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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of thrombin, D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone (PPACK)-inhibited thrombin, and thrombin receptor agonist peptide, SFLLRNPNDKYEPF (SFLL, a portion of the receptor unmasked after thrombin cleavage), on the expression of tissue factor (TF) and thrombomodulin by human saphenous vein endothelial cells (HSVECs) in culture were studied. Unstimulated cells contained very low amounts of TF mRNA as measured by the reverse transcriptase-PCR method. Thrombin treatment increased TF mRNA to 8.0 +/- 1.9 (n = 3) times the control level. The increase was detectable within 2 h and declined to near basal level by 6 h. Induction of TF mRNA was not blocked by cycloheximide, treatment with cycloheximide alone also increased TF mRNA levels, and thrombin in combination with cycloheximide further enhanced the accumulation of TF mRNA. Thrombin caused a 14.5 +/- 1.5-fold (n = 5) increase in TF activity on the surface of HSVECs and a 20.5 +/- 1.4-fold (mean +/- S.D., n = 2) increase in the extracellular matrix. The thrombin-induced effects on TF synthesis could be fully reproduced by the thrombin receptor agonist peptide, SFLL, whereas PPACK-inhibited thrombin did not influence TF expression. Thrombin increased thrombomodulin mRNA to 190 +/- 39% (n = 5) of control levels, whereas PPACK-inhibited thrombin or SFLL did not influence thrombomodulin mRNA levels. In contrast, surface-bound thrombomodulin cofactor activity and thrombomodulin antigen in the cell lysates did not change over 24 h of incubation with thrombin. However, thrombin caused a 2-fold increase in thrombomodulin antigen released into the conditioned medium, and immunoelectron microscopy of HSVECs also demonstrated the presence of thrombomodulin vesicles close to the luminal cell surface in thrombin-treated cultures. The Western blot pattern thrombomodulin in the conditioned medium of untreated and thrombin-treated cells was found to be similar, and soluble thrombomodulin occurred mainly as fragments of the cell-associated form. We conclude that the transcriptional control by thrombin causes an increase in both TF and thrombomodulin mRNA. The increase in TF mRNA levels is also paralleled by an increase in surface expression, is dependent on the proteolytic activity of thrombin, and is mediated by the same receptor as the recently cloned thrombin receptor in platelets. Up-regulation of thrombomodulin mRNA levels by thrombin is distinct from this pathway and is associated with unchanged expression on the cell surface.
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PMID:Thrombin regulates tissue factor and thrombomodulin mRNA levels and activities in human saphenous vein endothelial cells by distinct mechanisms. 767

The endothelial cell (EC) urokinase receptor plays an important role in the localization and receptor-mediated activation of EC-bound plasminogen and hence surface-localized fibrinolysis. Thrombin induced a rapid (< 5 minute), time- (0 to 30 minutes) and dose- (0.1 to 8 U/mL) dependent decrease in the specific binding of 125I-labeled two-chain urokinase-type plasminogen activator (tcu-PA) or diisopropylfluoro-phosphate-tcu-PA to urokinase-type plasminogen activator receptor (u-PAR) in cultured ECs from various sources (range, 21% to 50%). The thrombin receptor activation peptide but not control peptide showed a similar but reduced decrease in the specific binding of 125I-labeled tcu-PA to u-PAR. Incubation of thrombin-treated cultures (10 to 12 hours) in complete medium restored 125I-labeled tcu-PA ligand binding to normal levels. u-PAR mRNA levels rapidly (1 hour) increased and peaked 10 to 12 hours after thrombin treatment as analyzed by reverse transcriptase-polymerase chain reaction. Decreased thrombin-induced 125I-labeled tcu-PA binding correlated with the time-dependent decrease in surface-localized plasmin generation, as measured by the direct activation of 125I-labeled Glu-plasminogen and quantification of the 20-kD light chains of 125I-labeled plasmin. After incubation with thrombin, plasmin generation was decreased 50% to 56% (125 to 152 fmol/3 to 3.5 x 10(4) cells). Isolation of metabolically labeled 35S-labeled u-PAR from the media of thrombin and phospholipase C-treated human aortic cultures yielded approximately 10- and approximately 12-fold more 55-kD M(r) and approximately 6-fold more 35-kD M(r) 35S-labeled u-PAR forms than control cultures, respectively. The u-PAR antigen forms (M(r), 54 kD) and the glycosyl-phosphatidylinositol-anchored protein CD59 (M(r), 20 kD) were also simultaneously identified by immunoprecipitation in the media of thrombin-treated cultures. This suggests that thrombin may release u-PAR and decrease u-PA ligand binding through a common pathway involving phospholipase C. These results establish a novel interrelation between thrombin and EC fibrinolysis and suggest that thrombin may also have an additional regulatory role in the net expression of surface-localized EC fibrinolytic activity.
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PMID:Thrombin decreases the urokinase receptor and surface-localized fibrinolysis in cultured endothelial cells. 774 51

We addressed the balance between thrombin and its serpin protease nexin I (PNI) after sciatic nerve injury in the mouse. Prothrombin levels increased twofold 24 h after nerve crush, as measured by a specific chromogenic assay, and peaked at day 3. Thrombin activity also increased 2-4 days after injury in distal sciatic nerve segments. Nerve RNA analysis using reverse transcriptase--polymerase chain reaction (RT-PCR) assay confirmed that prothrombin was synthesized locally. We also monitored PNI levels in these injured nerve samples by complex formation with an 125I-labeled target protease and found peak activity occurring later, 6-9 days after the thrombin induction. These data indicate that nerve injury first induces the synthesis of prothrombin, which is subsequently converted to active thrombin. Nerve crush-induced thrombin is followed by the generation of functionally active PNI and may be directly responsible for its induction. By immunocytochemistry with anti-PNI antibody, we found that activated Schwann cells were the source of induced PNI. These results support the concept that the balance between serine proteases and their serpins is dysregulated during nerve injury and suggests a role for its reestablishment in nerve damage repair.
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PMID:Neural thrombin and protease nexin I kinetics after murine peripheral nerve injury. 886 30

Thrombin's potent effects on astrocytes are mediated by a specific receptor and inhibited by a serpin, protease nexin I (PNI). Thrombomodulin (TM), a membrane protein that forms complexes with thrombin, changing its enzymatic specificity, has not been studied in astrocytes. In primary astrocyte cultures, using Western blotting and immunocytochemistry, we found a 70 kDa TM band and TM localized to the surface with an anti-mouse TM monoclonal antibody. By reverse transcriptase coupled with polymerase chain reaction (RT-PCR), we found the correct sequence for mouse TM mRNA in astrocytes. Finally, we documented calcium-dependent activation of protein C by a thrombin:TM complex with thrombin added to the astrocytes. These results indicate the presence of functionally active TM at the astrocyte surface and add support to a role for thrombin signaling in the nervous system.
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PMID:Novel expression and localization of active thrombomodulin on the surface of mouse brain astrocytes. 906 32

The effect of methylmercury (MeHg; CH3HgCl) on the gene expression of monocyte chemotactic protein-1 (MCP-1) by human peripheral blood mononuclear cells (PBMC) was examined. PBMC were exposed with or without thrombin (1 U/ml) or MeHg (0.3 or 3.0 microM) for 24 hours. The total RNA was reverse transcribed and then amplified by the method of reverse transcriptase-polymerase chain reaction (RT-PCR). Thrombin enhanced MCP-1 mRNA expression in PBMC. MeHg inhibited thrombin-stimulated MCP-1 mRNA expression in a dose dependent manner. These findings suggest that MeHg affects the atherosclerotic process by changing MCP-1 mRNA expression in PBMC.
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PMID:Methylmercury modulation of monocyte chemotactic protein-1 mRNA expression in human peripheral blood mononuclear cells. 918 71

Thrombin acts on cells through the surface protease-activated receptor 1 (PAR-1), a G-protein-coupled member of the seven-transmembrane domain superfamily. On neural cells, thrombin has deleterious effects, killing neurons through apoptosis. Consequently, knowledge of PAR-1 expression in the nervous system may help to elucidate the role of thrombin in neurodegenerative disease. We developed a mimic construct to facilitate the highly sensitive technique of quantitative reverse transcriptase to PCR (qRT-PCR) to measure the differential expression of low copy number PAR-1 mRNA in neurodegenerative model systems. In this article, we report our results comparing homozygous wobbler (wr/wr) mice and normal littermates. By optimizing the transcription and quantitative PCR procedures to facilitate rapid copy number determination in small RNA samples, we documented a fivefold greater level of PAR-1 mRNA in the cervical spinal cord of wr/wr.
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PMID:Quantitative reverse transcriptase PCR to gauge increased protease-activated receptor 1 (PAR-1) mRNA copy numbers in the Wobbler mutant mouse. 969 52

Thrombin-treated tumor cells induce a metastatic phenotype in experimental pulmonary murine metastasis. Thrombin binds to a unique protease-activated receptor (PAR-1) that requires N-terminal proteolytic cleavage for activation by its tethered end. A 14-mer thrombin receptor activation peptide (TRAP) of the tethered end induces the same cellular changes as thrombin. Four murine tumor cells (Lewis lung, CT26 colon CA, B16F10 melanoma, and CCL163 fibroblasts) contain PAR-1, as detected by reverse transcriptase-polymerase chain reaction (RT-PCR). B16F10 cells did not contain the two other thrombin receptors, PAR-3 and glycoprotein Ib. TRAP-treated B16F10 tumor cells enhance pulmonary metastasis 41- to 48-fold (n = 17). Thrombin-treated B16F10 cells transfected with full-length murine PAR-1 sense cDNA (S6, S7, S14, and S22) enhanced their adhesion to fibronectin 1.5- to 2.4-fold (n = 5, P <.04), whereas thrombin-treated wild-type cells do not. S6 (adhesion index, 1.5-fold) and S14 (index, 2.4-fold) when examined by RT-PCR and Northern analysis showed minimal expression of PAR-1 for S6 over wild-type and considerable expression for S14. Immunohistochemistry showed greater expression of PAR-1 for S14 compared with wild-type or empty-plasmid transfected cells. In vivo experiments with the thrombin-treated S14 transfectant showed a fivefold to sixfold increase in metastases compared with empty-plasmid transfected thrombin-treated naive cells or S6 cells (n = 20, P =.0001 to .02). Antisense had no effect on thrombin-stimulated tumor mass. Thus, PAR-1 ligation and expression enhances and regulates tumor metastasis.
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PMID:Protease-activated receptor 1 (PAR-1) is required and rate-limiting for thrombin-enhanced experimental pulmonary metastasis. 980 63

Both thrombin and plasmin induce contraction of brain endothelial cells, which may increase capillary permeability thereby leading to disruption of the blood-brain barrier. Identification of thrombin receptors, as well as the influence of plasmin on their activation, in capillary endothelial cells and astrocytes are therefore essential for understanding injury-related actions of thrombin in the brain. Using the reverse transcriptase-polymerase chain reaction method, the present study shows that primary cultures of rat brain capillary endothelial (RBCE) cells and astrocytes derived from rat brain express two different thrombin receptors. The first is proteolytically activated receptor (PAR)-1, the receptor responsible for the vast majority of the thrombin's cellular activation functions; the second is PAR-3, a receptor described to be essential for normal responsiveness to thrombin in mouse platelets. In addition to these thrombin receptors, the mRNA (messenger RNA) for PAR-2, a possible trypsin receptor, was also identified. Functional significance of thrombin receptors was indicated by changes in [Ca2+]i in response to thrombin, as measured by FURA-2 fluorescence in RBCE cells. Thrombin as low as 4 nmol/L induced an abrupt increase in [Ca2+]i whereas, upon addition of active site-blocked thrombin or plasmin, [Ca2+]i remained unchanged. The [Ca2+]i signal attributable to thrombin was smaller in a low Ca2+-containing medium, indicating that an influx of Ca2+ from the extracellular medium makes a contribution to the overall [Ca2+]i rise. The amplitude of the transient [Ca2+]i signal was dependent on the concentration of thrombin, and repeated application of the enzyme caused an essentially complete and long-term desensitization of the receptor. The PAR-1 agonist peptide SFLLRN also elicited a transient increase in [Ca2+]i. After activation by SFLLRN, cells showed a diminished response to thrombin, but the response was not absent, indicating that PAR-3 might contribute to the generation of the [Ca2+]i signal. Pretreatment of RBCE cells with 100 nmol/L plasmin completely prevented [Ca2+]i rise attributable to thrombin. These data show that RBCE cells and astrocytes express at least two receptors for thrombin, PAR-1 and PAR-3, and probably both receptors are involved in thrombin-induced [Ca2+]i signals. Plasmin itself does not elevate [Ca2+]i but prevents the activation of receptors by thrombin.
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PMID:Identification of thrombin receptors in rat brain capillary endothelial cells. 1061 6

Thrombin appears to underlie myometrial contractions in response to intrauterine bleeding. In a similar fashion, thrombin generated within the uterus in the absence of active bleeding could also produce contractions. These studies sought to determine whether functionally active prothrombin is expressed in the pregnant and nonpregnant rat uterus. Uteri were obtained from proestrus/estrus and timed-pregnant Sprague-Dawley rats. Western blots were performed using antithrombin antibodies. Immunohistochemical studies were performed using the same antibodies along with the Vector Elite ABC kit. Qualitative reverse transcriptase-polymerase chain reaction studies were performed using rat prothrombin-specific oligonucleotide primers. In vitro uterine contraction studies were performed using Taipan snake venom (an exogenous prothrombinase) and components of the plasma prothrombinase complex (Factors Xa and V) with and without pretreatment with thrombin inhibitors (heparin or hirudin). The Western blots demonstrated prothrombin peptides in myometrial tissue from estrus and pregnant rats. The immunohistochemical studies confirmed prothrombin peptides in both the circular and longitudinal myometrium, along with the endometrium. The reverse transcriptase-polymerase chain reaction studies demonstrated prothrombin mRNA in the endometrium and placenta, but not in the myometrial smooth muscle. The Taipan snake venom stimulated a significant increase in contractions, which were suppressed by pretreatment with heparin and hirudin. The Factor Xa and V complex also significantly stimulated uterine contractions, which were likewise inhibited by hirudin. These studies provide evidence supporting the expression of functionally active prothrombin in the pregnant and nonpregnant rat uterus. Based on the presence of its mRNA, prothrombin appears to be synthesized in the endometrium and placenta; in contrast, the myometrial smooth-muscle cells appear to sequester preformed prothrombin. These results support the hypothesis that intrauterine thrombin could play an autocrine/paracrine role in the regulation of contractile activity.
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PMID:Intrauterine expression of prothrombin in the sprague-dawley rat. 1238 11

Fibrin deposition in the peritubular capillaries and along the tubular basement membrane is commonly observed in several renal diseases and suggests the involvement of blood coagulation in tubulointerstitial damage. It has been demonstrated that tissue factor (TF) is present in tubular epithelial cells of animal models of nephritis. Tissue factor pathway inhibitor (TFPI) regulates the extrinsic pathway of blood coagulation through its ability to inhibit TF activity and it is now thought to be produced mainly by the vascular endothelial cells. We examined whether human proximal tubular epithelial cells (PTEC) could produce TFPI and attempted to clarify the regulatory factors affecting TFPI production. Cultured human PTEC were used. The procoagulant activity (PCA) in PTEC lysate was quantified by measurement of the one-stage recalcification time. TFPI in the cell supernatants was measured by ELISA. The mRNA of TF and TFPI in PTEC was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). PCA which is compatible with TF activity was present in the PTEC lysate. TF mRNA and TFPI mRNA were detected in PTEC. The amount of TFPI increased over time in the cell supernatants. Immnoblot analysis revealed 40 kD protein of TFPI, and TFPI antigen was demonstrated in PTEC by immunofluorescence. The concentration of TFPI was significantly increased following incubation with thrombin and heparin in a dose- and time-dependent manner, although the amount of TFPI mRNA was not changed. Our study showed that TFPI is produced in cultured PTEC and added one more cell type that produced TFPI other than endothelial cells. Thrombin and heparin stimulated TFPI secretion from PTEC. TFPI of PTEC may act against generation of thrombin and tubular fibrin formation induced by tissue factor activation. The augmentation of TFPI secretion by heparin may play an important role in the modulation of anticoagulant properties of PTEC.
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PMID:Tissue factor pathway inhibitor production by human proximal tubular epithelial cells in culture. 1289 29


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