凝血酶調節素(Thrombomodulin, TM)是一種表現於血管內皮細胞表面的醣蛋白，是生理上重要的抗凝血因子，其蛋白質結構從氨基端依序被分為類外源凝集素(lectin-like)結構區(TMD1), EGF-like 結構區(TMD2), serine/ threonine-rich結構區(TMD3), transmembrane結構區(TMD4)及碳端的cytoplasmic結構區(TMD5)。其中，TM的氮端lectin-like結構區與調節免疫反應的C-type lectins具有序列相似性。本實驗的目的為探討TM在發炎反應扮演的角色，並且研究此分子是否擁有更多的未知的功能，進而思考TM分子在治療急性發炎，例如敗血症(sepsis)，其應用之價值。在首先利用酵母菌表現系統表現TMD1, TMD23以及TMD123重組蛋白質，經由鎳離子親和性樹脂管柱，可純化出高純度的重組蛋白。我們首先建立動物系統中發炎反應模式，將TMD1, TMD23以及TMD123以尾靜脈注射的方式注入體內，三十分鐘以後以腹腔注射的方式注入酯多醣(LPS, lipopolysaccharide)引起發炎反應；六小時後取心臟血測定血清中人類腫瘤壞死因子α (TNF-α)以及NO的濃度評估發炎反應的強弱。從實驗結果中我們發現，TMD1、 TMD23以及TMD123顯著的抑制由LPS引起之發炎反應以及肺部白血球浸潤現象。以相同濃度的TMD1, TMD23以及TMD123做比較，我們發現TMD123具有最強的抗發炎的效果。在胞外的實驗當中我們發現TMD1可以有效的抑制LPS對於單核白血球及巨噬細胞所引發的TNF-α以及NO的釋放。在TMD1的轉殖鼠當中，注入致死劑量的LPS之後，發現轉殖鼠血液中TNF-α量以及其死亡率遠比正常老鼠來的低，這個結果指出TMD1在抗發炎反應當中扮演相當重要的角色。然而因為TMD1具有與C-type lectins序列上以及結構上的相似性，我們推測TMD1擁有類似於C-type lectin所扮演的抗發炎功能。以酵素結合免疫吸附法的實驗結果得知TMD1會直接的與LPS作用；而這個作用會受到鈣離子, 過多的甘露糖(mannose)調節。另外，實驗發現TMD1除了會增加大腸桿菌(E.coli)聚集，更會加強巨嗜細胞的吞噬螢光標定膠乳珠 (latex bead)及大腸桿菌能力。從先前的文獻得知，TM會經由活化protein C的方式抑制發炎反應，此機制已經被研究得相當透徹。以上兩個TMD1完全新穎 (novel)的發現讓我們推測TMD1在先天免疫上扮演重要的地位。所以總括以上實驗數據以及參考文獻我們發現，TMD123會同時經由活化Protein C以及直接透過TMD1抑制發炎反應。實驗中許多新穎的發現值得使我們重新思考TM在急性發炎中扮演的角色以及其被開發成為藥物之應用價值。

　Thrombomodulin (TM) is a vascular endothelial cell receptor and cofactor in the clinically important protein C anticoagulant system. TM contains five structure domains: N-termianal lectin-like domain (TMD1), EGF-like domain (TMD2), serine and threonine-rich domain (TMD3), transmembrane domain (TMD4) and C-terminal cytoplasmic domain (TMD5). Recent studies show that TMD1 exhibits highly structural homology to rat AA4 antigen, human phagocytic C1q receptor (C1qRp) and other C-type lectins those are involved in immune regulation. In our study, we want to clarify the effect of TM domains in acute inflammation. Human tmd1, d23 and d123 gene were cloned into Pichia pastoris expression system. The proteins were purified with nickel-chelating Sepharose and then identified with specific antibody. Recombinant soluble TMD1, TMD23 and TMD123 were administrated intravenously in mice 30 min before infusing LPS (lipopolysaccharide). The results showed that TMD1, TMD23 and TMD123 dramatically inhibited LPS-induced serum TNF-α elevation and leukocyte accumulation in pulmonary alveolus. We further demonstrate that TMD123 was the most potent anti-inflammatory moiety. Moreover, we have prepared TMD1 transgenic mice. In exposure TMD1 transgenic mice to lethal dose of LPS, transgenic mice showed reduced cytokine release and exhibited reduced mortality in comparison with normal mice. Since TMD1 belongs to C-type lectin superfamily that may modulate LPS directly. In our binding assay, we showed that TMD1 bound to LPS and the binding ability was attenuated in the presence of Ca2+, EDTA or mannose. Furthermore, TMD1 not only agglutinated E. coli but also enhanced the macrophage phagocytosis of fluorescent latex bead and E. coli. Howerer, it is well-known that TM exhibits anti-inflammatory effect through the activation of protein C (APC), and the mechanism has already been fully elucidated. Here we showed TMD1 has two novel anti-inflammatory mechanisms of TMD1 against inflammation. Combining the previous studies and our results, we suggested that the anti-inflammatory ability of TM is both dependent and independent on the activation of protein C and the TMD1 has novel anti-inflammatory function. Furthermore, we suggest that TM domains have potential to become useful therapeutic agents in the future.

Bajzar L. Thrombin activatable fibrinolysis inhibitor and an antifibrinolytic pathway. Arterioscler Thromb Vasc Biol. 20:2511-2518, 2000.

Boffa MC, Burke B, Haudenschild CC. Preservation of thrombomodulin antigen on vascular and extravascular surfaces. J Histochem Cytochem. 35:1267-1276, 1987.

Broze GJ Jr, Higuchi DA. Coagulation-dependent inhibition of fibrinolysis: role of carboxypeptidase and the premature lysis of clots from hemophilic plasma. Blood 88:3815-3823, 1996.

Collins CL, Ordonez NG, Scharfer R, Cook CD, Xie SS, Granger J, Hsu PL, Fink L, Hsu SM. Thrombomodulin expression in malignant pleural mesothelioma and pulmonary adenocarcinoma. Am J Pathol. 141:827-833, 1992.

Conway EM, Boffa MC, Nowakowski B, Steiner-Mosonyi M. An ultrastructural study of thombomodulin endocytosis: internalization occurs via clathrin-coated and non-coated pits. J Cell Physiol. 151:604-612, 1992.

Conway EM, Nowakowski B, Steiner-Mosonyi M. Thrombomodulin lacking the cytoplasmic domain efficiently internalizes thrombin via nonclathrin-coated, pit-mediated endocytosis. J Cell Physiol. 158:285-298, 1994.

Conway EM, Pollefeyt S, Collen D, Steiner-Mosonyi M. The amino terminal lectin-like domain of thrombomodulin is required for constitutive endocytosis. Blood 89:652-661, 1997.

Conway EM, Van de Wouwer M, Pollefeyt S, Jurk K, Van Aken H, De Vriese A, Weitz JI, Weiler H, Hellings PW, Schaeffer P, Herbert JM, Collen D, Theilmeier G. The lectin-like domain of thrombomodulin confers protection from neutrophil-mediated tissue damage by suppressing adhesion molecule expression via nuclear factor kappaB and mitogen-activated protein kinase pathways. J Exp Med. 196:565-77, 2002.

Cunningham MA, Rondeau E, Chen X, Coughlin SR, Holdsworth SR, Tipping PG. Protease-activated receptor 1 mediates thrombin-dependent, cell-mediated renal inflammation in crescentic glomerulonephritis. J Exp Med. 2000 Feb 7;191(3):455-62.

Debault LE, Esmon NL, Olson JR, and Esmon CT. Distribution of the thrombomodulin antigen in the rabbit vasculature. Lab Invest. 54:172-178, 1986.

Dittman WA, Majerus PW. Structure and function of thrombomodulin: a natural anticoagulant. Blood. 1990 Jan 15; 75(2):329-36.

Devitt A, Moffatt OD, Raykundalia C, Capra JD, Simmons DL, Gregory CD. Human CD14 mediates recognition and phagocytosis of apoptotic cells. Nature. 1998 Apr 2; 392(6675):505-9.

Esmon CT. Coagulation inhibitors in inflammation. Biochem Soc Trans. 2005 Apr;33(Pt 2):401-5.

Esmon CT, Gu JM, Xu J, Qu D, Stearns-Kurosawa DJ, Kurosawa S. Regulation and functions of the protein C anticoagulant pathway. Haematologica. 1999 Apr;84(4):363-8.

Esmon CT, Esmon NL, Harris KW. Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation. J Biol Chem. 1982; 257:7944-7947.

Esmon CT, Esmon NL, Harris KW. Isolation of a membrane-bound cofactor for thrombincatalyzed activation of protein C. J Biol Chem 1982; 257:7944–7947

Esmon CT, Owen WG. Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. Proc Natl Acad Sci USA. 78:2249-2252, 1981.

Esmon CT, Stenflo J, Suttie JW. A new vitamin K–dependent protein: A phospholipidbinding zymogen of a serine esterase. J Biol Chem 1976; 251:3052–3056

Franco RF, Reitsma PH. Genetic risk factors of venous thrombosis. Hum Genet 2001; 109: 369–384

Fukudome K, Esmon C. Molecular cloning and expression of murine and bovine endothelial cell protein C/activated protein C receptor (EPCR). J Biol Chem. 1995; 270: 5571–5577.

Genovese T, Cuzzocrea S, Di Paola R, Failla M, Mazzon E, Sortino MA, Frasca G, Gili E, Crimi N, Caputi AP, Vancheri C. Inhibition or knock out of Inducible nitric oxide synthase result in resistance to bleomycin-induced lung injury. Respir Res. 2005 Jun 14;6(1):58

Hamada H, Ishii H, Sakyo K, Horie S, Nishiki K, Kazama M. The eqidermal growth factor-like domain of recombinant human thrombomodulin exhibits mitogenic activity for Swiss 3T3 cells. Blood. 86:225-233, 1995.

Hamatake M, Ishida T, Mitsudomi T, Akazawa K, Sugimachi K. Prognostic value and clinicopathological correlation of thrombomodulin in squamous cell carcinoma of the human lung. Clin Cancer Res. 2: 763-766, 1996.

Honda G, Masaki C, Zushi M, Tsuruta K, Sata M. The roles played by the D2 and D3 domains of recombinant human thrombomodulin in its function. J Biochem. 118:1030-1036, 1995.

Huang HC, Shi GY, Jiang SJ, Shi CS, Wu CM, Yang HY, Wu HL. Thrombomodulin–mediated cell adhesion: involvement of its lectin-like domain. J Biol Chem. 278:46750-46759, 2003.

Imada M, Imada S, Iwasaki H, Kume A, Yamaguchi H, Moore EE. Fetomodulin: marker surface protein of fetal development which is modulatable by cyclic AMP. Dev Biol. 122:483-491, 1987.

Imada S, Yamaguchi H, Nagumo M, Katayanagi S, Iwasaki H, Imada M. Identification of fetomodulin, a surface marker protein of fetal development, as thrombomodulin by gene cloning and functional assays. Dev Biol. 140:113-122, 1990.

Ishii H, Majerus PW. Thrombomodulin is present in human plasma and urine. J Clin Invest. 76:2178-2181, 1985.

Jagneaux T, Taylor DE, Kantrow SP. Coagulation in sepsis. Am J Med Sci. 2004 Oct;328(4):196-204.

Kennel SJ, Lankford T, Hughes B, and Hotchkiss JA. Quantitation of a murine lung endothelial cell protein, P112, with a double monoclonal antibody assay. Lab Invest. 59:692-701, 1988.

Kisiel W, Canfield WM, Ericsson EH. Anticoagulant properties of bovine plasma protein C following activation of thrombin. Biochem 1977; 16:5824–5831

Kurosawa S, Galvin JB, Esmon NL, Esmon CT. Proteolytic formation and properties of functional domains of thrombomodulin. J Biol Chem. 262:2206-2212, 1987.

Kokame K, Zheng X, Sadler JE. Activation of thrombin-activable fibrinolysis inhibitor requires epidermal growth factor-like domain 3 of thrombomodulin and is inhibited competitively by protein C. J Biol Chem. 273:12135-12139, 1998.

Koyama T, Parkinson JF, Aoki N, Bang NU, Muller-Berghaus G, Preissner KT. Relationship between post-translational glycosylation and anticoagulant function of secretable recombinant mutants of human thrombomodulin. British J Haematol. 78:515-522, 1991.

Lager DJ, Callaghan EJ, Worth SF, Raife TJ, Lentz SR. Cellular localization of thrombomodulin in human epithelium and squamous malignancies. Am J Pathol. 146:933-943, 1995.

Landry, D. W. & Oliver, J. A. The pathogenesis of vasodilatory shock. N. Engl. J. Med. 345, 588–595 (2001).

Landmann, R. Increased circulating soluble CD14 is associated with high mortality in Gramnegative septic shock. J. Infect. Dis. 171, 639–644 (1995).

Maruno M, Yoshimine T, kuroda R, Ishii H, Hayakawa T. Expression of thrombomodulin astrocytomas of various malignancy and in gliotic and normal brains. J Neurooncol. 19:155-160, 1994.

Maruyama I, Bell CE, Majerus PW. Thrombomodulin is found on endothelium of arteries, veins, capillaries and lymphatics, and on syncytiotrophoblast of human placenta. J Cell Biol. 101:363-371, 1985.

Maruyama I, Majerus PW. The turnover of thrombin-thrombomodulin complex in cultured human umbilical vein endothelial cells and A549 lung cancer cells. Endocytosis and degradation of thrombin. J Biol Chem. 260:15432-15438, 1985.

Martin D, Korthuis RJ, Perry M, Townsley MI, Taylor AE. Oxygen radical-mediated lung damage associated with alpha-naphthylthiourea. Acta Physiol Scand Suppl. 1986; 548:119-25.

Marumoto Y, Wakita K, Sugiyama N, Tsukioka K, Ogata M, Honda N. Characterization of glycosylation on recombinant soluble human thrombomodulin produced by silkworms. Biosci Biotechnol Biochem. 1993 May; 57(5):839-40.

Mccachren SS, Diggs J, Weinberg JB, Dittman WA. Thrombomodulin expression by human blood monocytes and by human synovial tissue lining macrophages. Blood 78:3128-3132, 1991.

McEver R. Leukocyte interactions mediated by selectins. Thromb Haemost. 66:80-87, 1991.

Mclean JS, Byrick RJ. ARDS and sepsis--definitions and new therapy. Can J Anaesth. 1993 Jul; 40(7):585-90.

Miyasaka N, Hirata Y. Nitric oxide and inflammatory arthritides, Life Sci 61 (1997), pp. 2073–2081.

Molinari A, Giorgetti C, Lansen J, Vaghi F, Orsini G, Faioni EM, Mannucci PM. Thrombomodulin is a cofactor for thrombin degradation of recombinant single-chain urokinase plasminogen activator in vitro and in a perfused rabbit heart model. Thromb Haemost. 69:226-232, 1992.

Munk GA, Parkinson JF, Groeneveld E, Bang NU, Rijken DC. Role of the glycosaminoglycan component of thrombomodulin in its acceleration of the inactivation of single-chain urokinase-type plasminogen activator by thrombin. Biochem J. 290:655-659, 1993.

Murakami S, Iwaki D, Mitsuzawa H, Sano H, Takahashi H, Voelker DR, Akino T, Kuroki Y. Surfactant protein A inhibits peptidoglycan-induced tumor necrosis fector-α secretion in U937 cells and alveolar macrophages by direct interaction with Toll-like receptor 2. J. Biol Chem. 277:6830-6837, 2002.

Nesheim M, Wang W, Boffa M, Nagashima M, Morser J, Bajzar L. Thrombin, thrombomodulin and TAFI in the molecular link between coagulation and fibrinolysis. Thromb Haemost. 78:386-391, 1997.

Osborn MJ, Rosen SM, Rothefield L, Zeleznick LD, Horecker BL. Lipopolysaccharide of the Gram-negative cell wall. Science. 1964 Aug 21; 145:783-9.

Owen WG, Esmon CT. Functional properties of an endothelial cell cofactor for thrombincatalyzed activation of protein C. J Biol Chem 1981; 256:5532–5535

Parkinson JF, Garcia JG, Bang NU. Decreased thrombin affinity of cell-surface thrombomodulin following treatment of cultured endothelial cells with beta-D-xyloside. Biochem Biophys Res Commun. 169:177-183, 1990.

Patthy L. Detecting distant homologies of mosaic proteins. Analysis of the sequences of thrombomodulin, thrombospondin complement components C9, C8 alpha and C8 beta, vitronectin and plasma cell membrane glycoprotein PC-1. J Mol Biol. 202:689-696, 1988.

Petersen T. The amino-terminal domain of thrombomodulin and pancreatic stone protein are homologous with lectins. FEBS Lett. 231:51-53, 1988.

Polgar J, Lerant I, Muszbek L, Machovich R. Thrombomodulin inhibits the activation of factor XIII by thrombin. Thromb Res. 43:685-690, 1986.

Pugin J, Heumann ID, Tomasz A, Kravchenko VV, Akamatsu Y, Nishijima M, Glauser MP, Tobias PS, Ulevitch RJ. CD14 is a pattern recognition receptor. Immunity. 1994 Sep;1(6):509-16

Raife TJ, Lager DJ, Madison KC, Piette WW, Howard EJ, Sturm MT, Chen Y, Lentz SR. Thrombomodulin expression by human keratinocytes. J Clin Invest. 93:1846-1851, 1994.

Reinhart K, Bayer O, Brunkhorst F, Meisner M. Markers of endothelial damage in organ dysfunction and sepsis. Crit Care Med. 2002 May;30(5 Suppl):S302-12.

Schenk-Braat EA, Morser J, Rijken DC. Identification of the epidermal growth factor-like domains of thrombomodulin essential for the acceleration of thrombin-mediated inactivation of single-chain urokinase-type plasminogen activator. Eur J Biochem. 268:5562-5569, 2001.

Sadler JE. Thrombomodulin structure and function. Thromb Haemost 1997; 78: 392 5.

Sakata Y, Curriden S, Lawrence D. Activated protein C stimulates the fibrinolytic activity of cultured endothelial cells and decreases antiactivator activity. Proc Nat Acad Sci U S A 1985; 82:1121–1125

Sano H, Sohma H, Muta T, Nomura S, Voelker DR, Kuroki Y. Pulmonary surfactant protein A modulates the cellular response to smooth and rough Lipopolysaccharides by interaction with CD14. J Immunol. 163:387-395, 1999.

Schutte H, Lohmeyer J, Rosseau S, Ziegler S, Siebert C, Kielisch H, Pralle H, Grimminger F, Morr H, Seeger W. Bronchoalveolar and systemic cytokine profiles in patients with ARDS, severe pneumonia and cardiogenic pulmonary oedema. Eur Respir J. 1996 Sep;9(9):1858-67.

Shirai T, Shiojiri S, Ito H, Yamamoto S, Kusumoto H, Deyashiki Y, Maruyama I, Suzuki K. Gene structure of human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C. J Biochem. 103:281-285, 1988.

Stenflo J. A new vitamin K-dependent protein: Purification from bovine plasma and preliminary characterization. J Biol Chem 1976; 251:355–363

Stenflo J, Fernlund P. Amino acid sequence of the heavy chain of bovine protein C. J Biol Chem 1982; 257:12180–12190

Suehiro T, Shimada M, Matsumata T, Taketomi A, Yamamoto K, Sugimachi K. Thrombomodulin inhibits intrahepatic spread in human hepato-cellular carcinoma. Hepatology 21:1285-1290, 1995.

Tamura A, Matsubara O, Hirokawa K, Aoki N. Detection of thrombomodulin in human lung cancer cells. Am J Pathol. 142: 79–85, 1993.

Taylor FB Jr, Peer GT, Lockhart MS. Endothelial cell protein C receptor plays an important role in protein C activation in vivo. Blood 2001; 97:1685–1688

Teasdale M, Bird C, Bird P. Internalization of the anticoagulant thrombomodulin is constitutive and does not require a signal in the cytoplasmic domain. Immunol Cell Biol. 72:480-488, 1994.

Tezuka Y, Yonezawa S, Maruyama I, Matsushita Y, Shimizu T, Obama H, Sagara M, Shirao K, Kusano C, Natsugoe S, et al. Expression of thrombomodulin in esophageal squamous cell carcinoma and its relationship to lymph node metastasis. Cancer Res. 55:4196-4200, 1995.

Tohda G, Oida K, Okada Y, Kosaka S, Okada E, Takahashi S, Ishii H, Miyamori I. Expression of thrombomodulin in atherosclerotic lesions and mitogenic activity of recombinant thrombomodulin in vascular in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 18: 1861-1869, 1998.

Ulutin ON, Seeger WH. autoprothrombin II and autoprothrombin II anticoagulant.Thromb Diath Haemorrh 1962; 7(Suppl): 256–263

Uronen Uronen H, Williams AJ, Dixon G, Andersen SR, Van Der Ley P, Van Deuren M, Callard RE, Klein N. Gram-negative bacteria induce proinflammatory cytokine production by monocytes in the absence of lipopolysaccharide (LPS). Clin Exp Immunol. 2000 Dec; 122(3):312-5.

Van de Wouwer M, Collen D, Conway EM. Thrombomodulin-protein C-EPCR system: integrated to regulate coagulation and inflammation. Arterioscler Thromb Vasc Biol. 2004 Aug;24(8):1374-83. Epub 2004 Jun 3.

Van de Wouwer M, Conway EM. Novel functions of thrombomodulin in inflammation. Crit Care Med. 32:S254-261, 2004.

Viles-Gonzalez JF, Fuster V, Badimon JJ. Thrombin/inflammation paradigms: a closer look at arterial and venous thrombosis. Am Heart J. 2005 Jan; 149(1 Suppl):S19-31.

Weiler-Guettler H, Aird WC, Rayburn H, Husain M, and Rosenberg RD. Developmentally regulated gene expression of thrombomodulin in postimplantation mouse embryos. Development 122:2271-2281, 1996.

Weiler H, Lindner V, Kerlin B, Isermann BH, Hendrickson SB, Cooley BC, Meh DA, Mosesson MW, Shworak NW, Post MJ, Conway EM, Ulfman LH, von Andrian UH, Weitz JI. Characterization of a mouse model for thrombomodulin deficiency. Arterioscler Thromb Vasc Biol. 21:1531-1537, 2001.

Wen DZ, Dittman WA, Ye RD, Deaven LL, Majerus PW, Sadler JE. Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene. Biochemistry 26:4350-4357, 1987.

Wilhelm S, Wilhelm O, Schmitt M, Graeff H. Inactivation of receptor bound pro-urokinase-type plasminogen activator (pro-UPA) by thrombin and thrombin/thrombomodulin complex. Biol Chem Hoppe Seyler. 375:603-608, 1994.

Wright, S. D, Ramos, R. A, Tobias, P. S, Ulevitch, R. J, Mathison, J. C. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249, 1431–1433 (1990).

Yu, B, Hailman, E, Wright, S. Lipopolysaccharide binding protein and soluble CD14 catalyze exchange of phospholipids. J. Clin. Invest. 99, 315–324 (1997).

Zhang Y, Weiler-Guettler H, Chen J, Wilhelm O, Deng Y, Qiu F, Nakagawa K, Klevesath M, Wilhelm S, Bohrer H, Nakagawa M, Graeff H, Martin E, Stern D, Rosenberg R, Ziegler R, Nawroth P. Thrombomodulin modulates growth of tumor cells independent of its anticoagulant activity. J Clin Invest. 101:1301-1309, 1998.

Zushi M, Gomi K, Yamamoto S, Maruyama I, Hayashi T, Suzuki K. The last three consecutive epidermal growth factor-like structures of human thrombomodulin comprise the minimum functional domain for protein C-activating cofactor activity and anticoagulant activity. J Biol Chem. 264:10351-10353, 1989.