Thrombin-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 48h 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.
Australian Centre for Blood Diseases, Monash University, Melbourne, Victoria 3004, Australia.
Be'eri
B
Niego
Andre L
AL
Samson
Karl-Uwe
KU
Petersen
Robert L
RL
Medcalf
D000704:Analysis of Variance; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D001792:Blood Platelets / Q000502:physiology; D015153:Blotting, Western; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010974:Platelet Aggregation / Q000187:drug effects / Q000502:physiology; D051381:Rats; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology*; D018180:Thrombomodulin / Q000378:metabolism*
D018180:Thrombomodulin; D013917:Thrombin