title: Toward safer thrombolytic agents in stroke: molecular requirements for NMDA receptor-mediated neurotoxicity.
abstract: Current thrombolytic therapy for acute ischemic stroke with tissue-type plasminogen activator (tPA) has clear global benefits. Nevertheless, evidences argue that in addition to its prohemorrhagic effect, tPA might enhance excitotoxic necrosis. In the brain parenchyma, tPA, by binding to and then cleaving the amino-terminal domain (ATD) of the NR1 subunit of N-methyl-D-aspartate (NMDA) glutamate receptors, increases calcium influx to toxic levels. We show here that tPA binds the ATD of the NR1 subunit by a two-sites system (K(D)=24 nmol/L). Although tenecteplase (TNK) and reteplase also display two-sites binding profiles, the catalytically inactive mutant TNKS478A displays a one-site binding profile and desmoteplase (DSPA), a kringle 2 (K2) domain-free plasminogen activator derived from vampire bat, does not interact with NR1. Moreover, we show that in contrast to tPA, DSPA does not promote excitotoxicity. These findings, together with three-dimensional (3D) modeling, show that a critical step for interaction of tPA with NR1 is the binding of its K2 domain, followed by the binding of its catalytic domain, which in turn cleaves the NR1 subunit at its ATD, leading to a subsequent potentiation of NMDA-induced calcium influx and neurotoxicity. This could help design safer new generation thrombolytic agents for stroke treatment.
authors.affiliation: INSERM, INSERM U919 'serine proteases and pathophysiology of the neurovascular unit', GIP Cyceron, Caen Cedex, France.
authors.forename: Jose P
authors.initials: JP
authors.lastname: Lopez-Atalaya
authors.forename: Benoit D
authors.initials: BD
authors.lastname: Roussel
authors.forename: Denis
authors.initials: D
authors.lastname: Levrat
authors.forename: Jérôme
authors.initials: J
authors.lastname: Parcq
authors.forename: Olivier
authors.initials: O
authors.lastname: Nicole
authors.forename: Yannick
authors.initials: Y
authors.lastname: Hommet
authors.forename: Karim
authors.initials: K
authors.lastname: Benchenane
authors.forename: Hervé
authors.initials: H
authors.lastname: Castel
authors.forename: Jérôme
authors.initials: J
authors.lastname: Leprince
authors.forename: Denis
authors.initials: D
authors.lastname: To Van
authors.forename: Ronan
authors.initials: R
authors.lastname: Bureau
authors.forename: Sylvain
authors.initials: S
authors.lastname: Rault
authors.forename: Hubert
authors.initials: H
authors.lastname: Vaudry
authors.forename: Karl-Uwe
authors.initials: KU
authors.lastname: Petersen
authors.forename: Jana Sopkova-de Oliveira
authors.initials: JS
authors.lastname: Santos
authors.forename: Carine
authors.initials: C
authors.lastname: Ali
authors.forename: Denis
authors.initials: D
authors.lastname: Vivien
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002478:Cells, Cultured; D064420:Drug-Related Side Effects and Adverse Reactions; D005343:Fibrinolytic Agents / Q000633:toxicity*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D010960:Plasminogen Activators / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D020521:Stroke / Q000378:metabolism*; D046509:Tissue Culture Techniques; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000378:metabolism
chemical_list: D005343:Fibrinolytic Agents; C409466:NR1 NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate; C069588:salivary plasminogen activator alpha 1, Desmodus rotundus; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator