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)

Lysophosphatidylcholine (LPC) increases extracellularly during ischemia in vivo in both animals and man as judged by measurements from venous effluents, but more recent studies have shown little or no increase in buffer-perfused, isolated heart preparations. The appearance of LPC in blood and lymph in animals and in venous effluents in man in response to ischemia suggests a vascular site for the production of LPC. The present study was performed to assess whether thrombin could stimulate phospholipase A2 in endothelial cells and whether this would evoke an increase in and release of LPC. Endothelial cells were disassociated from canine aortas by incubating with 0.1% collagenase for 20 min. Cells were plated and allowed to grow to confluence. Measurement of LPC was performed using Bligh and Dyer extraction of lipids, high performance liquid chromatography separation, and quantification of LPC using a recently developed radiometric assay employing [3H]acetic anhydride. Incubation of endothelial cells with thrombin (0.05 unit/ml) resulted in a 2.5-fold increase in LPC to 2.3 +/- 0.1 nmol/mg of protein at 2 min (p < 0.01) and returned to control levels within 20 min. The increase in LPC induced by thrombin exhibited a concentration-dependent response with an ED50 = 0.04 unit/ml. A concentration-dependent increase in LPC was also elicited by stimulation with the peptide portion of the thrombin receptor's tethered ligand SFLLRNPNDKYEPF with an ED50 = 8 microM. The LPC produced was rapidly and completely released into the surrounding media. Hirudin completely blocked the thrombin-induced increase in LPC. Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (0.1 microM), which rapidly inactivates thrombin's proteolytic activity in situ without impairing binding, or phenyl-prolyl-arginyl-chloromethyl ketone (PPACK, 5 nM), which inactivates thrombin due to chemical alteration of the proteolytic site, each prevented the increase in LPC in response to thrombin. Stimulation of protein kinase C with phorbol 12-myristate-13-acetate (PMA, 1 microM) enhanced the response to thrombin. In contrast, staurosporine (100 nM), H7 (15 microM), or chronic treatment with PMA for 20 h to down-regulate protein kinase C completely prevented the increase in LPC in response to thrombin. Thus, thrombin stimulation of endothelial cells in vivo during ischemia may be a primary mechanism contributing to the marked increase in LPC extracellularly during ischemia.
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PMID:Thrombin-induced release of lysophosphatidylcholine from endothelial cells. 839 49

Newborn rat brain astrocytes cultured in vitro in a chemically defined medium are shown to secrete enhanced levels of nerve growth factor (NGF) when they are exposed to various types of proteases. Proteolytic enzymes such as alpha-thrombin or collagenase induce a continuous, dose-dependent enhancement of the levels of cell-secreted NGF. Incubation of astrocytes for a 24-h period with 300 ng/ml of alpha-thrombin (approximately 9 nM, or 1 U/ml) results in an increase of the levels of cell-secreted NGF by a factor of three- to fourfold, and at doses 10 times higher, stimulation by a factor of up to four- to fivefold was observed. This phenomenon reflects an enhancement of the cellular pool of NGF mRNA, already noticeable after 3 h of treatment, which is preceded by a temporary activation of protooncogenes encoding transcription factors of the AP-1 family, such as c-fos, c-jun or junB. Trypsin, plasmin, alpha-chymotrypsin, or elastase also enhanced, to different extents, the levels of cell-secreted NGF. However, unlike alpha-thrombin or collagenase, these enzymes cause, above a critical concentration, an extensive cell detachment from the solid support, and this is accompanied by a decrease of their activity on the production of NGF, so that their dose-response curves are bell shaped. Stimulation was maximal at those concentrations that cause a limited loosening of the cell-substratum interactions, as evidenced by a retraction of some cell processes after 24 h of treatment. Studies of the effect of alpha-thrombin indicate that the proteolytic activity itself is required to enhance the production of NGF by astrocytes. Inactivation of alpha-thrombin with D-phenyl-alanyl-L-propyl-L-arginine chloromethyl ketone, phenylmethylsulfonyl fluoride, antithrombin III, or hirudin results in a marked decrease of the stimulatory effect. Furthermore, the prolonged presence of alpha-thrombin is required to elicit a maximal effect on the levels of extracellular NGF, which was observed after 48 h of treatment. It is known that some effects of alpha-thrombin require binding to the cell surface. We found that gamma-thrombin, which still has some proteolytic activity but has lost its ability to bind to the cell surface, is almost as potent as alpha-thrombin in promoting the release of NGF. It is concluded that the effect of thrombin on NGF synthesis is essentially mediated by its proteolytic activity.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Enhancement of the synthesis and secretion of nerve growth factor in primary cultures of glial cells by proteases: a possible involvement of thrombin. 843 76

Basic investigation of inhibitory effect on metastasis of nafamostat mesilate (FUT-175) which is a kind of serine protease inhibitors, was performed. Colon 26 cells were injected to the portal vein of CDF1 mice. FUT-175 at doses of 0.3, 1.0, 3.0, 10.0 mg/kg was injected intravenously every 7 day. Mice were sacrificed on day 21, and metastasis of liver surface were measured. The dose dependent reduction of metastasis was observed and reduction of metastasis of mice treated at a dose of 10.0 mg/kg was significant. FUT-175 showed no cytotoxicity at doses of 10(-5) M or less in vitro, and blood concentration of mice, treated at a dose of 10.0 mg/kg, was 2.67 x 10(-7) M. The results showed that inhibitory effect of FUT-175 on metastasis was not caused by direct cytotoxicity. FUT-175 at 2.67 x 10(-7) M in vitro can inhibit only thrombin and plasmin at nearly 50%, and can not inhibit platelet aggregation and collagenase directly. Possible mechanism of inhibition of metastasis is that FUT-175 inhibited both thrombin-mediated platelet aggregation and plasmin-mediated collagenase activation, that arrest and extravasation in cancer cells were inhibited. If protease inhibitor is administered continuously and immediately after surgery, liver metastasis may be prevented.
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PMID:[The inhibitory effect of nafamostat mesilate (FUT-175) on liver metastasis]. 843 54

The purpose of this study was to compare coronary and interstitial endothelin-1 (ET-1) levels in perfused rat hearts under several experimental conditions, because the cardiac tissue concentration of ET-1 is not clear. Hearts were perfused in an upside-down position with a colloid-free buffer at a constant flow rate of 9 ml/min/g heart wet weight, and immunoreactive ET-1 was determined in timed collections of coronary effluent and interstitial transudate produced by the ventricles and appearing on their surface. Basal ET-1 release into effluent was 0.26 +/- 0.007 pg/min/g, and 0.005 +/- 0.0012 pg/min/g in transudate. Basal ET-1 concentration was 0.11 +/- 0.005 pg/ml (transudate) and 0.03 +/- 0.002 pg/ml (effluent), indicating four-fold higher transudate than effluent levels (P < 0.05). Following perfusion of hearts with collagenase to remove endothelial cells, ET-1 release into effluent was reduced to one-third and completely abolished in transudate, indicating that the peptide originated from the vascular endothelium. Perfusion of hearts with angiotensin II (0.1 mumol/l) or thrombin (5 U/ml) increased coronary perfusion pressure and ET-1 secretion, but little affected the transudate/effluent ET-1 concentration ratio (5.5 and 3.2, respectively). When coronary flow was reduced to ischaemic level (1 ml/min/g over several hours), ET-1 secretion rates into effluent were decreased by 55-65%, but increased three- to four-fold on reperfusion at normal flow (P < 0.05). The ET-1 concentrations in both fluids were still always below 1 pg/ml. No change in coronary perfusion pressure compared to time-matched normoxic controls was observed. In the presence of the ET-1 converting enzyme inhibitor, phosphoramidon (1.7 mumol/l), ischaemia-induced increases of ET-1 secretion were attenuated, and this was accompanied by a time-dependent rise in coronary perfusion pressure up to 60% (P < 0.05). These are the first measurements of endogenous cardiac tissue ET-1 levels; they do not support a vasoconstrictor (pro-ischaemic) action of endogenous ET-1 in rat hearts following ischaemia/reperfusion, but rather point to a possible vasodilator role of the peptide under these conditions.
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PMID:Tissue endothelin-1 levels in perfused rat heart following stimulation with agonists and in ischaemia and reperfusion. 852 55

The aim of this study was to detect biologic factors in the structural deterioration of bioprosthetic heart valves. Prostheses were removed from patients after 4-8 years of implantation and submitted to biochemical and morphologic assays. Successive staining of biologic sections revealed colocalization of lipids and glycosaminoglycans underneath calcifications in the disintegrated extracellular matrix. On biochemical assays, the amidolysis of synthetic peptide substrates indicated thrombin, plasmin, and tissue plasminogen activator activities in the nonhemocompatible leaflets; 0.15 mol NaCl, 0.05 mol Tris, and 5 mmol CaCl2 extracts from the prostheses cleaved the peptide substrate for collagenase and lysed gelatin gels. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate disclosed the presence of low molecular mass polypeptides in extracts of the deteriorated prostheses. The detection of plasmin and collagenolytic enzyme(s), and the known broad proteolytic activity of plasmin, may point to the role of activation of the fibrinolytic system in the proteolytic degradation of bioprosthetic valves.
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PMID:Deterioration of bioprosthetic heart valves. 855 4

The tripeptide compounds, Glu-Arg-Pro-amide (ERPm), D-Pro-Thr-Trp-amide (dPTWm) and thioproline-Thr-Trp (tPTW), were obtained by screening of synthetic peptides for growth-inhibitory activity toward cultured transformed cells. The effects of these peptide compounds on proteases were investigated and the results showed that these compounds enhanced the amidolytic activity of serine proteases despite the fact that each reaction was carried out under optimal conditions. ERPm stimulated the activities of trypsin, chymotrypsin, thrombin, plasmin urokinase and elastase. dPTWm also showed similar effects except that toward chymotrypsin. tPTW elevated the activity only of trypsin, chymotrypsin and thrombin. Stimulation of trypsin activity by these compounds was also confirmed by using casein as a substrate. None of these compounds affected the amidolytic activities of metalloproteinases (MMP-1 and MMP-9), cysteine proteinases (m- and mu-calpains, cathepsin B and papain) or an exopeptidase (leucine aminopeptidase). The activation was at least partly due to the stabilization of the catalytic activity of proteases as well as prevention of autolysis.
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PMID:Enhancement of catalytic activities of serine proteases by tripeptides compounds. 863 1

The effect of methylmercury (CH3HgCl) on the production of endothelium-derived relaxing factor (EDRF) by cultured human umbilical vascular endothelial cells (HUVECs) based on its anti-aggregatory effect on human platelets was examined. HUVECs were harvested from umbilical veins by collagenase treatment. The platelet aggregation test was performed with cuvettes lined with HUVECs. Platelet aggregation induced by 0.05 units thrombin/ml was inhibited in the presence of HUVECs. This HUVEC-dependent anti-platelet aggregatory effect was enhanced by the addition of bradykinin (10 nmol/L), which stimulates the production of EDRF. Indomethacin (IND, 1 mumol/L) reduced the HUVEC-dependent anti-platelet aggregatory effect. The effect of NG-monomethyl-L-arginine L-NMMA, 100 mumol/L), an inhibitor of nitric oxide synthase (NOS) in endothelial cells, on HUVECs pretreated with IND showed almost complete platelet aggregation similar to results without HUVECs. The anti-platelet aggregatory effect of HUVECs pretreated with IND seemed to depend mainly on EDRF. Methylmercury (MeHg) (20-50 mumol/L) induced dose-dependent platelet aggregation in cuvettes, without HUVECs. Methylmercury (30 mumol/L) induced less platelet aggregation in the presence of HUVECs than in their absence. The degree of inhibitory effect by HUVECs on MeHg-induced platelet aggregation was reduced dose-dependently (30-50 mumol/L MeHg). Methylmercury-induced platelet aggregation at 50 mumol/L MeHg with or without HUVECs was similar. These findings suggest that this simple new experimental system is useful for assessing the production of EDRF by HUVECs, and show that MeHg inhibits the production of EDRF by HUVECs, which may be involved in the etiology of cardiovascular diseases such as hypertension and arteriosclerosis.
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PMID:The effect of methylmercury (CH3HgCl) on the production of endothelium-derived relaxing factor (EDRF) by cultured human umbilical vascular endothelial cells based on its anti-aggregatory effect on human platelets. 878 7

Prolonged bleeding by the host after the leech ceases to feed and several reports that the use of leeches restores blood flow in the microcirculation after plastic surgery led us to search for inhibitors of platelet aggregation in Hirudo medicinalis saliva. Dilute leech saliva was collected by phagostimulating starved leeches with a solution of arginine in saline. The saliva is shown to inhibit human platelet aggregation induced by thrombin, collagen, adenosine diphosphate (ADP), epinephrine, platelet activating factor (1-O-alkyl-2-acetyl-sn-3-glycerophophoryl choline [PAF]), and arachidonic acid. We have isolated the PAF inhibitor and found it to be an amphipathic phosphoglyceride. We have also purified apyrase adenosine triphosphate ([ATP] diphosphohydrolase), which inhibits ADP-induced platelet aggregation, and have described collagenase. Besides well-known hirudin, Hirudo saliva contains a potent inhibitor of coagulation factor Xa. We also report antiaggregant and anticoagulant activities in the crop content of the closely related Nile leech, Limnatis nilotica. Anticoagulants of hematophagous species are surveyed. We have used medicinal leeches in plastic surgery for decompression of skin flaps and in patients with postphlebitic syndrome and peripheral arterial occlusions. Preliminary results indicate certain beneficial effects of leech therapy.
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PMID:Platelet aggregation and coagulation inhibitors in leech saliva and their roles in leech therapy. 883 13

Surface coverage with autogeneous endothelial cells is effective in reducing thrombogenicity of an artificial vascular graft, but procedure for obtaining the cells is invasive for patients. The purpose of this study was to establish cultures of human endothelial cells separated from a small piece of subcutaneous fat tissue. A piece of tissue weighing about 10 mg was obtained from subcutaneous fat using a biopsy needle, and treated with collagenase and dispase. Microvascular endothelial cells were selected and other types of cells contaminating the cultures were eliminated by scraping with a needle under a microscope. The yield of the cells was 8362 +/- 4264/10 mg of subcutaneous fat (n = 7). The cultures reached confluence in about 2 weeks. The cells were positive for von Willebrand factor, P-selectin, and uptake of acetylated low density lipoprotein. The cells produced 15.9 +/- 3.3 ng/mg cell protein/h of 6-ketoprostaglandin F1 alpha (n = 5) when stimulated with thrombin. Thrombin also stimulated the production of platelet-activating factor: 7653 +/- 4297 dpm/10(6) cells (n = 5). Endothelin-1 accumulation in the medium of unstimulated endothelial cells was 0.54 +/- 0.16 ng/mg cell protein/10 h (n = 8). As a preliminary experiment for graft seeding, the cells were also cultured on pieces of a gelatin-coated Dacron graft, and scanning electron microscopy revealed the surface coverage of the graft. We herein described about successful culture of human microvascular endothelial cells from subcutaneous fat tissue obtained using a biopsy needle. The cultured cells may be applicable to a seeded vascular graft.
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PMID:Surface coverage of vascular grafts with cultured human endothelial cells from subcutaneous fat tissue obtained with a biopsy needle. 890 4

Beyond their critical role in thrombosis, platelets perform important functions in vascular remodeling, inflammation, and wound repair. Many of these functions are executed by molecules expressed by activated platelets. A novel molecule, activated-platelet protein-1 (APP-1), was identified by a monoclonal antibody against activated rabbit platelets. When platelets were stimulated by thrombin, A23187 or ADP, APP-I was expressed on the platelet surface. APP-1 was also detected in whole cell lysates of platelets, but not on the external surfaces of resting platelets. With maximal activation by thrombin, 15 900 +/- 2800 molecules APP-1 were expressed/platelet. A 2.3-kb cDNA fragment containing a partial coding sequence for APP-1 was isolated from a rabbit bone marrow library by expression cloning with the anti-APP-1 monoclonal antibody. When expressed as a recombinant fusion protein in bacteria, APP-1 bound specifically to poly(A)-Sepharose. The full-length cDNA coding for human APP-1, obtained by DNA hybridization techniques, showed 98.7% amino acid sequence identity with the rabbit protein. Northern analysis with human APP-1 identified a 3.7-kb mRNA transcript in megakaryocytic lines that express transcripts for platelet proteins. Human APP-1 has four ribonucleotide binding domains with ribonucleoprotein 1 and 2 motifs. By virtue of its ribonucleotide binding domains, APP-1 is structurally related to polyadenylate-binding protein, which regulates translation initiation and polyadenylate shortening, and to nucleolysin, a specific effector molecule found in the granules of cytotoxic T lymphocytes.
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PMID:Identification and structure of activated-platelet protein-1, a protein with RNA-binding domain motifs that is expressed by activated platelets. 903 Jul 41


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