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)

We investigated the effect of activated protein C (APC) on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats to investigate the possible usefulness of APC as a treatment for adult respiratory distress syndrome. Intravenously administered LPS (5 mg/kg) significantly increased pulmonary vascular permeability. APC prevented the LPS-induced increase in pulmonary vascular permeability observed at 6 hours. Heparin plus antithrombin III (ATIII) and active site-blocked factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, inhibited LPS-induced coagulopathy but did not prevent LPS-induced pulmonary vascular injury. LPS-induced pulmonary vascular injury was significantly attenuated in rats with nitrogen mustard-induced leukocytopenia and in rats treated with ONO-5046, a potent granulocyte elastase inhibitor. Administration of LPS also increased pulmonary accumulation of leukocytes, as evaluated by measurement of myeloperoxidase activity in the lungs. APC significantly reduced LPS-induced increases in pulmonary accumulation of leukocytes at 1 hour. Neither ATIII plus heparin nor DEGR-Xa inhibited leukocyte accumulation. Active site-blocked APC (DIP-APC) prevented neither the LPS-induced pulmonary accumulation of leukocytes nor the LPS-induced increase in pulmonary vascular permeability. These results suggest that the mechanism of APC inhibition of LPS-induced pulmonary vascular injury was independent of its anticoagulant activity and was related to its ability to inhibit accumulation of leukocytes. In addition, these findings suggest that the serine protease activity of APC may be essential to its inhibitory effect on LPS-induced pulmonary accumulation of leukocytes and subsequent pulmonary vascular injury.
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PMID:Activated protein C attenuates endotoxin-induced pulmonary vascular injury by inhibiting activated leukocytes in rats. 855 86

The cytochrome P450 responsible for the debrisoquine/sparteine polymorphism (P450 2D6) has been produced in large quantities by expression of a modified cDNA in baculovirus. A polyhistidine extension was incorporated at the C-terminus of the expressed protein, which, after purification of the protein on a nickel-agarose column, could be removed proteolytically by treatment with thrombin. Purified yields of P450 2D6 were 2.4 mg from 700 mL of cell culture. The protein had a greater than 90% heme content and was fully active, having no residual absorbance at 420 nm in the reduced CO complex. The quantities produced allowed direct study of the interaction of the substrate codeine with the enzyme by paramagnetic relaxation effects on the NMR spectrum of the substrate. Distances between the heme iron atom and substrate protons were calculated from these experiments, and the orientation of the substrate in the binding pocket was determined. This showed that codeine was bound with the methoxy group of the molecule closest to the heme iron (iron-methyl proton distance of 3.1 +/- 0.1 A), consistent with the observed O-demethylation to morphine. A model of the complex Of P450 2D6 with codeine was built from a multiple sequence and structure alignment of the known crystal structures for P450s, incorporating the experimental constraints derived from the NMR studies. This showed that the overall fold Of P450 2D6 is more similar to that of P450 BM3 than to either P450 cam or P450 terp. Codeine binds to P450 2D6 so that the methoxy group is directly above the A ring of the heme, while the basic nitrogen interacts with the carboxylate of aspartate 301.
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PMID:A model for human cytochrome P450 2D6 based on homology modeling and NMR studies of substrate binding. 860 4

Adult respiratory distress syndrome (ARDS) is a serious complication of disseminated intravascular coagulation (DIC) or multiple organ failure. To determine whether recombinant soluble human thrombomodulin (rsTM) may be useful in treating ARDS due to sepsis, we investigated the effect of rsTM on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats. The intravenous administration of rsTM prevented the increase in pulmonary vascular permeability induced by LPS. Neither heparin plus antithrombin III (AT III) nor dansyl Glu Gly Arg chloromethyl ketone-treated factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, prevented LPS-induced vascular injury. The agents rsTM, heparin plus AT III, and DEGR-Xa all significantly inhibited the LPS-induced intravascular coagulation. Recombinant soluble TM pretreated with a monoclonal antibody (moAb) that inhibits protein C activation by rsTM did not prevent the LPS-induced vascular injury; in contrast, rsTM pretreated with a moAb that does not affect thrombin binding or protein C activation by rsTM prevented vascular injury. Administration of activated protein C (APC) also prevented vascular injury. LPS-induced pulmonary vascular injury was significantly reduced in rats with leukopenia induced by nitrogen mustard and by ONO-5046, a potent inhibitor of granulocyte elastase. Results suggest that rsTM prevents LPS-induced pulmonary vascular injury via protein C activation and that the APC-induced prevention of vascular injury is independent of its anticoagulant activity, but dependent on its ability to inhibit leukocyte activation.
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PMID:Recombinant human soluble thrombomodulin reduces endotoxin-induced pulmonary vascular injury via protein C activation in rats. 860 7

The crystal structure of the noncovalent complex of bovine thrombin and a fibrinogen-A alpha tridecapeptide substrate analog, G17 psi, in which the scissile bond amide nitrogen of Gly-17f has been replaced by a methylene carbon, has been determined at 2.3 A resolution with an R factor of 17.1%. The geometry of the active site indicates that the crystal structure is a close model of the true Michaelis complex. The three independently determined thrombin/G17 psi complexes in the crystal asymmetric unit reveal novel interactions for the P2' and P3' residues-Pro-18f and Arg-19f, respectively-on the carboxyl-terminal side of the scissile bond and confirm previously observed interactions of the P1 (Arg-16f) through P10 (Asp-7f) positions on the amino-terminal side. The thrombin S2' binding site for Pro-18f, as observed in all three complexes, differs from that predicted by modeling studies and is notable for including two carbonyl oxygens of the thrombin main chain. Arg-19f occupies two binding sites on thrombin, S3'A and S3'B, which have dramatically different placements for the arginyl side chain and carboxyl terminus.
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PMID:Bovine thrombin complexed with an uncleavable analog of residues 7-19 of fibrinogen A alpha: geometry of the catalytic triad and interactions of the P1', P2', and P3' substrate residues. 885 38

Structures of the blood clotting enzyme thrombin complexed with hirugen and two active site inhibitors, RWJ-50353 10080(N-methyl-D-phenylalanyl-N-[5-[(aminoiminomethyl)amino]-1- [[(2-benzothiazolyl)carbonyl]butyl]-L-prolinamide trifluoroacetate hydrate) and RWJ-50215 (N-[4-(aminoiminomethyl)amino-1-[2- (thiazol-2-ylcarbonylethyl)piperidin- 1-ylcarbonyl]butyl]-5-(dimethylamino)naphthalenesulfonamide trifluoroacetate hydrate), were determined by x-ray crystallography. The refinements converged at R values of 0.158 in the 7.0-2.3-A range for RWJ-50353 and 0.155 in the 7.0-1.8-A range for RWJ-50215. Interactions between the protein and the thiazole rings of the two inhibitors provide new valuable information about the S1' binding site of thrombin. The RWJ-50353 inhibitor consists of an S1'-binding benzothiazole group linked to the D-Phe-Pro-Arg chloromethyl ketone motif. Interactions with the S1-S3 sites are similar to the D-phenylalanyl-prolyl-arginyl chloromethylketone structure. In RWJ-50215, a S1'-binding 2-ketothiazole group was added to the thrombin inhibitor-like framework of dansylarginine N-(3-ethyl-1,5-pentanediyl)amide. The geometry at the S1-S3 sites here is also similar to that of the parent compound. The benzothiazole and 2-ketothiazole groups bind in a cavity surrounded by His57, Tyr60A, Trp60D, and Lys60F. This location of the S1' binding site is consistent with previous structures of thrombin complexes with hirulog-3, CVS-995, and hirutonin-2 and -6. The ring nitrogen of the RWJ-50353 benzothiazole forms a hydrogen bond with His57, and Lys60F reorients because of close contacts. The oxygen and nitrogen of the ketothiazole of RWJ-50215 hydrogen bond with the NZ atom of Lys60F.
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PMID:Crystal structures of thrombin with thiazole-containing inhibitors: probes of the S1' binding site. 891 20

The objective of this study was to extend our understanding of the stability of heparin. Sodium heparin, derived from porcine intestinal mucosa, was first incubated in 0.1 N hydrochloric acid and 0.1 N sodium hydroxide at 30 and 60 degrees C and sampled at times ranging from 0 to 1000 h. The absorbance spectra of the products formed under basic conditions showed an ultraviolet maxima at 232 nm associated with chemically catalyzed beta-elimination at the uronic acid residues. The products formed under acidic conditions showed a decreased staining intensity consistent with desulfation and a decrease in molecular weight corresponding to hydrolysis of glycosidic linkages when analyzed by gradient polyacrylamide gel electrophoresis. Heparin samples were next prepared in 10 mM sodium phosphate buffer at pH 7.0 in sealed ampules that had been flushed with nitrogen and incubated at 100 degrees C. Samples taken at times ranging from 0 to 4000 h were then analyzed. Heparin was relatively stable over the first 500 h, after which it rapidly degraded. Heparin, assayed using both anti-factor Xa and anti-factor IIa amidolytic methods retained 80-90% of its activity over the first 500 h, but these activities dropped precipitously, to approximately 6% and approximately 0.5% of the initial activity at 1000 h and 2000 h, respectively. This rapid decomposition began only after the buffering capacity of the solution was overwhelmed by acidic degradants, which caused the pH to decrease. Decomposition processes observed under these conditions included the endolytic hydrolysis of glycosidic linkages and loss of sulfation, particularly N-sulfate groups, and were similar to the degradation processes observed in 0.1 N hydrochloric acid. This study provides initial observations on heparin degradation pathways. More complete, quantitative studies and studies leading to the isolation and characterization of specific degradants are still required.
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PMID:Accelerated stability studies of heparin. 892 82

The effect of oxidized starch (OS) which contained 15% of COOH groups and its nitroether (NOS) with 4% of nitrogen on coagulation properties of rat blood was studied in vitro and in vivo. The results of the study in vitro showed that OS did not affect the function of the coagulation system. In contrast to OS, a dose-dependent increase in prothrombin-, thrombin time, and activated partial thromboplastin time was observed for NOS. The activity of the components of the internal coagulation pathway changed when the NOS concentration reached 0.1 mg/ml. At a concentration of 0.6 mg/ml and higher this compound affect the external pathway and final stage of coagulation. According to the efficiency (in vitro) of the influence on the thrombine time I mg/ml NOS corresponded to 0.2 U/ml of heparine. The anticoagulant effect of NOS was also observed in vivo along with reliable changes in thromboplastin and thrombin time. Antithrombin activity of plasma remained the same. Standard test was negative and indicated to the absence of fibrin monomers. The pronounced anticoagulant effect of NOS in the experiments in vitro and quick response in the experiments in vivo make it possible to consider this compound as anticoagulant of direct action.
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PMID:[The anticoagulant action of the nitric acid ester of oxidized starch]. 897 59

The natural titanium oxide (TiO2) layer of commercial sheet titanium was dissolved in hydrofluoric acid. A new oxide layer was grown by oxidation in nitric acid or by annealing at 700 degrees C in air. At this temperature, reaction with nitrogen is unlikely. The purity of the oxidized sheet-titanium surfaces was investigated by Auger spectroscopy. The composition of both surfaces was TiO2 with carbon impurities. The carbon content of the acid-oxidized titanium was 20 +/- 2%, and the carbon content of the heat-oxidized titanium was 14 +/- 2% The initial reactions of the TiO2 surfaces with blood were investigated by short-time exposure to capillary blood and by detection of surface-adsorbed plasma proteins and cells with immunofluorescence. Antibodies specific to fibrinogen, complement factor C1q, prothrombin/thrombin, and platelet membrane antigen were used, and the fluorescence was quantitated by computer-aided image analysis. The results show that serine proteases are the dominating proteins adsorbed onto annealed titanium (C1q = 67 +/- 4.6; pt/t = 97 +/- 0.2; fib = 47 +/- 0.2). The adsorption of serine proteases was lower and the amount of fibrinogen was higher on the acid-oxidized surface (C1q = 46.3 +/- 2.6; pt/t = 25 +/- 2.9; fib = 64 +/- 0.7). Platelets adhered and spread on the annealed titanium surface within 5 sec of blood-material contact. The number of adhering platelets was higher on the acid-oxidized surface.
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PMID:The initial reactions of TiO2 with blood. 905 32

Thrombin is the key enzyme in the blood coagulation system, and inhibitors of its proteolytic activity are of therapeutic interest since they are potential anticoagulants. The most potent inhibitor of the benzamidine type is N alpha-[(2-naphthylsulfonyl)glycyl]-4-amidinophenylalanylpiperid ide (NAPAP). However, NAPAP and other benzamidine derivatives do not show favorable pharmacological properties; above all, they have very low systemic bioavailability after oral administration. The goal of designing new compounds was to obtain potent inhibitors with improved pharmacokinetic properties. Piperazide derivatives of 3-amidinophenylalanine as the key building block were synthesized. The piperazine moiety opened the possibility to introduce quite different substituents on the second nitrogen using common synthetic procedures. Some of the newly synthesized compounds are potent inhibitors of thrombin and offer an approach to study structure-function relationships for inhibition of thrombin and related enzymes and for the improvement of their pharmacokinetic properties.
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PMID:Synthesis and structure-activity relationships of potent thrombin inhibitors: piperazides of 3-amidinophenylalanine. 930 73

Phosphorylcholine groups attached to polymer surfaces are known to improve hemocompatibility. A photochemical method is presented to couple phosphorylcholine-containing aryl azides to poly(etherurethane) surfaces (PEUs). Two aryl azides that consist of a photoactivatable 4-azidobenzoyl group, a short spacer chain, and a phosphorylcholine endgroup were synthesized. The two compounds differ only in the type of spacer used: triethylene glycol for compound 1 and hexanediol for compound 2. These compounds were physically adsorbed to PEU surfaces. Upon UV irradiation, reactive intermediates are formed that react with nucleophilic groups on the polymer surface. The modified surfaces showed decreased underwater contact angles, indicating that hydrophilic phosphorylcholine groups are present at the surface. ESCA measurements showed the presence of phosphorus and positively charged nitrogen atoms in the outermost polymer layers (analyzed depth about 50 A), which is a strong indication of the presence of phosphorylcholine groups. Hemocompatibility in vitro was tested with thrombin generation assays and platelet adhesion tests. In thrombin generation assays the clotting time of platelet-rich plasma in contact with the polymer surface is determined. Clotting times were clearly prolonged for the modified surfaces. Surfaces modified with compound 2 showed slightly higher clotting times than those modified with compound 1. Repeated surface modification with compound 2 further increased the clotting time. For the tested surfaces an increase in the clotting time corresponds to an increase in the concentration of phosphorylcholine groups at the surface (as measured by ESCA and contact angle). Platelet adhesion studies with scanning electron microscopy demonstrated that fewer platelets (showing less activation) adhered to the modified surfaces than to the unmodified polyurethane.
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PMID:A photochemical method for the surface modification of poly(etherurethanes) with phosphorylcholine-containing compounds to improve hemocompatibility. 935 23


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