Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of the common lipid moiety of bacterial LPS on secretion from washed human platelets has been studied. The lipid A-rich LPS of S. minnesota R595 and a lipid A preparation both potentiated platelet serotonin secretion in response to IgG aggregates or immune complexes up to 50-fold but had little effect in the absence of IgG. Lipid A has been shown to bind immune aggregates, raising the possibility that its mechanism of action involved effective enlargement or insolubilization of the aggregates. IgG aggregates of dimer to tetramer size were shown to be platelet simuli, equivalent on a weight basis to larger soluble aggregates. The effect of both sizes of aggregates on platelets were equally enhanced by the LPS, indicating that increased size of aggregates alone could not account for the effect of LPS. Similarly, because lipid A-rich LPS enhanced platelet response to already insoluble immune complexes, its mechanism of action cannot simply be insolubilization of immune aggregates. These LPS did not enhance platelet stimulation by antiplatelet antibody, monosodium urate crystals, or thrombin and only slightly enhanced stimulation by insoluble human skin collagen. This indicates some stimulus specificity in the ability of LPS to increase platelet secretion. The enhancement of cell response to immune complexes by the common lipid region of LPS may represent a mechanism for the diverse effects of LPS in vivo and in vitro.
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PMID:Enhancement of platelet response to immune complexes and IgG aggregates by lipid A-rich bacterial lipopolysaccharides. 62 36

Gel-filtered human platelets exerted lytic activity on autologous red blood cells (RBC) when they were coincubated at 37 degrees C with platelet-activating agents, such as thrombin, collagen, ADP, LPS or PMA in the absence of plasma. Lysis of activated platelets themselves did not occur during the incubation period examined. Morphological observations showed that RBC exposed to thrombin-activated platelets were fragmented and/or transformed into spherocytes. This haemolytic reaction by thrombin-activated platelets did not occur at 4 degrees C, or in the presence of agents which inhibited glycolysis or elevated intracellular levels of cAMP, indicating that energy-dependent and cAMP-regulated platelet metabolism was required for this reaction. When platelets and RBC were incubated in the same vessel, but were prevented from coming into direct cell to cell contact by means of a membrane barrier, their cytotoxicity was reduced but not eliminated completely. No cytotoxic activity against RBC was detected in platelet-free supernatants obtained by centrifugation after activation of platelets with thrombin. On the contrary, activated and washed platelets retained the activity. These observations suggested that the cytotoxic activity was carried by some diffusible and easily inactivated factors, which were continuously produced and liberated from activated platelets. Cyclo-oxygenase inhibitors inhibited the haemolytic activity of thrombin-activated platelets, suggesting a role for some products of platelet-cyclo-oxygenase pathway in platelet-mediated haemolysis. These results provide the first evidence for a direct role of activated platelets in mediation of RBC-damage in the absence of any plasma factors.
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PMID:Cytotoxicity of activated platelets to autologous red blood cells. 132 17

We have characterized the mechanisms by which thrombin enhances neutrophil leukocyte (PMN) adhesion to human endothelial cells in vitro. Thrombin rapidly and transiently increased PMN adhesion by an action on the endothelial cells. The transience of the response was due to at least two factors: desensitization of the endothelial cell responsiveness to thrombin in the continued presence of the agonist; and the lability (t1/2 less than 15 min) of the effector molecules expressed by the endothelium. Experiments with exogenous platelet-activating factor (PAF) and with PAF antagonists demonstrated that PAF production, although it may facilitate the enhanced PMN adhesion seen in response to thrombin, is not sufficient to explain the reaction. By using a variety of antibodies directed against cell surface ligands, and comparing adhesion of PMN to endothelium and to protein-coated surfaces, we deduce that several endothelial ligands not previously reported as playing a role in PMN adhesion are involved in these interactions. Of particular interest was the finding that antibodies recognizing two thrombin-regulated endothelial cell surface ligands, GMP-140 and the CD63-related Ag, both inhibited adhesion of PMN to thrombin- or LPS-pretreated endothelium. We conclude that thrombin acts to enhance PMN adhesion to endothelium at least in part by transiently altering the conformation or level of expression of these ligands.
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PMID:Characterization of the enhanced adhesion of neutrophil leukocytes to thrombin-stimulated endothelial cells. 169 4

1. Incubation of smooth muscle cells (SMC) from bovine aorta for 3 min with human washed platelets treated with indomethacin (10 microM) promoted a cell number-related inhibition of platelet aggregation induced by thrombin (40 mu ml-1). This inhibition was not attributable to products of the cyclo-oxygenase pathway for the SMC were also treated with indomethacin (10 microM). 2. The inhibitory activity of the SMC on platelet aggregation was enhanced by incubating the SMC with E. coli lipopolysaccharide (LPS, 0.5 micrograms ml-1) for a period of 9 to 24 h. This effect was attenuated when cycloheximide (10 micrograms ml-1) was incubated together with LPS. Cycloheximide did not prevent the inhibitory activity of the non-treated cells. 3. The inhibition of platelet aggregation obtained with non-treated or LPS-treated SMC was potentiated by superoxide dismutase (SOD, 60 u ml-1) and ablated by oxyhaemoglobin (OxyHb, 10 microM). Preincubation of the SMC with NG-monomethyl-L-arginine (L-NMMA, 30-300 microM) for 60 min prevented their antiaggregatory activity. This effect was reversed by concurrent incubation with L-arginine (L-Arg, 100 microM) but not with D-arginine (D-Arg, 100 microM). 4. Exposure of the non-treated SMC (5 x 10(5) cells) to stirring (1000 r.p.m., 37 degrees C) for 10 min led to a significant increase in their levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP) but not adenosine 3':5'-cyclic monophosphate (cyclic AMP). L-NMMA (300 microM) attenuated the increase in cyclic GMP induced by stirring but did not affect the basal levels of cyclic GMP in the cells.5. These findings support the idea that non-treated or LPS-treated cultured SMC can produce an NO-like factor. Production by the latter requires protein synthesis as evidenced by blockade with cycloheximide. This NO-like factor may play a role in the auto-regulation of smooth muscle cell reactivity through a cyclic GMP-dependent mechanism.
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PMID:Nitric oxide from vascular smooth muscle cells: regulation of platelet reactivity and smooth muscle cell guanylate cyclase. 172 27

The mechanisms underlying the superinduction of procoagulant activity by cycloheximide (CHX) on LPS-activated human monocytes have been investigated. Tissue factor (TF) activity of intact, viable cells was quantitated with a plasma recalcification assay and assays using chromogenic substrates specific for thrombin and factor Xa (FXa). TF antigen was measured simultaneously by immunocytochemical staining and immunoblotting with an anti-TF monoclonal antibody (MAb). Peripheral blood mononuclear cells (PBMC) activated with LPS in the presence of low dose CHX expressed more TF activity (approx. 100% increase) than cells activated with LPS alone. However, TF antigen levels were decreased approximately 70% by CHX. This discordant relationship was due primarily to differences in rates of activation of factor X (FX); LPS/CHX-treated PBMC activated nearly twice as much FX as LPS-treated cells (2.19 +/- 0.37 versus 1.10 +/- 0.21 ng FXa/10(6) PBMC/min, respectively). These studies indicate that TF cofactor activity on LPS/CHX-treated monocytes was approximately 7 times greater than that present on LPS-treated cells. Increased TF functional activity may be due to CHX-induced alterations in the type and content of phospholipids (PL) in the cell membrane. Results showed that exogenous mixed PL markedly increased TF activity on LPS-activated monocytes, but not on LPS/CHX-activated cells, without increasing TF antigen levels or altering cell viability. Membrane alterations may occur on monocytes in certain pathological or iatrogenic conditions resulting in a highly active form of TF in vivo.
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PMID:Discordant expression of tissue factor antigen and procoagulant activity on human monocytes activated with LPS and low dose cycloheximide. 180 19

An experimental model incorporating cultured endothelial cells (EC) was used to study the "factor VIII bypassing" activity of prothrombin complex concentrates (PCC), a property exploited in the treatment of hemophiliacs with alloantibodies to factor VIII. Two PCC preparations were ineffective as stimuli of tissue factor expression by EC. However, incubation with a combination of PCC plus endotoxin (lipopolysaccharide, LPS) or tumor necrosis factor (TNF) induced much greater tissue factor expression than was seen in response to either substance alone. PCC expressed an additional direct procoagulant activity at the EC surface, which could not be attributed to either thrombin or factor Xa, and which was diminished by an anti-tissue factor antibody. Therefore factor VIIa, which was detectable in both PCC preparations, likely provided this additional direct procoagulant activity at the EC surface. We also excluded the possibility that coagulation proteases contained in or generated in the presence of PCC are protected from inactivation by AT III. Therefore, PCC can indirectly bypass factor VIII by enhancing induced endothelial tissue factor expression, and also possess direct procoagulant activity, probably mediated by factor VIIa.
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PMID:The factor VIII bypassing activity of prothrombin complex concentrates: the roles of factor VIIa and of endothelial cell tissue factor. 180 20

Incubation of human washed platelets with bovine aortic endothelial cells (ECs) treated with indomethacin resulted in an inhibition of thrombin-induced platelet aggregation that was dependent on the number of ECs added. Preincubation of ECs with Escherichia coli lipopolysaccharide (LPS; 0.5-2.0 micrograms/ml) for 1 min significantly enhanced their inhibitory activity. This effect was potentiated by superoxide dismutase (60 units/ml) and reversed by oxyhemoglobin (5-10 microM), indicating that the inhibition was due to the release of endothelium-derived relaxing factor (nitric oxide). When the ECs were pretreated with NG-monomethyl-L-arginine (30-300 microM) before LPS, the antiaggregatory activity was strongly reduced. The reduction of activity by NG-monomethyl-L-arginine was reversed by L-arginine (100 microM) but not by D-arginine (100 microM). Under similar conditions, LPS also enhanced the antiaggregatory activity of ECs grown on beads. The immediate enhancement by LPS of the release of endothelium-derived relaxing factor from endothelial cells may contribute to the rapid fall in blood pressure associated with endotoxin shock in vivo.
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PMID:Immediate release of a nitric oxide-like factor from bovine aortic endothelial cells by Escherichia coli lipopolysaccharide. 218 41

Expression of tumor necrosis factor (TNF alpha), tissue factor (TF), and interleukin 1-beta (IL-1 beta) mRNA was evaluated in monocytes isolated from patients infected with human immunodeficiency virus (HIV). There was a significant depression (66%) of the induced level of TF mRNA expression in response to lipopolysaccharide. Conversely, the response of TNF alpha and IL-1 beta, following LPS induction, was "normal." TF mRNA reduction was also observed to a lesser degree in AIDS-related complex patients (20%) but not in asymptomatic seropositives. TF is necessary for initiation of the coagulation protease cascade, leading to thrombin production and fibrin deposition, which play a role in inflammatory responses. Its selective reduction may be a factor in the diminished resistance to secondary infections observed in AIDS. Further, since the TF defect increases as patients progress toward AIDS, it may serve as a marker for disease progression.
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PMID:A selective defect in tissue factor mRNA expression in monocytes from AIDS patients. 229 2

Melanoma growth stimulatory activity factor (MGSA) is a polypeptide which was initially isolated from Hs294 human melanoma cells. Its sequence is identical to the deduced amino acid sequence of the human gro cDNA, isolated from a human tumor cell line. MGSA stimulates the proliferation of malignant melanoma cells, but its function for normal cells has not been defined. Here we report that human umbilical vein endothelial cells are capable of synthesizing and secreting MGSA. The expression and secretion of MGSA are strongly induced by factors often involved in inflammation such as IL-1, TNF, LPS and thrombin. The induction of MGSA mRNA is dose and time dependent and is independent of new protein synthesis. This stimulation could be mimicked by TPA, suggesting that the action could be mediated through activation of protein kinase C. Furthermore, addition of MGSA to the endothelial cell cultures induces gro/MGSA gene expression, implying that an autocrine mechanism exists. Our data suggest that the protein encoded by gro/MGSA mRNA may play a role in inflammation and exert its effects on endothelial cells in an autocrine fashion.
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PMID:Expression and secretion of gro/MGSA by stimulated human endothelial cells. 267 May 60

Among the effector molecules induced in monocytes by the cellular immune response is tissue factor (TF), the initiating receptor/cofactor of the extrinsic coagulation protease cascade that is also frequently observed on human tumor cells. Other cellular activators have also been described on monocytes and tumor cells. Analyses of the cellular immune procoagulant response would be aided by a simple and efficient form of quantitation. An assay for cellular procoagulant activity (PCA) induction and expression was developed utilizing the chromogenic thrombin substrate tosyl-Gly-Pro-Arg-p-nitroanilide acetate. The constitutive or induced PCA of a variety of cells was analyzed. Peripheral blood mononuclear cells, peritoneal exudate cells, 13762 Mat B (III) mammary carcinoma cells, 1591-RE fibrosarcoma cells, the macrophage cell line WEHI-265, a detector of PCA inducing lymphokines, or mixtures of these cells were incubated with or without stimuli, e.g., endotoxin, in 96-well microplates. After incubation the cells were assayed for PCA by addition of the chromogenic substrate for thrombin using fibrinogen depleted plasma as a source of the coagulation proteins factors VII, X, V and prothrombin. The absorbance at 405 nm was determined. Spontaneous cleavage of the chromogenic substrate restricted the assay to total analysis times of less than 14 min. The 13762 Mat B (III) rat tumor which constitutively expressed tissue factor-like procoagulant activity induced measurable substrate hydrolysis with as few as 100 cells/well. It was observed that the chromogenic substrate assay was approximately twice as sensitive as conventional clotting assays for procoagulant activity. Endotoxin stimulated human peripheral blood mononuclear cells and mouse peritoneal exudate cells were readily analyzed. The procoagulant activity of approximately 280 LPS-stimulated human monocytes generated sufficient thrombin to provide a significant measurable signal within 10 min. Also supernatants from mixed lymphocyte cultures as well as from immune lymphocyte responses to syngeneic tumor cell cultures induced procoagulant activity in the macrophage like cell line WEHI-265 as determined with the assay for thrombin generation. The hydrolysis of the substrate was attributed to thrombin formation since the induced cleavage was abolished by hirudin, the highly specific active site inhibitor of thrombin. This chromogenic thrombin assay can be used for measuring induction of viable cell expression or total cellular procoagulant activity rapidly and efficiently in large replicate numbers suitable for a variety of analyses of cellular immune responses including clonal analyses of gene induction.
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PMID:Amidolytic assay for procoagulant activity of lymphoid and tumor cells. 370 Oct 69


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