• L Gorbacheva, V Pinelis, S Ishiwata, S Strukova, and G Reiser.
• Lomonosov Moscow State University, Department of Human and Animal Physiology, Moscow, Russia.
• Neuroscience. 2010 Feb 17; 165 (4): 113811461138-46.

AbstractBrain injury is associated with neuroinflammation, neurodegeneration, and also blood coagulation with thrombin formation and generation of activated protein C (APC). We have previously shown that APC, a serine protease of hemostasis, at very low concentrations has protective effects in rat hippocampal and cortical neurons at glutamate-induced excitotoxicity through protease-activated receptor-1 (PAR-1) or endothelial receptor of protein C (EPCR)/PAR-1. The transcription factor nuclear factor kappaB (NF-kappaB) takes part in regulating neuronal survival in several pathological conditions. To elucidate the impact of NF-kappaB in APC-mediated cell survival, we investigated nuclear translocation of NF-kappaB p65 at glutamate- or thrombin-induced toxicity in hippocampal neurons. We used immunoassay and immunostaining with confocal microscopy with anti-NF-kappaBp65 antibody. We show that APC at concentrations as low as 1-2 nM inhibits translocation of NF-kappaB p65 into the nucleus of cultured rat hippocampal neurons, induced by 100 muM glutamate or 50 nM thrombin (but not 10 nM). The blocking effect of APC on NF-kappaB p65 translocation was observed at 1 and 4 h after treatment of neurons with glutamate, when the NF-kappaBp 65 level in the nucleus was significantly above the basal level. Then we investigated whether the binding of APC to EPCR/PAR-1 is required to control NF-kappaB activation. Antibodies blocking PAR-1 (ATAP2) or EPCR (P-20) abolished the APC-induced decrease of nuclear level of NF-kappaB p65 at glutamate-induced toxicity, whereas control antibodies to PAR-1 (S-19) and EPCR (IgG) exerted no effect. Thus, we suggest that the activation of NF-kappaB in rat hippocampal neurons mediates the glutamate- and thrombin-activated cell death program, which is reduced by exposure of cells to APC. APC induces the reduction of the nuclear level of NF-kappaB p65 in hippocampal neurons at glutamate-induced excitotoxicity via binding to EPCR and subsequent PAR-1 activation and signaling.

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