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)

Plasma fibronectin binds in a specific and saturable manner to thrombin-stimulated platelets. gamma-Thrombin stimulated 80% as much fibronectin binding to platelets as alpha-thrombin with conversion of less than or equal to 1% of platelet fibrinogen to fibrin. Afibrinogenemic and normal platelets bound similar quantities of fibronectin in the presence of calcium or magnesium-ethylene glycol tetra-acetic acid (EGTA). These observations indicate that fibronectin can interact with platelets without involvement of fibrin or fibrinogen. Nevertheless, two different effects of fibrin(ogen) on fibronectin binding were observed. First, exogenous fibrinogen inhibited fibronectin binding to thrombin-stimulated platelets. This inhibition was unidirectional, as fibronectin did not inhibit fibrinogen binding to ADP or thrombin-stimulated cells. Second, formaldehyde-fixed cells with surface-associated fibrin bound significant quantities of fibronectin. This interaction required calcium and did not occur on fixed cells with or without surface-bound fibrinogen. A portion of the ligand bound to fixed cells with surface-associated fibrin was modified to form a derivative with a molecular weight identical to that of the fibronectin subunit cross-linked to the alpha-chain of fibrin. This high mol wt derivative was also observed to a variable extent with living cells in the presence of magnesium or calcium but not in the presence of magnesium-EGTA. Thus, fibronectin binds to platelets by at least two mechanisms: (1) a fibrin(ogen)-independent pathway that requires divalent ions and is inhibited by exogenous fibrinogen; and (2) a fibrin-dependent pathway with an absolute calcium requirement. With nonaggregated, thrombin-stimulated platelets, the former pathway appears to predominate.
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PMID:Fibronectin binding to thrombin-stimulated platelets: evidence for fibrin(ogen) independent and dependent pathways. 400 31

Thrombospondin (TSP) is a glycoprotein secreted from the alpha-granules of platelets upon activation. In the presence of divalent cations, the secreted protein binds to the surface of the activated platelets and is responsible for the endogenous lectin-like activity associated with activated platelets. Platelets fixed with formaldehyde following activation by thrombin are agglutinated by exogenously added TSP. Fixed, nonactivated platelets are not agglutinated. The platelet agglutinating activity of TSP is optimally expressed in the presence of 2 mM each of Mg2+ and Ca2+. Reduction of the disulfide bonds within the TSP molecule inhibits its platelet agglutinating activity. TSP bound to the surface of fixed, activated platelets can be eluted by the addition of disodium ethylenediaminetetraacetate. This approach was exploited to identify the region of the TSP molecule containing the platelet binding site. The binding site resides within a thermolytic fragment of TSP with Mr 140 000 but is not present in the Mr 120 000 fragment derived from the polypeptide of Mr 140 000. Since both the Mr 140 000 and 120 000 fragments contain fibrinogen binding sites, this finding suggests that the binding of TSP to the platelet surface requires interaction with other platelet surface components in addition to fibrinogen. The observation that fibrinogen only partially inhibits the TSP-mediated agglutination of fixed, activated platelets is consistent with this interpretation.
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PMID:Characterization of the platelet agglutinating activity of thrombospondin. 402 36

The localization of thrombin receptors on mouse embryo (ME) cells was examined using electron microscope (EM) immunocytological techniques. ME cells were fixed with formaldehyde, prior to thrombin binding, and thrombin visualized on cell surfaces using affinity-purified antithrombin rabbit antibody and colloidal gold labeled anti-rabbit IgG. Colloidal gold particles were found in clusters on the surface of cells incubated with thrombin. There were approximately seven particles per cluster observed in thin sections with cluster diameters ranging from 70 to 200 nm. These clusters were not observed on cells incubated without thrombin. The total number of particles present on cells incubated with and without thrombin indicate that the colloidal gold labeling is approximately 98% specific for thrombin. Only four colloidal gold particles out of approximately 1,200 were associated with coated pits. Thus the thrombin receptor clusters do not appear to associate with coated membrane regions. To determine whether receptor-bound thrombin was internalized by receptor-mediated endocytosis, ME cells were incubated with 125I-thrombin and examined using EM autoradiography and the trypsin sensitivity of 125I-thrombin which was associated with the cells. In two types of experiments, where thrombin was incubated with cells at 4 degrees C and the temperature increased to 37 degrees C and where initial incubation was at 37 degrees C, the receptor-directed specific internalization proceeded at approximately the same rate as nonspecific internalization. These studies indicate that thrombin that binds to its receptors on ME cells is not rapidly internalized by receptor-mediated endocytosis.
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PMID:Receptor-bound thrombin is not internalized through coated pits in mouse embryo cells. 613 25

A method is described for the kinetic measurement of alpha-granule secretion by platelets. The method uses formaldehyde as a secretion-blocking reagent. This treatment alters the antigenecity of beta-thromboglobulin but not of Platelet Factor 4, both measured with commercially available reagents. Evidence is shown that this formaldehyde effect does not alter the secretion kinetics when the data are expressed as a percentage of a similarly treated reference sample. The method shows that following stimulation with thrombin or A 23187 alpha-granule secretion is much slower than dense granule secretion.
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PMID:Kinetic analysis of alpha-granule secretion by platelets. A methodological report. 621 67

To define better the role of the fibrinogen receptor in platelet physiology and to characterize it biochemically, a murine monoclonal antibody that completely blocks the binding of fibrinogen to the platelet surface was produced by the hybridoma technique with the aid of a functional screening assay. Purified F(ab')2 fragments and/or intact antibody completely blocked aggregation induced by ADP, thrombin, or epinephrine and the binding of radiolabeled fibrinogen to platelets induced by ADP. The antibody did not block agglutination of formaldehyde-fixed platelets by ristocetin or shape change induced by either ADP or thrombin. ADP- and epinephrine-induced release of ATP was completely inhibited by the antibody, but inhibition of release induced by collagen and thrombin was dose dependent and partial. The antibody also dramatically inhibited platelet retention in glass-bead columns, platelet adhesion to glass, and clot retraction. Thus, the antibody induced a thrombasthenic-like state. Immunofluorescent studies confirmed the specificity of the antibody for normal platelets and megakaryocytes and suggested that there is a marked decrease in detectable antigen in thrombasthenic platelets. Radiolabeled antibody bound to an average of approximately 40,000 sites on normal platelets but it bound to less than 2,000 sites on the platelets of a patient with thrombasthenia. The antibody immunoprecipitated both glycoproteins IIb and IIIa, and both glycoproteins bound to an affinity column of the antibody. These studies indicate that there is probably a single anatomic site that is crucial to the binding of all fibrinogen molecules and that this site is most likely on the glycoprotein IIb/IIIa complex. It also suggests that the thrombasthenic phenotype can be completely accounted for on the basis of the inhibition of fibrinogen binding to platelets.
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PMID:A murine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal platelets and binds to glycoproteins IIb and/or IIIa. 630 50

Thrombin binding to formaldehyde-fixed mouse embryo (ME) cells was visualized by indirect immunofluorescence as a dot-like pattern with dots of approximately 500 nm diameter located over the entire cell surface. Experiments comparing the binding of 125I-thrombin and dot appearance on parallel cultures indicate that the immunofluorescent pattern is specific for thrombin-binding to high-affinity receptors. Similar patterns were observed on cells fixed in ethanol or glutaraldehyde prior to thrombin binding and on cells maintained at 4 degrees C. These patterns were also observed in a number of established cell lines. Thus, thrombin receptors may be clustered prior to thrombin binding on all cells with these receptors. Comparing the amount of 125I-thrombin bound to CHO cells with the number of fluorescent dots per cell indicated that each dot represents a cluster of over 1000 receptors. On ME cells, the number of thrombin receptor clusters per cell ranged from fewer than 50 to over 5,000. Based on previous studies, this indicates that on ME cells each cluster contains an average of approximately 200 thrombin binding sites with some cells having few, if any, receptors and others having more than a million receptors per cell.
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PMID:Immunofluorescent visualization of specifically bound thrombin reveals cellular heterogeneity in number and density of preclustered receptors. 631 99

The effect of heparin on platelet aggregation was systematically examined on platelets in plasma (PRP), as well as on gel-filtered, washed, and formaldehyde-fixed platelets. Results indicate that, although heparin causes a mild potentiation of platelet aggregation in the PRP systems, a significant inhibitory activity is observed when heparin is added to isolated platelets. This inhibitory activity appears to be specific and not related to the impurities in the heparin preparations, as heparinase, as well as protamine, effectively neutralizes the heparin-mediated inhibitory activity on platelet aggregation. Although heparin-mediated inhibitory activity can be demonstrated in the presence of a number of different agonists (ADP, arachidonic acid, thrombin, Ionophore A23187, epinephrine, and ristocetin), the most pronounced inhibition is seen in the presence of ristocetin. Further studies show that heparin enhances thromboxane generation in isolated platelets. Platelets pretreated with heparin, however, fail to respond to preformed thromboxane. These findings suggest that, in addition to the potentiation of thromboxane production in platelets, heparin may also attribute some change(s) to the platelet(s)/platelet membrane, which interferes with their ability to respond to the agonists of platelet aggregation. This antiaggregatory activity of heparin was found to be inhibited by a factor(s) present in plasma but not in serum.
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PMID:Effect of heparin on platelet aggregation. 647 40

von Willebrand factor (VWF) functions in platelet aggregation, a form of cellular interaction. In vitro analysis of platelet aggregation, as measured by the platelet aggregometer, requires addition of a promoter such as the glycopeptide antibiotic ristocetin. Native multimeric VWF (Mr = 1-20 X 10(6)) can be reduced with sulfhydryl reagents to a monomeric state (Mr = 2 X 10(5)). In this study, the binding of bovine VWF and ristocetin to bovine platelets was investigated using fluorescence anisotropy of derivatized monomer protein and ristocetin and also by radioisotope methods using 125I-labeled monomer and native protein. Ristocetin bound to bovine platelets but not to VWF. VWF binding to formaldehyde-fixed platelets was dependent on the presence of a promoter such as ristocetin. The monomer and multimer VWF bound equally well in the presence of low ristocetin concentrations. Under these conditions, plots of VWF binding versus platelet concentration were sigmoidal, indicating positive cooperativity with respect to platelets. At higher (100 micrograms/ml) ristocetin concentrations, the binding curve was no longer sigmoidal. Ristocetin promoted the formation of small platelet aggregates, an effect that was amplified by the presence of VWF. In fact, all conditions which resulted in monomer or multimer VWF binding to platelets also caused formation of platelet aggregates observed by light microscopy. These combined results were consistent with VWF binding only to the interface between proximal platelets. High affinity binding could be provided by the presence of two cell surfaces and the resulting multiple binding interactions. Polycations, such as poly(L-lysine) and Polybrene, also promoted the formation of platelet aggregates and facilitated the binding of VWF to platelets. Physiological platelet activators such as thrombin, ADP, and collagen also facilitated VWF binding to native platelets and caused platelet aggregation. It appears possible that any process which causes the surface membranes of platelets to become spatially close will allow expression of VWF activity.
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PMID:von Willebrand factor. A protein which binds at the cell surface interface between platelets. 660 42

Dipeptidyl argininal (arginine aldehyde) affinity resins of general formula R-(X-Y-argininal) (where R = resin matrix and X, Y = amino acids of varied structure) are synthesized in a solid-phase procedure in which the dipeptide (-X-Y-) is first attached to the resin, followed by the joining of the Y amino acid to argininal semicarbazone, and decomposition of the semicarbazone in a methanol/acetic acid/formaldehyde reagent. An R-(Gly-Gly-argininal) resin binds urokinase tightly, but does not bind thrombin. However, thrombin binds strongly to R-(Phe-Pro-argininal), whereas urokinase does not bind. Accordingly, the X-Y-argininal ligands selectively bind proteinases of identical primary binding site specificity to arginine, but different secondary site specificity in -X-Y-. The selectivity is due to an amplification of peptide binding specificity caused by the transition-state analog properties of the ligands. While the affinity constants between peptide aldehyde and proteinase approach those of antibody-antigen interactions, the elution with semicarbazide (aldehyde-trapping reagent) buffers easily remove tightly bound proteinases without proteinase inhibitors or denaturation. Conditions for the binding and elution of proteinases, methods of regeneration and other characteristics of the resins are described.
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PMID:Transition-state affinity chromatography of trypsin-like proteinases with dipeptidyl argininal ligands. 662 59

The characteristics of spontaneous platelet aggregation (SPA) in a hereditary giant platelet syndrome (Montreal platelet syndrome, MPS) are examined. SPA was quantitated by microscopy from the decrease in single platelets in platelet-rich plasma (PRP). In contrast to normal donors, a significant proportion (20-50%) of platelets in MPS whole blood and PRP occurred in microaggregates typically containing 2-6 disk-shaped platelets. Stirring MPS-PRP at 1000 rpm for 10 minutes further increased the fraction of platelets in aggregates by 10-170%, the percentage increase not being correlated to the donor's platelet count (5000-220,000 microliters-1). Normal platelets resuspended in MPS platelet-poor plasma (PPP) did not undergo SPA, whereas MPS platelets resuspended in normal PPP or Ca2+-free, fibrinogen-free Tyrode's continued to show SPA. The increase in SPA could be inhibited by 10 microM prostaglandin (PG) E1, 150 mM ASA or glutaraldehyde or formaldehyde fixation; however, it was not inhibited by 10 nM PGI2 and was only partially inhibited by 1 microM 2-chloroadenosine and 1-10 units/ml apyrase. SPA in Acid-citrate-dextrose-PRP was much less than in PRP; however, SPA reoccurred on returning the platelets to platelet-free plasma or Tyrode's. Platelet aggregation (PA) could be increased over that due to SPA alone by the addition of adenosine diphosphate, adrenaline, collagen, ionophore A-23187, arachidonic acid and ristocetin, with results suggesting that the response to these agents is normal. The ristocetin-induced increase in PA was completely blocked by an IgG specific for Bernard-Soulier syndrome. In contrast, MPS platelets had a reduced sensitivity to thrombin, which appeared to be more pronounced at low platelet counts. There was no correlation between the thrombin insensitivity and the extent of SPA. Total adenosine triphosphate (ATP) and thrombin-induced release of ATP and platelet factor 4 appeared normal for MPS platelets. The ultrastructural features of MPS platelets were within normal limits except for an increased frequency of giant granules. SPA was observed for 5/5 MPS donors, but only one of three MPS donors' platelets evaluated for glycoprotein I and sialic acid content showed any measurable reduction as compared with normal controls. The above observations point to the existence of an as yet undetermined anomaly of MPS plasma membrane related to a fibrinogen and Ca2+ independent form of platelet aggregation.
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PMID:Spontaneous platelet aggregation in a hereditary giant platelet syndrome (MPS). 669 46


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