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)

Phospholipase A2 was solubilized from rat platelet membrane by 1 M KCl and purified to near homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC. The characteristics of the purified membrane-bound enzyme were compared with those of phospholipase A2 released from thrombin-stimulated rat platelets (Horigome, K., Hayakawa, M., Inoue, K., & Nojima, S. (1987) J. Biochem. 101, 625-631). The molecular weights, elution profiles on reversed-phase HPLC, and NH2-terminal sequences were identical for the two enzymes. Other characteristics of the two enzymes, such as specific activity, substrate specificity, pH optimum, Ca2+ requirement, heat lability, and sensitivity to p-bromophenacyl bromide were also indistinguishable. These findings suggest that both enzymes share a common structure.
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PMID:Purification and characterization of membrane-bound phospholipase A2 from rat platelets. 337 90

In contrast to fibroblasts, the exposure of G0/G1-arrested J774 cells, a murine macrophage-like tumor cell line, with either active or esterolytically inactive diisopropyl phosphorofluoridate-conjugated alpha-thrombin (the enzymatically active form of thrombin, EC 3.4.21.5) results in a mitogenic response as measured by increased [3H]thymidine incorporation. This response to thrombin is optimal at 10 nM and is specifically blocked by hirudin, a high-affinity thrombin inhibitor. When prethrombin 1 [a single-chain prothrombin derivative lacking fragment 1, resulting from the action of thrombin on prothrombin] is cleaved with cyanogen bromide, a fragment (peptide CB67-129) is produced that, like the parent thrombin molecule, is mitogenic for J774 cells but not for fibroblasts. Limited tryptic digests of this fragment retain the ability to stimulate macrophages--a function that can be mimicked by a synthetic tetradecapeptide homologue of CB67-129 (representing residues 367-380 of the human thrombin B chain sequence) but not by any of a series of well-known growth promoters, including platelet-derived growth factor, epidermal growth factor, nerve growth factor, and fibroblast epidermal growth factor, nerve growth factor, and fibroblast growth factor. The mitogenic effects of this peptide are not limited to J774 cells but can be expressed in other macrophage-like tumor cell lines, including P388D1, RAW, and PU5. In addition to increased [3H]thymidine incorporation, the synthetic B chain peptide stimulates cell proliferation as evidenced by a dose-dependent increase in total protein per culture well and cell number. We conclude that the thrombin molecule contains a macrophage growth factor domain that is separate and distinct from its active center. Thus, thrombin, in addition to its major role in hemostasis and thrombosis, may also have important functions in such basic processes as the inflammatory response and monocytopoiesis.
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PMID:Identification of a thrombin sequence with growth factor activity on macrophages. 345 76

The advent of sophisticated computer graphics systems that permit the representation of macromolecular structure has made it possible to examine protein structure in detail. We have used one aspect of this technology to develop a model of thrombin. The model is based on structural and functional similarities this enzyme exhibits with respect to proteins found in the family of serine proteinases. This review has covered interpretations of the structure of the model based on analyses of data that had been collected before and after the model was developed. On one hand, the conceptualization of primary and secondary features in the model of the active site of thrombin has for the most part been preceded by data from experiments on the interaction of thrombin with naturally occurring substrates and inhibitors. The features of the model explain these data adequately. On the other hand, the model has been more recently used in an interactive way to derive information about the bioregulatory aspects of thrombin. The realization that the amino-terminus portion of the cyanogen-bromide fragment was probably not part of the chemotactic activity, because it was probably internalized in the native protein, has suggested that synthetic analogs should focus more on the carboxyterminus of the peptide. It is hoped that in the future the model will continue to serve more in this function and that it can be used to explore further other aspects about the structural and functional relationships of this enzyme.
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PMID:Structure-function relationships of thrombin based on the computer-generated three-dimensional model of the B chain of bovine thrombin. 355 28

We report on three unrelated individuals with the same uncommon type of dysfibrinogenemia, originating from Bergamo, Essen and Perugia. None of them showed bleeding symptoms while the Bergamo patient and members of her family presented with a thrombotic tendency. The presence of a defective fibrinogen was suggested by prolonged thrombin and reptilase times. Furthermore, fibrinogen concentrations of less than 0.28 g/L were determined by the functional assay whereas values of 1.5-2.4 g/L were measured by heat precipitation or electroimmunoassay. Fibrinogen was isolated by affinity chromatography on insoluble fibrin monomer. The rate of fibrinopeptide release by thrombin was normal while the fibrin polymerization reaction was strongly delayed. An abnormal peptide (gamma 265-310) was isolated by high-performance liquid chromatography after cyanogen bromide cleavage of the purified gamma-chain of fibrinogen Bergamo II and Essen. The same peptide was also isolated following cyanogen bromide treatment of the intact fibrinogen Perugia. Sequence analyses of these peptides demonstrated the same amino acid exchange in all three fibrinogens: gamma 275 arginine----histidine. The described fibrinogen variants appear to possess a molecular defect which has thus far only been observed in fibrinogen Haifa.
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PMID:Three abnormal fibrinogen variants with the same amino acid substitution (gamma 275 Arg----His): fibrinogens Bergamo II, Essen and Perugia. 356 70

The role of antithrombin conformation in heparin-catalyzed inhibition of thrombin was investigated using antithrombins modified with the tryptophan reagent dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide (HNB). Affinity fractionation of HNB-labeled antithrombin (0.6-0.7 mol of HNB/mol of protein) on heparin-Sepharose using a linear salt gradient allowed separation of three singly labeled protein species and a fourth HNB-antithrombin species which co-eluted with unlabeled protein. Conformational alterations induced by heparin binding to each of the labeled antithrombins were assessed by spectroscopic techniques, including protein fluorescence, difference spectroscopy in the ultraviolet-visible range, and circular dichroism. Comparison of spectra of the labeled proteins in the presence and absence of added heparin indicated changes to occur in protein conformation at the sites of the bound HNB moieties and at aromatic amino acid residues within the protein matrix. These spectroscopic alterations mimicked changes induced by heparin in the native protein, but were reduced in magnitude. Rates of thrombin inactivation by the labeled antithrombins were measured over a wide range in both heparin concentration and inhibitor concentration to determine maximal rates of protease inactivation. The kinetic analysis indicated that each of these HNB-antithrombin derivatives, which undergo the heparin-induced changes to varying extents, can react with thrombin at the same maximal rate. Thus, this series of chemically modified antithrombin species demonstrated that the conformational change which is induced in antithrombin by heparin does not render the protein intrinsically more reactive toward thrombin.
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PMID:Antithrombin conformation and the catalytic role of heparin. II. Is the heparin-induced conformational change in antithrombin required for rapid inactivation of thrombin? 358 27

An abnormal fibrinogen was found in two asymptomatic members (father and daughter) of the same family, originating from northern Italy. Routine coagulation studies revealed prolonged thrombin and reptilase clotting times. Plasma fibrinogen levels, as determined by a functional assay, were markedly diminished, whereas the heat precipitation method indicated normal fibrinogen values. On the basis of these findings, a tentative diagnosis of dysfibrinogenemia was made, and according to the accepted nomenclature, this fibrinogen variant was called "fibrinogen Milano l." The time course of fibrinopeptide A and B release from fibrinogen Milano l was normal, but the aggregation of fibrin monomers was delayed. Two-dimensional electrophoresis of reduced variant fibrinogen chains showed a defective gamma-chain with increased cathodic mobility. An abnormal electrophoretic mobility was observed also for the gamma-chain remnants of fibrinogen fragments D1 and D2 derived from fibrinogen Milano l, whereas the charge anomaly was lost after a further digestion by plasmin to D3, suggesting that the structure abnormality of this variant is situated in the region gamma 303-356. An abnormal peptide was isolated after cyanogen bromide cleavage of intact fibrinogen Milano l. This fragment spans from position gamma 311 to gamma 336. Amino acid analysis of the abnormal peptide showed the presence of valine and a diminished content of aspartic acid. Sequence analysis demonstrated an amino acid exchange Asp----Val in the gamma-chain at position 330.
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PMID:Characterization of fibrinogen Milano I: amino acid exchange gamma 330 Asp----Val impairs fibrin polymerization. 370 59

Ficoll-thrombin purified suspensions of bovine, equine, ovine, and porcine peripheral blood lymphocytes were fractionated on nylon-wool columns. The percentages of surface immunoglobulin (SIg+)-bearing lymphocytes in the adherent (B-cell enriched) and nonadherent (T-cell enriched) fractions were determined for individual animals using fluorescein isothiocyanate conjugated species-specific anti-Ig sera. Subsequently, the human leukocyte antigen DR-specific monoclonal antibody, H4, was tested for its ability to recognize a cross-reactive antigen on the fractionated lymphocytes, using the microcytotoxicity technique. The H4 plus complement killed a percentage of lymphocytes equivalent to the percentage of SIg+ lymphocytes in the adherent and nonadherent fractions. In a parallel experiment, a 2 fluorochrome technique was used to visualize bovine lymphocytes that were SIg+ and H4+. Lymphocytes that were SIg+ also stained with ethidium bromide (orange fluorescence) after complement-mediated cytotoxicity. Seemingly, H4 recognizes an evolutionarily conserved major histocompatibility complex encoded class-II-like determinant on the B lymphocytes of cattle, horses, sheep, and swine.
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PMID:Cross-reactivity of a monoclonal antibody with bovine, equine, ovine, and porcine peripheral blood B lymphocytes. 389 40

The metabolism of [3H]PAF-acether ([1',2'-3H]alkyl-2-acetyl-sn-glycero-3-phosphorylcholine ([3H]alkylacetyl-GPC)) by rabbit platelets was investigated using thin-layer chromatography and high-performance liquid chromatography followed by radioactivity detection. After 2 h of incubation at 37 degrees C, 90 +/- 5.3% of [3H]PAF-acether taken up by the platelets were converted into a product identified as sn-2 long-chain acyl analogue ([3H]alkylacyl-GPC) which was incorporated in the membranes. This conversion was independent from extracellular calcium and was completely inhibited by platelet pre-exposure to 2 mM phenylmethylsulfonyl fluoride, a serine hydrolase inhibitor, which failed to inhibit the uptake of [3H]PAF-acether by the cells. The 2-deacetylated derivative, lyso-[3H]PAF-acether was found to be an intermediate of the conversion of [3H]PAF-acether into [3H]alkylacyl-GPC in platelet homogenates. Platelet stimulation with 2.5 U/ml of thrombin induced a reduction (16.5 +/- 2.2%) of its content of [3H]alkylacyl-GPC, accompanied by the release of [3H]PAF-acether and lyso-[3H]PAF-acether to the medium. These effects were suppressed by the phospholipase A2 inhibitor, p-bromophenacyl bromide. Our results demonstrate that intact platelets convert exogenous PAF-acether into alkylacyl-GPC, which can serve as the precursor of PAF-acether released during stimulation. The existence of a metabolic cycle for the uptake, the release and the inactivation of PAF-acether by platelets is suggested.
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PMID:1-O-alkyl-2-acyl-sn-glycero-3-phosphorylcholine is the precursor of platelet-activating factor in stimulated rabbit platelets. Evidence for an alkylacetyl-glycerophosphorylcholine cycle. 396 37

Heparin was covalently bound to solid substrate surfaces by means of four different chemistries. It was coupled to polymethylacrylate (PMA) beads with glutaraldehyde, carbodiimide, or radical polymerization initiated by Ce4+, or to agarose beads with cyanogen bromide. Each of these chemistries produced measurable amounts of surface-bound heparin, which was minimally elutable in contact with plasma. Antithrombin (AT) binding by heparinized PMA materials (compared with PMA control beads) ranged from no AT binding for the material heparinized with carbodiimide (PMA-Alb-Hep(EDC] to 3.6 micrograms/ml packed beads for the material heparinized by radical polymerization (PMA-MA-Hep). Heparin-like catalytic activity of these materials (assayed by measuring the generation of thrombin-antithrombin complex in plasma) correlated well with the amount of heparin bound, but not as well with AT binding capacity. Heparinized agarose, which exhibited a large AT binding capacity (2.2 mg AT per milliliter of packed gel), had virtually no catalytic activity because of its inability to release thrombin-antithrombin complex from the surface. Platelet interaction with heparinized materials that exhibit high AT binding capacity was reduced by pretreatment with normal plasma but not by pretreatment with AT-depleted plasma. Platelet interaction with heparinized materials with low AT binding capacities was not reduced by pretreatment with normal plasma. We conclude that AT binding by heparin reduces the platelet reactivity of heparinized surfaces.
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PMID:Catalytic activity and platelet reactivity of heparin covalently bonded to surfaces. 397 61

The mechanisms by which adenosine triphosphate, thrombin, and trypsin cause relaxation of vascular smooth muscle were investigated. Relaxation of the rat thoracic aorta with adenosine triphosphate, thrombin, and/or trypsin was associated with increased levels of cyclic guanosine monophosphate in both time- and concentration-dependent manners. Thrombin and trypsin did not alter cyclic adenosine monophosphate levels, whereas adenosine triphosphate increased cyclic adenosine monophosphate levels after significant relaxation occurred. Removal of the endothelium abolished adenosine triphosphate-, thrombin-, and trypsin-induced relaxation and the associated increased levels of cyclic nucleotides. Relaxation due to these agents was also inhibited by exposure to nordihydroguaiaretic acid, a lipoxygenase inhibitor, and eicosatetraynoic acid, a lipoxygenase and cyclooxygenase inhibitor. Indomethacin, a cyclooxygenase inhibitor, potentiated relaxation to these agents, whereas the increased levels of cyclic nucleotides due to adenosine triphosphate were unaltered. Bromophenacyl bromide, a phospholipase A2 inhibitor, decreased relaxation due to adenosine triphosphate, thrombin, and trypsin and the associated increased levels of cyclic nucleotides. Removal of extracellular calcium, which also presumably inhibits phospholipase A2, prevented the elevated levels of cyclic nucleotides and the inhibitory effects of adenosine triphosphate and trypsin on contraction. In contrast, sodium nitroprusside-induced relaxation and/or increased levels of cyclic guanosine monophosphate were unaltered by nordihydroguaiaretic acid, eicosatetraynoic acid, bromophenacyl bromide, and removal of extracellular calcium. After incubation of intact tissue with 32P-orthophosphate, the patterns of protein phosphorylation caused by adenosine triphosphate, thrombin, and trypsin were indistinguishable from those of acetylcholine, sodium nitroprusside and 8-bromo cyclic guanosine monophosphate. All these agents dephosphorylated myosin light chain. Thus, the present study supports the hypothesis that relaxation induced by adenosine triphosphate, thrombin, and trypsin is mediated through the formation of an endothelial factor which elevates cyclic guanosine monophosphate levels and causes cyclic guanosine monophosphate-dependent protein phosphorylation and dephosphorylation of myosin light chain.
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PMID:Mechanisms of adenosine triphosphate-, thrombin-, and trypsin-induced relaxation of rat thoracic aorta. 609 35


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