凝血酶調節素 (thrombomodulin，TM) 是一種廣泛表現的穿膜醣蛋白，具有多個功能區。除了抗凝血功能外，亦有研究指出凝血酶調節素參與調控發炎反應，但目前對於白血球上凝血酶調節素的功能尚未明瞭。本實驗室先前的研究顯示凝血酶調節素會透過其胺基端類凝集素功能區 (lectin-like domain) 特異性的結合醣類路易士Y (Lewis Y，Ley)，文獻指出路易士Y會透過醣化於黏附因子上，來調控細胞的黏附作用。因此，本論文主要探討在發炎時，白血球上凝血酶調節素是否會透過與內皮細胞上路易士Y的結合來調控白血球黏附。我們分析在多種發炎相關刺激物刺激情形下，人類單核球細胞 (THP-1) 上凝血酶調節素的表現量是否會受到影響，由實驗結果顯示經由巴豆醇-12-十四烷酸酯-13-乙酸酯 (phorbol 12-myristate 13-acetate)、腫瘤壞死因子α (tumor necrosis factor alpha) 及酯多醣體 (lipopolysaccharides) 刺激後，細胞表面的凝血酶調節素的表現量會上升。利用酵素連結免疫吸附法 (enzyme-linked immunosorbent assay)，發現游離態的路易士Y可以直接與細胞表面的凝血酶調節素結合。此外，滾動中的人類單核球細胞藉由凝血酶調節素黏附於流體腔上的路易士Y。我們更發現路易士Y和凝血酶調節素間的作用可以誘導p38的磷酸化，進一步促進β2整合素 (β2-integrin) 的活化。利用細胞穿孔實驗 (transwell assay) ，發現路易士Y和凝血酶調節素的結合可促進白血球細胞穿越活化的內皮細胞，並且此現象會被路易士Y和凝血酶調節素抗體所抑制。為了要觀察在動物模式中，白血球上凝血酶調節素是否會參與白血球趨化，我們利用頸動脈結紮手術誘發野生型(TMf/f)與白血球細胞上特異性凝血酶調節素剔除 (LysMCre/TMf/f) 小鼠的血管發炎反應，發現後者經由血管結紮後，白血球趨化和新生血管内膜增生較為減緩。綜合以上結果發現，在發炎情形下，白血球上凝血酶調節素會藉由與內皮細胞上路易士Y的結合來調控白血球和內皮細胞的交互作用。

Thrombomodulin (TM), a widely expressed transmembrane glycoprotein, has multiple functional domains. In addition to the well-known anticoagulant function, TM also regulates inflammatory responses. However, the role of TM on leukocyte still remains unclear. Our data previously showed that TM can specifically bind to the carbohydrate, Lewis Y (Ley), via its N-terminal lectin-like domain. Ley is found to be glycosylated on adhesion molecules and involved in cell adhesion. In this study, we intended to investigate whether leukocyte surface TM would participate in leukocyte adhesion by interacting with Ley on endothelial cells under inflammation. We analyzed membrane TM expression on THP-1 monocytic cells under various stimuli. The expression of TM on THP-1 monocytic cells was upregulated by monocyte activating agent, phorbol 12-myristate 13-acetate, as well as by inflammatory factors, tumor necrosis factor alpha and lipopolysaccharides. By using enzyme-linked immunosorbent assay, we found that soluble Ley could directly bind to TM on the surface of THP-1 monocytic cells. Moreover, the adhesion of rolling THP-1 monocytic cells to Ley-immobilized flow chamber was TM dependent. We further found that Ley could trigger p38 phosphorylation and was also TM dependent, which in turn led to β2-integrin activation, in THP-1 monocytic cells. By using transwell assay, the interaction of Ley and TM contributed to transendothelial migration, and which was abrogated by antibodies against either TM or Ley. To determine whether leukocyte TM would participate in leukocyte recruitment in vivo, we performed carotid artery ligation to induce vascular inflammation in wild-type (TMf/f) and myeloid-specific TM-deficient (LysMCre/TMf/f) mice. Reduced leukocyte recruitment and neointima formation were observed in myeloid-specific TM-deficient mice following carotid artery ligation. Our data provide a new insight showing that leukocyte TM could mediate leukocyte-endothelial interaction via binding to endothelial cell surface Ley under inflammation.

1. Davis, C., Fischer, J., Ley, K., and Sarembock, I. J. (2003) The role of inflammation in vascular injury and repair. Journal of thrombosis and haemostasis : JTH 1, 1699-1709
2. Tedgui, A., and Mallat, Z. (2006) Cytokines in atherosclerosis: pathogenic and regulatory pathways. Physiological reviews 86, 515-581
3. Boyce, J. A., Mellor, E. A., Perkins, B., Lim, Y. C., and Luscinskas, F. W. (2002) Human mast cell progenitors use alpha4-integrin, VCAM-1, and PSGL-1 E-selectin for adhesive interactions with human vascular endothelium under flow conditions. Blood 99, 2890-2896
4. Frangogiannis, N. G., Smith, C. W., and Entman, M. L. (2002) The inflammatory response in myocardial infarction. Cardiovascular research 53, 31-47
5. Rao, R. M., Yang, L., Garcia-Cardena, G., and Luscinskas, F. W. (2007) Endothelial-dependent mechanisms of leukocyte recruitment to the vascular wall. Circulation research 101, 234-247
6. Liu, Y., Shaw, S. K., Ma, S., Yang, L., Luscinskas, F. W., and Parkos, C. A. (2004) Regulation of leukocyte transmigration: cell surface interactions and signaling events. Journal of immunology (Baltimore, Md. : 1950) 172, 7-13
7. Sadler, J. E. (1997) Thrombomodulin structure and function. Thrombosis and haemostasis 78, 392-395
8. Stenflo, J. (1976) A new vitamin K-dependent protein. Purification from bovine plasma and preliminary characterization. The Journal of biological chemistry 251, 355-363
9. Raife, T. J., Lager, D. J., Madison, K. C., Piette, W. W., Howard, E. J., Sturm, M. T., Chen, Y., and Lentz, S. R. (1994) Thrombomodulin expression by human keratinocytes. Induction of cofactor activity during epidermal differentiation. The Journal of clinical investigation 93, 1846-1851
10. Maillard, C., Berruyer, M., Serre, C. M., Amiral, J., Dechavanne, M., and Delmas, P. D. (1993) Thrombomodulin is synthesized by osteoblasts, stimulated by 1,25-(OH)2D3 and activates protein C at their cell membrane. Endocrinology 133, 668-674
11. McCachren, S. S., Diggs, J., Weinberg, J. B., and Dittman, W. A. (1991) Thrombomodulin expression by human blood monocytes and by human synovial tissue lining macrophages. Blood 78, 3128-3132
12. Conway, E. M. (2012) Thrombomodulin and its role in inflammation. Seminars in immunopathology 34, 107-125
13. Lo, I. C., Lin, T. M., Chou, L. H., Liu, S. L., Wu, L. W., Shi, G. Y., Wu, H. L., and Jiang, M. J. (2009) Ets-1 mediates platelet-derived growth factor-BB-induced thrombomodulin expression in human vascular smooth muscle cells. Cardiovascular research 81, 771-779
14. Lager, D. J., Callaghan, E. J., Worth, S. F., Raife, T. J., and Lentz, S. R. (1995) Cellular localization of thrombomodulin in human epithelium and squamous malignancies. The American journal of pathology 146, 933-943
15. Cheng, T. L., Wu, Y. T., Lin, H. Y., Hsu, F. C., Liu, S. K., Chang, B. I., Chen, W. S., Lai, C. H., Shi, G. Y., and Wu, H. L. (2011) Functions of rhomboid family protease RHBDL2 and thrombomodulin in wound healing. The Journal of investigative dermatology 131, 2486-2494
16. Shi, C. S., Shi, G. Y., Hsiao, H. M., Kao, Y. C., Kuo, K. L., Ma, C. Y., Kuo, C. H., Chang, B. I., Chang, C. F., Lin, C. H., Wong, C. H., and Wu, H. L. (2008) Lectin-like domain of thrombomodulin binds to its specific ligand Lewis Y antigen and neutralizes lipopolysaccharide-induced inflammatory response. Blood 112, 3661-3670
17. Martin, F. A., Murphy, R. P., and Cummins, P. M. (2013) Thrombomodulin and the vascular endothelium: insights into functional, regulatory, and therapeutic aspects. American journal of physiology. Heart and circulatory physiology 304, H1585-1597
18. Grey, S. T., and Hancock, W. W. (1996) A physiologic anti-inflammatory pathway based on thrombomodulin expression and generation of activated protein C by human mononuclear phagocytes. Journal of immunology (Baltimore, Md. : 1950) 156, 2256-2263
19. Van de Wouwer, M., Plaisance, S., De Vriese, A., Waelkens, E., Collen, D., Persson, J., Daha, M. R., and Conway, E. M. (2006) The lectin-like domain of thrombomodulin interferes with complement activation and protects against arthritis. Journal of thrombosis and haemostasis : JTH 4, 1813-1824
20. Li, Y. H., Kuo, C. H., Shi, G. Y., and Wu, H. L. (2012) The role of thrombomodulin lectin-like domain in inflammation. Journal of biomedical science 19, 34
21. Varki, A. (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3, 97-130
22. Norden, R., Nystrom, K., Aurelius, J., Brisslert, M., and Olofsson, S. (2013) Virus-induced appearance of the selectin ligand sLeX in herpes simplex virus type 1-infected T-cells: involvement of host and viral factors. Glycobiology 23, 310-321
23. Garcia-Vallejo, J. J., van Liempt, E., da Costa Martins, P., Beckers, C., van het Hof, B., Gringhuis, S. I., Zwaginga, J. J., van Dijk, W., Geijtenbeek, T. B., van Kooyk, Y., and van Die, I. (2008) DC-SIGN mediates adhesion and rolling of dendritic cells on primary human umbilical vein endothelial cells through LewisY antigen expressed on ICAM-2. Molecular immunology 45, 2359-2369
24. Cao, Y., Merling, A., Karsten, U., and Schwartz-Albiez, R. (2001) The fucosylated histo-blood group antigens H type 2 (blood group O, CD173) and Lewis Y (CD174) are expressed on CD34+ hematopoietic progenitors but absent on mature lymphocytes. Glycobiology 11, 677-683
25. Halloran, M. M., Carley, W. W., Polverini, P. J., Haskell, C. J., Phan, S., Anderson, B. J., Woods, J. M., Campbell, P. L., Volin, M. V., Backer, A. E., and Koch, A. E. (2000) Ley/H: an endothelial-selective, cytokine-inducible, angiogenic mediator. Journal of immunology (Baltimore, Md. : 1950) 164, 4868-4877
26. Lin, W. L., Chang, C. F., Shi, C. S., Shi, G. Y., and Wu, H. L. (2013) Recombinant lectin-like domain of thrombomodulin suppresses vascular inflammation by reducing leukocyte recruitment via interacting with Lewis Y on endothelial cells. Arterioscler Thromb Vasc Biol 33, 2366-2373
27. Kuo, C. H., Chen, P. K., Chang, B. I., Sung, M. C., Shi, C. S., Lee, J. S., Chang, C. F., Shi, G. Y., and Wu, H. L. (2012) The recombinant lectin-like domain of thrombomodulin inhibits angiogenesis through interaction with Lewis Y antigen. Blood 119, 1302-1313
28. Ma, C. Y., Shi, G. Y., Shi, C. S., Kao, Y. C., Lin, S. W., and Wu, H. L. (2012) Monocytic thrombomodulin triggers LPS- and gram-negative bacteria-induced inflammatory response. Journal of immunology (Baltimore, Md. : 1950) 188, 6328-6337
29. Hidalgo, A., Peired, A. J., Wild, M. K., Vestweber, D., and Frenette, P. S. (2007) Complete identification of E-selectin ligands on neutrophils reveals distinct functions of PSGL-1, ESL-1, and CD44. Immunity 26, 477-489
30. Kumar, A., and Lindner, V. (1997) Remodeling with neointima formation in the mouse carotid artery after cessation of blood flow. Arterioscler Thromb Vasc Biol 17, 2238-2244
31. Daigneault, M., Preston, J. A., Marriott, H. M., Whyte, M. K., and Dockrell, D. H. (2010) The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages. PLoS One 5, e8668
32. Auwerx, J. (1991) The human leukemia cell line, THP-1: a multifacetted model for the study of monocyte-macrophage differentiation. Experientia 47, 22-31
33. Worley, J. R., Baugh, M. D., Hughes, D. A., Edwards, D. R., Hogan, A., Sampson, M. J., and Gavrilovic, J. (2003) Metalloproteinase expression in PMA-stimulated THP-1 cells. Effects of peroxisome proliferator-activated receptor-gamma (PPAR gamma) agonists and 9-cis-retinoic acid. The Journal of biological chemistry 278, 51340-51346
34. Crockett-Torabi, E., and Fantone, J. C. (1995) The selectins: insights into selectin-induced intracellular signaling in leukocytes. Immunologic research 14, 237-251
35. Blenis, J. (1993) Signal transduction via the MAP kinases: proceed at your own RSK. Proceedings of the National Academy of Sciences of the United States of America 90, 5889-5892
36. Crockett-Torabi, E. (1998) Selectins and mechanisms of signal transduction. Journal of leukocyte biology 63, 1-14
37. Lo, S. K., Lee, S., Ramos, R. A., Lobb, R., Rosa, M., Chi-Rosso, G., and Wright, S. D. (1991) Endothelial-leukocyte adhesion molecule 1 stimulates the adhesive activity of leukocyte integrin CR3 (CD11b/CD18, Mac-1, alpha m beta 2) on human neutrophils. The Journal of experimental medicine 173, 1493-1500
38. Kosako, H., Gotoh, Y., Matsuda, S., Ishikawa, M., and Nishida, E. (1992) Xenopus MAP kinase activator is a serine/threonine/tyrosine kinase activated by threonine phosphorylation. Embo j 11, 2903-2908
39. Owens, D. M., and Keyse, S. M. (2007) Differential regulation of MAP kinase signalling by dual-specificity protein phosphatases. Oncogene 26, 3203-3213
40. Simon, S. I., Hu, Y., Vestweber, D., and Smith, C. W. (2000) Neutrophil Tethering on E-Selectin Activates 2 Integrin Binding to ICAM-1 Through a Mitogen-Activated Protein Kinase Signal Transduction Pathway. The Journal of Immunology 164, 4348-4358
41. Boehme, M. W., Deng, Y., Raeth, U., Bierhaus, A., Ziegler, R., Stremmel, W., and Nawroth, P. P. (1996) Release of thrombomodulin from endothelial cells by concerted action of TNF-alpha and neutrophils: in vivo and in vitro studies. Immunology 87, 134-140
42. Rosenfeld, M. E. (2002) Leukocyte recruitment into developing atherosclerotic lesions: the complex interaction between multiple molecules keeps getting more complex. Arterioscler Thromb Vasc Biol 22, 361-363
43. Clausen, B. E., Burkhardt, C., Reith, W., Renkawitz, R., and Forster, I. (1999) Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic research 8, 265-277