Thrombin induced inhibition of neurite outgrowth from dorsal root ganglion neurons

Thrombin is a multifunctional protease. Recent studies on cultured neuronal cells have suggested a function for thrombin in the development and maintenance of the nervous system. Thrombin has been found to induce neurite retraction and reverse stellation in neuroblastoma cell lines and rat astrocytes, respectively. The major focus of our study was to investigate the potential role of thrombin in peripheral nervous system development using the rat embryonic dorsal root ganglion model. We found a dose dependent inhibition of neurite outgrowth from explant dorsal root ganglion cultures upon exposure to 2 to 200 nM thrombin. This effect was reversed by the specific thrombin inhibitor, hirudin. A synthetic peptide that imitates the fully active receptor, thrombin receptor activating peptide, was also found to inhibit neurite outgrowth from dorsal root ganglia. bis-Benzimide stained neuronal cultures did not show any evidence of cell death after exposure to thrombin or thrombin receptor activating peptides. Immunohistochemical studies revealed specific staining of the thrombin receptor on neurons, with intense labeling along neurites. Enriched neuronal cultures exposed to thrombin and thrombin receptor activating peptides revealed rapid activation of phospholipase Cγ-1, a second messenger associated with the thrombin receptor. These findings are the first to describe the localization of the thrombin receptor to dorsal root ganglion neurons. We propose that receptor activation is associated with thrombin induced inhibition of neurite outgrowth.