Modulation of Thrombin Signaling by Thrombomodulin Internalization Huey-Chun Huang (黃蕙君), Chun-Mei Wu, Guey-Yueh Shi, and Hua-Lin Wu. Department of Biochemistry, college of Medicine, National Cheng Kung University Department of Medical Technology, China Medical University, Taiwan Protease-activated receptors 1 (PAR 1) and thrombomodulin (TM) are two different types of thrombin receptors. The stimulating signal of thrombin mediated by PAR-1 was terminated by internalization of the activated receptor. TM was well studied for its anticoagulant activity by function as a cofactor for protein C activation. Internalization of thrombin-TM complex may regulate the concentration of thrombin and the duration of thrombin stimulation. In addition, the thrombin-TM complex may involve in the signal transduction pathway and the cross talk between the pathways induced by these two receptors may exist. Previously report d In this study HEK 293 cells were transfected with green fluorescent protein tagged- TM (TMG) and lectin-like domain-truncated TM (TMG(DL)) and the trafficking of TM molecules was observed with a confocal microscope. Thrombin induced internalization of TMG, but not TMG (DL). We also demonstrated that thrombin-TMG complex was internalized via caveolae-mediated pathway. TM was recycled to the plasma membrane while the thrombin was retained in the cytoplasm. Thrombin induced transient phosphorylation of extracellular signal regulated kinase (ERK) in HEK cells with a peak at 15 min. However, in the cells transfected with TMG and TMG(DL), thrombin induced a prolonged phosphorylation of ERK. Increased level of ERK phosphorylation appeared in the TMG-expressed cells and ERK phosphorylation remained on activation state at 4 hours after thrombin stimulation. Apparently, the cross talk occurred between these two kinds of thrombin receptors. These results indicated that internalization of thrombin is not essential for prolongation of ERK phosphorylation. T
