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)

The binding of type 1 plasminogen activator inhibitor (PAI-1) to the extracellular matrix (ECM) of cultured bovine aortic endothelial cells was investigated using purified 125I-labeled or L-[35S]methionine-labeled PAI-1 as probes. Little specific binding of latent PAI-1 to ECM previously depleted of endogenous PAI-1 could be demonstrated. In contrast, the guanidine-activated form of PAI-1 bound to ECM in a dose- and time-dependent manner, and binding was saturable. The dissociation constant (Kd) for this interaction was estimated to be 60 nM by Scatchard analysis, and approximately 6 pmol of activated PAI-1 was bound per cm2 of ECM. Binding was relatively specific since unlabeled, activated PAI-1 competed with 35S-labeled PAI-1 for binding to ECM, but latent PAI-1 did not. Moreover, PAI-2, protein C inhibitor (i.e. PAI-3), protease nexin-1, and alpha 2-antiplasmin were not able to compete. Tissue-type plasminogen activator (tPA) also inhibited binding, but diisopropyl fluorophosphate-inactivated tPA did not. Pretreatment of ECM with tPA, urokinase-type PA, or thrombin had no effect on its ability to subsequently bind PAI-1, whereas trypsin, plasmin, and elastase pretreatment greatly reduced its ability to bind PAI-1. Guanidine-activated, radiolabeled PAI-1 resembled active endogenous PAI-1 since it was unstable in solution but stable when bound to ECM. In addition, it formed complexes with tPA that had a relatively low affinity for ECM. These data suggest that ECM of bovine aortic endothelial cells contains a protease-sensitive structure that binds active PAI-1 tightly and relatively selectively and that this association stabilizes PAI-1 against the spontaneous loss of activity that occurs in solution.
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PMID:Binding of type 1 plasminogen activator inhibitor to the extracellular matrix of cultured bovine endothelial cells. 249 80

The techniques we developed to propagate HTM cells in serial cell culture have provided an opportunity to investigate the spectrum of endogenous PGs and other eicosanoids that are produced by these cells. PGE2 and PGF2 alpha were the major cyclooxygenase products detected by both radioimmunoassay and thin-layer chromatography. A small amount of 6-keto PGF1 alpha was also detected, indicating that these cells are able to produce prostacyclin. The observation of a substantial increase in the proportion of PGF2 alpha relative to PGE2 at later time periods after a media change suggests a metabolic conversion of PGE2 to PGF2 alpha by these cells. Bradykinin, thrombin, platelet activating factor, and serum were found to be effective stimulators of PG production by HTM cells, whereas calcium ionophore produced only a minor effect. Using high pressure liquid chromatography, elution profiles of radiolabeled metabolites of AA suggested the presence of certain lipoxygenase products, including LTB4, 12-HETE, 15-HETE, and a small amount of 5-HETE in HTM cells. The formation of these products was inhibited by both DEX and BW 755c, reinforcing the view that metabolic conversions of AA through the lipoxygenase pathway were possible in the trabecular meshwork. We also examined the effects of glucocorticoids on specific protein synthesis in the HTM cells, using 35S-methionine labeling and SDS-PAGE techniques. Short-term (1 day) DEX treatment revealed a major induction of a protein band at approximately 30 kDa. Longer treatments (1 to 3 weeks) resulted in major inductions at approximately 55 kDa inside the cells, with the presence of secreted forms (probably glycoproteins) between 55 and 72 kDa. The short-term DEX effect on protein synthesis a phospholipase inhibitor regulating eicosanoid production within the HTM. The longer-term induction may, on the other hand, be related more directly to the development of steroid glaucoma, based on our findings that the inductions of these proteins correlate with the observed time course and dose-dependence topical glucocorticoid effects on IOP. Continued in vitro and in vivo evaluations of the eicosanoid pathways in cultured HTM cells obtained from normal and glaucomatous human eyes may help to delineate their relationship to IOP regulation and the pathogenesis and treatment of glaucoma. Glucocorticoid-induced proteins may be key participants in the regulation of phospholipase activity and hence may represent a major control mechanism of the AA cascade.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Eicosanoid production and glucocorticoid regulatory mechanisms in cultured human trabecular meshwork cells. 250 21

The effect of ONO-3307 (4-sulfamoyl phenyl-4-guanidinobenzoate methanesulfonate), a new protease inhibitor, was studied on various proteases in vitro and in an experimental thrombosis model in vivo. ONO-3307 competitively inhibited trypsin, thrombin, plasma kallikrein, plasmin, pancreatic kallikrein and chymotrypsin; and their Ki values were 0.048 microM, 0.18 microM, 0.29 microM, 0.31 microM, 3.6 microM and 47 microM, respectively. In addition, ONO-3307 inhibited both elastase release from N-formyl-Met-Leu-Phe (fMLP)-stimulated leukocytes and tissue thromboplastin release from endotoxin-stimulated leukocytes. To examine the effects of ONO-3307 on disseminated intravascular coagulation (DIC), we developed an experimental thrombosis model. ONO-3307 (10 mg/kg/hr) completely inhibited the deposition of radioactive fibrin in kidney and lung. Gabexate mesilate (50 mg/kg/hr) was also effective in this model, but the effect of nafamostat mesilate was unclear. These results indicate that ONO-3307 exhibits a wide range of inhibitory effects on various proteases, and ONO-3307 may be useful for the treatment of protease-mediated diseases such as thrombosis and DIC.
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PMID:Inhibitory effects of ONO-3307 on various proteases and tissue thromboplastin in vitro and on experimental thrombosis in vivo. 251 29

Glucocorticoids have been shown to decrease prostaglandin I2 synthesis in human endothelial cells, suggesting the possible involvement of lipocortin in the inhibition of arachidonic acid liberation achieved by phospholipase A2 (De Caterina, R., and Weksler, B. B. (1986) Thromb. Haemostasis 55, 369-374). To test this hypothesis, human endothelial cells labeled with [14C]arachidonic acid were stimulated with thrombin (2 units/ml, 10 min), resulting in the secretion of free arachidonic acid together with various 14C-labeled metabolites, mainly 6-keto-prostaglandin F1 alpha, the stable derivative of prostaglandin I2. Under conditions where prior incubation of cells with dexamethasone reduced by 51% 6-keto-prostaglandin F1 alpha production, phospholipid hydrolysis induced by thrombin remained unaffected. Using three rabbit polyclonal antibodies directed against endonexin I, lipocortin I, and lipocortin II, evidence was obtained for the presence in human endothelial cells of equivalent amounts of lipocortin I and an immunologically unrelated 33-kDa protein, together with lower quantities of 67-kDa calelectrin/calcimedin. These Ca2+- and phospholipid-binding proteins were selectively extracted with [ethylene-bis(oxyethylene-nitrilo)]tetraacetic acid (EGTA) from cell membranes precipitated in the presence of Ca2+, and they displayed an inhibitory activity against pig pancreas phospholipase A2. However, the amounts of the three proteins were not changed by cell treatment with 2.5 microM dexamethasone, as detected upon polyacrylamide gel electrophoresis by silver staining, immunoblotting, or autoradiography following [35S]methionine in vivo labeling. Since the antiphospholipase A2 activity of EGTA extracts was hardly modified, it was concluded that an increased synthesis of lipocortin cannot account for the inhibition of prostaglandin synthesis brought about by dexamethasone, suggesting other biological functions for these proteins.
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PMID:Effect of dexamethasone on prostaglandin synthesis and on lipocortin status in human endothelial cells. Inhibition of prostaglandin I2 synthesis occurring without alteration of arachidonic acid liberation and of lipocortin synthesis. 252 36

When rat neutrophils were stimulated with chemotactic peptide N-formyl-Met-Leu-Phe; fMLP), phorbol myristate acetate (PMA), or immune complexes in the presence of homologous platelets, O2- responses were enhanced. Secretion products of thrombin-stimulated platelets as well as ATP, ATP gamma S, or ADP enhanced O2- responses of fMLP-stimulated neutrophils, although these nucleotides did not, by themselves, initiate an O2- response. Calcium transients were measured in neutrophils which were stimulated with fMLP under a variety of conditions (+/- ATP gamma S, +/- cytochalasin B) which enhance O2- responses. Neutrophils incubated with ATP gamma S alone developed brief calcium transients similar to those produced by fMLP. As compared to changes in intracellular calcium in fMLP-stimulated neutrophils, the copresence of ATP gamma S with fMLP did not cause a statistically significant difference in the calcium transients, even though O2- production was enhanced. In contrast, in the presence of cytochalasin B, the addition of ATP gamma S to fMLP-stimulated neutrophils produced greatly sustained and protracted elevations in intracellular calcium, correlating with further enhancement of O2- responses. In fMLP-stimulated neutrophils the latter phases of the calcium transients appeared to be dependent in part on the availability of extracellular calcium. These data suggest that ATP gamma S-induced enhancement of O2- responses in fMLP-stimulated rat neutrophils may be related to two mechanisms, one being independent of fMLP-induced intracellular calcium transients and the other, which is related to the presence of cytochalasin B, being linked to sustained elevations in intracellular calcium associated with mobilization of extracellular calcium.
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PMID:Signal transduction events in stimulated rat neutrophils: effects of adenine nucleotides. 253 77

We have shown previously that fluid phase platelet-activating factor (PAF) can enhance or "prime" polymorphonuclear (PMN) responses to subsequent stimulation with agonists such as formyl-methionine-leucine-phenylalanine (FMLP). Since thrombin induces PAF production in endothelial cells, we tested whether this thrombin-provoked endothelial PAF primes responses of marginated PMNs. Monolayers of human umbilical vein endothelial cells were exposed to either thrombin (0.5-5.0 units/ml) or buffer for up to 5 min and then PMNs were layered on top of the endothelial cells. After a further 5 min incubation, the PMNs were stimulated with a suboptimal concentration of FMLP (10(-7) M), and their superoxide production, elastase release, adhesion to endothelium, and capacity to cause endothelial cell lysis and detachment were assessed. Thrombin pretreatment significantly enhanced each of these FMLP-stimulated neutrophil responses. The extent of this enhancement correlated with both the dose and duration of thrombin treatment of endothelial cells and also the duration of PMN incubation with thrombin-exposed endothelium. Evidence that the augmentation was due to endothelial-derived PAF was obtained as follows: (1) thrombin induced [3H]acetate incorporation into endothelial PAF (assayed in lipid extracts); (2) antithrombin III conjointly inhibited this [3H]acetate uptake and prevented the priming effect of thrombin-treated endothelium on PMN responses; and (3) the PAF receptor antagonist BN52021, when preincubated with PMNs, also effectively blocked the enhancement of PMN responses. We conclude that thrombin stimulation of endothelial cells initiates a sequence of events culminating in the production of PAF--a membrane phospholipid capable of priming marginated PMNs. We suggest that this coagulation-fostered endothelial/PMN interaction may underlie a paracrine response that may potentiate PMN-mediated endothelial injury during sepsis and other thrombin-generating disorders.
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PMID:Thrombin-treated endothelium primes neutrophil functions: inhibition by platelet-activating factor receptor antagonists. 254 22

We have shown that the heavy chain of clathrin is phosphorylated in chicken embryo fibroblast cells transformed by Rous sarcoma virus, but not in normal cells. Approximately 1 mol of phosphate is bound for every 5 mol of heavy chain in the maximally phosphorylated transformed cells. Two-thirds of the phosphate is on serine and one-third on tyrosine residues. Clathrin heavy chain is a substrate for pp60v-src in vitro. Cleveland analysis of the in vivo and in vitro clathrin heavy chain phosphopeptides, generated by protease V8 digestion, show labeled proteolytic fragments of similar molecular weight, suggesting that pp60v-src could be directly responsible for the in vivo phosphorylation of clathrin. Phosphate is equally incorporated into clathrin in both the unassembled and the assembled clathrin pools, whereas [35S]methionine is preferentially incorporated into the assembled pool. In normal cells, clathrin visualized by immunofluorescent staining appears in a punctate pattern along the membrane surface and concentrated around the nucleus; in transformed cells the perinuclear staining is completely absent. The phosphorylation of clathrin heavy chain in transformed cells may be linked to previously observed transformation-dependent alterations in receptor-mediated endocytosis of ligands such as EGF and thrombin.
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PMID:Transformation by Rous sarcoma virus induces clathrin heavy chain phosphorylation. 254 3

We have measured mRNA levels for thrombomodulin, an endothelial membrane cofactor for the activation of protein C by thrombin, in a mouse hemangioma cell line. Cycloheximide, an inhibitor of protein synthesis, increased levels of thrombomodulin mRNA, as measured in an S1 nuclease protection assay, to 2.5-4.0 times control levels. Thrombomodulin transcription in response to cycloheximide treatment, as determined by nuclear run-on analysis, was 3.9 +/- 1.3 (mean +/- SD) times that found in untreated cells. Thrombin also increased thrombomodulin mRNA levels to 151 +/- 21% (mean +/- SD) of control levels after 2 hr. Transcription increased in response to thrombin by 2.1- to 7.3-fold. The combination of thrombin and cycloheximide had no additive effect on thrombomodulin mRNA levels. Thrombin treatment of hemangioma cells also caused an increase in thrombomodulin protein synthesis to 142 +/- 17% (mean +/- SD) of control levels as determined by immunoprecipitation of [32S]methionine-labeled thrombomodulin. We conclude that thrombomodulin expression is determined in part by the rate of transcription and that thrombomodulin mRNA levels in hemangioma cells are increased by treatment with cycloheximide or thrombin. The increased transcription in response to cycloheximide suggests the existence of a labile protein repressor of thrombomodulin transcription.
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PMID:Transcription of thrombomodulin mRNA in mouse hemangioma cells is increased by cycloheximide and thrombin. 255 Sep 31

Both congenital and acquired antithrombin-III (AT-III) deficiencies are amenable to replacement therapy. We describe two antithrombins produced by recombinant DNA techniques from human alpha 1-antitrypsin (alpha 1AT) cDNA in yeast. Alteration of the alpha 1AT active site, replacing methionine 358 with arginine, results in a thrombin inhibition rate similar to that of heparin-activated AT-III. Alteration of two further residues, to give a five-residue sequence identical to AT-III, does not increase this rate further. Neither antithrombin is activated by heparin; both are unglycosylated and have shorter in vivo half-lives (t1/2) than human alpha 1AT. These antithrombins should be suitable for therapeutic replacement of AT-III in cases of congenital deficiency and in conditions associated with acquired AT-III deficiency, such as disseminated intravascular coagulation.
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PMID:Characterization of antithrombins produced by active site mutagenesis of human alpha 1-antitrypsin expressed in yeast. 264 77

Monocytes produce thrombospondin (TSP), a trimeric glycoprotein whose primary function is not yet clear. Platelet-poor monocytes (less than 1 platelet/50 monocytes) cultured with [35S]methionine produced [35S] TSP barely detectable by immunoprecipitation with either monoclonal or polyclonal antibody to TSP. Platelet-poor monocytes that had not been so thoroughly depleted of platelets (6-12 platelets/50 monocytes) synthesized readily detectable amounts of [35S] TSP. Addition of increasing numbers of washed platelets to platelet-poor monocytes resulted in increasing synthesis of [35S]TSP. This monocyte-platelet interaction was specific for cell type; neither neutrophils nor red cells could substitute for platelets. The induction of synthesis was specific for TSP; monocyte synthesis of three other proteins was not induced upon the addition of platelets. Platelet lysate or thrombin-induced platelet releasate could not substitute for intact platelets. In fact, platelet lysate inhibited [35S]TSP synthesis by monocyte-platelet cultures. This inhibition was not due to endotoxin contamination, interference with immunoprecipitation, or dilution of the [35S]methionine pool. Platelets required contact with monocytes to exert their effect, as culturing the cell populations with a filter between them prevented increased [35S]TSP synthesis. Monocyte-platelet interactions may serve to specifically increase monocyte synthesis of the adhesive protein, TSP.
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PMID:Monocyte synthesis of thrombospondin. The role of platelets. 270 75


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