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)

Recent studies have shown that the synthetic peptides SFL LRN and SFL LRN PND KYEPF (thrombin receptor-activating peptides (TRAP)) derived from the deduced sequence of the new amino terminus of the cleaved thrombin receptor can mimic thrombin receptor activation, act as full agonists for platelet activation, and induce prostaglandin I2 production as well as cytosolic Ca2+ increase in human umbilical vein endothelial cells (HUVEC). Here, we have compared the ability of these synthetic peptide ligands and thrombin to stimulate platelet-derived growth factor (PDGF) production by, and monocyte adhesion to, HUVEC. Thrombin (50 units/ml) and TRAP (25 microM) maximally stimulated monocyte adhesion. Furthermore, the stimulation of E-selectin cell surface expression and the steady-state E-selectin mRNA levels by thrombin and TRAP were comparable. Thrombin (50 units/ml) stimulated PDGF production 400% above the basal level in 24 h, whereas the 6-mer and 14-mer TRAP, even at 200 microM, did not significantly stimulate PDGF production. Northern analysis, however, revealed that TRAP at 100 microM stimulated PDGF-A and -B chain mRNA expression to a level similar to that induced by thrombin. These results suggest that activation of cell signaling by TRAP can mimic thrombin and is sufficient for the stimulation of monocyte adhesion to HUVEC; however, thrombin-stimulated PDGF production by HUVEC may require mechanisms in addition to the signaling events initiated by TRAP or may require the participation of a novel thrombin receptor.
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PMID:Thrombin receptor-activating peptides differentially stimulate platelet-derived growth factor production, monocytic cell adhesion, and E-selectin expression in human umbilical vein endothelial cells. 751 96

Antioxidants have been proposed to be anti-atherosclerotic agents; however, the mechanisms underlying their beneficial effects are poorly understood. We have examined the effect of alpha-tocopherol (alpha-tcp) on one cellular event in atherosclerotic plaque development, monocyte adhesion to stimulated endothelial cells (ECs). Human umbilical vein ECs were pretreated with alpha-tcp before stimulation with known agonists of monocyte adhesion: IL-1 (10 ng/ml), LPS (10 ng/ml), thrombin (30 U/ml), or PMA (10 nM). Agonist-induced monocytic cell adhesion, but not basal adhesion, was inhibited in a time- and concentration-dependent manner by alpha-tcp. The IC50 of alpha-tcp on an IL-1-induced response was 45 microM. The inhibition correlated with a decrease in steady state levels of E-selectin mRNA and cell surface expression of E-selectin which is consistent with the ability of a monoclonal antibody to E-selectin to inhibit monocytic cell adhesion in this system. Probucol (50 microM) and N-acetylcysteine (20 mM) also inhibited agonist-induced monocytic cell adhesion; whereas, several other antioxidants had no significant effect. Protein kinase C (PKC) does not appear to play a role in the alpha-tcp effect since no suppression of phosphorylation of PKC substrates was observed. Activation of the transcription factor NF-kappa B is reported to be necessary but not sufficient for E-selectin expression in EC. Electrophoretic mobility shift assays failed to show an alpha-tcp-induced decrease in activation of this transcription factor after cytokine stimulation. It has been hypothesized that alpha-tcp acts as an anti-atherosclerotic molecule by inhibiting generation of oxidized LDL--a putative triggering molecule in the atherosclerotic process. Our results point to a novel alternative mechanism of action of alpha-tcp.
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PMID:Alpha-tocopherol inhibits agonist-induced monocytic cell adhesion to cultured human endothelial cells. 751 38

Human umbilical vein endothelial cells have recently been shown to respond to C5a with increases in intracellular Ca2+, production of D-myo-inositol 1,4,5-triphosphate and superoxide anion generation. In the current studies, C5a had been found to cause in a time- and dose-dependent manner rapid expression of endothelial P-selectin, secretion of von Willebrand factor, and adhesiveness for human neutrophils. The effects of C5a in P-selectin expression and adhesiveness of neutrophils were similar to the effects of histamine and thrombin on endothelial cells. The adhesiveness of C5a-stimulated endothelium for neutrophils was blocked by anti-P-selectin, but not by antibodies to intercellular adhesion molecule 1, E-selectin, or CD18. A cell-based ELISA technique has confirmed upregulation of P-selectin in endothelial cells exposed to C5a. Binding of C5a to endothelial cells has been demonstrated, with molecules bound being approximately 10% of those binding to neutrophils. By a reverse transcriptase-PCR technique, endothelial cells have been shown to contain mRNA for the C5a receptor. These data suggest that C5a may be an important inflammatory mediator for the early adhesive interactions between neutrophils and endothelial cells in the acute inflammatory response.
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PMID:C5a-induced expression of P-selectin in endothelial cells. 752 84

The vascular selectins P- and E-selectin are inducible adhesion proteins expressed by endothelial cells that have been shown to support shear-dependent rolling of myeloid cells. This interaction is thought to be a prerequisite event for subsequent steps, such as tight adhesion/aggregation and transendothelial cell migration, involved in the accumulation of leukocytes into tissues. Certain lymphocyte subsets have also been shown to bind the vascular selectins, but the importance of this interaction in mediating shear-dependent rolling, as described for myeloid cells, has not been demonstrated. We expand on our earlier observation that bovine gamma/delta T cells bind E-selectin by showing that this interaction leads to a reproducible rolling event in assays done under shear forces that approximate those that occur in vivo. E-selectin, expressed by L cell transfectants or cytokine-stimulated human and bovine endothelial cells, equally supports the shear-dependent rolling interaction. The lymphocyte adhesion proteins L-selectin, CD44, and CD2 do not contribute to this event. Neuraminidase treatment of the gamma/delta T cells or addition of EDTA to the assay completely blocks the rolling interaction. We further show for the first time that P-selectin expressed by thrombin-activated platelets or a soluble P-selectin/human Ig chimera specifically binds gamma/delta T cells. The P-selectin interaction is similar to the rolling event mediated by E-selectin--it requires divalent cations and sialic acid on the lymphocyte, it lacks involvement of L-selectin and CD44, and rolling occurs under physiologic shear conditions. These results provide the documentation that the vascular selectins can support shear-dependent rolling of a lymphocyte subset and that P-selectin mediates the adhesion of gamma/delta T cells.
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PMID:Cell surface P- and E-selectin support shear-dependent rolling of bovine gamma/delta T cells. 752 9

It is well known that granulocytes increase infarct size after reperfusion of the ischemic myocardium, and that monocytes promote atherogenesis. Those cells are also believed to play a contributory role in pathogenesis of coronary restenosis as response to arterial injury during balloon angioplasty. The adhesion of those leukocytes to the vascular endothelium is a prerequisite for their recruitment and accumulation in the lesion. Inflammatory mediators likely to occur under those conditions, e.g., histamine, thrombin, oxygen-derived free radicals (ODFR), interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and activated complement factors, induce in a distinct time course the (transient) expression of the leukocyte adhesion molecules P-selectin, E-selectin, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 on the endothelium. Only VCAM-1 is specific for monocytes; the others mediate the binding and subsequent extravasation of both monocytes and granulocytes. The response to the relevant inflammatory mediators, except for extracellularly produced ODFR, is coupled via specific receptors on the surface of the endothelium to specific signal transduction pathways and, except for P-selectin (early response), is directly dependent on protein synthesis (intermediate and late response). Protein kinase-C-induced phosphorylation of transcription factors is often shown to be involved. Protein synthesis is preceded by increased transcription of mRNA that is regulated in part by the transcription factor NF-kappa B. Indications have been obtained that intracellularly produced ODFR may be involved in the translocation of this transcription factor.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Leukocyte adhesion molecules on the vascular endothelium: their role in the pathogenesis of cardiovascular disease and the mechanisms underlying their expression. 752 71

P-selectin is an endothelial cell adhesion molecule which mediates the binding of neutrophils and monocytes. Its appearance at the cell surface can be induced within minutes by histamine and thrombin which rapidly stimulate the transport of P-selectin from intracellular storage granules to the plasma membrane. We have recently found a second regulation mechanism for P-selectin on mouse endothelioma cells. Like E-selectin, P-selectin is also regulated at the level of transcription. Both selectins are induced by LPS or TNF-alpha with a maximal expression level at the cell surface 3-4 h after stimulation. Here, we report that this up-regulation of the synthesis of P-selectin also occurs in vivo in endothelium of the mouse. Analysing brain tissue, which is devoid of constitutive expression of P-selectin, we found that LPS and also TNF-alpha strongly induce the expression of P-selectin on all venular endothelial cells of the leptomeninges and, at a weaker level, on some blood vessels of the brain parenchyma. Induction of P-selectin expression could also be observed in tissues, such as the tongue, where P-selectin is constitutively expressed on small venules but only rarely on larger venules. Strong staining for P-selectin on endothelium of all large venules was observed in tissues of LPS and TNF-alpha treated animals and staining for this newly synthesized P-selectin was enriched at the luminal surface of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression of P-selectin on endothelial cells is upregulated by LPS and TNF-alpha in vivo. 752 54

The migration of neutrophils from blood vessels to peripheral tissues is a key step of inflammation. This requires the formation of transient gaps between endothelial cells with concomitant leucocyte squeezing through these narrow apertures and immediate restoration of endothelium continuity. It is currently considered that the main role of selectins is to mediate the initial contact between flowing leucocytes and endothelial cells. We show here that the binding of E- or P-selectins by specific antibodies induces a marked 'rounding up' of interleukin-1- or thrombin-activated human endothelial cells, respectively. Also, anti-E-selectin antibodies trigger a transient increase in cytosolic calcium involving intracellular calcium stores. No such effect is observed when von Willebrand factor or intercellular adhesion molecule 1 are similarly bound. Thus, in addition to promoting the initial interaction between activated endothelium and moving leucocytes, selectins might play a role in the induction of subsequent endothelial deformation, which would facilitate leucocyte arrest and transmigration towards peripheral tissues, and enhance the diffusion of soluble molecules between intravascular and peripheral compartments. Our results are consistent with this hypothesis and demonstrate a new property of endothelial selectins.
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PMID:A novel role for E- and P-selectins: shape control of endothelial cell monolayers. 753 Dec

It is now well established that monocytes adhere to endothelial cells activated by oxidized low-density lipoproteins (LDL). However, the adhesive receptors on endothelial cells involved in binding monocytes, following an insult by oxidized LDL, remains to be elucidated. In this study we have looked at the effect of native or oxidized LDL on the expression of P-selectin. Native LDL (N-LDL) was oxidized by incubation with either endothelial cells (EC-LDL) or copper (Cu-LDL), or in culture medium as a control (C-LDL). Expression of P-selectin was assayed with an anti-P-selectin (CD62) monoclonal antibody (LYP20). Results show that EC-LDL and Cu-LDL, but not N-LDL or C-LDL, induce the expression of P-selectin by human umbilical-vein endothelial cells (HUVECs). Induction of P-selectin by low concentrations (20 micrograms/ml) of LDL is directly related to the state of oxidation of the LDL particles. In addition, high concentrations (100 micrograms/ml) of N-LDL also activate HUVECs by inducing P-selectin expression. This expression was sustained for a period of over 1 h on LDL-activated endothelial cells, in contrast with thrombin- or histamine-activated endothelial cells, whose P-selectin levels fall within 15-20 min after induction. E-selectin, in contrast with P-selectin, could not be induced by endothelial cells treated with low or high concentrations of oxidized LDL. Results in this study show that P-selectin expressed by oxidized-LDL-treated endothelial cells are involved in mediating the adhesion of a monocytic cell line (U937) or monocytes in peripheral-blood mononuclear cells. An anti-P-selectin monoclonal antibody (LYP20) inhibited the binding of U937 cells and monocytes. These results strongly suggest that P-selectin is involved in the early stages of atherogenesis.
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PMID:Oxidized low-density lipoprotein induces the expression of P-selectin (GMP140/PADGEM/CD62) on human endothelial cells. 753 99

An immunoconjugate was designed to target hirudin, a potent and specific inhibitor of thrombin, to the surface of activated endothelial cells. Hirudin was covalently cross-linked to the monoclonal antibody H18/7 that recognizes the extracellular domain of E-selectin (CD62E), an endothelium-leukocyte adhesion molecule that is expressed only on cytokine-activated endothelium. The hirudin-H18/7 immunoconjugate selectively bound to interleukin-1-activated but not to unactivated cultured human umbilical vein endothelial cells with a temporal profile similar to that of inducible cell-surface procoagulant activity. When bound to activated endothelial cells, the hirudin-H18/7 immunoconjugate significantly inhibited endogenous thrombin activity generated from coincubated human plasma and fibrin clot formation on the monolayer surface. Cellular responses that are mediated via the thrombin receptor, such as increases in cytoskeletal F-actin content, also were significantly downregulated, and monolayers were protected from thrombin-induced disruption by this treatment. The ability to selectively antagonize thrombin-dependent processes at the endothelium-blood interface may provide new insights into complex pathophysiological processes, such as thrombosis, inflammation, and atherogenesis. These studies also demonstrate the general feasibility of selective targeting of therapeutic agents to endothelial cells based on recognition of an activation-dependent surface phenotype.
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PMID:Immunoselective targeting of an anti-thrombin agent to the surface of cytokine-activated vascular endothelial cells. 754 99

Primary human vascular endothelial cells were immortalized by the integration of a single DNA copy of an amphotrophic, replication-deficient retrovirus containing the E6/E7 genes of human papilloma virus. To date, the resulting cell lines, designated EC-RF7 and EC-RF24, have been cultured for more than 1 year. The cell lines have retained a diploid karyotype, display no abnormalities, and are able to grow in a polar mode. Analysis of the EC-RF cell lines by indirect immunofluorescence, using an extensive panel of monoclonal antibodies, showed expression of endothelial cell-specific soluble (von Willebrand factor) and surface-bound antigens (endoglin, PCAM-1) indistinguishable from that of primary cells. In addition, the expression of the markers CD9, 13, 14, 29, 36, 40, 51, and 55 that are not restricted to endothelial cells was also similar for the immortalized and the primary endothelial cells. Immortalization did not alter the expression of the surface adhesion molecules E-selectin, VCAM-1, and ICAM-1 nor transmigration of neutrophils. The regulation of extracellular proteolytic activity by EC-RF24 was established by measuring both the induction of functional tissue factor (promotion of Factor Xa generation) and the functional deposition of plasminogen activator inhibitor 1 in the subendothelial matrix (SDS-resistant complex formation with thrombin). Finally, the biosynthesis of the endothelial cell-specific von Willebrand factor was studied in detail in the EC-RF24 cell line and the results were compared with those of primary endothelial cells.
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PMID:Maintenance of vascular endothelial cell-specific properties after immortalization with an amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7 DNA. 781 21


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