凝血酶調節素（thrombomodulin，TM）為一血管內皮細胞表面凝血酶（thrombin）受體，可抑制凝血酶之促凝活性並且當作凝血酶催化蛋白質C（protein C）活化過程的蛋白輔助因子。凝血酶調節素轉換凝血酶的促凝成抗凝活性，因而在蛋白質C抗凝途徑中扮演著中心的角色。由於TM在不同細胞中廣泛分佈的特性，近年的許多文獻指出凝血酶調節素不只是經由調節凝血酶而扮演抗凝因子角色，也參與其他重要生理功能的調節。在這個研究中，我們將凝血酶調節素不同的功能區域基因片段構築至酵母菌表現系統，進而以親和性鎳離子螯合管柱從酵母菌發酵液中純化出不同功能區片段的TM重組蛋白。已知的文獻指出，包含六個類表皮細胞生長因子結構區的TM重組蛋白具有刺激細胞分裂的能力。本研究利用試管內及活體模式，探究TM的不同功能區域片段對血管新生的新穎效應。精純過後的TM重組蛋白TMD123，TMD23，TMD2以及TMD2/EGF456等蛋白均可結合thrombin而有效活化protein C。在內皮細胞的實驗中，TMD23比TMD123，TMD2，TMD2/EGF123，TMD2/EFG456以及TMD1更具刺激DNA合成及細胞爬行的活性，此結果指出TMD23是活化血管內皮細胞最強的區域。此外，TMD23經由ERK1/2，p38以及PI-3激酶/AKT/內皮細胞一氧化氮合成酶的磷酸化而刺激細胞移動及類微血管樣結構生成。TMD23也促進間質金屬蛋白酶-3的表現及抑制血纖維蛋白溶酶原活化因子活性，可能進而調節細胞間質的分解而在血管新生過程導致內皮細胞的入侵以及移動。在活體實驗中，TMD23可誘發小鼠皮下Matrigel及嚴重免疫缺乏老鼠體內黑色素瘤的血管新生作用。在更進一步的Matrigel活體實驗，TMD23可誘發比TMD123更強之血管新生作用，而TMD1則無此活性。TMD23誘發之血管新生作用及HUVEC爬行能力會受到TMD1結構區域的干擾。此結果顯示，TMD23所具有的生物活性受到TMD1功能的調控。總結上述，TM不但是一種自然的抗凝固蛋白，也許經由自我調控的方式參與在調節血管新生的過程。此新發現暗示著生理TM片段也許在新血管生成扮演特定角色，TM片段在新血管生成的活性也提供缺血性疾病治療上的新方向。

　Thrombomodulin (TM), a thrombin receptor on endothelial cell surface, inhibits the procoagulant functions of thrombin and acts as a protein cofactor in thrombin-catalyzed activation of protein C. TM converts pro-coagulant thrombin into an anticoagulant and plays a central role in protein C anticoagulant pathways. Recent studies indicate that TM not only functions as an anticoagulant factor by modulating thrombin’s function. Owing to TM’s widely expression patterns on various cell types, TM may also participate in other physiological functions. In the present study, the DNA fragments of TM domain were subcloned into the Pichia pastoris expression system, and the TM domain (TMD) proteins from ferments were further purified by affinity nickel-chelating column chromatography. Previous study has shown that a recombinant TM domain containing six epidermal growth factor-like structures exhibits mitogenic activity. I explored the novel angiogenic effects of TMD proteins by using in vitro and in vivo models. When thrombin was combined with purified TMD123, TMD23, TMD2, and TMD2/EGF456, respectively, the complex could activate protein C. TMD23 had higher activity than the other TMD proteins in stimulation of DNA synthesis and migration in cultured human umbilical vein endothelial cells (HUVECs). These results suggest that TMD23 is the most potent region of TM for activation of HUVECs. Besides, TMD23 enhanced chemotactic motility and capillary-like tube formation in HUVECs, which was through an effect mediated by phosphorylation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase and the phosphatidylinositol-3 kinase/Akt/endothelial nitric oxide synthase pathway. TMD23 also stimulated the expression of matrix metalloproteinase-3 and inhibited plasminogen activator activity. These steps mediated extracellular proteolysis, which leads to endothelial-cell’s invasion and migration during angiogenesis. In murine angiogenic assays, TMD23 induced neovascularization in Matrigel and enhanced angiogenesis in Matrigel containing melanoma A2058 cells in severe combined immune deficiency mice. Furthermore, in Matrigel assay, TMD23 was a more potent angiogenic activator than TMD123 and TMD1. On the other hand, TMD1 diminished TMD23-induced angiogenic response and HUVEC migration. The results revealed that the biological function of TMD23 was modulated by TMD1 (lectin-like domain). In conclusion, TM not only acts as a natural anticoagulant, but also participates in regulation of angiogenesis by an auto-regulatory manner. TMD23 promoted the angiogenic activities in vitro and in vivo, suggesting that TM fragments may play a role in the formation of new vessels. These findings may provide a new therapeutic option for treating ischemia diseases.

1. Takano, S., et al., Plasma thrombomodulin in health and diseases. Blood, 1990. 76: p. 2024-9.
2. Esmon, C.T. and W.G. Owen, Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. Proc Natl Acad Sci USA, 1981. 78: p. 2249-52.
3. Esmon, C.T., N.L. Esmon, and K.W. Harris, Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation. J Biol Chem, 1982. 257: p. 7944-7.
4. 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: p. 859-64.
5. Suzuki, K., et al., Structure and expression of human TM, a thrombin receptor on endothelium acting as a cofactor for protein C activation. EMBO J, 1987. 6: p. 1891-7.
6. Petersen, T.E., The amino-terminal domain of thrombomodulin and pancreatic stone protein are homologous with lectins. FEBS Lett, 1988. 231: p. 51-3.
7. Drickamer, K., Two distinct classes of carbohydrate-recognition domains in animal lectins. J Biol Chem, 1988. 263: p. 9557-60.
8. Conway, E.M., et al., The amino terminal lectin-like domain of thrombomodulin is required for constitutive endocytosis. Blood, 1997. 89: p. 652-61.
9. Weisel, J.W., et al., The shape of thrombomodulin and interactions with thrombin as determined by electron microscopy. J Biol Chem, 1996. 271: p. 31485-90.
10.Villoutreix B, D.B., Molecular Model for the C-type Lectin Domain of Human Thrombomodulin. Journal of Molecular Modeling, 1998. 4: p. 310-22.
11.Zhang, Y., et al., Thrombomodulin modulates growth of tumor cells independent of its anticoagulant activity. J Clin Invest, 1998. 101: p. 1301-9.
12.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 kB and mitogen-activated protein kinase pathways. J Exp Med, 2002. 196: p. 565-77.
13.Huang, H.C., et al., Thrombomodulin-mediated cell adhesion: Involvement of its lectin-like domain. J Biol Chem, 2003. 278: p. 46750-9.
14.Bajzar, L., R. Manuel, and M.E. Nesheim, Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor. J Biol Chem, 1995. 270: p. 14477-84.
15.Kokame, K., X. Zheng, and J.E. Sadler, 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, 1998. 273: p. 12135-9.
16.Tsiang, M., S.R. Lentz, and J.E. Sadler, Functional domains of membrane-bound human thrombomodulin. EGF-like domains four to six and the serine/threonine-rich domain are required for cofactor activity. J Biol Chem, 1992. 267: p. 6164-70.
17.Zushi, M., et al., Aspartic acid 349 in the fourth epidermal growth factor-like structure of human thrombomodulin plays a role in its Ca2+-mediating binding to protein C. J Biol Chem, 1991. 266: p. 19886-9.
18.Slungaard, A., et al., Platelet factor 4 enhances generation of activated protein C in vitro and in vivo. Blood, 2003. 102: p. 146-51.
19.Schenk-Braat, E.A., J. Morser, and D.C. Rijken, 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, 2001. 268: p. 5562-9.
20.de Munk, G.A., E. Groeneveld, and D.C. Rijken, Acceleration of the thrombin inactivation of single chain urokinase-type plasminogen activator (pro-urokinase) by thrombomodulin. J Clin Invest, 1991. 88: p. 1680-4.
21.Molinari, A., et al., 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, 1992. 67: p. 226-32.
22.Hamada, H., et al., The epidermal growth factor-like domain of recombinant human thrombomodulin exhibits mitogenic activity for Swiss 3T3 cells. Blood, 1995. 86: p. 225-233.
23.Tohda, G., et al., Expression of thrombomodulin in atherosclerotic lesions and mitogenic acitvity of recombinant thrombomodulin in vasular smooth muscle cells. Arterioscler. Throm Vasc Biol, 1998. 18: p. 1861-9.
24.Preissner, K.T., et al., Domain structure of the endothelial cell receptor thrombomodulin as deduced from modulation of its anticoagulant functions. Evidence for a glycosaminoglycan-dependent secondary binding site for thrombin. J Biol Chem, 1990. 265: p. 4915-22.
25.Ye, J., C.T. Esmon, and A.E. Johnson, The chondroitin sulfate moiety of thrombomodulin binds a second molecule of thrombin. J Biol Chem, 1993. 268: p. 2373-9.
26.Koyama, T., et al., Different glycoforms of human thrombomodulin. Their glycosaminoglycan-dependent modulatory effects on thrombin inactivation by heparin cofactor II and antithrombin III. Eur J Biochem, 1991. 198: p. 563-70.
27.Koyama, T., et al., Relationship between post-translational glycosylation and anticoagulant function of secretable recombinant mutants of human thrombomodulin. Br J Haematol, 1991. 78: p. 515-22.
28.Dudek, A.Z., et al., Platelet factor 4 binds to glycanated forms of thrombomodulin and to protein C. A potential mechanism for enhancing generation of activated protein C. J Biol Chem, 1997. 272: p. 31785-92.
29.Conway, E.M., et al., Structure-function analyses of thrombomodulin by gene-targeting in mice: the cytoplasmic domain is not required for normal fetal development. Blood, 1999. 93: p. 3442-50.
30.Conway, E.M., B. Nowakowski, and M. Steiner-Mosonyi, Thrombomodulin lacking the cytoplasmic domain efficiently internalizes thrombin via nonclathrin-coated, pit-mediated endocytosis. J Cell Physiol, 1994. 158: p. 285-98.
31.Teasdale, M.S., C.H. Bird, and P. Bird, Internalization of the anticoagulant thrombomodulin is constitutive and does not require a signal in the cytoplasmic domain. Immunol Cell Biol, 1994. 72: p. 480-8.
32.Wen, D.Z., et al., Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene. Biochemistry, 1987. 26: p. 4350-7.
33.Jackman, R.W., et al., Human thrombomodulin gene is intron depleted: nucleic acid sequences of the cDNA and gene predict protein structure and suggest sites of regulatory control. Proc Natl Acad Sci U S A, 1987. 84: p. 6425-9.
34.Tazawa, R., et al., Functional characterization of the 5'-regulatory region of the human thrombomodulin gene. J. Biol. Chem., 1993. 113: p. 600-6.
35.Dittman, W.A., T. Kumada, and P.W. Majerus, Transcription of thrombomodulin mRNA in mouse hemangioma cells is increased by cycloheximide and thrombin. Proc Natl Acad Sci U S A, 1989. 86: p. 7179-82.
36.Ohashi, K., et al., The biosynthesis of thrombomodulin and its enhancement by dibutyryl cAMP in a human megakaryoblastic cell line, UT-7. Int J Hematol, 1991. 54: p. 341-9.
37.Raife, T.J., E.M. Demetroulis, and S.R. Lentz, Regulation of thrombomodulin expression by all-trans retinoic acid and tumor necrosis factor-alpha: differential responses in keratinocytes and endothelial cells. Blood, 1996. 88: p. 2043-9.
38.Oida, K., et al., Effect of a protein phosphatase inhibitor, okadaic acid, on thrombomodulin expression in cultured human umbilical vein endothelial cells. Thromb Res, 1997. 85: p. 169-76.
39.Hirokawa, K. and N. Aoki, Up-regulation of thrombomodulin by activation of histamine H1-receptors in human umbilical-vein endothelial cells in vitro. Biochem J, 1991. 276: p. 739-43.
40.Shi, J., et al., Statins increase thrombomodulin expression and function in human endothelial cells by a nitric oxide-dependent mechanism and counteract tumor necrosis factor alpha-induced thrombomodulin downregulation. Blood Coagul Fibrinolysis, 2003. 14: p. 575-85.
41.Calnek, D.S. and B.W. Grinnell, Thrombomodulin-dependent anticoagulant activity is regulated by vascular endothelial growth factor. Exp Cell Res, 1998. 238: p. 294-8.
42.Lentz, S.R., et al., Consequences of hyperhomocyst(e)inemia on vascular function in atherosclerotic monkeys. Arterioscler Thromb Vasc Biol, 1997. 17: p. 2930-4.
43.Ishii, H., et al., Oxidized phospholipids in oxidized low-density lipoprotein down-regulate thrombomodulin transcription in vascular endothelial cells through a decrease in the binding of RARbeta-RXRalpha heterodimers and Sp1 and Sp3 to their binding sequences in the TM promoter. Blood, 2003. 101: p. 4765-74.
44.Moore, K.L., et al., Endotoxin enhances tissue factor and suppresses thrombomodulin expression of human vascular endothelium in vitro. J Clin Invest, 1987. 79: p. 124-30.
45.Moore, K.L., C.T. Esmon, and N.L. Esmon, Tumor necrosis factor leads to the internalization and degradation of thrombomodulin from the surface of bovine aortic endothelial cells in culture. Blood, 1989. 73: p. 159-65.
46.Kapiotis, S., et al., Interleukin-4 counteracts pyrogen-induced downregulation of thrombomodulin in cultured human vascular endothelial cells. Blood, 1991. 78: p. 410-5.
47.Dittman, W.A., et al., The structure and function of mouse thrombomodulin. Phorbol myristate acetate stimulates degradation and synthesis of thrombomodulin without affecting mRNA levels in hemangioma cells. J Biol Chem, 1988. 263: p. 15815-22.
48.van der Velden, P.A., et al., A frequent thrombomodulin amino acid dimorphism is not associated with thrombophilia. Thromb Haemost, 1991. 65: p. 511-3.
49.Norlund, L., et al., A common thrombomodulin amino acid dimorphism is associated with myocardial infarction. Thromb Haemost, 1997. 77: p. 248-51.
50.Ohlin, A.K., L. Norlund, and R.A. Marlar, Thrombomodulin gene variations and thromboembolic disease. Thromb Haemost, 1997. 78: p. 396-400.
51.Ohlin, A.K. and R.A. Marlar, The first mutation identified in the thrombomodulin gene in a 45-year-old man presenting with thromboembolic disease. Blood, 1995. 85: p. 330-6.
52.Ireland, H., et al., Thrombomodulin gene mutations associated with myocardial infarction. Circulation, 1997. 96: p. 15-8.
53.Li, Y.H., et al., G-33A mutation inthe promoter region of thrombomodulin gene and its association with coronary artery disease and plama slouble thrombomodulin levels. Am. J Cardiol, 2000. 85: p. 8-12.
54.Kunz, G., et al., Naturally occurring mutations in the thrombomodulin gene leading to impaired expression and function. Blood, 2002. 99: p. 3646-53.
55.Norlund, L., B. Zoller, and A.K. Ohlin, A novel thrombomodulin gene mutation in a patient suffering from sagittal sinus thrombosis. Thromb Haemost, 1997. 78: p. 1164-6.
56.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: p. 743-8.
57.Salomaa, V., et al., Soluble thrombomodulin as a predictor of incident coronary heart disease and symptom less carotid artery atherosclerosis in the Atherosclerosis Risk in Communities (ARIC) Study: a case-cohort study. Lancet, 1999. 353: p. 1729-34.
58.Healy, A.M., et al., Absence of the blood-clotting regulator thrombomodulin causes embryonic lethality in mice before development of a functional cardiovascular system. Proc Natl Acad Sci USA, 1995. 92: p. 850-4.
59.Healy, A.M., et al., Intravascular coagulation activation in a murine model of thrombomodulin deficiency: effects of lesion size, age, and hypoxia on fibrin deposition. Blood, 1998. 92: p. 4188-97.
60.Weiler-Guettler, H., et al., A targeted point mutation in thrombomodulin generates viable mice with a prethrombotic state. J Clin Invest, 1998. 101: p. 1983-91.
61.Christie, P.D., et al., A murine model of myocardial microvascular thrombosis. J Clin Invest, 1999. 104: p. 533-9.
62.Isermann, B., et al., Endothelium-specific loss of murine thrombomodulin disrupts the protein C anticoagulant pathway and causes juvenile-onset thrombosis. J Clin Invest, 2001. 108: p. 537-46.
63.Uchiba, M., et al., Effect of human urinary thrombomodulin on endotoxin-induced intravascular coagulation and pulmonary vascular injury in rats. Am J Hematol, 1997. 54: p. 118-23.
64.Uchiba, M., et al., Recombinant thrombomodulin prevents endotoxin-induced lung injury in rats by inhibiting leukocyte activation. Am J Physiol, 1996. 271): p. L470-5.
65.Uchiba, M., et al., Recombinant human soluble thrombomodulin reduces endotoxin-induced pulmonary vascular injury via protein C activation in rats. Thromb Haemost, 1995. 74: p. 1265-70.
66.Bernard, G.R., et al., Safety and dose relationship of recombinant human activated protein C for coagulopathy in severe sepsis. Crit Care Med, 2001. 29: p. 2051-9.
67.Bernard, G.R., et al., Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med, 2001. 344: p. 699-709.
68.Brueckmann, M., et al., Activated protein C inhibits the release of macrophage inflammatory protein-1-alpha from THP-1 cells and from human monocytes. Cytokine, 2004. 26: p. 106-13.
69.Joyce, D.E., et al., Gene expression profile of antithrombotic protein c defines new mechanisms modulating inflammation and apoptosis. J Biol Chem, 2001. 276: p. 11199-203.
70.Riewald, M., et al., Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science, 2002. 296: p. 1880-2.
71.Bajzar, L., et al., Thrombin activatable fibrinolysis inhibitor: not just an inhibitor of fibrinolysis. Crit Care Med, 2004. 32: p. S320-4.
72.Asai, S., et al., Absence of procarboxypeptidase R induces complement-mediated lethal inflammation in lipopolysaccharide-primed mice. J Immunol, 2004. 173: p. 4669-74.
73.Waugh, J.M., et al., Thrombomodulin overexpression to limit neointima formation. Circulation, 2000. 102: p. 332-7.
74.Waugh, J.M., et al., Local overexpression of thrombomodulin for in vivo prevention of arterial thrombosis in a rabbit model. Circ Res, 1999. 84: p. 84-92.
75.Weiler, H., et al., Characterization of a mouse model for thrombomodulin deficiency. Arterioscler Thromb Vasc Biol, 2001. 21: p. 1531-7.
76.Rijneveld, A.W., et al., Thrombomodulin mutant mice with a strongly reduced capacity to generate activated protein C have an unaltered pulmonary immune response to respiratory pathogens and lipopolysaccharide. Blood, 2004. 103: p. 1702-9.
77.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: p. 1267-74.
78.Wang, H., et al., HMG-1 as a late mediator of endotoxin lethality in mice. Science, 1999. 285: p. 248-51.
79.Taniguchi, N., et al., High mobility group box chromosomal protein 1 plays a role in the pathogenesis of rheumatoid arthritis as a novel cytokine. Arthritis Rheum, 2003. 48: p. 971-81.
80.Isermann, B., et al., The thrombomodulin-protein C system is essential for the maintenance of pregnancy. Nat Med, 2003. 9: p. 331-7.
81.Boffa, M.C., B. Burke, and C.C. Haudenschild, Preservation of thrombomodulin antigen on vascular and extravascular surfaces. J Histochem Cytochem, 1987. 35: p. 1267-76.
82.Wong, V.L., et al., Regional distribution of thrombomodulin in human brain. Brain Res, 1991. 556: p. 1-5.
83.Raife, T.J., et al., Thrombomodulin expression by human keratinocytes: induction of cofactor activity during epidermal differentiation. J Clin Invest, 1994. 93: p. 1846-51.
84.Conway, E.M., B. Nowakowski, and M. Steiner-Mosonyi, Human neutrophils synthesize thrombomodulin that does not promote thrombin-dependent protein C activation. Blood, 1992. 80: p. 1254-63.
85.McCachren, S.S., et al., Thrombomodulin expression by human blood monocytes and by human synovial tissue lining macrophages. Blood, 1991. 78: p. 3128-32.
86.Suzuki, K., et al., Functionally active thrombomodulin is present in human platelets. J Biochem, 1988. 104: p. 628-32.
87.Tezuka, Y., et al., Expression of thrombomodulin in esophageal squamous cell carcinoma and its relationship to lymph node metastasis. Cancer Res., 1995. 55: p. 4196-200.
88.Suehiro, T., et al., Thrombomodulin inhibits intrahepatic spread in human hepato-cellular carcinoma. Hepatology, 1995. 21: p. 1285-90.
89.Tamura, A., et al., Prognostic significance of thrombomodulin expression and vascular invasion in stage I squamous cell carcinoma of the lung. Lung Cancer, 2001. 34: p. 375-82.
90.Fujiwara, M., et al., Antisense oligodeoxynucleotides against thrombomodulin suppress the cell growth of lung adenocarcinoma cell line A549. Pathol Int, 2002. 52: p. 204-13.
91.Yamamoto, S., et al., Urinary thrombomodulin, its isolation and characterization. J. Biochem. (Tokyo). 1993. 113: p. 433-40.
92.Boehme, M.W.J., P. Galle, and W. Stremmel, Kinetics of thrombomodulin release and endothelial cell injury by neutrophil-derived proteases and oxygen radicals. Immunology, 2002. 107: p. 340-9.
93.Hosaka, Y., et al., Inhibition of invasion and experimental metastasis of murine melanoma cells by human soluble thrombomodulin. Cancer Lett, 2000. 161: p. 231-40.
94.Tonini, T., F. Rossi, and P.P. Claudio, Molecular basis of angiogenesis and cancer. Oncogene, 2003. 22: p. 6549-56.
95.Folkman, J., Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med, 1995. 1: p. 27-31.
96.Liekens, S., E. De Clercq, and J. Neyts, Angiogenesis: regulators and clinical applications. Biochem Pharmacol, 2001. 61: p. 253-70.
97.Folkman, J., et al., Isolation of a tumor factor responsible for angiogenesis. J Exp Med, 1971. 133: p. 275-88.
98.Han, H.S., et al., Effect of thrombomodulin on plasminogen activation. Fibrinolysis and Proteolysis, 2000. 14: p. 221-8.
99.Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970. 227: p. 680-5.
100.Shi, G.Y., et al., Plasmin and the regulation of tissue-type plasminogen activator biosynthesis in human endothelial cells. J Biol Chem, 1992. 267: p. 19363-8.
101.Lee, O.H., et al., Identification of angiogenic properties of insulin-like growth factor II in in vitro angiogenesis models. Brit J Cancer, 2000. 82: p. 385-91.
102.Passaniti, A., et al., A simple quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor. Lab Invest, 1992. 67: p. 519-28.
103.Shirai, T., et al., Gene structure of human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C. J Biochem, 1988. 103: p. 281-5.
104.Suzuki, K., H. Kusumoto, and S. Hashimoto, Isolation and characterization of thrombomodulin from bovine lung. Biochim. Biophys. Acta., 1986. 882: p. 343-52.
105.Rousseau, S., et al., p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelilal cells. Oncogene, 1997. 15: p. 2169-77.
106.Kawasaki, K., et al., Activation of the phosphatidylinositol 3-kinase/protein kinase Akt pathway mediates nitric oxide-induced endothelial cell migration and angiogenesis. Mol Cell Biol, 2003. 23: p. 5726-37.
107.Pepper, M.S., Role of the matrix metalloproteinase and plasminogen activator-plasmin systems in angiogenesis. Arterioscler. Thromb Vasc Biol, 2001. 21: p. 1104-17.
108.Kumar, R., et al., Regulation of distinct steps of angiogenesis by different angiogenic molecules. Int J Oncol, 1998. 12: p. 749-57.
109.Pepper, M.S., et al., Synergistic induction of t-PA by vascular endothelial growth factor and basic fibroblast growth factor and localization of t-PA to Weibel-Palade bodies in bovine microvascular endothelial cells. Thromb Haemost, 2001. 86: p. 702-9.
110.Carmeliet, P., Angiogenesis in health and disease. Nat Med, 2003. 9: p. 653-660.
111.Folkman, J. and Y. Shing, Angiogenesis. J Biol Chem, 1992. 267: p. 10931-4.
112.Yancopoulos, G.D., et al., Vascular-specific growth factors and blood vessel formation. Nature, 2000. 407: p. 242-8.
113.Cao, R., et al., Suppression of angiogenesis and tumor growth by the inhibitor K1-5 generated by plasmin-mediated proteolysis. Proc. Natl. Acad. Sci. USA, 1999. 96: p. 5728-33.
114.Jimenez, B., et al., Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1. Nat Med, 2000. 6: p. 41-8.
115.O'Reilly, M.S., et al., Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell, 1997. 88: p. 277-85.
116.Corbacho, A.M., G. Martinez de la Escalera, and C. Clapp, Roles of prolactin and related members of the prolactin/growth hormone/placental lactogen family in angiogenesis. J Endocrin, 2002. 173: p. 219-38.
117.Bourin, M.C., A.E. Lundgren, and U. Lindahl, Isolation and characterization of the glycosaminoglycan component of rabbit thrombomodulin proteoglycan. J Biol Chem, 1990. 265: p. 15424-31.
118.Bechard, D., et al., Endocan is a novel chondroitin sulfate/dermatan sulfate proteoglycan that promotes hepatocyte growth factor/scatter factor mitogenic activity. J Biol Chem, 2001. 276(51): p. 48341-9.
119.Tapon-Bretaudiere, J., et al., A fucosylated chondroitin sulfate from echinoderm modulats in vitro fibroblast growth factor 2-depedent angiogenesis. Mol Cancer Res, 2002. 1: p. 96-102.
120.Pieper, J.S., et al., Loading of collagen-heparan sulfate matrices with bFGF promotes angiogenesis and tissue generation in rats. J. Biomed. Mater Res, 2002. 62: p. 185-94.
121.Alexander, C.M., et al., Syndecan-1 is required for Wnt-1-induced mammary tumorigenesis in mice. Nat Genet, 2000. 25: p. 329-32.
122.Horowitz, A., E. Tkachenko, and M. Simons, Fibroblast growth factor-specific modulation of cellular response by syndecan-4. J Cell Biol, 2002. 157: p. 715-25.
123.Miettinen, M. and M. Sarlomo-Rikala, Expression of calretinin, thrombomdulin, keratin 5, and mesothelin in lung carcinomas of different types: an immunohistochemical analysis of 596 tumors in comparison with epithelioid mesotheliomas of the pleura. Am. J Surg Pathol, 2003. 27: p. 150-8.
124.Wilhelm, S., et al., Thrombomodulin, a receptor for the serine protease thrombin, is decreased in primary tumors and metastases but increased in ascitic fluids of patients with advanced ovarian cancer FIGO IIIc. Int J Oncol, 1998. 13: p. 645-51.
125.Ishii, H. and P.W. Majerus, Thrombomodulin is present in human plasma and urine. J Clin Invest, 1985. 76: p. 2178-81.
126.Boehme, M.W., et al., Release of thrombomodulin from endothelial cells by concerted action of TNF-alpha and neutrophils: in vivo and in vitro studies. Immunology, 1996. 87: p. 134-40.
127.Lohi, O., S. Urban, and M. Freeman, Diverse substrate recognition mechanisms for rhomboids; thrombomodulin is cleaved by mammalian rhomboids. Curr Biol, 2004. 14: p. 236-41.
128.Iwamoto, R. and E. Mekaka, Heparin-binding EGF-like growth factor: a juxtacrine growth factor. Cytokine & Growth Factor Reviews, 2000. 11: p. 335-44.
129.Iwamoto, R. and E. Mekada, Heparin-binding EGF-like growth factor: a juxtacrine growth factor. Cytokine Growth Factor Rev, 2000. 11: p. 335-44.
130.Morbidelli, L., et al., Angiosuppressive and angiostimulatory effects exerted by synthetic partial sequences of endostatin. Clin Cancer Res, 2003. 9: p. 5358-69.
131.Van de Wouwer, M., D. Collen, and E.M. Conway, Thrombomodulin-protein C-EPCR system integrated to regulate coagulation and inflammation. Arterioscler Thromb Vasc Biol, 2004. 24: p. 1-10.