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 effects of lysoPC, four other amphiphiles containing a linear 16 carbon alkane tail and chlorpromazine on platelet cytoskeletal assembly were compared. LysoPC and nonmetabolized amphiphiles all caused time-dependent inhibition followed by potentiation of thrombin-induced aggregation, serotonin secretion and cytoskeletal assembly in gel-filtered platelets, a result which ruled out hydrolysis of the amphiphiles as the mechanism of the time dependence. Hexadecanesulfonate was superior as a potentiator and cetyltrimethyl ammonium bromide (CTAB) was a better inhibitor. On the contrary, inhibition of platelet activation by arachidonate was not effected in a time-dependent manner and the actin-crosslinking proteins, actin-binding protein and myosin, were selectively prevented from incorporation into cytoskeletal cores, although protein phosphorylation and actin polymerization still occurred. Chlorpromazine also showed this selective inhibition of cytoskeletal assembly. LysoPC at concentrations which have been reported to cause development of filopodia did increase slightly the amount of actin present in Triton X-100-insoluble cores but not protein phosphorylation or incorporation of actin-crosslinking proteins. The effective concentrations of lysoPC and chlorpromazine can be predicted from the Meyer-Overton-Mullins rule of anesthesia which indicates their general effectiveness, but their specific effects only partially overlap.
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PMID:The effects of lysophosphatidylcholine and related amphiphiles on platelet cytoskeletal assembly. 609 54

Aggregation of washed rabbit platelets by thrombin and by carrageenan is accompanied by the activation of phospholipase A2 and by the synthesis of thromboxanes. Accordingly, aggregation, the accompanying release reaction and the activation of phospholipase are blocked by p-bromophenacyl bromide and by CB 874 (2,3-dibromo (4'-cyclohexyl-3'-chloro)-phenyl-4-oxo-butyric acid), two recognized inhibitors of the enzyme. Since these two reagents also inhibit aggregation and the release reaction induced by thrombin and by carrageenan on washed human platelets, it might have been anticipated that the mechanisms of aggregation of the platelets from the two species are similar. Nevertheless, no thromboxanes A2 or B2, nor activation of phospholipase A2 could be demonstrated with the use of carrageenan on human platelets, under conditions where thrombin was effective. It is concluded that carrageenan activates the human platelets by phospholipase A2- and thromboxane A2-independent mechanisms, and that the inhibitors of phospholipase A2 may block platelet functions by mechanisms other than inhibition of the expected enzyme.
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PMID:Carrageenan-induced activation of human platelets is independent of phospholipase A2 and of formation of thromboxanes. 611 Jul 20

External stimulate Na+ efflux from platelet plasma membrane vesicles. Efflux is apparently electrogenic since K+ diffusion potentials induced with valinomycin (interior positive) accelerate and potentials of the opposite polarity (interior negative) inhibit. In the presence of stimulatory anions, voltage-dependent Na+ efflux is much faster than Na+-Na+ exchange in the absence of an induced membrane potential. Anions stimulate voltage-dependent efflux in the following order: SCN- greater than I- greater than NO3- greater than Br- approximately acetate approximately Cl- greater than F- approximately SO42- greater than HPO42-, gluconate, and isethionate. Thiocyanate, the most stimulatory anion, increases Na+ efflux 20-fold in the presence of a membrane potential (interior positive). Stimulation of efflux by Cl- is a saturable phenomenon with a K0.5 of 41 mM and a maximal 2-3-fold stimulation over the basal level of efflux. Neither basal nor valinomycin-stimulated efflux was influenced by the presence of the platelet-aggregating agents thrombin, epinephrine, or ADP in the presence of fibrinogen.
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PMID:Anion-dependent sodium ion conductance of platelet plasma membranes. 626 84

An inactive derivative of wheat germ agglutinin, which is a strong activator of blood platelets, was prepared by selective chemical modification of the lectin with cyanogen bromide at acid pH. The derivative was then used as a probe to learn about the initial events in platelet stimulation by physiological agents. Amino acid analysis of the modified lectin confirmed specific cleavage of a methionine residue. Gel filtration studies indicated a molecular weight for the lectin derivative similar to the unmodified lectin. In gel electrophoresis in the presence of sodium dodecyl sulfate, reduced samples of the derivative showed two bands and the main component migrated slightly faster than the native lectin. The derivative retained the capacity to precipitate an antibody to the lectin although at least one of the antigenic sites was lost due to chemical modification. The derivative did not compete with the unmodified lectin for binding to platelets. Unlike the parent lectin, the derivative did not aggregate platelets even at a ten fold higher concentration. Under similar conditions, there were about 1.0 X 10(5) binding sites/platelet for the lectin derivative with an apparent dissociation constant of 1.7 microM compared to 5 X 10(5) sites/cell and a dissociation constant of 0.4 microM for the native lectin. Overnight incubation of platelets or red cells with the derivative in microtiter plates showed about 2-5% agglutinating activity for the derivative compared to the unmodified lectin. Incubation of platelets with the lectin derivative inhibited platelet aggregation by thrombin while aggregation induced by a number of other agents was not significantly affected. This inhibitory effect of the lectin derivative on thrombin-induced platelet aggregation could be readily reversed with GlcNAc. The lectin derivative may be a useful tool to explore the structure-function relationship of cell surface components.
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PMID:Preparation and some properties of a derivative of wheat germ agglutinin with altered biological activity. 641 Nov 30

Cardiotoxin, isolated from Naja naja atra snake venom, potentiates platelet aggregation induced by ADP, thrombin, collagen and venom phospholipase A2. The malondialdehyde formation caused by ADP, thrombin and venom phospholipase A2 were also increased in the presence of cardiotoxin. Both potentiation of aggregation and increase in malondialdehyde were blocked by indomethacin or Ca2+ (5 mM or 0.05 mM). Cardiotoxin did not potentiate thrombin-induced aggregation of p-bromophenacyl bromide-modified platelets. Thromboxane B2 formation induced by thrombin or collagen was also increased by cardiotoxin, while that by arachidonate was not affected. As a membrane-active polypeptide, cardiotoxin might augment the Ca2+-flux during the activation of the platelet membrane by aggregation inducers and then increase the activation of endogenous phospholipase A2.
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PMID:Cardiotoxin from Naja naja atra snake venom: a potentiator of platelet aggregation. 647 95

A single tryptophan residue on antithrombin has been modified with dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide. This alteration led to a 500-fold reduction in the heparin-dependent acceleration of thrombin-modified antithrombin interactions, as well as a 10-fold decrease in the avidity of the modified protease inhibitor for mucopolysaccharide. Preincubation of antithrombin with the octasaccharide binding domain of heparin prior to treatment with dimethyl-(2-hydroxy-5-nitrobenzyl) sulfonium bromide was able to suppress modification of the critical tryptophan and preserve the functional capacities of the protease inhibitor. Fluorescence quenching experiments indicated that the modifiable tryptophan groups of antithrombin were exposed to the solvent environment. Based upon these data, it was proposed that the loss of "heparin cofactor" activity of antithrombin must be predominantly due to an inability of the modified protease inhibitor to undergo a conformational transition required for mucopolysaccharide-dependent "activation" of the macromolecule.
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PMID:The role of tryptophan residues in heparin-antithrombin interactions. 648 8

It is known that storage at pH 6 stabilizes thrombin against inactivation. In order to determine whether structural changes accompany this stabilization, the conformation of human alpha-thrombin at pH 6.0 and 7.5 was investigated by chemical modification, difference spectroscopy, circular dichrosim, and thermal stability. It was shown that the CD spectra at the 230-200 nm peptide transition were indistinguishable at the two pH values, indicating no differences in the secondary structure as also indicated by the thermal stability of the enzyme at pH 6.0, 7.4 and 8.3. However, differences were observed in the 300-250 nm aromatic transition suggesting some changes in the microenvironment of the aromatic chromophores. Solvent perturbation in 20% ethylene glycol and 20% dimethylsulfoxide showed that at pH 7.5, 4.3 +/- 0.3 tryptophan and 8.6 +/- 0.4 tyrosine residues were exposed and accessible to the solvent whereas at pH 6.0 these values were 3.6 +/- 0.1 tryptophan and 7.8 +/- 0.4 tyrosine residues. At pH 7.5, 6.0 +/- 0.5 tryptophan residues were found reactive toward dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide while 2.5 +/- 0.3 were found reactive at pH 6.0. Accompanying these structural changes were ultraviolet absorption and CD spectral changes with transition midpoints at pH 6.45 characteristic of histidine ionization. These spectral changes were lost when alpha-thrombin was modified by diethylpyrocarbonate but not by N-alpha-tosyl-L-Lysinechloro-methyl ketone. It is concluded that a second histidine residue, not the active site His-43, is associated with the pH dependent conformational changes at pH 6.0. The ionization of this histidine residue and the accompanying conformational changes could explain the reduced catalytic efficiency and stability of alpha-thrombin at pH 6.
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PMID:Conformational integrity of human alpha-thrombin. 652 46

Collagens were obtained from decalcified human embryonic calvaria by pepsin digestion. After removal of the type I collagen, the more soluble collagens were precipitated at 1.2 M NaCl (acid pH), followed by a selective precipitation step at neutral pH, using a NaCl concentration of 4.5 M. Analysis of this latter precipitate by polyacrylamide slab gel electrophoresis and ion-exchange chromatography revealed the presence of a heterogeneous group of proteins ranging in size from approximately 10 000 daltons to over 120 000 daltons. Proteolysis, as a source for these diverse components, was ruled out both by studies employing protease inhibitors and experiments employing thrombin which indicated that no helical denaturation had occurred during extraction. A comparison with standard collagen preparations suggested that the major bands present in these samples corresponded to the alpha 1-, alpha 2-, and alpha 3-chains of type V collagen. The data also showed that the major helical organization of these chains was [alpha 1(V)]2 alpha 2(V). Data suggesting that proteolysis can occur during the chromatographic separation of the individual alpha-chains are presented. This proteolysis was sensitive to inhibitors and its possible role in modifying the chain composition of type from calvaria and other tissues is discussed. Unique cyanogen bromide peptides distinguishable from those of type I and type V were present in these precipitates and suggests the presence of novel collagen types. The small amounts of these collagens precluded a determination of the exact nature of these components.
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PMID:Heterogeneity in the collagens extracted from human embryonic calvaria. 662 4

Chemical modification of a single tryptophan residue in antithrombin III with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide blocks heparin binding and the heparin-enhanced inhibition of thrombin without altering the heparin-independent rate of thrombin inhibition (Blackburn, M. N., and Sibley, C. C. (1980) J. Biol. Chem. 255, 824-826). The labeled protein was reduced and carboxymethylated and then cleaved with cyanogen bromide. The peptide containing the hydroxynitrobenzyl-labeled tryptophan was isolated by gel filtration and ion exchange chromatography. Amino acid analysis of the labeled peptide indicates that it corresponds to residues 21 through 89 of human antithrombin III. The site of labeling corresponds to Trp 49, which is located within the disulfide-stabilized loops near the NH2-terminal end of the antithrombin III molecule.
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PMID:The heparin-binding site of antithrombin III. Identification of a critical tryptophan in the amino acid sequence. 669 5

Thrombospondin (TS), a protein first described in platelets, was recently shown to be synthesized and secreted by endothelial cells, fibroblasts, and smooth muscle cells. The presence of TS in the extracellular matrix of cultured cells has prompted us to examine the associations of this protein with matrix macromolecules. Interactions of TS with both matrix and serum proteins were tested using an enzyme-linked immunosorbent assay. With this assay we assessed the binding of TS in solution to proteins adsorbed to polystyrene microtiter plates. Among collagens, platelet TS bound to type V but not to types I, III, or IV. This selective interaction was confirmed in experiments using proteins linked to cyanogen bromide-activated Sepharose. TS released from platelets in response to thrombin activation, as well as that secreted by endothelial cells in culture, bound to type V but not to type I collagen-Sepharose. No binding was observed to denatured type V collagen-Sepharose. The binding region for type V collagen was located in a chymotrypsin-produced fragment of TS with chains of Mr = 70,000, after reduction. Interactions of TS with a number of other proteins, including fibronectin, fibrinogen, and laminin, could be demonstrated using the enzyme-linked immunosorbent assay technique but the interpretation of these findings is difficult since comparable binding to protein-Sepharose was not always observed. Our findings suggest that both the extravascular distribution and function of TS in vivo may involve an interaction with type V collagen.
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PMID:Interactions of thrombospondin with extracellular matrix proteins: selective binding to type V collagen. 669 1


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