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)

Antithrombin-III Denver is a mutant protein which differs from the normal in being defective in serine protease binding (Sambrano, J. E., Jacobson, L. J., Reeve, E. B., Manco-Johnson, M. J., and Hathaway, W. E. (1986) J. Clin. Invest. 77, 887-893). It was isolated from the blood of an individual heterozygous for the abnormal gene by: affinity separation on heparin-Sepharose to obtain an antithrombin fraction, and gel filtration of the species present following complexing of the antithrombin fraction with a small excess of thrombin. The reduced, S-carboxymethylated protein formed a mixture of soluble tryptic peptides which was fractionated on Vydac C18. A single, unique peptide not present in a parallel experiment with normal antithrombin-III was isolated. This peptide was identified by sequence analysis and synthesis to correspond to residues 394-399 in the known sequence of the inhibitor, with leucine replacing reactive site P'1 residue Ser394. Although chromatograms of the tryptic peptides from the normal and mutant proteins were otherwise indistinguishable, the existence of additional residue replacements is not excluded. Measurements of the rate of thrombin binding by the mutant protein with p-aminobenzamidine as a fluorescent indicator showed that the second-order rate constant is reduced drastically. Meaningful measurements with the mutant protein could only be made in the presence of heparin and revealed a reduction of about 4000-fold in the rate constant.
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PMID:Antithrombin-III Denver, a reactive site variant. 380 13

It has been recognized for many years that alpha-thrombin, like other better known mitogens (eg, PDGF, EGF, etc) is capable of initiating proliferation in quiescent cells belonging to the fibroblast family. However, unlike these other peptides, thrombin is a serine protease whose function as a growth stimulator for fibroblasts is intimately linked to its esterolytic activity. Thus, while native alpha-thrombin is capable of evoking DNA synthesis in G0/G1-arrested cells, neither enzymatically inactive thrombin (eg, iPR2P-alpha-thrombin) nor partially degraded thrombin (eg, gamma-thrombin) shares in this capability. Data from our laboratory have shown that thrombin is chemotactic for peripheral blood monocytes and for cells belonging to the monocyte/macrophage family and that this activity is not dependent upon thrombin's enzymatic properties. Our recent findings demonstrate that thrombin also serves as a growth factor for these cells, and this mitogenic capability is independent of esterolytic function and resides in the same region of the molecule as that responsible for chemotaxis. Additionally, by means of techniques such as computer modeling and peptide synthesis, we have now been able to delineate a distinct mitogenic subsite within this chemotactic thrombin sequence. Thus, the sequence in the thrombin B chain that mediates chemotaxis represents a true cell interactive exosite additionally capable of stimulating growth and possibly other biological functions in cells of macrophage/monocyte lineage.
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PMID:Growth-promoting effects of esterolytically inactive thrombin on macrophages. 380 33

Ancrod is a thrombinlike enzyme from Malayan pit viper (Agkistrodon rhodostoma) venom that has a selective enzyme substrate specificity for fibrinogen. Unlike thrombin, it splits only fibrinopeptide A from the fibrinogen molecule and does not activate factor XIII. Simultaneously with the occurrence of hypofibrinogenemia there is a reduction of plasma plasminogen and a rise in fibrin degradation products, suggesting secondary recruitment of the fibrinolytic enzyme system. Ancrod was given to 18 patients with systemic lupus erythematosus and glomerular and vascular microthrombi. Before treatment vascular plasminogen activator (VPA) was low or unmeasurable in 14, an inhibitor of urokinase-induced plasminogen activation (IPA) was elevated in 18, and an inhibitor of plasmin (PI) was elevated in five. Ancrod treatment resulted in prompt normalization of IPA levels in 13 patients; they were classified as fibrinolysis responders. In five patients IPA levels remained elevated throughout treatment with ancrod; they were classified as fibrinolysis nonresponders. In these five the PI level was elevated before treatment and decreased slowly toward the normal range during ancrod administration. The PI did not appear related to the nonspecific serine protease inhibitors, and was shown to be identical with alpha 2-antiplasmin. In the fibrinolysis responders serial histologic studies showed a striking decrease of disappearance of microvascular thrombosis; in the fibrinolysis nonresponders microvascular thrombosis persisted. The action of ancrod is discussed.
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PMID:Ancrod: normalization of fibrinolytic enzyme abnormalities in patients with systemic lupus erythematosus and lupus nephritis. 387 65

In vitro effects of S-2441, H-D-Pro-Phe-Arg-NH-Heptyl, include potent anti-bradykinin activity and broad-spectrum inhibition of serine proteases involved in the coagulation cascade. In this study, rats infused with 7.8 X 10(8) viable Escherichia coli were treated either with saline (group A) or with intravenous (0.1 mg) and intraperitoneal (0.4 mg) doses of S-2441 (group B). Survival rates for groups A and B were 68% and 98%, at 12 hours (P less than 0.001), and 37% and 73% at 24 hours (P less than 0.001), respectively. Hematologic studies revealed that S-2441 significantly inhibited E. coli-induced prolongation of prothrombin time and partial thromboplastin time as well as a rapid decrease in the values of factor X, anti-thrombin III, and fibrinogen. In addition, S-2441 attenuated E. coli-induced hypoglycemia and a marked reduction of serum complement level. Ultrastructural evaluation of the liver demonstrated that S-2441 prevented the development of extensive sinusosoidal microthrombosis and hepatocellular necrosis. The results indicate that S-2441 affords protection in lethal gram-negative bacteremia owing in part to attenuation of disseminated intravascular coagulation and complement-mediated reactions. The findings are consistent with the concept that S-2441 and related oligopeptides modulate serine protease-mediated responses involving inhibition of active enzymes with competitive antagonism of pharmcologically active products formed during the activation of coagulation, fibrinolytic, kallikrein, and complement systems.
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PMID:Efficacy of S-2441, a synthetic oligopeptide, in a rat model for gram-negative bacteremia. 388 74

A new enzyme which hydrolyzes anilide substrates of p-guanidino-L-phenylalanine in preference to those of arginine was found in the ascitic plasma from Ehrlich ascites tumor-bearing mice. The activity of this enzyme on N alpha-benzyloxycarbonyl-p-guanidino-L-phenylalanine p-nitroanilide was strongly inhibited by diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride but not by sulfhydryl-reactive reagents and metal chelating agents. Peptide substrates containing p-guanidino-L-phenylalanine were hydrolyzed by this enzyme much faster than those containing arginine. These results suggest that this enzyme is a different type of serine protease from trypsin and thrombin. This enzyme was also found in the human gastric and colon cancer cells and their surrounding ascitic plasmas.
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PMID:A new serine protease which preferentially recognizes p-guanidino-L-phenylalanyl residue in ascitic plasma from Ehrlich ascites tumor-bearing mice. 389 Aug 49

The time-dependent inactivation of several serine proteases including human leukocyte elastase, cathepsin G, rat mast cell proteases I and II, and human skin chymase by a number of 3-alkoxy-4-chloroisocoumarins, 3-alkoxy-4-chloro-7-nitroisocoumarins, and 3-alkoxy-7-amino-4-chloroisocoumarins at pH 7.5 and the inactivation of several trypsin-like enzymes including human thrombin and factor XIIa by 7-amino-4-chloro-3-ethoxyisocoumarin and 4-chloro-3-ethoxyisocoumarin are reported. The 3-alkoxy substituent of the isocoumarin is likely interacting with the S1 subsite of the enzyme since the most reactive inhibitor for a particular enzyme had a 3-substituent complementary to the enzyme's primary substrate specificity site (S1). Inactivation of several enzymes including human leukocyte elastase by the 3-alkoxy-7-amino-4-chlorisocoumarins is irreversible, and less than 3% activity is regained upon extensive dialysis of the inactivated enzyme. Addition of hydroxylamine to enzymes inactivated by the 3-alkoxy-7-amino-4-chloroisocoumarins results in a slow (t1/2 greater than 6.7 h) and incomplete (32-57%) regain in enzymatic activity at pH 7.5. Inactivation by the 3-alkoxy-4-chloroisocoumarins and 3-alkoxy-4-chloro-7-nitroisocoumarins on the other hand is transient, and full enzyme activity is regained rapidly either upon standing, after dialysis, or upon the addition of buffered hydroxylamine. The rate of inactivation by the substituted isocoumarins is decreased when substrates or reversible inhibitors are present in the incubation mixture, which indicates active site involvement. The inactivation rates are dependent upon the pH of the reaction mixture, the isocoumarin ring system is opened concurrently with inactivation, and the reaction of 3-alkoxy-7-amino-4-chloroisocoumarins with porcine pancreatic elastase is shown to be stoichiometric. The results are consistent with a scheme where 3-alkoxy-7-amino-4-chloroisocoumarins react with the active site serine of a serine protease to give an acyl enzyme in which a reactive quinone imine methide can be released. Irreversible inactivation could then occur upon alkylation of an active site nucleophile (probably histidine-57) by the acyl quinone imine methide. The finding that hydroxylamine slowly catalyzes partial reactivation indicates that several inactivated enzyme species may exist. The 3-alkoxy-substituted 4-chloroisocoumarins and 4-chloro-7-nitroisocoumarins are simple acylating agents and do not give stable inactivated enzyme structures.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Reaction of serine proteases with substituted 3-alkoxy-4-chloroisocoumarins and 3-alkoxy-7-amino-4-chloroisocoumarins: new reactive mechanism-based inhibitors. 391 97

An enzyme bearing thrombin-like specificity has been purified to homogeneity from the venom of Trimeresurus flavoviridis (the Habu snake). The enzyme is a monomer with a molecular weight of 23,500 as determined by analytical gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein contains approximately 210 amino acid residues and has a relatively high content of aspartic acid and glutamic acid. The isoelectric point was 4.8 and the extinction coefficient at 280 nm for a 1% solution was 11.5. The enzyme acted directly on fibrinogen to form a fibrin clot with 2.0 NIH units. Analysis by high performance liquid chromatography of enzyme-treated fibrinogen revealed the release of a peptide identical in composition to thrombin-induced fibrinopeptide A, but no peptide corresponding to fibrinopeptide B was detected. The enzyme showed esterase and amidase activities on synthetic substrates containing arginine. The enzyme exhibited higher activity toward tosyl-L-arginine methyl ester (TAME) but 6-times lower activity toward benzoyl-L-arginine p-nitroanilide when compared with bovin thrombin. The esterase activity was inhibited by diisopropylfluorophosphate and at a slower rate by phenylmethanesulfonyl fluoride, but was least affected by tosyl-L-lysine chloromethyl ketone, showing that the enzyme is a serine protease like thrombin. The enzyme showed a bell-shaped pH dependence of kcat/Km for hydrolysis of TAME, with a maximum around pH 8.5.
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PMID:Purification and characterization of a coagulant enzyme from Trimeresurus flavoviridis venom. 391 Jun 43

Bovine corneal endothelial cells in culture bind internalize and degrade [125I]-trypsin. Binding involves the active site of trypsin and increases as a function of [125I]-trypsin concentration. Saturation is observed at a concentration of 0.5-1.0 micrograms ml-1. The cell surface binding of [125I]-trypsin is specific: a seven-fold excess of unlabeled trypsin abolishes about 60% of the total cell surface-associated radioactivity. In addition, thrombin competes poorly with [125I]-trypsin cell surface binding and only 20% of the specific cell surface binding of [125I]-trypsin is subjected to competition with thrombin. This fraction of the cell surface-bound [125I]-trypsin which is accessible to competition with thrombin appears in a covalent complex of [125I]-trypsin X protease-nexin with a molecular weight of 64000 daltons. The cells, when incubated at 37 degrees C, appear to internalize the cell surface-bound [125I]-trypsin at a rate of 0.15-0.25 ng (10(6) cells)-1 min-1. Both the non-covalently cell surface-bound and the protease-nexin (PN) mediated-bound [125I]-trypsin are internalized by the cells, but the [125I]-trypsin X PN complexes contribute about 75% of the total amount of [125I]-trypsin internalized by the cells. The internalized [125I]-trypsin is degraded by the cultures at a rate of about 0.05 ng (10(6) cells)-1 min-1 and the degradation products are released by the cells into the incubation medium as a trichloroacetic acid non-precipitable material. Chloroquine inhibits about 60% of the internalization of [125I]-trypsin by the cells, and inhibits more than 80% of the degradation process of [125I]-trypsin, which indicates that the degradation of the ligand is taking place in lysosomes. Bovine corneal endothelial cells in culture have demonstrated the binding and metabolism of the serine protease trypsin. This described process may indicate the ability of corneal endothelial cells to control the activity of serine proteases in their microenvironment.
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PMID:Binding and processing of trypsin by cultured bovine corneal endothelial cells. 400 80

Coagulation factor IX is a vitamin K-dependent glycoprotein that circulates in blood as a precursor of a serine protease. Incubation of human factor IX with human alpha-thrombin resulted in a time and enzyme concentration-dependent cleavage of factor IX yielding a molecule composed of a heavy chain (mol wt 50,000) and a doublet light chain (mol wt 10,000). The proteolysis of factor IX by thrombin was significantly inhibited by physiological levels of calcium ions. Under nondenaturing conditions, the heavy and light chains of thrombin-cleaved factor IX remained strongly associated, but these chains were readily separated by gel filtration in the presence of denaturants. Amino-terminal sequence analyses of the isolated heavy and light chains of thrombin-cleaved human factor IX indicated that thrombin cleaved peptide bonds at Arg327-Val328 and Arg338-Ser339 in this molecule. Comparable cleavages were observed in bovine factor IX by bovine thrombin and occurred at Arg319-Ser320 and Arg339-Ser340. Essentially, a complete loss of factor IX procoagulant activity was associated with its cleavage by thrombin. Furthermore, thrombin-cleaved factor IX neither developed coagulant activity after treatment with factor XIa nor inhibited the coagulant activity of native factor IX. These data indicate that thrombin cleaves factor IX near its active site serine residue, rendering it incapable of activating factor X. Whether or not this reaction occurs in vivo is unknown.
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PMID:Proteolytic inactivation of blood coagulation factor IX by thrombin. 406 23

The significance of baseline coagulation times and plasma concentrations of serine protease inhibitors as determinants of the relationship between heparin activity and its anticoagulant effect has been investigated in vitro. Citrated plasma was prepared from blood obtained from 20 normal subjects, and heparin added to yield concentrations ranging from 0.05 to 1.5 units/ml. The anticoagulant effect was determined by the activated partial thromboplastin time (APTT) and thrombin time (TT). An excellent linear relationship was observed between the natural logarithms (ln) of the coagulation times and heparin activity. Baseline APTT values ranged from 28.4 to 59.7 s and the slope values for the ln APTT vs heparin curves ranged from 1.488 to 3.427 ml/unit. Similar range was observed in the slope values for the ln TT vs heparin curves. There was a highly significant positive correlation between the ln APTT vs heparin slope values and the baseline APTT values (r: 0.905; p less than 0.001). There was also a weak but statistically significant positive correlation between plasma concentrations of alpha 2 macroglobulin and baseline APTT values (0.02 greater than 0 greater than 0.01) and slope values of the ln APTT vs heparin curves (0.02 greater than p greater than 0.01). Furthermore, there was a statistically significant positive correlation between plasma concentrations of alpha 1 antitrypsin and baseline TT values (0.05 greater than p greater than 0.02) and slope values of the 1n TT vs heparin (0.02 greater than p greater than 0.01). Neither baseline APTT and TT values nor slope values of the ln APTT and TT vs heparin curves were statistically significantly related to plasma concentrations of antithrombin III, fibrinogen, or alpha 1 acid glycoprotein. This study has demonstrated that baseline APTT is a major determinant of the anticoagulant response to heparin in vitro, as determined by that same coagulation test, and it illustrates that there is a wide intersubject variation in the anticoagulant response to heparin in vitro.
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PMID:Intersubject variability in the anticoagulant response to heparin in vitro. 617 54


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