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)

Tritiated N alpha-acetyl-L-lysine chloromethyl ketone (ALCK) was synthesized on a laboratory scale for use as an active-site-directed affinity label in the fluorographic detection of proteases after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The synthesis involved acetylation of N epsilon-benzyloxycarbonyl-L-lysine chloromethyl ketone with [3H]acetic anhydride just before the removal of the benzyloxycarbonyl group. By this method, [3H]ALCK with a specific activity of 250 mCi/mmol was obtained as a crystal. Trypsin, thrombin, plasmin, papain, and clostripain were inactivated by ALCK according to first-order kinetics. For fluorographic detection of proteases, enzyme samples were allowed to react with [3H]ALCK and then resolved by SDS-PAGE. Proteases that reacted with [3H]ALCK could be detected with a sensitivity equivalent to or higher than that of Coomassie brilliant blue R-250 staining. A trypsin-like protease in Pronase, clostripain as a contaminant in a commercial preparation of Clostridium histolyticum collagenase, and cysteine proteases in Porphyromonas gingivalis could be detected.
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PMID:Synthesis of N alpha-[3H]acetyl-L-lysine chloromethyl ketone and its use in the fluorographic detection of proteases. 825 Feb 26

The aim of the present study was to clarify the control of Na+/H+ exchange in platelets activated via the thrombin receptor. When human BCECF-loaded platelets were stimulated with the thrombin-receptor-activating peptide (TRAP; amino acid sequence SFLLRN), which activates the receptor independently of proteolysis, the cytosolic pH (pHi) rose from 7.13 +/- 0.04 (n = 6) to 7.27 +/- 0.04 (n = 5), followed by a rapid decrease to resting values. Trypsin, which cleaves the receptor, induced a rapid and irreversible rise in pHi to 7.31 +/- 0.06 (n = 5). gamma-Thrombin, which cleaves the receptor but is unable to bind to the hirudin-like domain, induced a slow and irreversible rise in pHi to 7.31 +/- 0.04 (n = 14). alpha-Thrombin, which cleaves the receptor and binds to its hirudin-like domain, induced a rapid and irreversible rise in pHi to 7.31 +/- 0.04 (n = 22). Changes in pHi induced by TRAP, trypsin, gamma- and alpha-thrombin were accompanied by similar changes in cytosolic Ca2+ concentration ([Ca2+]i) and 32P-pleckstrin, a substrate of protein kinase C (PKC). The separate chelation of Ca2+i (30 microM BAPTA-AM) or inhibition of PKC (1 microM staurosporine) induced about 50% inhibition of the pHi responses triggered by TRAP, trypsin, gamma- and alpha-thrombin, but the combination induced complete inhibition. Thus the different types of activation of the thrombin receptor control Na+/H+ exchange via the same mechanism. Binding of thrombin to the hirudin-like domain accelerates exchange activation, whereas proteolysis of the receptor is essential for a sustained increase in pHi.
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PMID:Different pathways for control of Na+/H+ exchange via activation of the thrombin receptor. 828 Jan 10

The role of matrix metalloproteinase-9 (MMP-9, 92 kDa gelatinase/type IV collagenase) in invasion of mononuclear phagocytes was studied with U937 monoblastoid cells. 12-o-tetradecanoyl 13-phorbol acetate (TPA) differentiated them to macrophage-like cells with induction of MMP-9, and tumor necrosis factor alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) stimulated the production of MMP-9 by TPA-treated cells. TNF alpha also induced the production of MMP-9 by TPA-untreated U937 cells without morphological differentiation. Other agents including dimethyl sulfoxide (DMSO), all-trans-retinoic acid (all-trans-RA), platelet-derived growth factor and 3';5'-cyclic monophosphate had no effects on MMP-9 production by TPA-treated or -untreated cells, but all-trans-RA and DMSO did have a morphological effect on the differentiation of the cells. These data suggest that MMP-9 production by U937 cells is regulated by a mechanism independent of the differentiation to macrophage-like cells. MMP-9 was purified to homogeneity as an inactive zymogen with M(r) 92,000 (proMMP-9) from TPA-differentiated U937 cells treated with TNF alpha. ProMMP-9 was activated by p-aminophenylmercuric acetate (APMA) generating an active species of M(r) 67,000. Trypsin and cathepsin G also attained activation of the zymogen to its full activity obtained by APMA activation, but plasmin, leukocyte elastase, thrombin and plasma kallikrein had no ability to activate it. APMA-activated MMP-9 degraded type I gelatin readily and cleaved native collagen types III, IV and V. Invasion assays using reconstituted basement membrane coupled with a type IV collagenolysis assay showed good correlations between invasiveness, type IV collagenolysis and proMMP-9 production. Invasion was significantly inhibited by EDTA, alpha 2-macroglobulin and tissue inhibitor of metalloproteinases-1, but not by inhibitors of cathepsin G and leukocyte elastase. These data suggest that MMP-9 plays an important role in the invasion of mononuclear phagocytes through basement membranes.
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PMID:Matrix metalloproteinase-9 (92 kDa gelatinase/type IV collagenase) from U937 monoblastoid cells: correlation with cellular invasion. 831 9

The platelet glycoprotein Ib-IX complex (GP Ib-IX) is essential for the initial attachment of platelets to the wall of damaged arteries. In this study, an N-terminal fragment of human GP Ib alpha (residues 1-318), containing the ligand binding sites for von Willebrand factor (vWF) and thrombin, as well as the entire human GP Ib alpha were expressed in Chinese hamster ovary cells. The transfected cells secreted a 48- and a 110-kDa protein, respectively, into the supernatant. Both recombinant proteins were purified by immunoaffinity chromatography. The purified proteins bound soluble vWF in the presence of botrocetin as demonstrated in solid-phase binding assays. The dissociation constant (Kd) for 125I-vWF binding to the recombinant 110-kDa protein was 1.2 +/- 0.2 nM as compared with 1.0 +/- 0.3 nM for vWF binding to purified platelet GP Ib-IX. Both recombinant proteins were also retained on thrombin-Sepharose 4B. The 48-kDa protein contained two N-linked oligosaccharide chains. A 125-kDa protein was identified in the lysate of cells transfected with the coding sequence for the entire GP Ib alpha. Trypsin treatment of this protein generated a 110-kDa fragment, whereas the secreted 110-kDa protein remained unchanged. Post-translational removal of the C-terminal transmembrane domain of recombinant GP Ib alpha might have facilitated the secretion of the soluble glycocalicin-like 110-kDa fragment. In addition, flow cytometry and immunofluorescence microscopy demonstrated that the expression of GP Ib alpha alone is sufficient for its incorporation into the cell surface membrane. These data indicate that two soluble fragments of human GP Ib alpha with binding activity for vWF and thrombin can be expressed in mammalian cells and that the incorporation of GP Ib alpha into the surface membrane does not depend on co-expression with GP Ib beta and/or GP IX.
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PMID:Expression and characterization of functionally active fragments of the platelet glycoprotein (GP) Ib-IX complex in mammalian cells. Incorporation of GP Ib alpha into the cell surface membrane. 837 10

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

Trypsin, thrombin, and peptide analogues of the new amino terminus of the proteolyzed thrombin receptor, SFLLRN and SFLLRNPNDKYEPF, stimulated embryonic fibroblasts cultured as 3-dimensional tissue-like aggregates to elaborate a fibronectin-rich extracellular matrix. Enzymatically inactive thrombin and the control peptide FLLRN failed to stimulate matrix production. The induction of cell proliferation correlated with production of the fibronectin matrix. The regions of active cell proliferation in the fibroblast aggregates co-localized with the matrix and peptide analogues of the RGD cell-adhesion site of fibronectin reversibly inhibited the accumulation of the fibronectin matrix and the stimulation of cell proliferation by SFLLRN. Two different preparations of the fibronectin matrix stimulated cell proliferation in aggregates cultured in growth factor-free medium. We suggest that the stimulation of matrix production is a necessary event for mitogenic signaling in mesenchymal tissue. The tight coupling between the matrigenic and mitogenic activities of growth factors was absent in monolayer cultures of chick embryonic fibroblasts since thrombin and trypsin induced proliferation of monolayer-cultured cells without inducing the production of a fibronectin matrix.
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PMID:Thrombin stimulation of matrix fibronectin. 855 59

The Erythrina variegata Kunitz family trypsin inhibitors, ETIa and ETIb, prolonged the activated partial thromboplastin time (APTT) and also the prothrombin time (PT) of human plasma, but the Kunitz family chymotrypsin inhibitor, ECI, and Bowman-Birk family inhibitor, EBI, from E. variegata hardly prolonged these times. Trypsin inhibitors ETIa and ETIb inhibited the amidolytic activity of factor Xa, and ETIb but not ETIA inhibited plasma kallikrein. Neither ETIa nor ETIb exhibited any inhibitory activity toward beta-factor XIIa and thrombin. Furthermore, trypsin inhibitors ETIa and ETIb inhibited plasmin, a serine proteinase in the fibrinolytic system, whereas ECI and EBI did not. These results indicate that Erythrina Kunitz proteinase inhibitors possess different potency toward serine proteinases in the blood coagulation and fibrinolytic systems, in spite of their high similarity in amino acid sequence.
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PMID:Inhibitory potency of Erythrina variegata proteinase inhibitors toward serine proteinases in the blood coagulation and fibrinolytic systems. 898 61

We have examined protease-mediated activation of the mitogen-activated protein (MAP) kinase cascade in rat aortic smooth-muscle cells and bovine pulmonary arterial fibroblasts. Exposure of smooth-muscle cells to trypsin evoked rapid and transient activation of c-Raf-1, MAP kinase kinase 1 and 2 and MAP kinase that was sensitive to inhibition by soybean trypsin inhibitor. The actions of trypsin were closely mimicked by the proteinase-activated receptor 2 (PAR-2)-activating peptide sequence SLIGRL but not LSIGRL. Peak MAP kinase activation in response to both trypsin and SLIGRL was also dependent on concentration, with EC50 values of 12.1 +/- 3.4 nM and 62.5 +/- 4.5 microM respectively. Under conditions where MAP kinase activation by SLIGRL was completely desensitized by prior exposure of smooth-muscle cells to the peptide, trypsin-stimulated MAP kinase activity was markedly attenuated (78.9 +/- 15.1% desensitization), whereas the response to thrombin was only marginally affected (16.6 +/- 12.1% desensitization). Trypsin and SLIGRL also weakly stimulated the activation of the MAP kinase homologue p38 in smooth-muscle cells without any detectable activation of c-Jun N-terminal kinase. Strong activation of the MAP kinase cascade and modest activation of p38 by trypsin were also observed in fibroblasts, although in this cell type these effects were not mimicked by SLIGRL nor by the thrombin receptor-activating peptide SFLLRNPNDKYEPF. Reverse transcriptase-PCR analysis confirmed the presence of PAR-2 mRNA in smooth-muscle cells but not fibroblasts. Our results suggest that in vascular smooth-muscle cells, trypsin stimulates the activation of the MAP kinase cascade relatively selectively, in a manner consistent with an interaction with the recently described PAR-2. Activation of MAP kinase by trypsin in vascular fibroblasts, however, seems to be independent of PAR-2 and occurs by an undefined mechanism possibly involving novel receptor species.
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PMID:Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases. 900 84

We studied the effects of serine proteases on cytokine gene expression by cultured normal human keratinocytes. In resting keratinocytes, steady-state mRNA levels for interleukins IL-1 alpha, IL-1 beta, IL-7, and IL-8, transforming growth factors alpha and beta, and tumor necrosis alpha were sufficient to be detected by our reverse transcriptase-polymerase clozin reaction method. Incubation of keratinocytes with 25 nM trypsin or 1 unit/ml thrombin for 24 hr selectively upregulated mRNA levels for granulocyte-macrophage colony-stimulating factor (GM-CSF) and Il-6 to detectable levels. Keratinocytes secreted GM-CSF and IL-6 protein in response to these proteases. Monensin did not inhibit the gene expression for the cytokines, thereby excluding the possibility of intervention by secreted molecules. Aprotinin and argatroban inhibited the effects of the proteases. SFLLRN and SLIGRL, tethered ligand receptor peptides for thrombin receptor and for proteinase-activated receptor 2 (PAR-2), respectively, duplicated the effects of the proteases on keratinocytes, which expressed mRNA for both receptors. Trypsin increased tyrosine phosphorylated proteins and intracellular free calcium concentrations. Tyrphostin, pertussis toxin, or H-7 suppressed trypsin- and thrombin-induced GM-CSF gene expression. Our results demonstrate that the serine proteases activate thrombin receptors and PAR-2 on keratinocytes, triggering intracellular signaling and then inducing the synthesis of GM-CSF. We speculate that serine proteases modulate the course of physiological and pathological processes in the skin by stimulating keratinocytes to produce the cytokines.
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PMID:Thrombin and trypsin induce granulocyte-macrophage colony-stimulating factor and interleukin-6 gene expression in cultured normal human keratinocytes. 906 88

Serine proteinases are involved in several physiological processes and elicit profound cellular effects in a variety of tissues. Besides the thrombin receptor a second receptor, activated by trypsin, the proteinase-activated receptor 2 (PAR-2), was cloned and characterized. Both enzymes generate a new extracellular N-terminus by limited proteolytic cleavage which functions as tethered ligand to activate the receptor. Synthetic peptides corresponding to the sequences of the newly generated N-terminus are able to mimic the effects of the enzymes. In porcine pulmonary arteries trypsin and the receptor-derived peptide SLIGRL elicited an endothelium-dependent transient relaxation of PGF2alpha-precontracted vessels. The EC50 values for trypsin and SLIGRL amounted to 1.1 +/- 0.2 nM and 5.4 +/- 0.6 microM, respectively. Trypsin and SLIGRL caused a homologous desensitization but thrombin and the thrombin receptor-activating peptide SFLLRN were still able to elicit pronounced relaxant effects. The trypsin- and SLIGRL-induced relaxant responses were markedly diminished after blockade of the nitric oxide synthesis by N(G)-nitro-L-arginine methyl ester (200 microM) and were absent in endothelium-denuded vessels. Indomethacin and hirudin did not influence the relaxant effects. The effect of trypsin but not that of SLIGRL was blocked by the proteinase inhibitor aprotinin suggesting that only proteolytically active trypsin activates the receptor. Benzamidine derivatives of the 3-amidinophenylalanine type with different affinity for trypsin and thrombin inhibited the vascular effects of trypsin (IC50 0.007-0.7 microM) correlating with its antitrypsin activity. The data suggest that the vascular effects of trypsin and SLIGRL are mediated through activation of PAR-2 which differs from the thrombin receptor.
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PMID:Trypsin- and SLIGRL-induced vascular relaxation and the inhibition by benzamidine derivatives. 940 26


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