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 ( KD=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.
Background and Objective:We have previously shown that tissue type plasminogen activator (t-PA) potentiates NMDA receptor (NMDAR) signaling by proteolysis at arginine 260 of the amino terminal domain (ATD) of the NR1 subunit (NR1). The molecular background of this action has been elucidated using recombinant rt-PA and the structurally-related plasminogen activators, desmoteplase, reteplase, and tenecteplase. Methods: Cleavage of ATD-NR1 was examined using rATD-NR1, a His-tagged recombinant form, as an enzyme substrate, immunoblot analysis being performed post-incubation of rATD-NR1 with a plasminogen activator. Additionally, the binding of plasminogen activators to rATD-NR1 was analyzed using surface plasmon resonance (SPR). Results: Cleavage of the rATD-NR1 was observed with rt-PA, reteplase and tenecteplase, but not desmoteplase, which was the only compound tested that possesses no kringle2 domain. Rt-PA bound to rATD-NR1 in a concentration-dependent and reversible manner consistent with a two-site kinetic model (KD = 24 nM). Analysis indicated that two binding steps are required for cleavage of the NR1 subunit, i.e. kringle2 binding as the first step and binding of the catalytic domain as the second step. Tenecteplase bound to rATD-NR1 with characteristics similar to those of rt-PA and with a reliable fit to two-site kinetics. Binding of reteplase occurred with a pattern similar to that of tPA and TNK, but with a lower apparent affinity. By contrast, no binding was obtained when desmoteplase was tested. Summary: We have found that rt-PA binds ATD-NR1 by a two-site system involving sequential binding of its kringle2 and catalytic domains. Only plasminogen activators carrying a kringle2 domain were able to cleave the rATD-NR1. Thus, this domain appears to be critical for t-PA-mediated enhancement of NMDAR signaling. Our data suggest that strategies able to prevent or avoid kringle2-dependent enhancement of NMDAR signaling may allow for thrombolysis without neurotoxicity.
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