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)

Purified thrombin from an exogenous source is a hemostatic agent commonly used in neurosurgical procedures. The toxicity of thrombin in the brain, however, has not been examined. This study was performed to assess the effect of thrombin on brain parenchyma, using the formation of brain edema as an indicator of injury. Ten microliters of test solution was infused stereotactically into the right basal ganglia of rats. The animals were sacrificed 24 hours later, and the extent of brain edema and ion content were measured. Concentrations of human thrombin as low as 1 U/microliter resulted in a significant increase in brain water content. Rats receiving 10 U/microliters had a mortality rate of 33% compared to no mortality in the groups receiving smaller doses. Thrombin-induced brain edema was inhibited by a specific and potent thrombin inhibitor, hirudin. A medical grade of bovine thrombin commonly used in surgery also caused brain edema when injected at a concentration of 2 U/microliters. Edema formation was prevented by another highly specific thrombin inhibitor, N alpha-(2-Naphthalenesulfonylglycyl)-4-DL-phenylalaninepiperidid e (alpha-NAPAP). Thrombin-induced brain edema was accompanied by increases in brain sodium and chloride contents and a decrease in brain potassium content. Changes in brain ions were inhibited by both hirudin and alpha-NAPAP, corresponding to the inhibition of brain water accumulation. This study shows that thrombin causes brain edema when infused into the brain at concentrations as low as 1 U/microliter, an amount within the range of concentrations used for topical hemostasis in neurosurgery.
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PMID:Intracerebral infusion of thrombin as a cause of brain edema. 749 Jun 19

The effect of ibuprofen on thrombin-induced pulmonary edema was studied in rats. Thrombin infusion produced a significant increase in lung weight, wet weight/dry weight ratio and relative lung water content, a rise in mean pulmonary arterial pressure and a fall in mean systemic arterial pressure. It also caused a progressive decrease in PaO2 and a continuous increase in pH and PaCO2. Administration of either the S-isomer or R-isomer of ibuprofen at doses of 5 mg/kg body weight prior to thrombin infusion resulted in significant reduction in lung weight, wet weight/dry weight ratio and water content. The wet weight/dry weight ratio and the water content were somewhat lower after infusion of the S-isomer than of the R-isomer. Ibuprofen diminished the thrombin-induced increase in mean pulmonary arterial pressure and attenuated the early and late decrease in mean systemic arterial pressure caused by thrombin. Ibuprofen also stabilized thrombin-induced impairments in PaO2, PaCO2 and pH. The results thus indicate that ibuprofen effectively counteracts hemodynamic changes, stabilizes impairments in arterial blood gas variables and attenuates the increase in lung vascular permeability to protein with pulmonary edema caused by thrombin. The results also indicate a substantial R to S chiral inversion of ibuprofen in vivo in the rat.
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PMID:Effect of ibuprofen on thrombin-induced pulmonary edema in the rat. 754 27

The preventive effect of indomethacin on thrombin-induced pulmonary edema was studied in rats. Administration of thrombin caused a significant increase in lung weight, wet weight to dry weight ratio (WW/DW), and relative lung water content. During infusion of thrombin, mean pulmonary artery pressure rose and mean systemic artery pressure fell, PaO2 decreased progressively and there was a continuous rise in pH and PaCO2. An inhibitor of cyclooxygenase, indomethacin, at a dose of 1 mg/kg body weight, induced a significant further increase in lung weight (p < 0.05), and a tendency towards an increase in WW/DW and water content compared with animals given thrombin alone. Treatment with indomethacin, however, counteracted the elevated pulmonary artery pressure occurring in the early phase after thrombin infusion, but not that in the late phase. Systemic artery pressure was not affected by indomethacin. The increases in pH and PaCO2 after thrombin infusion were attenuated and remained stable almost at baseline level after indomethacin administration. Indomethacin did not prevent the hypoxemia induced by thrombin infusion. In conclusion, although indomethacin prevented the early increase in pulmonary artery pressure due to thrombin and the decrease in pH and the increase in PaCO2, it caused lung vascular permeability to protein to increase more than with thrombin alone.
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PMID:Effect of indomethacin on thrombin-induced pulmonary edema in the rat. 757 Nov 66

Intra-alveolar clot formation is a common finding in acute and chronic inflammatory lung diseases. Incorporation of lipophilic surfactant components into a growing fibrin clot has recently been reported (Am. J. Respir. Cell Mol. Biol. 1993; 9:213-220). In the present study, we investigated the influence of such surfactant incorporation on the elastic properties and water permeability of the fibrin polymer. Thrombelastography and compaction experiments were employed for assessment of the elastic properties, and the permeability characteristics of the clot material were addressed in fibrin-packed columns. Two calf lung surfactant extracts (CLSE and Alveofact), Curosurf, and a synthetic phospholipid mixture (dipalmitoylphosphatidylcholine, phosphatidylglycerol, and palmitic acid at a ratio of 68.5:22.5:9 [wt/wt]) were used. The presence of surfactant did not affect the cleavage of fibrinopeptide A upon incubation of fibrinogen with thrombin (enzyme-linked immunosorbent assay technique). Similarly, kinetics and extent of factor XIII-induced covalent crosslinkage of the fibrin network remained unchanged in the presence of surfactant (sodium dodecyl sulfate polyacrylamide gel electrophoresis and D-Dimer quantification upon subsequent clot lysis). All surfactants, however, dose-dependently decreased the elastic modulus of the arising fibrin polymer. The maximal amplitude in thrombelastography was reduced, and the recovery of fluid after centrifugation of the fibrin clot increased. Fibrin clots embedding natural surfactant material displayed reduced permeability for saline as compared with control fibrin polymers. Subsequent washout of lipids from these clots with Triton X-100 resulted in increased hydraulic conductivity. This was accompanied by an increase in pore size, suggesting altered architecture of the fibrin matrix generated in the presence of surfactant.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Surfactant incorporation markedly alters mechanical properties of a fibrin clot. 757 9

The ligand-binding domain of the low-density lipoprotein receptor comprises seven cysteine-rich repeats, which have been highly conserved through evolution. This domain mediates interactions of the receptor with two lipoprotein apoproteins, apo E and apo B-100, putatively through a calcium-dependent association of the ligands with a cluster of acidic residues on the receptor. The second repeat (rLB2) of the receptor binding domain has been expressed as a thrombin-cleavable GST fusion protein, cleaved, and purified. On oxidation the protein refolded to give a single peak on reverse-phase HPLC. The aqueous solution structure of rLB2 has been determined using two-dimensional 1H NMR spectroscopy. In contrast to the amino-terminal repeat, rLB1, rLB2 has a very flexible structure in water. However, the conformation of rLB2 is markedly more ordered in the presence of a 4-fold molar excess of calcium chloride; the proton resonance dispersion and the number of NOESY cross-peaks are greatly enhanced. The three-dimensional structure of rLB2, obtained from the NMR data by molecular geometry and restrained molecular dynamics methods, parallels that of rLB1, with an amino-terminal hairpin structure followed by a succession of turns. However, there are clear differences in the backbone topology and structural flexibility. As for rLB1, the acidic residues are clustered on one face of the module. The side chain of Asp 37, which is part of a completely conserved SDE sequence thought to be involved in ligand binding, is buried, as is its counterpart (Asp 36) in rLB1. These results provide the first experimental support for the hypothesis that each of the repeats in the ligand-binding domain has a similar global fold but also highlight significant differences in structure and internal dynamics.
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PMID:Three-dimensional structure of the second cysteine-rich repeat from the human low-density lipoprotein receptor. 757 52

The interaction of rabbit lung thrombomodulin (TM) and C-terminal hirudin 54-65 fragment (Hir54-65) with human alpha-thrombin were investigated by exploiting their competitive inhibition of thrombin-fibrinogen interaction. Measurements of Ki values for TM and Hir54-65 interactions with human alpha-thrombin performed over a temperature range spanning from 10 to 40 degrees C showed a constant enthalpy for both ligands. The enthalpic and entropic contributions to the free energy of binding, however, are different for TM and the hirudin peptide. The calculated values of delta H and delta S, in fact, were -47.3 +/- 2.51 kJ (-11.3 +/- 0.6 kcal)/mol and -42.7 +/- 7.9 J (-10.2 +/- 1.9 cal)/mol.K for the hirudin peptide, while being -22.9 +/- 2.09 kJ (-5.47 +/- 0.5 kcal)/mol and 102.50 +/- 6.69 J (24.5 +/- 1.6 cal)/mol.K respectively for TM binding. These findings indicate that the interaction between thrombin and Hir54-65 is largely driven by the enthalpic contribution, whereas the positive entropy change is the driving force for the formation of the thrombin-TM complex. In other experiments performed in the presence of various concentrations of either sorbitol or sucrose it could be demonstrated that the value of the equilibrium association constant for thrombin-TM interaction increases as a function of the osmotic pressure, while the thrombin-Hir54-65 interaction was not affected by the same conditions. Moreover, control experiments showed that no major conformational changes are produced on TM by osmotic pressures used in the present study. From these experiments it was calculated that roughly 35 water molecules are released into the bulk water upon TM binding. Such a phenomenon, which is likely to be responsible for the entropic change described above, indicates the relevance of hydration processes for the formation of the thrombin-TM adduct.
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PMID:Thrombin-thrombomodulin interaction: energetics and potential role of water as an allosteric effector. 764 71

Thrombin is an allosteric serine protease existing in two forms, slow and fast, targeted toward anticoagulant and procoagulant activities. The slow --> fast transition is induced by Na+ binding to a site contained within a cylindrical cavity formed by three antiparallel beta-strands of the B-chain (Met180-Tyr184a, Lys224-Tyr228, and Val213-Gly219) diagonally crossed by the Glu188-Glu192 strand. The site is shaped further by the loop connecting the last two beta-strands and is located more than 15 A away from the catalytic triad. The cavity traverses through thrombin from the active site to the opposite surface and contains Asp189 of the primary specificity site near its midpoint. The bound Na+ is coordinated octahedrally by the carbonyl oxygen atoms of Tyr184a, Arg221a, and Lys224, and by three highly conserved water molecules in the D-Phe-Pro-Arg chloromethylketone thrombin. The sequence in the Na+ binding loop is highly conserved in thrombin from 11 different species and is homologous to that found in other serine proteases involved in blood coagulation. Mutation of two Asp residues flanking Arg221a (D221A/D222K) almost abolishes the allosteric properties of thrombin and shows that the Na+ binding loop is also involved in direct recognition of protein C and antithrombin.
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PMID:The Na+ binding site of thrombin. 767 82

A new nonbiologic photopolymerizable glue, polyethyleneglycol 400 diacrylate, was studied with respect to its mechanical and biochemical interaction with human blood vessels. Using the human placental artery model, we tested the ability of polyethyleneglycol 400 diacrylate to prevent leakage of blood at the site of vascular anastomoses, which are made porous by the presence of tissue gaps and suture puncture sites. Fibrin glue is known to augment local vessel thrombogenicity through the presence of the coagulation enzyme thrombin. We tested the effect of externally applied polyethyleneglycol 400 diacrylate (which does not contain thrombin) on luminal thrombin activity and platelet deposition from flowing human blood. At a shear rate of 312 per second and a transmural pressure of 80 cm H2O, the leakage rate of saline from human placental artery anastomoses was 1.0 +/- 1.2 ml/min (n = 8). When the same anastomoses were then glued, 7 of 8 of the anastomoses leaked less than 0.05 ml/min (p < 0.05). Platelet deposition to human vessels was not influenced by the external application of polyethyleneglycol 400 diacrylate either on intact vessels (no polyethyleneglycol 400 diacrylate, 0.51 +/- 0.28 x 10(6) platelets/cm2; with polyethyleneglycol 400 diacrylate, 0.47 +/- 0.26 x 10(6) platelets/cm2; n = 7) or at anastomoses (no polyethyleneglycol 400 diacrylate, 0.69 +/- 0.36 x 10(6) platelets/cm2; with polyethyleneglycol 400 diacrylate, 0.53 +/- 0.33 x 10(6) platelets/cm2; n = 8), p > 0.05.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A new photopolymerizable blood vessel glue that seals human vessel anastomoses without augmenting thrombogenicity. 770 75

Anhydrothrombin, a catalytically inactive derivative of thrombin in which dehydroalanine replaces the active-site serine, was prepared by a novel method. The active-site serine of thrombin was modified to dehydroalanine by promoting the beta-elimination of phenylmethylsulfonic acid from phenylmethylsulfonyl fluoride-inactivated thrombin under conditions in which the enzyme is unfolded. After the elimination reaction was quenched, the resulting anhydrothrombin was folded by diluting the denaturant, Gdn.HCl, to nondenaturing concentrations. Anhydrothrombin was purified by PAB affinity chromatography. Both native thrombin and anhydrothrombin were digested by cyanogen bromide, and the peptides from the region of the active-site serine (S205) were isolated by reverse-phase high-pressure liquid chromatography. Serine was present in the native thrombin peptide but absent from the anhydrothrombin peptide, as shown by amino acid analysis. This anhydrothrombin peptide was found to be 18.7 +/- 1.6 lower in mass units than the native peptide by electrospray mass spectrometry, in accord with the elimination of a water molecule. The anhydrothrombin preparation was monomeric, as determined by sedimentation equilibrium. Anhydrothrombin was used in a competitive titration of the complex of native thrombin with the leech saliva protein hirudin, a potent thrombin inhibitor, as measured by the recovery of thrombin amidolytic activity. This demonstrated that anhydrothrombin is capable of nativelike binding interactions with macromolecular ligands.
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PMID:Preparation and characterization of anhydrothrombin. 775 77

The X-ray crystal structure of prethrombin2 (pre2), the immediate inactive precursor of alpha-thrombin, has been determined at 2.0 A resolution complexed with hirugen. The structure has been refined to a final R-value of 0.169 using 14,211 observed reflections in the resolution range 8.0-2.0 A. A total of 202 water molecules have also been located in the structure. Comparison with the hirugen-thrombin complex showed that, apart from the flexible beginning and terminal regions of the molecule, there are 4 polypeptide segments in pre2 differing in conformation from the active enzyme (Pro 186-Asp 194, Gly 216-Gly 223, Gly 142-Pro 152, and the Arg 15-Ile 16 cleavage region). The formation of the Ile 16-Asp 194 ion pair and the specificity pocket are characteristic of serine protease activation with the conformation of the catalytic triad being conserved. With the determination of isomorphous structures of hirugen-thrombin and D-Phe-Pro-Arg chloromethyl ketone (PPACK)-thrombin, the changes that occur in the active site that affect the kinetics of chromogenic substrate hydrolysis on binding to the fibrinogen recognition exosite have been determined. The backbone of the Ala 190-Gly 197 segment in the active site has an average RMS difference of 0.55 A between the 2 structures (about 3.7 sigma compared to the bulk structure). This segment has 2 type II beta-bends, the first bend showing the largest shift due to hirugen binding. Another important feature was the 2 different conformations of the side chain of Glu 192. The side chain extends to solvent in hirugen-thrombin, which is compatible with the binding of substrates having an acidic residue in the P3 position (protein-C, thrombin platelet receptor). In PPACK-thrombin, the side chain of Asp 189 and the segment Arg 221A-Gly 223 move to provide space for the inhibitor, whereas in hirugen-thrombin, the Ala 190-Gly 197 movement expands the active site region. Although 8 water molecules are expelled from the active site with PPACK binding, the inhibitor complex is resolvated with 5 other water molecules.
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PMID:The isomorphous structures of prethrombin2, hirugen-, and PPACK-thrombin: changes accompanying activation and exosite binding to thrombin. 775 83


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