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)

We have identified and biochemically characterized an antigen, 8A3, which is expressed on activated T lymphoblasts and activated platelets. Monoclonal antibodies to 8A3 were raised against the primitive lymphoid/myeloid cell line KG1a and additionally bound to the erythroleukemia-derived cell line HEL, whilst exhibiting little or no reactivity with a panel of other hematopoietic cell lines. The 8A3 antigen was expressed on poorly differentiated T-cell leukemias and on phytohemagglutinin-activated T-cells maintained in interleukin-2 (7,000 sites/cell). This antigen, though not detected on resting platelets, was expressed on thrombin-activated platelets (2,000 sites/platelet). Antibodies to 8A3 identified polypeptides of Mr 170,000 and 150,000 in lysates of surface-iodinated KG1a cells, T lymphoblasts, and activated platelets under both reducing and nonreducing conditions. However, peptide mapping and susceptibily to glycosidases indicated that the 8A3 antigen was a monomeric glycoprotein of Mr 170,000 which contained two N-linked endoglycosidase H-sensitive glycans, and that the Mr 150,000 structure was derived from it by proteolytic degradation. The 8A3 antigen was not detectably phosphorylated in KG1a cells in vivo, nor did immune complexes containing it exhibit kinase activity in vitro. Structural and serologic characteristics of the 8A3 antigen indicate that it is different from other previously described leukocyte activation antigens including transferrin receptors, interleukin-2 receptors, members of the integrin family of adhesion molecules, or "restricted" members of the leukocyte-common antigen/CD45 cluster. Furthermore, the 8A3 antigen does not appear to be related to the other previously described activation-specific platelet molecule, GMP140/PADGEM. This antibody may be useful in monitoring T-cell activation status in some clinical situations and in characterizing clinically relevant activation-associated platelet membrane alterations.
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PMID:Identification of a cell-surface antigen associated with activated T lymphoblasts and activated platelets. 198 5

Located close to the crown of the V3 type-specific neutralization loop of the human immunodeficiency virus type 1 (HIV-1) (IIIB) SU glycoprotein gp120, are several potential sites that should be susceptible to proteolytic cleavage by enzymes of trypsinlike or chymotrypsinlike specificity, or by aspartic proteinases. The linkages potentially sensitive to chymotryptic/aspartic proteinase cleavage are retained also within the equivalent domain of HIV-2 (ROD) gp105. We show that thrombin and tryptase cleave HIV-1 gp120 specifically at the tryptic site (GPGR decreases AFVT), and that cathepsin E, an endosomal aspartic proteinase, cleaves at the chymotrypsinlike site (GPGRAF decreases VT). HIV-2 gp105 is also cut by cathepsin E at a site (QIML decreases MSGH) in its V3 loop. Cleavage of HIV-1 gp120 by thrombin is enhanced by sCD4 binding, but is prevented by transient exposure of gp120 to nonionic detergent. Thrombin treatment of HIV-1 gp120 destroys the binding sites for some neutralizing monoclonal antibodies (MAbs) on the V3 loop, but does not affect the affinity of gp120 for sCD4. Conversely, binding of neutralizing MAbs to the HIV-1 V3 loop prior to addition of thrombin or cathepsin E blocks the cleavage reactions, and the binding of some HIV-positive sera to gp120 blocks thrombin cleavage. Analysis of published sequences suggests that all HIV-1, HIV-2, and simian immunovirus (SIV) isolates contain potential proteolytic cleavage sites at similar positions in their V3 loops or equivalent domains. We suggest that cleavage of the V3 loop by a cell surface or endosomal proteinase occurs during the HIV-cell fusion reaction, and that neutralizing antibodies directed against the V3 loop might act by inhibition of this reaction.
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PMID:The V3 loops of the HIV-1 and HIV-2 surface glycoproteins contain proteolytic cleavage sites: a possible function in viral fusion? 201 14

In this study, the question of whether glycoprotein Ib (GPIb) mediates both high and moderate affinity pathways of alpha-thrombin-induced platelet activation was examined. Flow cytometric studies, using a panel of monoclonal antibodies (MoAbs), showed that Serratia marcescens protease treatment removed greater than 97% of the glycocalicin portion of GPIb but did not affect the changes in the expression of GPIX or GMP-140 that were induced by high concentrations of alpha-thrombin (10 nmol/L). However, Serratia treatment almost completely abolished the increase in platelet surface GMP-140 induced by low concentrations of alpha-thrombin (0.5 nmol/L) and diminished the downregulation of platelet surface GPIX by 60.9% +/- 5.6% (mean +/- SEM, n = 3). When present in 20-fold molar excess, an MoAb directed against the alpha-thrombin/von Willebrand factor (vWf) binding domains of GPIb completely blocked the ristocetin-dependent binding of vWf to platelets but inhibited only to about 50% the binding of alpha-thrombin and the activation-dependent binding of vWf. In platelets treated with Serratia marcescens protease to remove GPIb, a concentration of this MoAb 16,000-fold in excess of the maximum possible remaining copies of GPIb failed to inhibit platelet activation by alpha-thrombin. These studies demonstrate that activation of intact platelets by alpha-thrombin proceeds by both GPIb-dependent and GPIb-independent mechanisms.
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PMID:Glycoprotein Ib (GPIb)-dependent and GPIb-independent pathways of thrombin-induced platelet activation. 201

The role of glycoprotein (GP) IIb-IIIa complexes and of adhesive proteins in mediating platelet aggregation is now well defined. However, less is known of the changes that occur once aggregation has begun. We report immunogold staining of thin sections of platelets or platelet aggregates, embedded in Lowicryl K4M, after the use of polyclonal antibodies to GP IIb or GP IIIa, fibrinogen (Fg), von Willebrand factor (vWF), and thrombospondin (TSP). Bound immunoglobulin G (IgG) was located by species-specific anti-IgG coupled to 5-nm gold particles and by electron microscopy. Initial experiments with platelet-rich plasma confirmed the feasibility of visualizing adhesive proteins between platelets in aggregates. Experiments then continued, using stirred suspensions of washed platelets incubated with alpha-thrombin. After 20 seconds, platelets were in contact without detectable release, although giant secretory vesicles containing adhesive proteins were seen. Internal pools of GP IIb-IIIa were progressively externalized within the aggregate. Secreted Fg was readily detected between platelets at 40 seconds. After 3 minutes, when most of the secretion had occurred, Fg had a patchwork-like distribution within the aggregate. After 6 minutes, zones with closely interspaced surface membranes, usually representing pseudopods, were dominant and Fg free. Results for vWF and TSP were similar to those for Fg. Nonetheless, GP IIb-IIIa complexes continued to be located between adjacent surface membranes throughout the aggregate. Thrombin-induced platelet aggregates were isolated, and sodium dodecyl sulfate-soluble extracts were obtained. Western blot experiments showed that, although fibrinopeptide A had been cleaved, degradation of adhesive proteins by platelet proteases had not occurred. These results emphasize that a platelet aggregate is a dynamic structure and suggest that not all surface-contact interactions are mediated by Fg or the other adhesive proteins tested in this study.
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PMID:Thrombin-induced platelet aggregates have a dynamic structure. Time-dependent redistribution of glycoprotein IIb-IIIa complexes and secreted adhesive proteins. 202 7

In the presence of extracellular Ca2+, epinephrine induces a rise in cytoplasmic Ca2+ ([Ca2+]i) that is associated with fibrinogen binding to the platelet surface, platelet aggregation, and enhancement of the thrombin-stimulated [Ca2+]i rise and protein phosphorylation. Whether the [Ca2+]i rise induced by epinephrine results from Ca2+ entry associated with fibrinogen binding to its receptor on the platelet surface, the glycoprotein (gp) IIb-IIIa complex, is unknown. To determine the importance of the occupancy of the gp IIb-IIIa receptor on platelet function after epinephrine administration, we studied the effects of two monoclonal antibodies (M-148 and 7E3) and two synthetic peptide analogues to fibrinogen (synthetic tetrapeptides Arg-Gly-Asp-Ser (RGDS) and dodecapeptide His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val [gamma-(400-411)]), all of which bind to gp IIb-IIIa and inhibit fibrinogen binding and platelet aggregation on the epinephrine-induced rise in [Ca2+]i and enhancement of thrombin's phosphorylation of the 47-kDa substrate of protein kinase C (p47). None of the gp IIb-IIIa ligands significantly enhanced or inhibited the epinephrine-induced [Ca2+]i rise or its augmentation of p47 phosphorylation after thrombin administration; however, the synergistic [Ca2+]i rise that follows addition of both epinephrine and thrombin was reduced by both antibodies and both peptides. Thus ligand binding of gp IIb-IIIa does not influence the epinephrine-induced [Ca2+]i rise or its promotion of protein kinase C activation by thrombin; these events can be dissociated from the synergistic [Ca2+]i rise.
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PMID:Calcium mobilization and glycoprotein IIb-IIIa complex ligands in epinephrine-stimulated platelets. 203 81

The glycoprotein Ib/IX complex on platelets is responsible for the first stage of haemostasis as an essential component in the primary adhesion of platelets to damaged vessel walls. Glycocalicin is the extracellular part of platelet glycoprotein Ib alpha and contains the von Willebrand factor and thrombin binding sites. Disulphide bonds are implicated in the von Willebrand binding site and studies with peptides point towards a region of glycocalicin with four cysteines as containing the binding sites for both von Willebrand factor and thrombin. The position and linkage of these two disulphide bonds are now determined to be 209-248 and 211-264 and the relevance of this double-loop structure for glycoprotein Ib/IX function is discussed.
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PMID:Identification of the disulphide bonds in human platelet glycocalicin. 207 Jul 94

A previously described in-vitro rat granulosa cell plasminogen activator bioassay for FSH has been modified and applied in the assay of human serum. This modified method consists of exposing the diethylstilboestrol-stimulated granulosa cells from 25- to 26-day-old rats to FSH or test substance for 3.5 h in wells coated with 125I-labelled fibrinogen and treated with thrombin. Following stimulation with FSH, the dose-related production of plasminogen activator was measured as the degree of 125I-labelled fibrinolysis in the presence of added plasminogen. Using the urinary FSH/LH bioassay reference preparation as the assay standard, the useful range of the assay was 0.3-15 IU/l, with an assay sensitivity of 0.3 IU/l. As determined using purified glycoprotein hormone preparations, the assay was highly specific for FSH. The minor degree of FSH bioactivity measured in some of the hormone preparations was accounted for by the amount of FSH contamination in these preparations. To abolish interference caused by unknown serum factors, we heat-treated the serum samples for 15 min at 56 degrees C before the assay. The results indicated that neither immunoreactivity nor bioactivity was affected by this treatment. Furthermore, heat-treated human sera gave responses parallel to the standard curve at the three dose levels (2, 4 and 8 microliters) studied. We used this bioassay to estimate the FSH-like bioactivity in 15 human serum samples. The estimates of immunoreactive FSH in these samples correlated well with the corresponding FSH bioactivity (r = 0.745, n = 15 and P less than 0.05). The results indicate that with this sensitive and rapid (completed within 24 h) bioassay, it should be possible to measure FSH bioactivity in heat-treated human serum samples.
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PMID:A rat granulosa cell plasminogen activator bioassay for FSH in human serum. 211 93

Blood coagulation factor VIII is a large glycoprotein that circulates in plasma at relative low concentration (0.1 microgram/ml). It consists of a heterogeneous mixture of a series heavy-chain peptides (90-200 kDa), each associated with a light chain of 80 kDa. To gain insight into the physical properties of the protein, we have characterized purified human factor VIII by electron microscopy and rotary shadowing. Electron microscopy of rotary shadowed factor VIII molecules showed predominantly a single globular domain structure, with a somewhat asymmetric shape, while two-domain structures were also encountered. The overall dimensions of the globular domains ranged from 4 x 6 nm to 8 x 12 nm. EDTA treatment of factor VIII reduced the overall dimensions (2.5 x 5 nm to 6 x 10 nm) while treatment with thrombin reduced the dimensions to a small extent. In complexes with von Willebrand factor, factor VIII appeared localized at the globular domains of von Willebrand factor multimers. In addition, incubation of factor VIII with Staphylococcus aureus V8 protease fragments SpII and SpIII revealed only binding to the globular domains of SpIII. In this study, the first morphological characterization of human factor VIII is presented, together with its direct localization on von Willebrand factor multimers.
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PMID:Characterization of human factor VIII and interaction with von Willebrand factor. An electron microscopic study. 212 68

Clinical, experimental and ultrastructural studies strongly suggest a role for platelets in metastatic dissemination. Several mechanisms have been proposed to explain the potential contribution of blood platelets to the metastatic cascade. Experimentally, many tumour cells of either animal or human origin have the capacity to activate platelets, although the mechanisms by which malignant cells exert this effect is not yet fully understood. Possible mechanisms include: (1) generation of thrombin; (2) activation by ADP; (3) release of cathepsin B; (4) eicosanoid metabolism. A number of observations also indicated that tumour-cell-induced platelet aggregation required specific receptor sites. We have shown that platelet glycoprotein GPIb and the complex GPIIb/IIIa are necessary for tumour-cell-induced platelet aggregation. We and others reported the isolation of a microparticulate aggregating material from different types of tumour cell lines. This material has been identified as a sialolipoprotein complex which possesses tissue-factor-like activity. The role of sialic acid in the metastatic potential of cells is also believed to be important and may partly modulate their interactions with platelets. In vivo, rheological factors may also regulate the interactions of tumour cells with blood and vascular structures and an alternative approach to the evaluation of platelet-tumour-cell interaction under dynamic conditions has been the use of perfusion systems. Thus, we have established the crucial role of Ca2+ in supporting tumour-cell-platelet activation and subsequent thrombus formation. More recently we investigated the patterns of adhesion of a highly metastatic human adenocarcinoma of the lung to exposed extracellular matrix generated by human vascular endothelial cells in culture.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The role of platelets in cancer metastasis. 213 51

The Na+/H+ antiporter, which regulates intracellular pH in virtually all cells, is one of the best examples of a mitogen- and oncogene-activated membrane target whose activity rapidly changes on stimulation. The activating mechanism is unknown. A Na+/H+ antiporter complementary DNA fragment was expressed in Escherichia coli as a beta-galactosidase fusion protein, and a specific antibody to the fusion protein was prepared. Use of this antibody revealed that the Na+/H+ antiporter is a 110-kilodalton glycoprotein that is phosphorylated in growing cells. Mitogenic activation of resting hamster fibroblasts and A431 human epidermoid cells with epidermal growth factor, thrombin, phorbol esters, or serum, stimulated phosphorylation of the Na+/H+ antiporter with a time course similar to that of the rise in intracellular pH.
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PMID:Growth factors induce phosphorylation of the Na+/H+ antiporter, glycoprotein of 110 kD. 215 36


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