engineered Plasminogen activator inhibitor 1 (PAI-1) mutants with enhanced functional stability by disrupting key ion pairs. These stable PAI-1 variants show extended half-lives while retaining normal activity against tPA and uPA.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Correction: TRAF2 is a biologically important necroptosis suppressor.

Cell death and differentiation·2025
Same author

A novel cell-permeable peptide prevents protein SUMOylation and supports the mislocalization and aggregation of TDP-43.

Neurobiology of disease·2023
Same author

DOSE DEPENDENCE OF RADIATION INDUCED DAMAGE IN THE THERMOLUMINESCENT RESPONSE OF LIF:Mg,Ti (TLD-100).

Radiation protection dosimetry·2020
Same author

Newborn adipokines and early childhood growth.

Pediatric obesity·2018
Same author

Autologous mononuclear cells from different sources are seen to improve wound healing in patients with haematological malignancies.

The Malaysian journal of pathology·2018
Same author

Oral application of a periodontal pathogen impacts SerpinE1 expression and pancreatic islet architecture in prediabetes.

Journal of periodontal research·2017

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Plasminogen activator inhibitor 1 (PAI-1) is a critical regulator of fibrinolysis, belonging to the serine protease inhibitor (Serpin) superfamily.
  • PAI-1 naturally transitions from an active to an inactive latent form, with a short half-life under physiological conditions.
  • Understanding PAI-1 stability is crucial for therapeutic applications targeting thrombosis and related disorders.

Purpose of the Study:

  • To engineer and characterize PAI-1 mutants with significantly increased functional stability.
  • To investigate the role of specific ion pairs in the conformational stability of PAI-1.
  • To assess the impact of mutations on PAI-1 activity and half-life.

Main Methods:

  • Site-directed mutagenesis was employed to introduce mutations disrupting the Arg-30/Glu-350 ion pair in recombinant PAI-1.

Related Experiment Videos

  • Functional activity of PAI-1 mutants against tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) was evaluated.
  • Half-life determination, circular dichroism spectroscopy, and thermal stability assays were performed to characterize mutant protein properties.

    • Main Results:

    • Three PAI-1 mutants exhibited extended functional half-lives compared to wild-type PAI-1 (wtPAI-1), ranging from 1.2 to 2.1 hours.
    • All engineered mutants retained normal inhibitory activity against both tPA and uPA.
    • Structural analyses indicated similar secondary structures across active and latent forms, but latent mutants showed reduced thermal stability compared to latent wtPAI-1.

    Conclusions:

    • Specific mutations disrupting the Arg-30/Glu-350 ion pair successfully enhance the functional stability of PAI-1.
    • These findings represent the first report of engineered PAI-1 mutants with demonstrably improved functional half-lives.
    • The study provides insights into PAI-1 conformational dynamics and offers potential for developing more stable PAI-1-based therapeutics.