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)

A monoclonal antibody (Mab) has been raised against native thrombospondin (TSP), the endogenous lectin of human platelets, that inhibits the hemagglutination of trypsinized, glutaraldehyde-fixed human erythrocytes by purified TSP. This Mab, designated A2.5, also inhibits the agglutination of fixed, activated platelets by TSP. Mab A2.5 immunoprecipitates a 25-kilodalton (kDa) peptide from chymotryptic digests of TSP that is not disulfide bonded to any other region of the TSP molecule. This fragment represents the previously characterized heparin binding domain of TSP [Dixit, V.M., Grant, G.A., Santoro, S.A., & Frazier, W.A. (1984) J. Biol. Chem. 259, 10100-10105]. In agreement with this assignment, heparin inhibits the binding of Mab A2.5 to TSP. Another Mab, designated C6.7, also blocks TSP-mediated hemagglutination, yet has no effect on the agglutination of fixed, activated platelets by TSP. This Mab has been shown to inhibit the thrombin-stimulated aggregation of live platelets and to immunoprecipitate an 18-kDa fragment from chymotryptic digests, which is distinct from the heparin binding domain [Dixit, V.M., Haverstick, D.M., O'Rourke, K.M., Hennessy, S.W., Grant, G.A., Santoro, S.A., & Frazier, W.A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3472-3476].
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PMID:Effects of anti-thrombospondin monoclonal antibodies on the agglutination of erythrocytes and fixed, activated platelets by purified thrombospondin. 405 97

Using an enzyme-linked immunosorbent assay, we have demonstrated that purified human fibrinogen forms a complex with adsorbed platelet thrombospondin. The formation of the fibrinogen-thrombospondin complex was specific, saturable, and partially inhibited by mannosamine, glucosamine, and arginine. These same inhibitors have been previously shown to block thrombin-induced platelet lectin activity and platelet thrombospondin lectin activity. Adsorbed thrombospondin also formed a complex with fibronectin, although the extent of complex formation was significantly less than the extent of formation of the fibrinogen-thrombospondin complex. Platelet membrane glycoproteins IIb and IIIa, which have been previously shown to bind fibrinogen, did not inhibit the formation of the fibrinogen-thrombospondin complex. The present study supports the hypothesis that the interaction of fibrinogen with thrombospondin on the activated platelet surface may be an important step in the platelet aggregation process.
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PMID:Complex formation of platelet thrombospondin with fibrinogen. 621 38

A possible receptor for thrombin on the platelet membrane has been identified. Whole platelets were treated with 125I-labelled thrombin followed by washing of the platelets, solubilization in Triton X-100, crossed immunoelectrophoresis and autoradiography. A heavily labelled antigen which migrated slightly more slowly than albumin was observed. No corresponding arc was seen on the same immunoplate when stained with Coomassie brilliant blue, indicating that the antigen possessed weak antigenic properties and/or was present in very small amounts. When 125I-labelled thrombin that had been inactivated by phenylmethylsulphonyl fluoride was used, no such labelled arc was seen. The radiolabelled immunoprecipitate does not represent any of the antigens identified hitherto in the immunoelectrophoretic patterns obtained with platelets or platelet material. The electrophoretic mobility of the antigen was influenced neither by neuraminidase treatment of the platelets prior to the 125I-labelled thrombin exposure nor by inclusion of concanavalin A, wheat-germ lectin or lentil lectin in the gel during the first-dimension electrophoresis. This suggests that the antigen does not represent a glycoprotein. Upon subcellular fractionation the radioactively labelled arc was observed in the cytosol fraction following crossed immunoelectrophoresis and autoradiography. Analysis of the secreted proteins after induction of the release reaction with 125I-labelled thrombin revealed labelling of immunoprecipitates representing thrombospondin, albumin and the 'line' form of platelet factor 4. This confirms that stable complexes of 125I-labelled thrombin and platelet proteins can exist in the presence of Triton X-100 and during electrophoresis.
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PMID:Demonstration of 125I-labelled thrombin binding platelet proteins by use of crossed immunoelectrophoresis and autoradiography. 630 75

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

Washed human platelets suspended in Ca2+-free buffer bind thrombospondin secreted in response to stimulation by the calcium ionophore A23187. Under these conditions, the secreted thrombospondin binds to the surface of the platelets monovalently, that is, the thrombospondin does not agglutinate the platelets. In addition, the secreted thrombospondin can bind monovalently to the surface of the erythrocytes used to assay the endogenous lectin of human platelets. These findings resolve the contradictions resulting from the apparent requirement for free Ca2+ in the binding of secreted thrombospondin to the plasma membranes of platelets, the behavior of purified thrombospondin in hemagglutination assays and the characteristics of the endogenous lectin (thrombospondin) expressed by platelets stimulated with A23187 or gamma-thrombin.
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PMID:Secreted platelet thrombospondin binds monovalently to platelets and erythrocytes in the absence of free Ca2+. 642 Sep 29

Depending on cell type and mode of growth stimulation, an intact cytoplasmic microtubule system may either support or suppress passage through the prereplicative G1 phase (growth and maturation) and entrance into the S phase (DNA synthesis) of the cell cycle. In peripheral blood lymphocytes exposed to mitogenic lectins, colchicine and other antimicrotubular drugs inhibit blast transformation and initiation of DNA synthesis. The inhibitory effect is not due to decreased cellular binding of lectin or lack of generation of a stimulatory signal. Rather, it can be explained by an inability of the cells to pass through the G1 phase at a normal rate in the absence of cytoplasmic microtubules. The formation of new organelles and the growth in cell size that occur during this phase is markedly delayed by the drugs. For example, the Golgi complex, an organelle system that participates in membrane biogenesis and other basic cellular functions, is reduced in size and structurally disorganized. In cells with a shorter prereplicative phase, such as fibroblasts and smooth muscle cells, antimicrotubular drugs inhibit DNA synthesis in growth-arrested cultures exposed to optimal concentrations of serum, thrombin or platelet-derived growth factor (PDGF). On the other hand, antimicrotubular drugs stimulate DNA replication in serum-free cultures and enhance the stimulatory effect of insulin, epidermal growth factor (EGF), fibroblast growth factor (FGF), and prostaglandin F2 alpha on entrance into S phase. Moreover, stabilization of cytoplasmic microtubules with taxol has been found to block microtubule disassembly and initiation of DNA synthesis by colchicine and to inhibit thrombin- and EGF-stimulated DNA synthesis under serum-free conditions. These findings suggest that partial microtubule disassembly is an inherent step in the reactions that precede DNA replication and mitosis. However, the cell biological and molecular details of these reactions and the exact role of microtubules remain enigmatic.
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PMID:The microtubular cytoskeleton and the initiation of DNA synthesis. 648 51

The amino sugars glucosamine, galactosamine and mannosamine (30 mM) inhibited aggregation of human or rabbit platelets induced by ADP, collagen, thrombin, PAF or high concentrations of sodium arachidonate. 125I-fibrinogen binding during ADP-induced aggregation, and release of amine storage granule contents were also inhibited. Increasing the calcium concentration of the suspending medium to 5 mM did not overcome the inhibitory effect on the release reaction. The amino sugars deaggregated rabbit platelets that had been aggregated by ADP, collagen or thrombin, but deaggregated human platelets readily only when ADP was used as the aggregating agent. Fibrinogen-induced aggregation of chymotrypsin-treated platelets was blocked by the amino sugars. They did not inhibit platelet adherence to a collagen-coated glass surface, nor affect release of granule contents from the adherent platelets. Aggregation and release induced by low concentrations of sodium arachidonate or the divalent cation ionophore A23187 were potentiated, indicating that the effects of the amino sugars on platelets are more complex than simple inhibition of the lectin-like activity that becomes available on the surface of platelets that have undergone the release reaction. One of the effects of the amino sugars, however, is interference with the binding of fibrinogen to platelets. The effects of the amino sugars are shared by other primary amines.
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PMID:Effect of amino sugars on platelet aggregation and on fibrinogen binding. 649 68

Antiserum against a 23Kd heparin binding fragment of thrombospondin inhibits the aggregation of platelets in response to ADP, collagen or thrombin. The antiserum inhibits the secretion-dependent second phase, but not the primary phase of aggregation of platelets responding to ADP. Although immune serum added during the second phase of ADP-induced aggregation causes some inhibition of secretion, it also causes reversal of aggregation to the level produced during primary aggregation. Since thrombospondin is the endogenous lectin of human platelets, these results support the conclusion that the endogenous lectin mediates, at least in part, the secretion-dependent aggregation of platelets. Our data suggest that the region of thrombospondin which contains the heparin binding domain(s) present in the 23Kd fragment play(s) a critical role in secretion-dependent aggregation of platelets.
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PMID:Antibodies against a 23Kd heparin binding fragment of thrombospondin inhibit platelet aggregation. 649 83

Thrombospondin (TSP), the major alpha-granule protein of human platelets, binds to the activated platelet surface upon platelet stimulation. TSP has hemagglutinating (lectin-like) activity and forms a specific complex with fibrinogen. Based on these observations, it was postulated that the interaction of TSP and fibrinogen on the activated platelet surface may be an important step in the platelet aggregation process. To test this hypothesis, monospecific, affinity-purified anti-TSP Fab fragments were prepared and their effects on platelet aggregation and platelet fibrinogen binding were studied. Anti-TSP Fab caused significant interference with thrombin- and collagen-induced platelet aggregation, as monitored by both turbidometric aggregometry and particle counting measuring the disappearance of single platelets. Phase-contrast microscopy revealed that anti-TSP Fab caused a marked decrease in platelet macroaggregates and an increase in microaggregates and nonaggregated single platelets. Anti-TSP Fab did not affect the initial phase of ADP-induced platelet aggregation but caused rapid platelet disaggregation with the abolition of the secondary phase of aggregation. The effect of anti-TSP Fab was not mediated by a direct inhibition of platelet secretion. The effect of anti-TSP Fab on specific binding of labeled fibrinogen to thrombin-stimulated platelets was also studied. Anti-TSP Fab caused a marked decrease in the affinity of fibrinogen binding to the receptors on the activated platelet surface. Kinetic analyses revealed significant displacement of labeled fibrinogen by unlabeled fibrinogen in the presence of anti-TSP Fab, suggesting that TSP serves to stabilize fibrinogen binding to the activated platelet surface and reinforces the strength of interplatelet interactions. It is proposed that platelet aggregation is a dynamic, multistep process, governed initially by the platelet membrane glycoprotein IIb/IIIa-fibrinogen interaction, with the TSP-fibrinogen interaction playing an important role in determining the size and reversibility of platelet aggregates.
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PMID:Role of thrombospondin in platelet aggregation. 650 68

Concanavalin A dimer interacts with fibrinogen and soluble fibrin at pH 5.2 Analysis of the binding data shows that there are in both cases four binding sites per molecule and that the dissociation constant does not change by removal of fibrinopeptides A and B. Ultracentrifugal studies show that no aggregates of fibrinogen or fibrin are formed through concanavalin A binding and that up to four molecules of concanavalin A dimer can bind to one molecule of fibrinogen or fibrin. These results imply that the four carbohydrate chains in the molecule are accessible to concanavalin A dimer. There is a diminution in the coagulation of fibrinogen by thrombin at low relative lectin concentrations and an increase at high concentrations. However, the lectin always favours the aggregation of fibrin monomers and does not have any inhibitory effect on the release of fibrinopeptides. We conclude that the electric charge in the neighbourhood of the carbohydrate in both chains, B beta and gamma plays an important role in the attraction between monomeric fibrin and fibrinogen-monomeric fibrin. The different effect of concanavalin A on the coagulation, depending on the relative concentration of the lectin, would be the result of the screening of this electric charge favouring either the interaction of fibrinogen-monomeric fibrin or the polymerization of monomeric fibrin.
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PMID:Fibrinogen and fibrin interaction with concanavalin A dimer and its influence on coagulation. 672 79


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