Urinary Thrombomodulin, Its Isolation and Characterization1

Yamamoto, Sachiko; Mizoguchi, Toshimi; Tamaki, Takashi; Ohkuchi, Masao; Kimura, Shigeru; Aoki, Nobuo

doi:10.1093/oxfordjournals.jbchem.a124063

Cited by 21 publications

(22 citation statements)

References 2 publications

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“…While this manuscript was in preparation, Yamamoto et al [24] reported the isolation and characterization of two active forms of urinary thrombomodulin which appear to share some features of the 63157-kDa doublet described in this study. These workers did not observe the N-terminal heterogeneity reported in this study.…”

Section: Discussionmentioning

confidence: 65%

Purification and characterization of two forms of soluble thrombomodulin from human urine

Jackson

Tetaz

Salem

et al. 1994

European Journal of Biochemistry

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We have isolated and characterized two forms of soluble thrombomodulin from human urine. The purification procedure consisted of ultrafiltration, chromatography on DEAE-Sepharose, affinity chromatography on diisopropyl-phosphate -thrombin and/or monoclonal anti-thrombomodulin IgG affigel followed by reverse-phase HPLC. An active soluble form of thrombomodulin was purified 1600-fold from 34-1 urine. The purified protein migrated as a doublet, with molecular mass 761 72 kDa under reducing conditions and 63/57 kDa under non-reducing conditions as determined by SDSPAGE. Amino acid analysis of the 63/57-kDa soluble thrombomodulin confirmed sequence identity with human thrombomodulin and demonstrated N-terminal heterogeneity. Compared to membrane-type thrombomodulin, the purified 63/57-kDa soluble thrombomodulin was more active as a cofactor for protein-C activation. The second major thrombomodulin fragment in urine is an inactive 35-kDa thrombomodulin polypeptide derived from the N-terminal extracellular region of thrombomodulin.Thrombomodulin is an endothelial cell-surface glycoprotein which forms a high-affinity non-covalent complex with thrombin. In this complex, thrombomodulin acts as a cofactor for thrombin in the activation of protein C, enhancing the reaction rate by greater than 1000-fold to form activated protein C. Activated protein C acts as a natural anticoagulant by proteolytic degradation of factors Va and VIIIa [ l , 21. Thrombomodulin is a single-chain 554-residue integral membrane protein comprising a signal peptide, an N-terminal domain, six epidermal-growth-factor-like repeats, an O-glycosylation-rich domain followed by a transmembrane segment and a short cytoplasmic tail [3]. The protein has been isolated from rabbit [2], bovine lungs [4] and human placenta [5]. The molecular and functional characteristics of membrane thrombomodulin have been well delineated [6, 71. Cell-surface expression of thrombomodulin is regulated by a number of mechanisms including a tumour-necrosisfactor-mediated process which regulates transcription of the thrombomodulin gene [ 81, internalization by receptor-mediated endocytosis [9] and proteolytic cleavage from the endothelial cell membrane [lo]. The relative role of each of these mechanisms in the surface expression of thrombomodulin has yet to be defined.Previous studies have identified the presence of molecules in plasma and urine that are immunologically indistinguishable from membrane thrombomodulin [ 111. Some of these molecules act as thrombin cofactors in the activation

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Section: Discussionmentioning

confidence: 65%

Purification and characterization of two forms of soluble thrombomodulin from human urine

Jackson

Tetaz

Salem

et al. 1994

European Journal of Biochemistry

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“…To assess TMD23 effects on tumor neoangiogenesis, 1ϫ10 6 human melanoma A2058 cells (ATCC CRL-11147) were mixed with Matrigel (0.5 mL) containing heparin (60 U/mL) and various concentrations of TMD23 and subcutaneously injected into athymic nude mice. Matrigel plugs were harvested after 18 days and frozen in liquid nitrogen.…”

Section: Effect Of Tmd23 On Tumor Neoangiogenesismentioning

confidence: 99%

“…3 Ablation of the TM gene in mouse embryos causes early postimplantation death before a functional cardiovascular system is established. 4 Soluble forms of TM derived from extracellular TM domains found in plasma and urine 5,6 have been recognized as markers of endothelial cell injury. 7 Indeed, clinical studies have demonstrated an inverse correlation between plasma levels of soluble TM and coronary artery disease and atherosclerosis.…”

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confidence: 99%

Evidence of Human Thrombomodulin Domain as a Novel Angiogenic Factor

Shi

Chang

et al. 2005

Circulation

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Background-Thrombomodulin is an anticoagulant, endothelial-cell-membrane glycoprotein. A recombinant thrombomodulin domain containing 6 epidermal growth factor-like structures exhibits mitogenic activity. This study explored the novel angiogenic effects of the recombinant domain using in vitro and in vivo models. Methods and Results-Human recombinant thrombomodulin containing 6 epidermal growth factor-like structures (TMD2) and TMD2 plus a serine and threonine-rich domain (TMD23) were prepared using the Pichia pastoris expression system. Combined with purified TMD2 or TMD23, thrombin effectively activated protein C. TMD23 had higher activity than TMD2 in stimulating DNA synthesis in cultured human umbilical vein endothelial cells. Additionally, TMD23 stimulated chemotactic motility and capillarylike tube formation in human umbilical vein endothelial cells, an effect mediated through 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 endothelial cell expression of matrix metalloproteinases and plasminogen activators, which mediated extracellular proteolysis, leading to endothelial cell invasion and migration during angiogenesis. Furthermore, TMD23-containing implants in rat cornea induced ingrowth of new blood vessels from the limbus. With the murine angiogenesis assay, TMD23 not only induced neovascularization coinjected with Matrigel and heparin but also enhanced angiogenesis in Matrigel containing melanoma A2058 cells in nude mice. Conclusions-The

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“…Thrombomodulin is a thrombin receptor expressed on the endothelium which acts as a cofactor for thrombin-catalyzed activation of protein C [6,8]. The presence of thrombomodulin was demonstrated throughout the vascular and lymphatic endothelium, and syncytio-trophoblast [16].…”

Section: Introductionmentioning

confidence: 99%

Urinary thrombomodulin is down regulated in schistosomiasis associated bladder cancer

Refaat

Ali

Hassan

et al. 2012

Journal of Genetic Engineering and Biotechnology

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Background: Thrombomodulin (TM) is a surface glycoprotein and expressed in many cancers. The aim of the present study was to detect the expression levels of TM in plasma and urine of bladder cancer patients and to compare these levels to the clinicopathological data of the patients as well as the ploidy status of their exfoliated urinary cells. We studied the levels of TM in plasma and urine samples of 57 bladder cancer patients and 10 controls using the (ELISA) assay and compared the results to the ploidy status of the cells taken from the patents urine samples as well as their clinicopathological profile.Results: Urinary TM was significantly down regulated while plasma TM was significantly up regulated in bladder cancer patients. Plasma TM was significantly higher in SCC than TCC patients. The sensitivity and specificity of urinary TM were 90% and 86%, respectively. While the sensitivity and specificity of plasma TM were 76% and 80%, respectively.Conclusion: Urinary TM is significantly down regulated, while plasma TM is significantly up regulated in bladder cancer as compared to the control group. Urinary TM has superior sensitivity and specificity over plasma TM. Urinary TM could be used as a predictive marker in bladder cancer. Further studies are needed to detect the prognosis significance of thrombomodulin in schistosomiasis associated bladder cancer.

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Urinary Thrombomodulin, Its Isolation and Characterization1

Cited by 21 publications

References 2 publications

Purification and characterization of two forms of soluble thrombomodulin from human urine

Purification and characterization of two forms of soluble thrombomodulin from human urine

Evidence of Human Thrombomodulin Domain as a Novel Angiogenic Factor

Urinary thrombomodulin is down regulated in schistosomiasis associated bladder cancer

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