Novel expression and localization of active thrombomodulin on the surface of mouse brain astrocytes

Abstract

Thrombin's potent effects on astrocytes are mediated by a specific receptor and inhibited by a serpin, protease nexin I (PNI). Thrombomodulin (TM), a membrane protein that forms complexes with thrombin, changing its enzymatic specificity, has not been studied in astrocytes. In primary astrocyte cultures, using Western blotting and immunocytochemistry, we found a 70 kDa TM band and TM localized to the surface with an anti-mouse TM monoclonal antibody. By reverse transcriptase coupled with polymerase chain reaction (RT-PCR), we found the correct sequence for mouse TM mRNA in astrocytes. Finally, we documented calcium-dependent activation of protein C by a thrombin:TM complex with thrombin added to the astrocytes. These results indicate the presence of functionally active TM at the astrocyte surface and add support to a role for thrombin signaling in the nervous system.

Localization of thrombomodulin in pericryptal fibroblasts of the rat duodenum

“…TM binds thrombin and changes the substrate specificity of thrombin toward protein C. The thrombin-TM complex activates protein C. Activated protein C in turn inactivates clotting factors Va and VIIa and plasminogen activator inhibitor type I, thus shifting the hemostatic balance to anticoagulation (Lentz and Sadler 1994). However, TM has not only been found in the vascular endothelial cells but also in some types of non-vascular cells, including the mesothelia of the pleura, pericardium, and synovial membrane (Boffa et al 1987), brain astrocytes (Pindon et al 1997), ciliary epithelia (Daimon et al 1997), and keratinocytes (Daimon and Nakano 1999). The function of TM outside vessels is not known, although this protein was postulated to play roles during embryogenesis in addition to anticoagulation (Imada et al 1990;Healy et al 1995).…”