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)

Human plasma fibronectin was denatured with 8 M urea and reduced with dithiothreitol. Dialysis or dilution of the solution led to formation of fibronectin dimers which migrated in non-reducing SDS/PAGE similarly to untreated control protein. When the redimerized fibronectin was reduced and re-electrophoresed it formed a doublet of alpha and beta chains of equal intensity indicating that it was a heterodimer. Low concentrations (less than 1 mM) of Fe3+ enhanced the redimerization of fibronectin, suggesting that metal ions may mediate oxidative reactions in the formation of the disulfides. Consequently, redimerization of fibronectin was completely prevented by deferoxamine, an iron chelator. Dimerization of fibronectin took place most effectively at pH greater than or equal to 8.8 but decreased strongly at lower pH, representing more unfavourable conditions for the action of the thiolate anion in the thiol/disulfide exchange reaction. Redimerized fibronectin, however, lost many of its binding properties to macromolecular ligands, suggesting that the disulfide bonding did not entirely regenerate the proper conformation of the protein. Pulse/chase experiments of fibroblast cultures showed that the initially monomeric fibronectin was rapidly and quantitatively dimerized under conditions representing natural pH and environment. SDS/PAGE analysis of the dialyzed urea-denatured/reduced thrombin and plasmin digests of fibronectin revealed that the NH2-terminal 30-kDa fragment and other fragments that contained intrachain disulfides quantitatively regained their non-reduced electrophoretic mobility. The results show that the dimerization and formation of intrachain disulfides of fibronectin may occur, in part, spontaneously, based on the amino acid sequence information of the protein. However, complete disulfide formation may also need other factors, present only in living cells, as suggested by pulse/chase experiments in fibroblasts.
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PMID:Disulfide-bonded dimerization of fibronectin in vitro. 176 Oct 59

Recent studies have revealed a role for platelets and the platelet-adhesive proteins, fibronectin and von Willebrand factor (vWF) in platelet-tumor cell interaction in vitro and metastasis in vivo. The present report documents the effect of thrombin treatment of platelets on this interaction in vitro and in vivo. In vitro, thrombin at 100-1,000 mU/ml maximally stimulated the adhesion of six different tumor cell lines from three different species two- to fivefold. As little as 1-10 mU/ml was effective. The effect of thrombin was specific (inhibitable by hirudin, dansyl-arginine N-(3-ethyl-1,5 pentanediyl) amide and unreactive with the inactive thrombin analogue N-P-tosyl-L-phenylchloromethylketone-thrombin and D-phenylalanyl-L-propyl-L-arginine chloromethylketone-thrombin (PPACK-thrombin), and required high-affinity thrombin receptors (competition with PPACK-thrombin but not with N-P-tosyl-L-lysine-chloromethyl-ketone-thrombin). Functionally active thrombin was required on the platelet surface. Binding of tumor cells to thrombin-activated platelets was inhibitable by agents known to interfere with the platelet GPIIb-GPIIIa integrin: monoclonal antibody 10E5, tetrapeptide RGDS and gamma chain fibrinogen decapeptide LGGAKQAGDV, as well as polyclonal antibodies against the platelet adhesive ligands, fibronectin and vWF. In vivo, thrombin at 250-500 mU per animal increased murine pulmonary metastases fourfold with CT26 colon carcinoma cells and 68-413-fold with B16 amelanotic melanoma cells. Thus, thrombin amplifies tumor-platelet adhesion in vitro two- to fivefold via occupancy of high-affinity platelet thrombin receptors, and modulation of GPIIb-GPIIIa adhesion via an RGD-dependent mechanism. In vivo, thrombin enhances tumor metastases 4-413-fold with two different tumor cell lines.
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PMID:Thrombin stimulates tumor-platelet adhesion in vitro and metastasis in vivo. 184 69

Substantial amounts of macromolecular activators of phagocytosis from platelets (MAPPs) were released in response to exposure of platelets to the specific agonists thrombin and collagen and to calcium ionophore A23187. Centrifugation of the platelets in culture medium also resulted in a release of MAPPs, but not when the platelets were frozen and thawed prior to centrifugation. In an experiment using outdated platelet concentrates, larger and smaller MAPPs (1-MAPP and s-MAPP, respectively) were purified from the thrombin stimulated supernatant (PRPr-plasma) by sequential steps of ammonium sulfate precipitation, delipidation with ethylacetate, ConA-Sepharose affinity chromatography, MONO Q anion exchange chromatography, and Superose 12 gel filtration. This procedure yielded 59,500-fold and 63,600-fold purified 1-MAPP (0.95 mg) and s-MAPP (0.41 mg), respectively, from 1,320 ml PRPr-plasma containing 84,500 mg protein. By gradient polyacrylamide gel electrophoresis the respective molecular weights (MW) of 1-MAPP and s-MAPP were 290-320 kd and 140-160 kd; isoelectric points were 5.6 and 4.6. Both MAPPs activated neutrophil phagocytosis of IgG-SRBC at concentrations in the range of 1 pM-100 nM. Indirect enzyme-linked immunosorbent assay (ELISA) and comparisons of the concentrations required for phagocytosis activation suggested that the MAPPs were not derived from fibronectin.
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PMID:Purification and characterization of macromolecular phagocytosis activators released from platelets. 191 63

Essential mixed cryoglobulinemia (EMC) is a rheumatic disorder characterized by widespread vasculitis. To better define the nature of the vasculitic process and to possibly outline assessment methods reliable for using in a clinical context, we studied plasma levels of three endothelial related peptides: fibronectin (FN), von Willebrand factor (vWF) and tissue plasminogen activator (t-PA), and those of thrombin-antithrombin III complexes (TAT) as markers of activation of the coagulation in 21 patients and in 16 controls. In EMC we found a picture consisting of reduced FN and increased vWF, t-PA, and TAT levels, suggesting a condition of endothelial cell damage with thrombin formation in vivo. Since we previously demonstrated the presence of chronic disseminated intravascular coagulation in these patients, we may assume that endothelial cells stressed by cryoprecipitation or stimulated by soluble mediators may be actively involved in the vasculitic process and possibly express procoagulant properties. This is a good example of the complex interplay existing between autoimmunity and coagulation mechanisms. We also suggest that FN, vWF, t-PA and TAT should be considered as additional clinical parameters when evaluating patients with EMC.
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PMID:Clinical significance of endothelial damage markers in essential mixed cryoglobulinemia. 195 Mar 76

The elastic modulus (G') of factor XIIIa induced fibrinogen gels was found to be substantially lower than the G' of fibrin gels that were formed by clotting fibrinogen with thrombin. The addition of fibronectin and/or the reducing reagent dithiothreitol (DTT) to the factor XIIIa coagulation mixture led to the formation of a weaker gel structure, while the rigidity of thrombin induced clots was not appreciably affected by the inclusion of the DTT but increased somewhat in the presence of fibronectin. The reasons for the differing clot rigidities are discussed in terms of biochemical mechanisms.
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PMID:The elastic modulus of fibrin clots and fibrinogen gels: the effect of fibronectin and dithiothreitol. 197 Apr 86

The interaction of Factor XIII with cultured fibroblasts was examined using 125I-labeled protein and immunofluorescence. Platelet or plasma Factor XIII bound to confluent cell layers. Binding reached an apparent steady state after 8 h. Activation with thrombin increased the binding of both the platelet and plasma forms of the enzyme. After a 1-2 h lag, a chloroquine-inhibitable increase in trichloroacetic acid-soluble radioactivity was detected in the medium. Gel electrophoresis in sodium dodecyl sulfate indicated that approximately 16-fold more a subunit (catalytic) of 125I-plasma Factor XIII bound to the cell layer than b subunit (carrier) and that some large complexes containing Factor XIII were formed with the cell layer. Factor XIII binding increased linearly with concentrations of Factor XIII up to 230 micrograms/ml, whereas a component of the degradation of Factor XIII was saturable at about 20 micrograms/ml. Factor XIII associated with cell layers was catalytically active since it could cross-link fibronectin. By immunofluorescence the a subunit of Factor XIII was localized to fibronectin-containing extracellular fibrils and, in the presence of chloroquine, to intracellular granules. These results indicate that the a subunit of Factor XIII binds to the fibroblast extracellular matrix and matrix assembly sites, where it remains active, and to a putative cell-surface receptor which mediates its internalization and degradation.
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PMID:Binding and degradation of blood coagulation factor XIII by cultured fibroblasts. 197 26

C-reactive protein (CRP) is an acute phase inflammatory protein in man which binds to phosphocholine, chromatin, histones, and the 70-kDa protein of the U1 small nuclear ribonucleoprotein particle in a calcium-dependent, phosphocholine-inhibitable manner. CRP also binds to other proteins including fibronectin. The determinants involved in CRP binding to these diverse proteins have not been identified. The binding of CRP to histones was examined as these proteins are available in large quantity at high purity and subject to protease digestion with well characterized products. Histone H1 was digested with thrombin and trypsin to produce three distinct fragments, N-terminal, central globular, and C-terminal. CRP was shown only to bind to the C-terminal fragment. Binding to histone H2A was also examined. CRP binding was not diminished by cleavage of the C-terminal fragment but was greatly decreased when the central globular region of H2A was tested. Peptides were prepared to be identical to the N- and C-terminal fragments of H2A. The N-terminal (15 amino acid) fragment of H2A blocked CRP-induced precipitation of phosphocholine-coupled bovine serum albumin and histone H2A, whereas the C-terminal fragment showed no inhibition. Thus we have defined the first reported CRP binding determinant on a protein.
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PMID:Definition of a C-reactive protein binding determinant on histones. 198 77

Thrombin, a serine protease generated at wound sites, takes part in multiple biological functions, including wound healing. The present report elucidates the effect of thrombin on fibronectin (FN) synthesis and secretion in fibroblasts and epithelial cells. Subconfluent cultures of mouse LB fibroblasts and human A549 epithelial cells were exposed to various concentrations of bovine plasma thrombin at 37 degrees C for 16 hr. After exposure, cells were processed for determination of cell-associated and secreted FN by metabolic labeling, immunoprecipitation, immunofluorescence, and peroxidase immunocytochemistry. The correlation of FN production with cell growth was studied by a combined procedure of peroxidase immunocytochemistry and light microscopic autoradiography. The amounts of cell-associated and secreted FN were significantly increased with dose increments of thrombin. The increases were most evident in secreted FN. The increase of cell-associated FN was also evidenced by results from immunofluorescence and immunocytochemical studies. Ultrastructurally, the intracellular FN was localized in rough endoplasmic reticulum, Golgi complexes, and secretory granules, whereas non-released extracellular FN was localized in the plasma membrane of cell-to-cell contacts and in the extracellular fibrils. More intense cytoplasmic FN staining was observed in cells that were not labeled with [3H]-thymidine, indicating that FN production may vary with different phases of cell growth. The results imply that thrombin may play an important role in the early phases of wound healing.
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PMID:Thrombin stimulation of synthesis and secretion of fibronectin by human A549 epithelial cells and mouse LB fibroblasts. 200 71

In order to investigate whether a fibrin-fibronectin-containing matrix of a peripheral regeneration chamber could promote the growth of central nervous system neurons, hippocampal and septal slices were co-cultured in the presence of this acellular substrate. In introducing the peripheral matrix into a 2-mm-long tube between hippocampal and septal slices, a spatio-temporal sequence of cell migration and axonal growth was described by light and electron microscopy. Axons were able to elongate directly into the flocculent material constituting the matrix and a possible neurite-promoting activity was implicated in this process as axonal growth was not detected in direct contact with rat plasma coagulated with calcium, or chicken plasma coagulated with thrombin, used as control matrices. However, in the 3 different substrates tested, astrocytes were able to migrate and dilated astroglial processes containing intermediate filaments were detected. Axonal processes were observed growing on the glial cell surface. GFAP-positive phagocytic cells, that could be of the same origin as astrocytes, were involved in matrix removing. Neuronal growth and glial migration arose from hippocampal and septum slices and acetylcholinesterase-containing fibers were seen in the bridging structure suggesting that cholinergic axons were able to progress to the hippocampal slice. This technique appeared to provide a model in which axonal growth and cell migration can be studied 'in vitro' in a 3-dimensional environment.
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PMID:Axonal growth and glial migration from co-cultured hippocampal and septal slices into fibrin-fibronectin-containing matrix of peripheral regeneration chambers: a light and electron microscope study. 205 10

A method has been developed for preparing suspensions of washed human platelets that have lost as much as 90% of their dense granule and alpha granule contents as a result of stimulation by thrombin (0.9 U/ml for 3 min at 37 degrees C), and recovering the platelets without using a proteolytic enzyme. Glycyl-L-prolyl-L-arginyl-L-proline (GPRP) was used to prevent polymerization of released fibrinogen and arginyl-glycyl-aspartyl-serine (RGDS) to block the interaction of released fibrinogen, vWf or fibronectin with the glycoprotein IIb/IIIa complex. The thrombin used to degranulate the platelets was neutralized with D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (FPRCH2Cl) and prostaglandin E1 was added to return the platelets towards a disc shape. The degranulated platelets aggregated in response to ADP, platelet activating factor, arachidonate and the thromboxane A2 mimetic, U46619 in the presence of added fibrinogen; the platelets changed shape but did not aggregate in response to collagen. Thrombin and the calcium ionophore, A23187, caused aggregation without added fibrinogen. Synergism between pairs of aggregating agents at low concentrations was observed. Little TXB2 was formed when the platelets were reaggregated by thrombin. RGDS and F(ab')2 fragments of an antibody to fibrinogen inhibited reaggregation induced by thrombin and A23187 indicating that small amounts of fibrinogen at the platelet surface may support aggregation by strong agonists. Adherence of thrombin-degranulated platelets to a collagen-coated surface was less than for controls, but spreading was more extensive. Electron-microscopic immunogold cytochemistry with anti-human fibrinogen IgG showed numerous gold particles in platelet vacuoles.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characteristics of thrombin-degranulated human platelets: development of a method that does not use proteolytic enzymes for deaggregation. 205 23


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