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)

The effect of human platelets on the adhesion of polymorphonuclear leukocytes (PMNs) to cultured endothelial cells was investigated. Resting platelets inhibited the adhesion of PMNs stimulated by N-formyl-methionyl-leucyl-phenylalanine (fMLP), leukotriene B4 (LTB4), and tumor necrosis factor-alpha (TNF-alpha). Platelets similarly inhibited PMN adhesion induced by endothelial cell activation with TNF-alpha. The inhibitory effect depended on platelet number, was not associated with detectable platelet activation, and was also exerted by paraformaldehyde-fixed platelets. Moreover, supernatants of U46619- or thrombin-stimulated platelets were ineffective, thus excluding a role for constituents released as a result of the platelet-release reaction. Strong inhibition of PMN adhesion was exerted by platelet lysates. The inhibitory activity associated with lysates was sedimentable, heat sensitive, and not dialyzable through a membrane with a molecular-weight cutoff of 8,000; it was directed toward PMNs and was not due to cytotoxic effects or a general inhibition of PMN responsiveness to stimulation, since enzymatic release from activated PMNs was unaffected by platelet lysates. Finally, the activity was not prevented by specific adenosine inhibitors and anti-P-selectin monoclonal antibody. These data suggest that resting platelets can exert an inhibitory effect on PMN adhesion to the vessel wall during inflammatory and thrombotic conditions.
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PMID:Polymorphonuclear leukocyte adhesion to endothelial cells is inhibited by resting platelets. 128 Apr 66

Previous studies have shown that thrombin-activated platelets interact through the P-selectin with neutrophils and monocytes. To identify other types of leukocytes capable of such an interaction, eosinophils, basophils, and lymphocytes were isolated from whole blood. Binding of these cells to activated platelets was examined in a double immunofluorescence assay and the results show that activated platelets not only bind to neutrophils and monocytes, but also to eosinophils, basophils, and subpopulations of T lymphocytes. Using monoclonal antibodies (MoAbs) specific for subsets of T cells, we could further demonstrate that the T cells which bind activated platelets are natural killer (NK) cells and an undefined subpopulation of CD4+ and CD8+ cells. All these interactions were dependent on divalent cations and were completely inhibited by an MoAb against P-selectin. Thus, P-selectin mediates the binding of activated platelets to many different types of leukocytes. Studies with leukocytes treated with proteases or neuraminidase have shown that the structures recognized by P-selectin are glycoproteins carrying sialic acid residues. Because the loss of binding of activated platelets to neuraminidase-treated neutrophils was almost complete, but only partial to treated eosinophils, basophils, and monocytes, the latter cell types may have different P-selectin ligands in addition to those present on neutrophils. We found that two previously identified ligands for P-selectin, the oligosaccharides Le(x) and sialyl-Le(x), had little or no inhibitory effect on adhesion of activated platelets to leukocytes and that binding was not inhibited by MoAbs against these oligosaccharides. In addition, there was no correlation between the expression of Le(x) on several cell types and their capacity to bind activated platelets. In contrast, the expression of sialyl-Le(x) on cells was almost perfectly correlated with their ability to bind activated platelets. Thus, while Le(x) cannot be a major ligand for P-selectin, a possible role for sialyl-Le(x) in P-selectin-mediated adhesion processes cannot be dismissed. Finally, activated platelets were found to bind normally to monocytes and neutrophils of patients with paroxysmal nocturnal hemoglobulinuria (PNH) and to neutrophils from which phosphatidyl inositol (PI)-linked proteins had been removed by glycosylphosphatidyl inositol-specific phospholipase C (GPI-PLC) digestion. This suggests that at least part of the P-selectin ligands on these cells are not GPI-anchored.
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PMID:P-selectin mediates Ca(2+)-dependent adhesion of activated platelets to many different types of leukocytes: detection by flow cytometry. 137 47

We recently reported the characterization of a platelet granule membrane protein of molecular weight (mol wt) 40,000 called granulophysin (Gerrard et al: Blood 77:101, 1991), identified by a monoclonal antibody (MoAb D545) raised to purified dense granule membranes. Using immunoelectron-microscopic techniques on frozen thin sections, this protein was localized in resting and thrombin-stimulated platelets. In resting platelets, labeled with antigranulophysin antibodies and immunogold probes, label was localized to the membranes of one or two clear granules per platelet thin section. D545 also labeled dense granules in permeabilized whole platelets and isolated dense granule preparations examined by whole-mount techniques. Expression of granulophysin on the platelet surface paralleled dense granule secretion as measured by 14C-serotonin release under conditions in which lysosomal granule release, as measured by beta-glucuronidase secretion, was less than 5%. After thrombin stimulation, both the surface-connected canalicular system and the plasma membrane were labeled, demonstrating redistribution of granulophysin associated with degranulation. Double labeling experiments with D545 and antibodies to the alpha-granule membrane protein, P-selectin, demonstrated labeling of both P-selectin and granulophysin on dense granule membranes. Distribution of both proteins on the plasma membrane after platelet stimulation was similar. The results demonstrate that granulophysin is localized to the dense granules of platelets and is redistributed to the plasma membrane after platelet activation.
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PMID:Platelet dense granule membranes contain both granulophysin and P-selectin (GMP-140). 137 48

Thrombin cleaves its receptor at arginine-41, resulting in the generation of a new receptor NH2-terminus with the sequence SFLLRNPNDKYEPF. This peptide (TRP-14) may signal a variety of thrombin's responses. We examined the effects of TRP-14 in inducing endothelial cell hyperadhesivity and neutrophil (PMN) adhesion to endothelial cell monolayers. Human umbilical vein endothelial cells (HUVECs) challenged with TRP-14 (10(-4) to 10(-5) M) produced concentration-dependent increases in endothelial adhesivity to PMN. In contrast, position 1 to 2 inverted peptide (FSLLRNPNDKYEPF) did not induce the response. The adhesion response was transient; that is, PMN adhesion increased within 15 minutes and decreased by 75 minutes after TRP-14 challenge of HUVECs. The transient endothelial adhesiveness paralleled the time course of P-selectin expression. TRP-14-induced release of P-selectin from intracellular stores may be a critical determinant of the response since treatment of endothelial cells with anti-P-selectin monoclonal antibody (mAb) G1 prevented the increase in PMN adhesion. Control nonneutralizing anti-P-selectin mAb S12 and mAb RR1/1 directed against intercellular adhesion molecule-1 (ICAM-1) on HUVECs were ineffective. The results indicate that the "tethered ligand" of the thrombin receptor created by the proteolytic action of thrombin on its receptor (i.e., TRP-14) signals increased endothelial adhesiveness by a P-selectin-dependent mechanism. Thrombin-induced PMN adhesion may involve formation of a new NH2-terminus of the endothelial thrombin receptor with the sequence SFLLRNPNDKYEPF followed by activation of endothelial second messenger pathways and the transient expression of P-selectin.
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PMID:Thrombin receptor 14-amino acid peptide mediates endothelial hyperadhesivity and neutrophil adhesion by P-selectin-dependent mechanism. 138 Dec 92

Rapid translocation of P-selectin (GMP-140) from cytoplasmic granules to the cell membrane of endothelial cells promotes adhesive interactions with neutrophils which, when activated, damage the endothelium. The role of P-selectin in lung vascular endothelial injury in rats after systemic activation of complement by intravenous infusion of cobra venom factor has been assessed. Within 5-10 min after cobra venom factor infusion, the pulmonary vasculature demonstrated immunohistochemical expression of an epitope that reacts with anti-human P-selectin. Monoclonal antibody to human P-selectin blocked in vitro adherence of rat or human platelets (activated with thrombin) to neutrophils and was demonstrated to react with thrombin-activated rat platelets. The antibody did not react with rat neutrophils. In vivo, the antibody had strongly protective effects against cobra venom factor-induced pulmonary vascular injury as determined by permeability changes and hemorrhage. In parallel, lung myeloperoxidase content was greatly reduced and, by transmission electron microscopy, there was markedly diminished adherence of neutrophils to the pulmonary vascular endothelium and much diminished injury of endothelial cells, as defined by hemorrhage. These data indicate that anti-human P-selectin reacts with a pulmonary vascular antigen in rats and that this antigen is essential for the full expression of lung injury.
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PMID:Neutrophil-dependent acute lung injury. Requirement for P-selectin (GMP-140). 138 77

Thrombin-induced expression of endothelial adhesivity toward neutrophils (PMN) was studied using human umbilical vein endothelial cells (HUVEC). HUVEC were challenged with human alpha-thrombin for varying durations up to 120 min, after which the cells were fixed with 1% paraformaldehyde and 51Cr-labeled human PMN were added to determine PMN adhesion. Endothelial adhesivity increased within 15 min after alpha-thrombin exposure, and the response persisted up to 120 min. Expression of endothelial adhesion proteins, P-selectin (GMP-140, PADGEM, CD62), and intercellular adhesion molecule-1 (ICAM-1; CD54) on the endothelial surface was quantitated by increase in the specific binding of anti-P-selectin mAb G1 and anti-ICAM-1 mAb RR1/1 labeled with 125I. P-selectin expression was maximal at 5-15 min alpha-thrombin exposure and decayed to basal levels within 90 min. In contrast, ICAM-1 activity increased at 30 min and remained elevated for 120 min after alpha-thrombin challenge. The initial endothelial adhesivity was dependent on P-selectin expression since PMN adhesion occurring within the first 30 min after alpha-thrombin challenge was inhibited by mAb G1. The later prolonged PMN adhesion was ICAM-1 dependent since this response was inhibited by mAb RR1/1 and to the same degree by the anti-CD18 mAb IB4. Anti-ELAM-1 mAb BB11 had no effect on adhesion of PMN to the alpha-thrombin-challenged cells. The initial P-selectin expression and PMN adhesion responses were reproduced by the 14-amino peptide (SFLLRNPNDKYEPF) (thrombin-receptor activity peptide; TRP-14) which comprised the NH2 terminus created by thrombin's proteolytic action on its receptors. However, TRP-14-induced PMN adhesion was transient, and TRP-14 did not cause ICAM-1 expression. The ICAM-1-dependent PMN adhesion mediated by alpha-thrombin was protein synthesis independent since ICAM-1 expression and PMN adhesion were not inhibited by cycloheximide pretreatment of HUVEC. Moreover, Northern blot analysis indicated absence of ICAM-1 mRNA signal up to 180 min after alpha-thrombin challenge. In conclusion, thrombin-induced endothelial adhesivity involves early- and late-phase responses. The initial reversible PMN adhesion is mediated by rapid P-selectin expression via TRP-14 generation. Thrombin-induced PMN adhesion is stabilized by a protein synthesis-independent upregulation of the constitutive ICAM-1 activity which enables the interaction of ICAM-1 with the CD18 beta 2 integrin on PMN.
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PMID:Thrombin-induced expression of endothelial P-selectin and intercellular adhesion molecule-1: a mechanism for stabilizing neutrophil adhesion. 138 47

CD62, also called PADGEM protein, GMP-140, or P-selectin, is a granule membrane protein of endothelial cells and platelets that is mobilized to the plasma membrane following exposure to mediators such as thrombin, histamine, complement components, or peroxides. Data presented to date suggest that one ligand of CD62 includes CD15 (Lewis x determinant) and sialic acid. We show here that sulfatides, heterogeneous 3-sulfated galactosyl ceramides, are an apparently unrelated ligand of CD62. Sulfatides are expressed on the plasma membrane of, and are excreted by, granulocytes, and constitute the principal ligand for CD62 on the plasma membrane of some tumor cells. CD62 binds to sulfatides adsorbed to plastic as avidly as it binds to myeloid or tumor cells. We find that granulocytes excrete sulfatides at a rate predicted to allow them to be rapidly released from CD62 once they have exited the bloodstream.
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PMID:CD62/P-selectin recognition of myeloid and tumor cell sulfatides. 171 59

After the intravenous infusion of N-hydroxysuccinimido biotin into dogs, 80.6% +/- 9.7% (n = 5) of platelets were covalently labeled with biotin. The in vivo survival of the biotinylated platelets was monitored by flow cytometry and was normal as compared with previous reports for dog platelets. The ability of the biotinylated platelets to be activated was analyzed by measuring the expression of cell-surface P-selectin after incubation with graded concentrations of thrombin. When P-selectin expression was examined 3 hours after labeling, biotinylated platelets were indistinguishable from the nonlabeled population of platelets, indicating that biotinylation did not adversely affect the cells. On consecutive days after biotinylation, the thrombin dose-response curves for biotinylated and nonbiotinylated platelets were repeated, and as the biotinylated-platelets aged, they became less responsive to thrombin. On days 3, 4, and 5, the thrombin EC50 for the aged, biotinylated platelets as compared with the total population of platelets was 136%, 150%, and 178%, respectively. Increasing age clearly impairs the reactivity of platelets towards thrombin as quantitated by the expression of cell-surface P-selectin.
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PMID:Aged platelets have an impaired response to thrombin as quantitated by P-selectin expression. 750 77

To determine if interleukin-6 (IL-6) administration influences platelet function, platelet activation was analyzed sequentially in IL-6-treated (80 micrograms/kg/d) and control dogs. Platelet activation was determined in whole blood by flow cytometry by quantitating the binding of a monoclonal antibody to platelet surface P-selectin after stimulation with graded doses of thrombin. Administration of IL-6 resulted in a twofold decrease in the thrombin concentration required for induction of half-maximal P-selectin expression (ED50) compared with control animals. The ED50 returned to normal after cessation of IL-6 administration. As measured by P-selectin expression, enhanced responsiveness to the strong agonist platelet activating factor (PAF) was also observed in the IL-6-treated dogs. IL-6 had no effect on the susceptibility of platelets to thrombin activation when incubated with anticoagulated dog blood. The data show that, in addition to augmenting the platelet count in normal dogs, IL-6 enhances the sensitivity of platelets to activation in response to thrombin and PAF.
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PMID:Alteration of platelet function in dogs mediated by interleukin-6. 750 49

P-Selectin (CD62/GMP140/PADGEM) is an inducible cell-surface glycoprotein expressed by endothelial cells and platelets following stimulation by inflammatory mediators such as thrombin, histamine, or peroxides. P-Selectin mediates the binding of leukocytes to activated vascular endothelium at sites of inflammation and plays a role in mediating the binding of activated platelets to leukocytes and the vascular cell wall. The adhesive function of P-selectin is mediated by its calcium-dependent (or C-type) lectin domain, which is known to bind to carbohydrate ligands including fucosyl-N-acetyllactosamine (Lex, CD15), sialyl-Lex, and 3-sulfated galactosylceramides (sulfatides). Sulfatides can efficiently block P-selectin/myeloid cell binding in vitro and are excreted at high levels by activated granulocytes. These observations led to the hypothesis that sulfatide may play a role in facilitating the disengagement of CD62, allowing the efficient exit of granulocytes from the blood stream at sites of inflammation. In this report, we extend our previous mutagenesis analysis of the P-selectin binding site [Hollenbaugh, D., Bajorath, J., Stenkamp, R., & Aruffo, A. (1993) Biochemistry 32, 2960] and show that replacement of Tyr48 with Ser or Lys113 with Arg results in P-selectin mutants that, although correctly folded, do not bind to HL60 cells. These results suggest that the conservation of charged and hydrogen-bonding site chains is not sufficient to maintain the P-selectin function and that the exact stereochemistry provided by the side chains of residues lining the P-selectin binding pocket is critical for P-selectin binding.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:CD62/P-selectin binding sites for myeloid cells and sulfatides are overlapping. 750 45


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