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 ability of heparin fractions of different molecular weight to potentiate the action of antithrombin III against the coagulation factors thrombin and Xa has been examined in purified reaction mixtures and in plasma. Residual thrombin and Xa have been determined by their peptidase activities against the synthetic peptide substrates H-D-Phe-Pip-Arg-pNA and Bz-Ile-Gly-Arg-pNA. High molecular weight heparin fractions were found to have higher anticoagulant activities than low molecular weight heparin when studied with both thrombin and Xa incubation mixtures in purified mixtures and in plasma. The inhibition of thrombin by heparin fractions and antithrombin III was unaffected by other plasma components. However, normal human plasma contained a component that inhibited the heparin and antithrombin III inhibition of Xa particularly when the high molecular weight heparin fraction was used. Experiments using a purified preparation of platelet factor 4 suggested that the platelet-derived heparin-neutralizing protein was not responsible for the inhibition.
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PMID:Evidence for a plasma inhibitor of the heparin accelerated inhibition of factor Xa by antithrombin III. 47 56

We discovered an enzyme in human platelets that deamidates substance P and other tachykinins. Because an amidated carboxyl terminus is important for biological activity, we purified and characterized this deamidase. The enzyme, released from human platelets by thrombin, was purified to homogeneity by ammonium sulfate precipitation, followed by chromatography on an octyl-Sepharose column and chromatofocusing on PBE 94. The purified enzyme exhibits esterase, peptidase, and deamidase activities. The peptidase activity (with furylacryloyl-Phe-Phe) is optimal at pH 5.0 while the esterase (benzoyl-tyrosine ethyl ester) and deamidase (D-Ala2-Leu5-enkephalinamide) activities are optimal at pH 7.0. With biologically important peptides, the enzyme acts both as a deamidase (substance P, neurokinin A, and eledoisin) and a carboxy-peptidase (with bradykinin, angiotensin I, substance P-free acid, oxytocin-free acid) at neutrality, although the carboxypeptidase action is faster at pH 5.5. Enkephalins, released upon deamidation of enkephalinamides, were not cleaved. Gly9-NH2 of oxytocin was released without deamidation. Peptides with a penultimate Arg residue were not hydrolyzed. Some properties of the deamidase are similar to those reported for cathepsin A. The deamidase is inhibited by diisopropylfluorophosphate, inhibitors of chymotrypsin-type enzymes, and mercury compounds while other inhibitors of catheptic enzymes, trypsin-like enzymes, and metalloproteases were ineffective. In gel filtration, the native enzyme has an Mr = 94,000 while in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr = 52,000 indicating it exists as a dimer. After reduction, deamidase dissociates into two chains of Mr = 33,000 and 21,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [3H]diisopropylfluorophosphate labeled the active site serine in the Mr = 33,000 chain. The first 25 amino acids of both chains were sequenced. They are identical with the sequences of the two chains of lysosomal "protective protein" which, in turn, has sequence similarity to the KEX1 gene product and carboxypeptidase Y of yeast. This protective protein complexes with beta-galactosidase and neuraminidase in lysosomes and is vitally important in maintaining their activity and stability. A defect in this protein is the cause of galactosialidosis, a severe genetic disorder. The ability of physiological stimuli (e.g. thrombin or collagen) to release the deamidase from platelets indicates that it may also be involved in the local metabolism of bioactive peptides.
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PMID:A peptidase in human platelets that deamidates tachykinins. Probable identity with the lysosomal "protective protein". 169 76

We compared the in vitro degradation of porcine and human insulin in the subcutaneous tissue of rat. The insulin degrading activity was largely confined to the 160000 X g supernatant fraction of subcutaneous tissue. The degradation of human insulin was approximately half that of porcine insulin in the supernatant fraction. The degradation of porcine insulin in subcutaneous tissue was inhibited by bacitracin, leupeptin, phosphoramidon, and Z-Gly-Pro-Leu-Gly, though the human insulin degradation was not. The degradation of both insulins was accelerated by glutathione. While the proteolytic enzyme activities of cathepsin-B and collagenase-like peptidase were detectable in subcutaneous tissue, chymotrypsin, elastase, kallikrein, alpha-thrombin, and trypsin activities were almost negligible. These in vitro studies suggest that human insulin is comparatively stable against proteolytic enzymes, probably collagenase-like peptidase or cathepsin-B, in the subcutaneous tissue, which support the in vivo evidence.
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PMID:Fate of porcine and human insulin at the subcutaneous injection site. II. In vitro degradation of insulins in the subcutaneous tissue of the rat. 240 62

Endothelial injury has been proposed as a feature of a wide variety of vascular diseases, and release of endothelial lysosomal hydrolases could contribute to the pathological changes seen. We have determined the relative activities of 14 glycosidases, two esterases and four peptide hydrolases in human umbilical vein endothelial cells and investigated whether known agonists of endothelial function, or materials known to modulate hydrolase secretion in other phagocytic cells, influenced the activity or secretion of these enzymes by human umbilical vein endothelial cells. Hexosaminidase, beta-galactosidase, beta-glucuronidase and alpha-iduronidase accounted for most of the measured glycosidase activity. Acid phosphatase activity greatly exceeded arylsulphatase activity, and most of the measured peptidase activity was due to acid peptidases. Optimum pH and apparent Km values were determined for the most abundant hydrolases. Exposure of human umbilical vein endothelial cells to bradykinin, thrombin or interleukin-1 resulted in negligible release of either hexosaminidase or lactate dehydrogenase (LDH), in contrast to phorbol myristate acetate, which caused a parallel, dose-dependent release of both enzymes. Treatment of these cells with calcium ionophore A23187, trypsin or platelet-activating factor, caused less than 10% release of either hexosaminidase or LDH. Agents known to modulate lysosomal enzyme secretion by other phagocytic cells failed to induce selective secretion of lysosomal enzymes by human umbilical vein endothelial cells.
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PMID:Lysosomal hydrolases of human vascular cells: response to agonists of endothelial function. 264 39

1 The effect of perturbation of intact blood platelets with proteolytic enzymes was studied with respect to 5-hydroxytryptamine (5-HT) transport, adenine transport and intracellular Na(+) and K(+) levels.2 Leucine aminopeptidase and thrombin reduced 5-HT transport, released 5-HT from pre-labelled platelets and disturbed the gradient to monovalent cations. Leucine amino-peptidase, but not thrombin, inhibited adenine transport.3 alpha-Chymotrypsin and carboxypeptidases A and B were without effect on the parameters studied.4 Trypsin selectively reduced 5-HT transport. It did not release 5-HT from blood platelets or inhibit adenine transport.5 The action of proteolytic enzymes is discussed in terms of proteins localized in the external membrane that may function in part as membrane carriers.
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PMID:Effect of selective proteolysis on the accumulation of 5-hydroxytryptamine by intact rat blood platelets. 445 19

Trypomastigotes, the infective stages of the intracellular parasite Trypanosoma cruzi, induce rapid and repetitive cytosolic free Ca2+ transients in fibroblasts. Buffering or depletion of intracellular free Ca2+ inhibits cell entry by trypomastigotes, indicating a role for this signaling event in invasion. We show here that the majority of the Ca(2+)-signaling activity is associated with the soluble fraction of parasites disrupted by sonication. Distinct cell types from different species are responsive to this soluble factor, and intracellular free Ca2+ transients occur rapidly and reach concentrations comparable to responses induced by thrombin and bombesin. The Ca(2+)-signaling activity does not bind concanavalin A and is strongly inhibited by a specific subset of protease inhibitors. The only detectable protease in the fractions with Ca(2+)-signaling activity is an unusual alkaline peptidase of 120 kDa, to which no function had been previously assigned. The activity of the protease and cell invasion by trypomastigotes are blocked by the same specific inhibitors that impair Ca(2+)-signaling, suggesting that the enzyme is required for generating the response leading to infection. We demonstrate that the 120-kDa peptidase is not sufficient for triggering Ca(2+)-signaling, possibly being involved in the processing of precursors present only in infective trypomastigotes. These findings indicate a biological function for a previously identified unusual protozoan protease and provide the first example of a proteolytically generated parasite factor with characteristics of a mammalian hormone.
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PMID:A 120-kDa alkaline peptidase from Trypanosoma cruzi is involved in the generation of a novel Ca(2+)-signaling factor for mammalian cells. 789 Jun 27

We measured the ability of the thrombin receptor activating peptide, SFLLR-NH2 (P5A) to stimulate 3H-thymidine incorporation in hamster CCL-39 fibroblasts either alone or in combination with the thrombin-derived polypeptides, YPPWNKNFTENDLL (TDP-1) and AGYKPDEGKRGDACEGDSGGPFV (TDP-2). In the presence (but not absence) of the amino peptidase inhibitor amastatin (10 microM), P5A alone (7.5 to 100 microM) caused a 1.5- to 2-fold stimulation of thymidine incorporation above basal, even though this inhibitor did not abrogate the degradation of P5A by other peptidases present in the assay medium. Neither TDP-1 nor TDP-2 alone had any effect on thymidine incorporation. However, TDP-1 (30 to 90 microM) considerably augmented P5A-mediated thymidine incorporation at low P5A concentrations (7.5 to 30 microM), shifting the P5A concentration-effect curve to the left. TDP-2 was inactive in this regard. The EC50 for this potentiating action of TDP-1 was approximately 40 microM. Further, thrombin, rendered proteolytically inactive by a low-molecular-weight bifunctional inhibitor, hirutonin-6, also acted synergistically with P5A to stimulate CCL-39 cell thymidine incorporation. We hypothesize that thrombin can cause its cellular effects, such as thymidine incorporation, not only via the proteolytic activation of its G-protein-coupled receptor, but also via the concurrent and synergistic interaction of its TDP-1 peptide domain with a separate cell surface docking site.
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PMID:Synergistic actions of a thrombin-derived synthetic peptide and a thrombin receptor-activating peptide in stimulating fibroblast mitogenesis. 895 98

The characteristics of endothelin (ET) release from guinea-pig tracheal epithelium were investigated, including examination of the effects of several pro-inflammatory mediators. In confluent cultured guinea-pig tracheal epithelial cells (GPTECs) there was a time-dependent basal release of immunoreactive ET (ir-ET) from 4-48 h. Basal ir-ET release from GPTECs was unaffected by the peptidase inhibitors, thiorphan (10 microM), benzamidine (1 mM), pepstatin-A (30 microM), aprotinin (1 microgram/ml), bacitracin (20 micrograms/ml) or leupetin (50 microM), but was inhibited by phosphoramidon, the neutral metalloprotease inhibitor (IC50 = 16.8 microM), or the calcium chelator, EGTA (10 mM). There was little ir-ET release 1 day after placing GPTECs in culture, although appreciable release (> 10-fold higher) was detected on days 5 and 7. No significant release of ir-ET was demonstrated from intact guinea-pig trachea. Human thrombin (0.1-10 U/ml), LPS (0.3-10 ng/ml) and the phorbol ester, phorbol 12-myristate-13-acetate (0.1 nM-1 microM), significantly increased ir-ET release, whereas TNF-alpha (0.1-10 ng/ml), RANTES (0.1-100 nM), IL-1 (0.01-10 ng/ml), bradykinin (1 nM-10 microM), CGRP (0.01 nM-1 microM), PDGF (0.1-3 ng/ml), Sar9, Met(O2)11-Sub P, Nle10-NKA 4-10 and senktide (selective NK-1, NK-2 and NK-3 receptor agonists, respectively; 1 nM-10 microM), LTD4 (1 nM-10 microM) or major basic protein (10 nM-1 microM) were without stimulatory effect. The results indicate that the enzyme responsible for the basal release of ET from cultured GPTECs is a Ca(2+)-dependent, phosphoramidon-sensitive, neutral metalloprotease. Furthermore, normally there is minimal ET release from guinea-pig airway epithelium but this can be increased markedly by culturing the cells to confluence, and by select pro-inflammatory mediators.
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PMID:Characterization of endothelin release from guinea-pig tracheal epithelium: influence of proinflammatory mediators including major basic protein. 969 42

The synthesis of cell-associated and secreted proteins by Streptococcus gordonii FSS2, an infective endocarditis (IE) isolate, was influenced by both environmental pH and carbon source. Controlling the pH at 7.5 in stirred batch cultures showed that cell-associated and secreted protein concentrations were increased during late exponential and stationary phase by 68% and 125%, respectively, compared with similar cultures without pH control. The expression of five glycosidase and eight peptidase activities were examined using fluorogen-labelled synthetic substrates. Enzyme activities were significantly down-regulated during exponential growth, increasing during stationary phase (P<0.01) whether the culture pH was controlled at pH 7.5 or allowed to fall naturally to pH 4.4. Culture-supernatant activities were significantly increased (P<0.05) when the pH was maintained at 6.0 or 7.5, indicating modulation of enzyme activity by pH. Growth under nitrogen-limitation/glucose-excess conditions resulted in a significant repression of cell-associated glycosidase activities (P<0.01), whilst in the supernatant, activities were generally reduced. The expression of peptidase activities in the culture supernatant did not significantly change. The results suggest a possible role for catabolite repression by glucose in regulating enzyme expression. When S. gordonii FSS2 was cultured with 50% (v/v) added heat-inactivated foetal bovine serum, several cell-associated enzyme activities increased initially but were then reduced as the culture time was extended to 116 h. Culture-supernatant enzyme activities (N-acetyl-beta-D-glucosaminidase, N-acetyl-beta-D-galactosaminidase, thrombin, Hageman factor, collagenase and chymotrypsin), however, were significantly increased (P<0.01) over the same time period. The findings indicated that most of the important glycosidases synthesized by S. gordonii FSS2 were down-regulated by acid growth conditions and may also be subject to catabolite repression by glucose but conversely may be up-regulated by growth in serum. These results may have implications for streptococcal growth in an IE vegetation and in the mouth between meals or during sleep.
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PMID:Environmental regulation of glycosidase and peptidase production by Streptococcus gordonii FSS2. 1093 96

TAFI (thrombin activatable fibrinolysis inhibitor) is a plasma procarboxypeptidase that upon activation inhibits the fibrinolytic process by removing the C-terminal lysines from partially degraded fibrin. The generation of activated TAFI (TAFIa) has been suggested to represent a mechanism of thrombus resistance to thrombolytic therapy. However, the ability of TAFI to inhibit fibrinolysis by pharmacological concentrations of t-PA has not been properly investigated. We used an in vitro model consisting of 125I-fibrin blood clots submerged in autologous defibrinated plasma. Upon addition of t-PA (125-5,000 ng/ml) and CaCl2 (25 mM), samples were incubated at 37 degrees C, and clot lysis was measured at intervals from the radioactivity released into solution. The role of TAFI was assessed either by neutralizing the generated TAFIa with the specific inhibitor PTI (50 microg/ml) or by enhancing TAFI activation through the addition of recombinant soluble thrombomodulin (solulin, 1 microg/ml). In our clot lysis model, activation of TAFI amounted to about 20% of inducible carboxypeptidase activity. Addition of PTI, however, produced a significant increase in the extent of lysis only at concentrations of t-PA equal to or lower than 250 ng/ml. When solulin was added to the plasma surrounding the clot, about 70% of TAFI was activated within 15 min. Under these conditions, inhibition of clot lysis was very marked in samples containing 125 or 250 ng/ml of t-PA, but negligible in those containing pharmacological concentrations of the activator (1,000 and 5,000 ng/ml). Additional experiments suggest that loss of fibrin-dependence by elevated concentrations of t-PA may be one of the mechanisms explaining the lack of effect of TAFIa. Our data indicate that, under our experimental conditions, clot lysis by pharmacological concentrations of t-PA is not influenced by TAFIa even after maximal activation of this procarboxy-peptidase.
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PMID:Thrombin activatable fibrinolysis inhibitor (TAFI) does not inhibit in vitro thrombolysis by pharmacological concentrations of t-PA. 1134 2


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