Australian Centre for Blood Diseases, Monash University, Melbourne, Victoria 3004, Australia.
Brain Res. 2011 Mar 24;1381:38-51. doi: 10.1016/j.brainres.2011.01.016. Epub 2011 Jan 15.
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.
凝血酶，一种以其在凝血中的作用而闻名的丝氨酸蛋白酶，也在中枢神经系统中诱导多种蛋白酶激活受体 (PAR) 介导的反应，这些反应有助于许多脑部疾病。由于凝血酶的蛋白水解特异性是由血栓调节蛋白 (TM) 独特控制的，因此我们研究了凝血酶和重组可溶性人 TM 形式 (Solulin，INN：sothrombomodulin alpha; rhsTM) 如何影响大鼠海马培养物的机制。用凝血酶处理海马培养物长达 48 小时会导致明显的形态重排，形成扩张的无细胞区 (CFA)，并降低细胞活力；这两种作用都被 rhsTM 阻断。用选择性 PAR-1 激动剂 TRAP (SFLLRN) 处理会导致 CFA 的形成，表明 CFA 的形成涉及 PAR-1 信号。来自 PAR-1(-/-) 小鼠的星形胶质细胞也对凝血酶的 CFA 反应减弱。凝血酶诱导的 CFA 形成是细胞运动和细胞形态发生重大变化的结果，而不是由于细胞脱落。免疫细胞化学和功能分析表明，这些海马培养物中对凝血酶敏感的细胞是星形胶质细胞。rhsTM 能够减弱凝血酶诱导的 ERK1/2 磷酸化。rhsTM 能够减弱凝血酶诱导的 ERK1/2 磷酸化。最后，星形胶质细胞在 NMDA 耗尽神经元后仍保持对凝血酶的敏感性，这一结果在纯星形胶质细胞培养物中得到了证实。因此，凝血酶通过 PAR-1 诱导海马星形胶质细胞的激活，但不诱导神经元激活，这一过程可以通过 rhsTM 进行调节。
