Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thrombomodulin is an endothelial cell surface glycoprotein that forms a 1:1 complex with thrombin. In this form, thrombin can activate approximately 1,000-fold more protein C than thrombin alone and does not activate coagulation factors, V and VIII, and platelets. Activated protein C inactivates factors Va and VIIIa. Thus thrombomodulin converts thrombin from a procoagulant protease to an anticoagulant. The soluble thrombomodulin present in human urine and plasma appears to represent a truncated form that lacks the transmembrane and cytoplasmic domains of tissue thrombomodulin. The plasma level of thrombomodulin has been used as a marker for endothelial injury in vivo. Elevated levels of soluble thrombomodulin were reported in the plasma from the patients with disseminated intravascular coagulation, adult respiratory distress syndrome (ARDS), and diabetes mellitus retinopathy.
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PMID:[Soluble thrombomodulin: as a marker of endothelial injury]. 805 97

Thrombomodulin is a vascular endothelial cell transmembrane protein that forms a 1:1 complex with thrombin, this interaction product forming the basis of a physiologically important natural anticoagulant system. Transcriptional down-regulation of thrombomodulin occurs following exposure of cultured endothelial cells to cytokines, while up-regulation is induced by retinoic acid and dibutyryl cyclic AMP. Thrombomodulin is also regulated developmentally, appearing in the parietal endoderm of 7.5-day-old mouse embryos. We determined that cell surface functional thrombomodulin in cultured human umbilical vein endothelial cells (HUVEC) and A549 cells increased 3.2- and 6.7-fold, respectively, in response to 24 h of continuous 42 degrees C heat shock stress. Northern analyses of thrombomodulin mRNA accumulation also showed a delayed response that was characterized by an augmentation in mRNA levels that started 12-18 h after the initiation of the stress, and continued to rise, without attenuation, during 48 h of continuous heat shock. Nuclear run-on studies confirmed that the predominant mechanism of augmentation was transcriptional. Furthermore, the heat shock-induced up-regulation of thrombomodulin in HUVEC abrogated the suppressive effect of tumor necrosis factor. Analysis of the 5' region of the thrombomodulin gene revealed six highly conserved tandem copies of the five base pair recognition unit that is the consensus sequence for a heat shock element. We hypothesize that the stress-induced augmentation in thrombomodulin gene transcription is mediated via heat shock factors binding to the heat shock element and that the stress response of thrombomodulin may have a biological role to protect the vascular endothelium during a variety of stresses, including inflammation, infection, and/or development.
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PMID:Heat shock of vascular endothelial cells induces an up-regulatory transcriptional response of the thrombomodulin gene that is delayed in onset and does not attenuate. 807 33

Thrombomodulin (TM) is a transmembrane vascular endothelial cell receptor that is a cofactor in a major physiologically relevant natural anticoagulant system. We recently developed a cell model to examine one mechanism of regulation of TM cell surface expression and visually demonstrated that the receptor undergoes internalization predominantly via noncoated pits (Conway et al., 1992, J. Cell. Phys., 151:604-612). We have extended these studies to examine the role of the cytoplasmic domain of TM by deleting this region and expressing the truncated version of the molecule in COS cells (COS.Cyto.Del cells). Electron microscopy demonstrated internalization of gold-labeled anti-TM antibody or thrombin in a time- and temperature-dependent manner, similar to that seen with the wild-type transfected cells (COS.TM-CR). Endocytosis was characterized by initial surface clustering of gold particles, followed by aggregation into noncoated pits, early endosome formation, and, finally, entry into multivesicular bodies and lysosomes. There was a notable absence of gold particles in clathrin-coated pits and vesicles. The kinetics of binding and internalization of 125I-labeled ligand in COS.Cyto.Del cells was compared with that of COS.TM-CR cells and was not significantly different. These studies provide ultrastructural and quantitative data to indicate that TM efficiently undergoes endocytosis via nonclathrin-coated pits when the receptor is lacking the cytoplasmic domain. This finding suggests that there may be alternative regions of the molecule that mediate those signals necessary for internalization.
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PMID:Thrombomodulin lacking the cytoplasmic domain efficiently internalizes thrombin via nonclathrin-coated, pit-mediated endocytosis. 810 66

The vascular endothelium, by virtue of its position at the interface between blood and the vessel wall, is known to play a critical role in the control of thrombosis and fibrinolysis. Thrombomodulin (TM) is a surface receptor that binds thrombin and is a potent activator of the protein C anticoagulant pathway. Although TM expression is known to be regulated by various cytokines, little is known about its response to ever-present biomechanical stimuli. We have explored the role of fluid shear stress, imparted on the luminal surface of the endothelial cell as a result of blood flow, on the expression of TM mRNA and protein in both bovine aortic endothelial (BAE) and bovine smooth muscle (BSM) cells in an in vitro system. We report in the present study that TM expression is regulated by flow. Subjecting BAE cells to fluid shear stress in the physiological range of magnitude of 15 (moderate shear stress) and 36 (elevated shear stress) dynes/cm2 resulted in a mild transient increase followed by a significant decrease in TM mRNA to 37% and 16% of its resting level, respectively, by 9 hours after the onset of flow. In contrast, shear stress at the low magnitude of 4 dynes/cm2 did not affect TM mRNA levels. The sensitivity of TM mRNA expression by flow was found to be specific to endothelium, since it was not observed in BSM cells exposed to steady laminar shear stress of 15 dynes/cm2. Furthermore, unlike BAE cells, BSM cells did not exhibit altered cell shape nor align in the direction of flow after 24 hours of shear stress at 15 dynes/cm2.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Endothelial expression of thrombomodulin is reversibly regulated by fluid shear stress. 815 32

Thrombomodulin (TM) binds thrombin to form a complex that activates the plasma anticoagulant zymogen protein C. TM is an integral membrane glycoprotein that contains a chondroitin sulfate moiety. Interaction with thrombin involves both the protein component of TM, specifically the growth factor-like repeats 4-6 (TM 4-6), and chondroitin sulfate. Removal of chondroitin sulfate decreases the affinity for thrombin approximately 10-fold and shifts the Ca2+ dependence of protein C activation from simple saturation at > or = 500 microM Ca2+ to a distinct optimum at approximately 100 microM Ca2+. Thrombin possesses two regions of high positive charge, anion binding exosites 1 and 2. Anion binding exosite 1 interacts with the growth factor region of TM while exosite 2 is involved in binding prothrombin activation fragment 2 or heparin. We demonstrate that recombinant TM, truncated at the membrane-spanning domain, or TM 4-6 can bind thrombin when fragment 2 is present either covalently attached (meizothrombin des-fragment 1) or in reversible association. With meizothrombin des-fragment 1, the Ca2+ dependence of protein C activation is independent of the presence of the chondroitin sulfate on TM. At 0.27 mM Ca2+, TM containing chondroitin sulfate binds thrombin (Kd(app) = 0.3 nM) approximately 45 times tighter than meizothrombin des-fragment 1 (Kd(app) = 14 nM). However, the chondroitin-free form binds thrombin (Kd(app) = 2.4 nM) only approximately 4 times tighter than meizothrombin des-fragment 1 (Kd(app) = 9.4 nM). These studies suggest that occupancy of anion binding exosite 2 by either chondroitin sulfate or fragment 2 alters thrombin conformation resulting in the altered Ca2+ dependence of protein C activation.
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PMID:Occupancy of anion binding exosite 2 on thrombin determines Ca2+ dependence of protein C activation. 816 79

Thrombomodulin is an endothelial cell surface glycoprotein that inhibits the procoagulant activities of thrombin and accelerates activation of the anticoagulant protein C. Because protein C deficiency is associated with cutaneous thrombosis, we investigated the expression of thrombomodulin in human skin. Thrombomodulin was detected by immunohistochemical staining both in dermal endothelial cells and in epidermal keratinocytes. Within the epidermis, thrombomodulin staining was limited to keratinocytes of the spinous layer, suggesting that thrombomodulin is induced when basal keratinocytes begin to terminally differentiate. Thrombomodulin expression also correlated with squamous differentiation in epidermal malignancies; little or no thrombomodulin staining was seen in five basal cell carcinomas, whereas strong thrombomodulin staining was observed in each of five squamous cell carcinomas. Human foreskin keratinocytes cultured in medium containing 0.07 mM calcium chloride synthesized functional thrombomodulin with cofactor activity comparable to thrombomodulin in human umbilical vein endothelial cells. Stimulation of keratinocyte differentiation with 1.4 mM calcium chloride for 48 h produced 3.5-, 3.2-, and 5.6-fold increases in thrombomodulin cofactor activity, antigen, and mRNA, respectively. These observations suggest that thrombin is regulated by keratinocyte thrombomodulin at sites of cutaneous injury, and indicate a potential role for thrombomodulin in epidermal differentiation.
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PMID:Thrombomodulin expression by human keratinocytes. Induction of cofactor activity during epidermal differentiation. 816 84

Recent advances in determining anti-thrombogenic functions of vascular endothelial cells are reviewed. The following anticoagulant and fibrinolytic systems of endothelial cells are physiologically important; (1) Endothelial cell-derived metabolites including prostacyclin and nitric oxide (NO) support platelet inactivity. (2) Antithrombin III and tissue factor pathway inhibitor (TFPI) bound to heparin-like proteoglycans on endothelial cell membrane inhibit activated serine protease coagulation factors such as thrombin, factor Xa and factor VIIa-tissue factor complex. (3) Thrombomodulin converts thrombin from procoagulant into anticoagulant. Thrombin associated to thrombomodulin on endothelial cells activates protein C. Activated protein C in concert with protein S bound to endothelial cell membrane inactivates factors Va and VIIIa. (4) A receptor for both tissue plasminogen activator and plasminogen on endothelial cells provides an efficient plasmin generating system. Perturbation of these anti-thrombogenic systems of endothelial cells is caused by endotoxin (LPS), cytokines such as interleukin-1 and tumor necrosis factor (TNF), and risk factors for atherogenesis including lipoprotein(a) and homocysteine may result in arterial or venous thrombosis with subsequent development of atherosclerosis.
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PMID:[Anticoagulant and fibrinolytic systems of the injured vascular endothelial cells]. 817 40

Thrombomodulin (TM) is an essential cofactor for the physiologic activation of the anticoagulant protein C by thrombin. We have observed that the expression of TM mRNA in response to retinoic acid was markedly increased in human U937 monoblast-like cells, and human MEG01 megakaryocyte-like cells, but not in human umbilical vein cells, murine hemangioma cells, human K562 erythroblast-like cells, and murine HSD fibroblast-like cells. TM activity in U937 cells and MEG01 cells was not detectable in untreated cells, but developed rapidly after treatment with retinoic acid. In endothelial cells there was minimal change in TM activity in response to retinoic acid treatment. We have isolated clones for the genes for murine and human TM and have identified potential retinoic acid response elements in the 5'-flanking region of the human gene. In U937 cells the increase in mRNA levels was associated with increased transcription, and transient transfection studies with reporter plasmids demonstrate functional retinoic acid response elements present in the 5'-flanking region of the gene. Deletion of, and mutations introduced into, the potential retinoic acid response element confirm the functional response in transient transfections.
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PMID:Characterization of thrombomodulin expression in response to retinoic acid and identification of a retinoic acid response element in the human thrombomodulin gene. 820 15

Thrombomodulin (TM) is an endothelial cell thrombin receptor that converts thrombin from a procoagulant to an anticoagulant enzyme. It has previously been shown that TM is expressed in both a high-M(r) form containing chondroitin sulphate and a low-M(r) form lacking this modification. Site-directed mutagenesis of a soluble human TM derivative (TMD1) was employed to determine the attachment site(s) of this functionally important oligosaccharide on the core protein. Although there are four serine residues within the Ser/Thr-rich domain of TMD1 that might support glycosaminoglycan assembly, our analysis demonstrates that the primary site of attachment is at Ser474, and evidence is presented for low levels of attachment at Ser472. It was possible to improve the overall degree of attachment by mutating Ser472 to glutamic acid (so as to conform Ser474 to the xylosyltransferase acceptor consensus acidic-Gly-Ser-Gly-acidic); however, a significant proportion (approx. 35%) of the total TM still lacked a glycosaminoglycan moiety. Mutants that possess a substitution for Ser474 show an increased mobility of their low-M(r) form on SDS/PAGE compared with native TMD1. Isolation and sequencing of a C-terminal peptide demonstrated that this serine is modified in the low-M(r) form of native TMD1. An apparent 'acceptor consensus overlap' at Ser474 suggests that the mechanism behind the glycosaminoglycan split of TM may involve a competition for substrate between xylosyltransferase and N-acetylgalactosaminyltransferase.
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PMID:Identification of the predominant glycosaminoglycan-attachment site in soluble recombinant human thrombomodulin: potential regulation of functionality by glycosyltransferase competition for serine474. 821 7

Despite its rather recent identification, the protein C activation system has afforded many investigators with unique opportunities to probe the molecular basis by which cofactors function. Thrombomodulin clearly exerts its specificity switch both by interacting directly with the fibrinogen binding site on thrombin (exosite 1) and by altering the conformation within the enzyme center. At least in the case of thrombomodulin, these conformational changes appear to overcome repulsive interactions between acidic residues in the substrate and the enzyme. To determine whether the models derived from attempts at the molecular analysis of the protein C activation complex are at all relevant to the other coagulation complexes will require further examination, but the concept that residues near the cleavage site contact residues in the free enzyme in an unfavorable fashion, and that the cofactors overcome these inhibitory interactions is a hypothesis that is directly testable for all of the complexes. The availability of crystal structures for the coagulation enzymes, coupled with the capacity to mutagenize both the substrate and the enzyme, promises to provide new insights into molecular events that control coagulation.
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PMID:Molecular events that control the protein C anticoagulant pathway. 823 11


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