A long-acting PAI-1 inhibitor reduces thrombus formation

Thromb Haemost. 2017 Jun 28;117(7):1338-1347. doi: 10.1160/TH16-11-0891. Epub 2017 Apr 13.


Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of tissue-type and urokinase-type plasminogen activators (t/uPA) and plays an important role in fibrinolysis. Inhibition of PAI-1 activity prevents thrombosis and accelerates fibrinolysis, indicating that PAI-1 inhibitors may be used as effective antithrombotic agents. We previously designed a PAI-1 inhibitor (PAItrap) which is a variant of inactivated urokinase protease domain. In the present study, we fused PAItrap with human serum albumin (HSA) to develop a long-acting PAI-1 inhibitor. Unfortunately, the fusion protein PAItrap-HSA lost some potency compared to PAItrap (33 nM vs 10 nM). Guided by computational method, we carried out further optimisation to enhance inhibitory potency for PAI-1. The new PAItrap, denominated PAItrap(H37R)-HSA, which was the H37R variant of PAItrap fused to HSA, gave a six-fold improvement of IC50 (5 nM) for human active PAI-1 compared to PAItrap-HSA, and showed much longer plasma half-life (200-fold) compared to PAItrap. We further demonstrated that the PAItrap(H37R)-HSA inhibited exogenous or endogenous PAI-1 to promote fibrinolysis in fibrin-clot lysis assay. PAItrap(H37R)-HSA inhibits murine PAI-1 with IC50 value of 12 nM, allowing the inhibitor to be evaluated in murine models. Using an intravital microscopy, we demonstrated that PAItrap(H37R)-HSA blocks thrombus formation and platelet accumulation in vivo in a laser-induced vascular injury mouse model. Additionally, mouse tail bleeding assay showed that PAItrap(H37R)-HSA did not affect the global haemostasis. These results suggest that PAItrap(H37R)-HSA have the potential benefit to prevent thrombosis and accelerates fibrinolysis.

Keywords: PAI-1 inhibitor; clot lysis; human serum albumin (HSA); thrombosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bleeding Time
  • Disease Models, Animal
  • Drug Design
  • Fibrinolysis / drug effects
  • Fibrinolytic Agents / blood
  • Fibrinolytic Agents / chemistry
  • Fibrinolytic Agents / pharmacology*
  • Half-Life
  • Humans
  • In Vitro Techniques
  • Mice
  • Mice, Inbred C57BL
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Peptide Fragments / pharmacology
  • Plasminogen Activator Inhibitor 1 / blood
  • Plasminogen Activator Inhibitor 1 / chemistry
  • Plasminogen Activator Inhibitor 1 / pharmacology*
  • Protein Engineering
  • Recombinant Fusion Proteins / blood
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / pharmacology
  • Serine Proteinase Inhibitors / blood
  • Serine Proteinase Inhibitors / chemistry
  • Serine Proteinase Inhibitors / pharmacology
  • Serpin E2 / antagonists & inhibitors
  • Thrombosis / prevention & control*
  • Urokinase-Type Plasminogen Activator / pharmacology


  • Fibrinolytic Agents
  • PAItrap peptide
  • Peptide Fragments
  • Plasminogen Activator Inhibitor 1
  • Recombinant Fusion Proteins
  • SERPINE1 protein, human
  • Serine Proteinase Inhibitors
  • Serpin E2
  • Serpine2 protein, mouse
  • Urokinase-Type Plasminogen Activator