Research ReportThrombin-induced activation of astrocytes in mixed rat hippocampal cultures is inhibited by soluble thrombomodulin

Thrombin, a serine protease known for its role in coagulation, also induces a variety of protease activated receptor (PAR)-mediated responses in the central nervous system that contribute to many brain pathologies. Since the proteolytic specificity of thrombin is uniquely controlled by thrombomodulin (TM), we investigated the mechanisms by which thrombin and a recombinant soluble form of human TM (Solulin, INN: sothrombomodulin alpha; rhsTM) could influence rat hippocampal cultures. Treatment of hippocampal cultures with thrombin for up to 48 h resulted in a significant morphological rearrangement with the formation of expansive cell-free areas (CFAs) and a reduction in cell viability; both effects were blocked by rhsTM. Treatment with the selective PAR-1 agonist, TRAP (SFLLRN) caused the formation of CFAs, suggesting that CFA formation involved PAR-1 signaling. Astrocytes prepared from PAR-1−/− mice also had an attenuated CFA response to thrombin. Thrombin-induced CFA formation was a consequence of cell movement and substantial changes in cell morphology, rather than due to cell detachment. Immunocytochemical and functional analyses revealed that the thrombin-sensitive cells within these hippocampal cultures were astrocytes. The effects of thrombin on CFA development were mediated by astrocyte-specific release of intracellular calcium and signalling through ERK1/2. rhsTM was able to attenuate thrombin-induced ERK1/2 phosphorylation. Finally, astrocytes were shown to maintain thrombin-sensitivity following neuronal depletion with NMDA, a result which was confirmed with pure astrocyte cultures. Hence thrombin mediates PAR-1-induced activation of hippocampal astrocytes, but not neurons, in a process that can be modulated by rhsTM.

Research HighlightsThe work described in this manuscript presents novel data on the role of thrombin on astrocyte function in the hippocampus, and the modulating effect of soluble thrombomodulin. The key results obtained are as follows: ► Thrombin induces astrocyte-specific changes in free calcium and ERK signalling ► The effect of thrombin on calcium levels occurs in a subpopulation of astrocytes ► Thrombin causes novel astrocyte driven alteration of culture morphology via PAR-1 ► The thrombin effect on astrocytes does not require neurons. ► Thrombomodulin blocks thrombin signalling and astrocyte rearrangement