凝血酶調節素（thrombomodulin, TM）為最先發現其表現於內皮細胞上的一種醣蛋白，且具有抗凝血功能。TM透過與凝血酶的作用活化protein C來達到抗凝血作用。TM除了具有抗凝血功能外，也有研究指出TM的胺基端類凝集素功能區（TMD1）具有抗發炎的特性。本實驗室先前的研究發現TMD1可以抑制由酯多醣體（lipopolysaccaride）所引起的急性發炎反應。然而，TMD1對於調控粥狀動脈硬化之慢性發炎的機制，仍是未知。為了探討TMD1在抑制粥狀動脈硬化的功能，本實驗利用酵母菌表現系統來製備重組TMD1wild-type 及兩個突變蛋白，包括TMD1的第25個胺基酸由alanine改變為threonine及第61個胺基酸由glycine改變為alanine。在臨床研究中已發現，TMD1的胺基酸序列帶有這兩種點突變的人，其產生心肌梗塞或腦靜脈竇栓塞等血栓相關疾病機率較大。首先我們在細胞實驗中，利用動脈硬化中會產生的人類腫瘤壞死因子α 刺激人類臍靜脈內皮細胞，發現在外加TMD1並不影響內皮細胞上黏著因子的表現。但我們卻發現TMD1可以抑制單核球細胞黏附及穿越內皮細胞層，並且此作用可能是透過TMD1結合於修飾黏附因子的醣類Lewisy 。然而，帶有TMD1點突變的蛋白其抑制效果則較差。另外，利用載體蛋白E基因剔除老鼠（Apo E-/- mice）做為粥狀動脈硬化的動物模式，將不同劑量的TMD1以腹腔注射至小鼠中。結果顯示小鼠主動脈斑塊產生隨著TMD1濃度增加而有減少的趨勢；反之，帶有點突變的TMD1抑制動脈硬化的效果則較TMD1 wild-type來的差。綜合以上結果發現TMD1對於粥狀動脈硬化的產生有抑制的效果，且是透過與表現在內皮細胞上的醣類Lewisy作用，進而干擾單核球細胞的行為，使之不能黏附並穿越內皮細胞，而引發發炎反應。

Thrombomodulin (TM) is a glycoprotein which is first identified on the surface of vascular endothelial cells (ECs). TM activates anticoagulation through thrombin-mediated formation of activated protein C. It has recently been reported that the extracellular N-terminal lectin-like domain (D1) of TM possesses anti-inflammatory properties. Our previous results show that TMD1 could suppress lipopolysaccaride-induced acute inflammatory responses. However, the detailed mechanisms of TMD1 in chronic inflammation, such as atherosclerosis, are still unknown. To further investigate the role of TMD1 in atherosclerosis, we prepared and purified recombinant TMD1 wild-type protein and its mutants, TMD1 Ala25Thr and Gly61Ala, using Pichia pastoris expression system. These two mutants are naturally occurring polymorphisms in TMD1 and significantly correlated with thromboembolic diseases, such as sagittal sinus thrombosis or myocardial infarction. In vitro study showed that TMD1 had no significant effect on the expression of cell adhesion molecules on tumor necrosis factor-α-stimulated human umbilical vein endothelial cells. However, TMD1 blocked the adhesion and the transendothelial migration of monocytes by binding to Lewisy which decorates adhesion molecules on ECs. Besides, the inhibitory effects of TMD1 mutants on adhesion and migration of monocytes were reduced. In vivo, the apolipoprotein E knockout mice were injected intraperitoneally with different doses of recombinant TMD1 protein. The data showed that the area of atherosclerotic plaque in mice aortas were reduced in the TMD1-treated groups in a dose-dependent manner. We also found that the effects of TMD1 mutants on atherosclerotic plaque formation were partially reduced compared with wild-type. In conclusion, TMD1 may suppress atherosclerosis by inhibiting the interaction between monocytes and ECs through binding to Lewisy.

1. Nawa, K., et al., Presence and function of chondroitin-4-sulfate on recombinant human soluble thrombomodulin. Biochem Biophys Res Commun, 1990. 171(2): p. 729-37.
2. Esmon, N.L., W.G. Owen, and C.T. Esmon, Isolation of a membrane-bound cofactor for thrombin-catalyzed activation of protein C. J Biol Chem, 1982. 257(2): p. 859-64.
3. Sakata, Y., et al., Activated protein C stimulates the fibrinolytic activity of cultured endothelial cells and decreases antiactivator activity. Proc Natl Acad Sci U S A, 1985. 82(4): p. 1121-5.
4. Esmon, C.T., Crosstalk between inflammation and thrombosis. Maturitas, 2004. 47(4): p. 305-14.
5. Van de Wouwer, M. and E.M. Conway, Novel functions of thrombomodulin in inflammation. Crit Care Med, 2004. 32(5 Suppl): p. S254-61.
6. Riewald, M., et al., Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science, 2002. 296(5574): p. 1880-2.
7. Murakami, K., et al., Activated protein C attenuates endotoxin-induced pulmonary vascular injury by inhibiting activated leukocytes in rats. Blood, 1996. 87(2): p. 642-7.
8. Grinnell, B.W., R.B. Hermann, and S.B. Yan, Human protein C inhibits selectin-mediated cell adhesion: role of unique fucosylated oligosaccharide. Glycobiology, 1994. 4(2): p. 221-5.
9. Drake, W.T., et al., Thrombin enhancement of interleukin-1 and tumor necrosis factor-alpha induced polymorphonuclear leukocyte migration. Lab Invest, 1992. 67(5): p. 617-27.
10. Bizios, R., et al., Thrombin-induced chemotaxis and aggregation of neutrophils. J Cell Physiol, 1986. 128(3): p. 485-90.
11. Laterre, P.F., et al., Hospital mortality and resource use in subgroups of the Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) trial. Crit Care Med, 2004. 32(11): p. 2207-18.
12. Myles, T., et al., Thrombin activatable fibrinolysis inhibitor, a potential regulator of vascular inflammation. J Biol Chem, 2003. 278(51): p. 51059-67.
13. Campbell, W.D., et al., Inactivation of C3a and C5a octapeptides by carboxypeptidase R and carboxypeptidase N. Microbiol Immunol, 2002. 46(2): p. 131-4.
14. Conway, E.M., et al., 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, 2002. 196(5): p. 565-77.
15. Van de Wouwer, M., et al., The lectin-like domain of thrombomodulin interferes with complement activation and protects against arthritis. J Thromb Haemost, 2006. 4(8): p. 1813-24.
16. Abeyama, K., et al., The N-terminal domain of thrombomodulin sequesters high-mobility group-B1 protein, a novel antiinflammatory mechanism. J Clin Invest, 2005. 115(5): p. 1267-74.
17. Breslow, J.L., Cardiovascular disease burden increases, NIH funding decreases. Nat Med, 1997. 3(6): p. 600-1.
18. Dong, Z.M., et al., The combined role of P- and E-selectins in atherosclerosis. J Clin Invest, 1998. 102(1): p. 145-52.
19. Collins, R.G., et al., P-Selectin or intercellular adhesion molecule (ICAM)-1 deficiency substantially protects against atherosclerosis in apolipoprotein E-deficient mice. J Exp Med, 2000. 191(1): p. 189-94.
20. Nakashima, Y., et al., Upregulation of VCAM-1 and ICAM-1 at atherosclerosis-prone sites on the endothelium in the ApoE-deficient mouse. Arterioscler Thromb Vasc Biol, 1998. 18(5): p. 842-51.
21. Ross, R., The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature, 1993. 362(6423): p. 801-9.
22. Wu, K.K., Soluble thrombomodulin and coronary heart disease. Curr Opin Lipidol, 2003. 14(4): p. 373-5.
23. Salomaa, V., et al., Soluble thrombomodulin as a predictor of incident coronary heart disease and symptomless carotid artery atherosclerosis in the Atherosclerosis Risk in Communities (ARIC) Study: a case-cohort study. Lancet, 1999. 353(9166): p. 1729-34.
24. Wu, K.K., et al., Interaction between soluble thrombomodulin and intercellular adhesion molecule-1 in predicting risk of coronary heart disease. Circulation, 2003. 107(13): p. 1729-32.
25. Kunz, G., et al., Naturally occurring mutations in the thrombomodulin gene leading to impaired expression and function. Blood, 2002. 99(10): p. 3646-53.
26. Doggen, C.J., et al., A mutation in the thrombomodulin gene, 127G to A coding for Ala25Thr, and the risk of myocardial infarction in men. Thromb Haemost, 1998. 80(5): p. 743-8.
27. Thude, H., et al., Analysis of the thrombomodulin gene in patients with venous thrombosis. Thromb Res, 2002. 107(3-4): p. 109-14.
28. Varki, A., Biological roles of oligosaccharides: all of the theories are correct. Glycobiology, 1993. 3(2): p. 97-130.
29. Holgersson, J., M.E. Breimer, and B.E. Samuelsson, Basic biochemistry of cell surface carbohydrates and aspects of the tissue distribution of histo-blood group ABH and related glycosphingolipids. APMIS Suppl, 1992. 27: p. 18-27.
30. Yuriev, E., et al., Three-dimensional structures of carbohydrate determinants of Lewis system antigens: implications for effective antibody targeting of cancer. Immunol Cell Biol, 2005. 83(6): p. 709-17.
31. Zhu, K., et al., A novel function for a glucose analog of blood group H antigen as a mediator of leukocyte-endothelial adhesion via intracellular adhesion molecule 1. J Biol Chem, 2003. 278(24): p. 21869-77.
32. Zen, K., et al., Critical role of mac-1 sialyl lewis x moieties in regulating neutrophil degranulation and transmigration. J Mol Biol, 2007. 374(1): p. 54-63.
33. Halloran, M.M., et al., Ley/H: an endothelial-selective, cytokine-inducible, angiogenic mediator. J Immunol, 2000. 164(9): p. 4868-77.
34. Garcia-Vallejo, J.J., et al., DC-SIGN mediates adhesion and rolling of dendritic cells on primary human umbilical vein endothelial cells through Lewis(Y) antigen expressed on ICAM-2. Mol Immunol, 2008. 45(8): p. 2359-69.
35. Braunersreuther, V. and F. Mach, Leukocyte recruitment in atherosclerosis: potential targets for therapeutic approaches? Cell Mol Life Sci, 2006. 63(18): p. 2079-88.
36. Vasta, G.R., et al., C-type lectins and galectins mediate innate and adaptive immune functions: their roles in the complement activation pathway. Dev Comp Immunol, 1999. 23(4-5): p. 401-20.
37. Drickamer, K., Two distinct classes of carbohydrate-recognition domains in animal lectins. J Biol Chem, 1988. 263(20): p. 9557-60.
38. Huang, H.C., et al., Thrombomodulin-mediated cell adhesion: involvement of its lectin-like domain. J Biol Chem, 2003. 278(47): p. 46750-9.
39. Young, J.L., P. Libby, and U. Schonbeck, Cytokines in the pathogenesis of atherosclerosis. Thromb Haemost, 2002. 88(4): p. 554-67.
40. Nan, B., et al., Effects of TNF-alpha and curcumin on the expression of thrombomodulin and endothelial protein C receptor in human endothelial cells. Thromb Res, 2005. 115(5): p. 417-26.
41. Fan, J. and T. Watanabe, Inflammatory reactions in the pathogenesis of atherosclerosis. J Atheroscler Thromb, 2003. 10(2): p. 63-71.
42. Moriwaki, H., et al., Ligand specificity of LOX-1, a novel endothelial receptor for oxidized low density lipoprotein. Arterioscler Thromb Vasc Biol, 1998. 18(10): p. 1541-7.
43. Kume, N., et al., Inducible expression of lectin-like oxidized LDL receptor-1 in vascular endothelial cells. Circ Res, 1998. 83(3): p. 322-7.
44. Chen, M., T. Masaki, and T. Sawamura, LOX-1, the receptor for oxidized low-density lipoprotein identified from endothelial cells: implications in endothelial dysfunction and atherosclerosis. Pharmacol Ther, 2002. 95(1): p. 89-100.
45. Ohlin, A.K. and R.A. Marlar, Thrombomodulin gene defects in families with thromboembolic disease--a report on four families. Thromb Haemost, 1999. 81(3): p. 338-44.
46. Treutiger, C.J., et al., High mobility group 1 B-box mediates activation of human endothelium. J Intern Med, 2003. 254(4): p. 375-85.
47. Fiuza, C., et al., Inflammation-promoting activity of HMGB1 on human microvascular endothelial cells. Blood, 2003. 101(7): p. 2652-60.
48. Kalinina, N., et al., Increased expression of the DNA-binding cytokine HMGB1 in human atherosclerotic lesions: role of activated macrophages and cytokines. Arterioscler Thromb Vasc Biol, 2004. 24(12): p. 2320-5.