Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thrombomodulin (TM), the endothelial cell surface receptor for thrombin-mediated activation of protein C and of its anticoagulant system, is involved in maintaining vascular nonthrombogenicity, and depressed TM activity may induce intravascular fibrin formation. TM antigen was previously found by immunohistochemical methods in rabbit glomeruli. We therefore attempted to identify the corresponding TM activity in isolated detergent-solubilized rat and human glomeruli. Like purified lung TM, rat glomeruli extracts accelerated the hydrolysis by activated protein C of the chromogenic substrate S-2238 in the presence of 10 nM thrombin, as determined by spectrophotometry. One mg glomerular protein promoted the formation of 681 +/- 115 nmol activated protein C, the equivalent of the amount generated by 845 ng of purified rabbit TM. TM activity correlated with the protein content of the glomerular extracts (r = 0.94). These extracts prolonged rat plasma activated partial thromboplastin time. Incubation of glomeruli with tumor necrosis factor-alpha (TNF) or E. coli lipopolysaccharide depressed their TM-like activity in a dose and time dependent manner. Incubation with TNF suppressed their anticoagulant activity. In human glomeruli, TM activity was also found at a level which corresponded to their TM antigen content, and was determined by ELISA with mouse monoclonal antibody. These results indicate that measurement of glomerular TM activity might help to clarify the mechanisms of intraglomerular fibrin deposition in renal diseases.
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PMID:Quantification and modulation of thrombomodulin activity in isolated rat and human glomeruli. 131 19

The endothelial cell surface receptor thrombomodulin (TM) displays various anticoagulant functions: it acts as a cofactor for the activation of protein C (PC) by thrombin, prevents the activation of fibrinogen, platelets and Factor V by thrombin. TM was also shown to accelerate the inhibition of thrombin by its physiological inhibitor antithrombin III (ATIII). The studies performed on rabbit lung TM were undertaken in order to provide better understanding, along with the identification and the characterization of functional domains, to the mechanism of action of TM. On the basis of the physical and chemical properties of TM, which were compatible with those of a proteoglycan, the presence of a sulfated polysaccharide chain covalently bound to TM, constituting an acidic domain independent of the protein C activation cofactor site, was suggested. Further enzymatic and chemical characterization showed that rabbit TM was in fact a chondroitin sulfate proteoglycan. Monoclonal antibodies raised against rabbit TM and proteins known for their ability to neutralize the activity of heparin, as well as TM submitted to chondroitinase digestion were used in order to identify the role of the different structural domains of TM. Binding of thrombin to TM at a primary site on the protein part is a prerequisite for all the biological activities of TM. However, while this binding is sufficient for TM to promote the activation of PC by thrombin, the inhibition by TM of thrombin-induced fibrinogen clotting and factor V activation requires the interaction of thrombin at a secondary site with the polysaccharide chain of TM. This interaction with the polysaccharide chain (which carries a highly sulfated trisaccharide at the non-reducing terminus) leads to the inhibition of the procoagulant functions of TM-bound thrombin towards fibrinogen and factor V as well as an increased reactivity of the enzyme with ATIII. These results were rationalized in the functional model proposed for the rabbit TM-proteoglycan. An original aspect of the TM-proteoglycan resides in the fact that the chondroitin sulfate side chain brings new anticoagulant activities, in addition to the PC activation cofactor activity, to the molecule. TM is a new type of proteoglycan with important regulatory function in hemostasis, which anticoagulant properties depend on both the protein core and the polysaccharide chain.
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PMID:[Thrombomodulin: a new proteoglycan. Structure-function relation]. 165 16

The association of thrombin with thrombomodulin, a non-enzymatic endothelial cell surface receptor, alters the substrate specificity of thrombin. Complex formation converts thrombin from a procoagulant to an anticoagulant enzyme. Structure-function analysis of this change in specificity is facilitated by the availability of two soluble proteolytic derivatives of thrombomodulin, one consisting of the six repeated growth factor-like domains of thrombomodulin (GF1-6) and the other containing only the fifth and sixth such domains (GF5-6). Both derivatives can bind to thrombin and block fibrinogen clotting activity, though only the larger GF1-6 can stimulate the activation of protein C. To ascertain whether the substrate specificity change from fibrinogen to protein C is accompanied by structural changes in the active site of the enzyme, fluorescent dyes were positioned at different locations within the active site. A 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) dye was covalently attached to the active site serine to form dansyl-thrombin, while either a fluorescein or an anilinonaphthalene-6-sulfonic acid (ANS) dye was attached covalently to the active site histidine of thrombin via a D-Phe-Pro-Arg linkage. The environment of the dansyl dye was altered in a similar fashion when either GF1-6 or GF5-6 bound to thrombin, since a similar reduction in dansyl emission intensity was elicited by these two thrombomodulin derivatives (25 and 32%, respectively). These spectral changes, and all others in this study, were saturable and reached a maximum when the ratio of thrombomodulin derivative to thrombin was close to 1. The environments of the fluorescein and ANS dyes were also altered when GF1-6 bound to thrombin because binding resulted in emission intensity changes of -13% and +18%, respectively. In contrast, no fluorescence changes were observed when the fluorescein and ANS thrombin derivatives were titrated with GF5-6. Thus, the structure of the active site was altered by thrombomodulin both immediately adjacent to the active site serine and also more than 15 A away from it. However, the structural change far from Ser-195 was only elicited by thrombomodulin species that stimulate thrombin-dependent activation of protein C.
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PMID:The active site of thrombin is altered upon binding to thrombomodulin. Two distinct structural changes are detected by fluorescence, but only one correlates with protein C activation. 166 Apr 64

Thrombomodulin is an endothelial cell surface receptor for thrombin that acts as a physiological anticoagulant. The properties of recombinant human thrombomodulin were studied in COS-7, CHO, CV-1, and K562 cell lines. Thrombomodulin was expressed on the cell surface as shown by the acquisition of thrombin-dependent protein C activation. Like native thrombomodulin, recombinant thrombomodulin contained N-linked oligosaccharides, had Mr approximately 100,000, and was inhibited or immunoprecipitated by anti-thrombomodulin antibodies. Binding studies demonstrated that nonrecombinant thrombomodulin expressed by A549 carcinoma cells and recombinant thrombomodulin expressed by CV-1 and K562 cells had similar Kd's for thrombin of 1.3 nM, 3.3 nM, and 4.7 nM, respectively. The Kd for DIP-thrombin binding to recombinant thrombomodulin on CV-1(18A) cells was identical with that of thrombin. Increasing concentrations of hirudin or fibrinogen progressively inhibited the binding of 125I-DIP-thrombin, while factor Va did not inhibit binding. Three synthetic peptides were tested for ability to inhibit DIP-thrombin binding. Both the hirudin peptide Hir53-64 and the thrombomodulin fifth-EGF-domain peptide Tm426-444 displaced DIP-thrombin from thrombomodulin, but the factor V peptide FacV30-43 which is similar in composition and charge to Hir53-64 showed no binding inhibition. The data exclude the significant formation of a ternary complex consisting of thrombin, thrombomodulin, and hirudin. These studies are consistent with a model in which thrombomodulin, hirudin, and fibrinogen compete for binding to DIP-thrombin at the same site.
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PMID:Equilibrium binding of thrombin to recombinant human thrombomodulin: effect of hirudin, fibrinogen, factor Va, and peptide analogues. 217 73

The present study was undertaken to investigate the role of plasminogen activator inhibitor type 1 (PAI-1) and activated protein C (APC) in the regulation of tumor cell invasion. PAI-1 was purified in active form from conditioned medium of human umbilical vein endothelial cells under denaturing conditions (4 M guanidine-HCl). The purified inhibitor reacts with urokinase-type plasminogen activator (uPA) and APC. Two selected human lines, HOC-I (ovarian cancer cells) and SMT-ccl (choriocarcinoma cells), preferentially invaded through reconstituted basement membranes in an in vitro invasion assay using a modified Boyden chamber. The present study determined the efficacy of these two agents (PAI-1 and APC) used alone or in combination in inhibiting or facilitating tumor cell invasion. Active PAI-1 inhibited the tumor cell surface receptor-bound uPA activity. In an in vitro invasion assay, active PAI-1 reduced tumor cell invasive potential in a dose-dependent manner. When SMT-ccl cells saturated with uPA-PAI-1 complexes were treated with a 50-fold molar excess of APC, PAI-1-APC complex was demonstrated in conditioned medium, indicating that PAI-1 was dissociated from receptor-bound uPA on tumor cells and that tumor cell-associated uPA restored its enzymatic activity. Although APC alone had no effect on tumor cell invasion, the addition of APC to the cells saturated with uPA-PAI-1 complexes showed regeneration of tumor cell surface receptor-bound uPA activity and produced substantial and efficient invading effects. These data suggest that PAI-1 activity may be neutralized by APC or that APC may promote tumor cell invasion via inactivation of PAI-1 by formation of a stable PAI-1-APC complex. These observations suggest that APC may play a critical role in the initiation of a hematogenous metastatic process (extravasation step).
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PMID:Role of activated protein C in facilitating basement membrane invasion by tumor cells. 826 50

The interaction of the T cell surface protein CD28 with its ligand, B7-1 or B7-2, provides a critical costimulatory signal for T cell activation. T cells from CD28- mice are deficient in a variety of responses, including those to lectins and allogeneic spleen cells. However, some immune responses do occur in CD28- mice, suggesting the existence of alternate costimulatory pathways. In this work, we show that T cells purified from CD28- mice respond to B lymphomas expressing 4-1BB ligand (4-1BBL), a member of the TNF gene family. This response is inhibited by a soluble form of 4-1BB, the T cell surface receptor for 4-1BBL. Thus, 4-1BBL/4-1BB interaction provides costimulatory signals to T cells independent of signaling through the CD28 receptor. We find that 4-1BBL is inducible on splenic B cells by CD40 ligand/CD40 interaction or by culturing of splenic dendritic cells, treatments that also induce B7 family molecules. CD28- T cells fail to respond in an MLR to resting allogeneic spleen cells. However, treatment of spleen cells with CD40 ligand renders them competent in activation of CD28- T cells. In contrast to results using B lymphomas as APC, soluble 4-1BB fails to inhibit the T cell response to activated spleen cells. This failure of soluble 4-1BB to block an MLR between CD28+ or CD28- T cells and allogeneic spleen cells is in contrast to a previous report with CD28+ cells.
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PMID:Costimulation of CD28- T lymphocytes by 4-1BB ligand. 899 67

Protein C is an important regulatory mechanism of blood coagulation. Protein C functions as an anticoagulant when converted to the active serine protease form on the endothelial cell surface. Thrombomodulin (TM), an endothelial cell surface receptor specific for thrombin, has been identified as an essential component for protein C activation. Although protein C can be activated directly by the thrombin-TM complex, the conversion is known as a relatively low-affinity reaction. Therefore, protein C activation has been believed to occur only in microcirculation. On the other hand, we have identified and cloned a novel endothelial cell surface receptor (EPCR) that is capable of high-affinity binding of protein C and activated protein C. In this study, we demonstrate the constitutive, endothelial cell-specific expression of EPCR in vivo. Abundant expression was particularly detected in the aorta and large arteries. In vitro cultured, arterial endothelial cells were also found to express abundant EPCR and were capable of promoting significant levels of protein C activation. EPCR was found to greatly accelerate protein C activation by examining functional activity in transfected cell lines expressing EPCR and/or TM. EPCR decreased the dissociation constant and increased the maximum velocity for protein C activation mediated by the thrombin-TM complex. By these mechanisms, EPCR appears to enable significant levels of protein C activation in large vessels. These results suggest that the protein C anticoagulation pathway is important for the regulation of blood coagulation not only in microvessels but also in large vessels.
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PMID:Activation mechanism of anticoagulant protein C in large blood vessels involving the endothelial cell protein C receptor. 952 19

The human ovarian cancer cell line OV-MZ-19, established from a patient with cystadenocarcinoma of the ovary, expressing thrombomodulin (TM), a cell surface receptor for the serine protease thrombin, interacts with monoclonal and polyclonal antibodies having different specificity for TM. These antibodies detect TM antigen by means of flow cytofluorometry, laser scanning microscopy, immunocytochemistry, and ELISA. Therefore a highly sensitive ELISA for TM antigen was established using two different monoclonal antibodies to quantify TM in tissue extracts and biological fluids, e.g. peritoneal malignant ascites. Primary malignant ovarian tumors and metastases of the omentum and intestine contain TM antigen as determined by ELISA but in significantly lower concentrations than benign ovarian tumors (p=0.0056). In contrast, malignant ascitic fluid of patients with advanced ovarian cancer (FIGO IIIc) contain significantly elevated concentrations of soluble TM than benign peritoneal exudates (p=0.0003). Immunoaffinity purified ascites-derived TM efficiently activates protein C. Protein C activation of ascites-derived TM as well as TM expressed by the tumor cells is inhibited by the monoclonal antibodies. TM abrogates the procoagulant activity of thrombin, reduces pericellular thrombin via internalization, accelerates the thrombin-mediated inactivation of pro-uPA, and the EGF domains of TM exhibit mitogenic activity towards fibroblasts and tumor cells. Both, thrombin and pro-uPA play important roles in tumor invasion and metastasis. Therefore, downregulation and/or release of TM into ascitic fluid may play an important role in the malignant behavior of tumor cells.
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PMID: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. 973 90

The endothelial cell surface receptor thrombomodulin (TM) inhibits blood coagulation by forming a complex with thrombin, which then converts protein C into the natural anticoagulant, activated protein C. In mice, a loss of TM function causes embryonic lethality at day 8.5 p.c. (post coitum) before establishment of a functional cardiovascular system. At this developmental stage, TM is expressed in the developing vasculature of the embryo proper, as well as in non-endothelial cells of the early placenta, giant trophoblast and parietal endoderm. Here, we show that reconstitution of TM expression in extraembryonic tissue by aggregation of tetraploid wild-type embryos with TM-null embryonic stem cells rescues TM-null embryos from early lethality. TM-null tetraploid embryos develop normally during midgestation, but encounter a secondary developmental block between days 12.5 and 16.5 p.c. Embryos lacking TM develop lethal consumptive coagulopathy during this period, and no live embryos are retrieved at term. Morphogenesis of embryonic blood vessels and other organs appears normal before E15. These findings demonstrate a dual role of TM in development, and that a loss of TM function disrupts mouse embryogenesis at two different stages. These two functions of TM are exerted in two distinct tissues: expression of TM in non-endothelial extraembryonic tissues is required for proper function of the early placenta, while the absence of TM from embryonic blood vessel endothelium causes lethal consumptive coagulopathy.
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PMID:Tissue-restricted expression of thrombomodulin in the placenta rescues thrombomodulin-deficient mice from early lethality and reveals a secondary developmental block. 1122 38

Thrombomodulin is an endothelial cell surface receptor for thrombin. It plays an important role in the regulation of blood coagulation by decreasing thrombin activity and activating protein C. This study examined the possible association between the thrombomodulin -33G/A polymorphism and acute myocardial infarction. We recruited 278 patients (mean age 57.5 years, 241 men) with documented myocardial infarction and 450 age- and sex-matched control subjects. Polymerase chain reaction and single-strand conformation polymorphism was used to define the thrombomodulin -33G/A polymorphism. The frequency of the thrombomodulin GA+AA genotype among patients with myocardial infarction was higher than that in control subjects (22.7% vs. 16.2%, odds ratio [OR] 1.5, 95% confidence interval [CI] 1.0 to 2.2). The -33G/A polymorphism (GA+AA genotype) was significantly associated with myocardial infarction (OR 1.6, 95% CI 1.1 to 2.5) as was hypertension, diabetes mellitus and smoking. Among young myocardial infarction patients (age < or =45 years, n = 72), the frequency of -33G/A polymorphism was more significantly higher than that in control subjects (29.2% vs. 16.2%, OR 2.1, 95% CI 1.2 to 3.8). The -33G/A polymorphism (OR 2.3, 95% CI 1.3 to 4.1) and smoking (OR 4.5, 95% CI 2.5 to 7.9) were the only independent risk factors for young myocardial infarction. Furthermore, among patients who did not smoke, the polymorphism was associated with a nonsignificant increase in the risk of young myocardial infarction (OR 1.9, 95% CI 0.6 to 5.6); whereas, in the presence of smoking, the increase was statistically significant (OR 2.3, 95% CI 1.2 to 4.7). Smoking carriers of the thrombomodulin -33G/A polymorphism had a nearly 10-fold increased risk of young myocardial infarction (OR 9.8, 95% CI 4.3 to 22.4) when compared with nonsmoking non-carriers. We concluded that there was a significant association between the thrombomodulin -33G/A polymorphism and myocardial infarction in our population, especially in young patients. The clinical effect of this genetic factor was enhanced by smoking.
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PMID:Synergistic effect of thrombomodulin promoter -33G/A polymorphism and smoking on the onset of acute myocardial infarction. 1184 62


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