Molecular requirements for safer generation of thrombolytics by bioengineering the tissue-type plasminogen activator A chain

J Thromb Haemost. 2013 Mar;11(3):539-46. doi: 10.1111/jth.12128.


Background: Thrombolysis with tissue-type plasminogen activator (t-PA) is the only treatment approved for acute ischemic stroke. Although t-PA is an efficient clot lysis enzyme, it also causes damage to the neurovascular unit, including hemorrhagic transformations and neurotoxicity.

Objectives: On the basis of the mechanism of action of t-PA on neurotoxicity, we aimed at studying the molecular requirements to generate safer thrombolytics.

Methods: We produced original t-PA-related mutants, including a non-cleavable single-chain form with restored zymogenicity (sc*-t-PA) and a t-PA modified in the kringle 2 lysine-binding site (K2*-t-PA). Both sc*-t-PA and K2*-t-PA showed fibrinolytic activities similar to that of wild-type t-PA on both euglobulin-containing and plasma-containing clots. In contrast to wild-type t-PA, the two mutants did not promote N-methyl-d-aspartate receptor-mediated neurotoxicity.

Conclusions: We designed t-PA mutants with molecular properties that, in contrast to t-PA, do not induce neurotoxicity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Bioengineering* / instrumentation
  • Bioengineering* / methods
  • Bioreactors
  • Cell Death / drug effects
  • Drug Design
  • Fibrinolysis / drug effects*
  • Fibrinolytic Agents / metabolism
  • Fibrinolytic Agents / pharmacology*
  • Fibrinolytic Agents / toxicity
  • HEK293 Cells
  • Humans
  • Kringles
  • Mice
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Neurons / drug effects
  • Neurons / metabolism
  • Neurons / pathology
  • Neurotoxicity Syndromes / etiology
  • Neurotoxicity Syndromes / metabolism
  • Neurotoxicity Syndromes / pathology
  • Neurotoxicity Syndromes / prevention & control*
  • Point Mutation
  • Rats
  • Receptors, N-Methyl-D-Aspartate / drug effects
  • Receptors, N-Methyl-D-Aspartate / metabolism
  • Recombinant Proteins / pharmacology
  • Structure-Activity Relationship
  • Thrombolytic Therapy* / adverse effects
  • Tissue Plasminogen Activator / biosynthesis
  • Tissue Plasminogen Activator / genetics
  • Tissue Plasminogen Activator / pharmacology*
  • Tissue Plasminogen Activator / toxicity
  • Transfection


  • Fibrinolytic Agents
  • Receptors, N-Methyl-D-Aspartate
  • Recombinant Proteins
  • Tissue Plasminogen Activator