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)

P-selectin is an adhesion receptor for leukocytes on thrombin-activated platelets and endothelial cells. It contains a NH2-terminal carbohydrate-recognition domain, an epidermal growth factor motif, nine consensus repeats, a transmembrane domain, and a cytoplasmic tail. We expressed two soluble forms of P-selectin, one truncated after the ninth repeat (tPS) and the other lacking the transmembrane domain due to alternative RNA splicing (asPS). When visualized by electron microscopy, each was a monomeric rod-like structure with a globular domain at one end, whereas membrane P-selectin (mPS) from platelets formed rosettes with the globular domains facing outward. Sedimentation velocity and equilibrium studies confirmed that tPS and asPS were asymmetric monomers, whereas mPS was oligomeric. HL-60 cells adhered to immobilized tPS and asPS, although less efficiently than to mPS. 125I-Labeled tPS and asPS bound to approximately 25,000 sites/neutrophil and approximately 36,000 sites/HL-60 cell with an apparent Kd of 70 nM. Treatment of HL-60 cells with O-sialoglycoprotease eliminated the binding sites for asPS. We conclude that 1) P-selectin is a rigid, asymmetric protein; 2) monomeric soluble P-selectin binds to high affinity ligands with sialylated O-linked oligosaccharides on leukocytes; and 3) oligomerization of mPS enhances its avidity for leukocytes.
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PMID:Structural and functional characterization of monomeric soluble P-selectin and comparison with membrane P-selectin. 768 12

Rapid upregulation of the adhesion molecule GMP-140 (P-selectin) on endothelial cells is believed to play an important role in the initial binding of leukocytes to endothelium, a very early step in the inflammatory response. Activated platelets that are involved in the coagulation system and in inflammatory processes also express GMP-140 on their surfaces. The objectives of the present study were to develop a monoclonal antibody against this adhesion molecule in the dog and to use this antibody to study platelet-neutrophil interactions in whole blood and to characterize the in vivo localization of GMP-140 in canine tissues. Five Balb/c mice were immunized with thrombin-stimulated dog platelets, and clones were screened using an enzyme-linked immunosorbent assay. The clone MD3 (IgG1) showed preferential binding to activated as compared with resting platelets. Flow cytometric analysis using MD3 revealed that 27% of circulating neutrophils in unstimulated blood had platelets bound to their surfaces; stimulation with platelet activating factor increased this percentage to 85%. Immunoblot analysis of solubilized dog platelets resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the antibody MD3 recognized an approximately 140-kd protein. Immunohistochemical study of normal dog tissues with MD3 revealed that the antigen was present in endothelial cells of arteries, capillaries, and veins, depending on the specific tissue examined. Blood vessels staining positively with MD3 were most abundant in the digestive system (liver, stomach, small and large intestines), moderate in the lungs, kidneys, spleen, lymph nodes, and endocrine glands, and minimal in the brain, myocardium, skeletal system, and skin. Based on its presence on stimulated but not resting platelets, its molecular weight, and its vascular distribution, the antigen recognized by MD3 appears to be the selectin GMP-140 of the dog. This study documents that the cellular and tissue distribution of GMP-140 in dogs is very similar to that in human beings.
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PMID:Production of a monoclonal antibody against canine GMP-140 (P-selectin) and studies of its vascular distribution in canine tissues. 768 99

1. P-selectin (PADGEM, GMP140, or CD62) a member of lectin-like adhesive proteins is expressed on the surface of activated degranulated canine platelets and is the calcium-dependent receptor for leukocyte adhesion. 2. The electrophoretic mobility of P-selectin, by Western blot analysis and immunoprecipitation from radiolabeled membranes of canine and human platelets, was similar or identical and immunocytochemical studies localized P-selectin in internal vesicles similar to the alpha granule localization in human platelets. 3. Two antibodies to human P-selectin KC4.1 and AC1.2 crossreacted with canine platelets whose surface binding, in response to agonists thrombin, calcium ionophore (A23187), phorbol esters and ADP, was similar. 4. Anti-P-selectin antibodies in conjunction with crossreacting anti-GPIIb/IIIa antibodies (A2A9, 7E3, RUU-PL7F12) enables the analysis of activated platelets, platelet-derived microparticles and platelet-leukocyte interactions in canine models by flow cytometry.
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PMID:Characterization of canine platelet P-selectin (CD 62) and its utility in flow cytometry platelet studies. 768 39

Unstimulated neutrophils inhibited activation and recruitment of thrombin- or collagen-stimulated platelets in an agonist-specific manner. This occurred under conditions of close physical cell-cell contact, although biochemical adhesion between the cells as mediated by P-selectin was not required. Moreover, in the presence of monoclonal P-selectin antibodies that blocked biochemical platelet-neutrophil adhesion, thrombin-stimulated platelets were more efficiently downregulated by neutrophils. This suggested a prothrombotic role for P-selectin under these circumstances. The neutrophil downregulatory effect on thrombin-stimulated platelets was amplified by lipoxygenase inhibition with 5,8,11,14-eicosatetraynoic acid. In contrast, the neutrophil inhibitory effect on platelets was markedly reduced by platelet-derived 12S-hydroxy-5,8-cis, 10-trans, 14-cis-eicosatetraenoic acid (12S-HETE), as well as by the platelet-neutrophil transcellular product, 12S,20-dihydroxy-5,8,10,14-eicosatetraenoic acid (12S,20-DiHETE), but not by another comparable metabolite, 5S,12S-dihydroxy-6-trans, 8-cis, 10-trans, 14-cis-eicosatetraenoic acid (5S,12S-DiHETE), or the neutrophil-derived hydroxy acid leukotriene B4. The neutrophil downregulatory effect on thrombin-induced platelet reactivity was enhanced by aspirin treatment. This may represent a novel action of aspirin as an inhibitor of platelet function. These results provide in vitro biochemical and functional evidence for the thromboregulatory role of neutrophils and emphasize the multicellular aspect of hemostasis and thrombosis.
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PMID:Downregulation of human platelet reactivity by neutrophils. Participation of lipoxygenase derivatives and adhesive proteins. 769 Jul 78

Activated platelets expressing P-selectin in their surfaces are known to adhere to monocytes and neutrophils. We examined the possibility that the leukocytes are functionally modified by their adhesion to activated platelets. We used human peripheral blood monocytes and neutrophils and measured superoxide anion generation by these cells cultured with platelets. The levels of superoxide anion production were found to be markedly elevated when thrombin-activated platelets were used. The extent of this enhancement was much smaller when leukocytes were cultured with resting platelets than activated platelets. The increase depended on incubation time and platelet concentration. The membranes prepared from activated platelets also induced superoxide anion production, but the culture supernatant of activated platelets did not. The enhanced superoxide anion production was inhibited by anti-P-selectin antibody, anti-sialyl-Lewis X antibody, or a soluble recombinant P-selectin fusion protein. These results indicate that the adhesion of activated platelets to the leukocytes through P-selectin was a crucial step for the activation of leukocyte function, and support the idea that activated platelets are actively involved in inflammation processes.
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PMID:Activated platelets induce superoxide anion release by monocytes and neutrophils through P-selectin (CD62). 769 Jul 99

P-selectin is an adhesion receptor for leukocytes that is redistributed from secretory granule membranes to the surfaces of activated platelets and endothelial cells. The cytoplasmic domain of P-selectin contains two serines, two threonines, and one tyrosine that could potentially be phosphorylated. We found that P-selectin was phosphorylated in both platelets and endothelial cells and that phosphorylation rapidly increased after cell activation. Approximately 0.02, 0.05, and 0.08 mol of phosphate/mol of P-selectin were incorporated, respectively, into resting, thrombin-activated, and phorbol ester-activated platelets. Phosphorylation was completely inhibited by the protein kinase C inhibitors, staurosporine, H-7, and chelerythrine, and was enhanced by the phosphatase inhibitors, okadaic acid and calyculin-A. Phosphoamino acid analysis of 32P-labeled P-selectin showed that phosphorylation occurred predominantly on serine with lesser amounts on threonine. When expressed in transfected Chinese hamster ovary cells, P-selectin was also phosphorylated. Mutagenesis studies showed that Ser788 was the principal site of phosphorylation, with minor sites on the other serine and threonine residues of the cytoplasmic domain. Phosphorylation may regulate membrane trafficking or other functions of P-selectin.
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PMID:The cytoplasmic domain of P-selectin is phosphorylated on serine and threonine residues. 769 Dec 35

The stimulated release of von Willebrand factor (vWF) from endothelial cells by secretagogues such as thrombin is associated with the translocation of Weibel-Palade bodies to the cell membrane and the surface expression of P-selectin (also known as GMP 140, PADGEM and CD 62). P-selectin, which is stored in Weibel-Palade bodies, is a neutrophil and monocyte adhesion molecule important in the initiation of inflammation. We have developed a simple assay for the detection of P-selectin on endothelial cells using indirect immunofluorescence and flow cytometry and have confirmed that this is temporally related to vWF release. The assay has been used to demonstrate that IL-1 does not cause Weibel-Palade body degranulation but that trypsin does. This has implications for the use of passaged endothelial cells in the study of vWF release and the assay has numerous possible applications in study of mechanisms of stimulated vWF release.
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PMID:von Willebrand factor release and P-selectin expression is stimulated by thrombin and trypsin but not IL-1 in cultured human endothelial cells. 769 90

This study characterizes a new congenital thrombocytopenia with mild hemorrhagic tendency occurring in a woman and her child with the following features. We found a deletion of the distal part of one chromosome 11 [del(11)q23.3-->qter] that was detected by cytogenetic analysis and confirmed by chromosome painting in the two patients and also an increased number of bone marrow megakaryocytes (MKs), including numerous micromegakaryocytes (mMKs) associated with a normal platelet life span. A normal number of MK colonies in culture was observed with one third of them containing a few large MKs; however, these were always associated with mMKs identified by immunologic staining. A massive cell lysis was observed at the end of the maturation. Fifteen percent of the platelets in the peripheral blood showed giant alpha-granules resulting from the fusion of alpha-granules. These giant granules, which appeared in red on giemsa stain, had a mean diameter of 1.5 microns and showed all markers (detected at electron microscopy by immunogold method) of matrix and alpha-granule membrane, ie, von Willebrand factor, fibrinogen, CD41, CD62P (P-selectin); however, they differed from lysosomes because acid phosphatases were not present. These giant alpha-granules were unable to release their contents after stimulation by thrombin, in contrast to platelets with normal morphology. Abnormalities in bone marrow MK maturation that were detected at the electron microscopic level and that led to lysis of numerous MKs were responsible for thrombocytopenia and were similar in both patients. MK abnormalities are probably the consequence of the chromosome aberration. ETS 1 and FLI, two proto-oncogenes that appear to be essential with GATA1 for the normal expression of MK-specific genes, map to 11q23-q24 and are, thus, deleted in this thrombocytopenia. In conclusion, the association of all these abnormalities constitutes a new familial platelet disorder and may present a valuable model for exploring the role of some genes involved in the regulation of thrombopoiesis.
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PMID:A new congenital dysmegakaryopoietic thrombocytopenia (Paris-Trousseau) associated with giant platelet alpha-granules and chromosome 11 deletion at 11q23. 863 71

Previous studies have reported that the platelets of healthy term neonates have either diminished or normal reactivity compared to the platelets of adults. To circumvent the methodologic problems of previous studies, we used a whole blood flow cytometric method to study neonatal platelet reactivity to thrombin, a combination of ADP and epinephrine, and U46619 (a stable thromboxane A2 analogue). Inclusion in the assay of the peptide GPRP (an inhibitor of fibrin polymerization) enabled us to study platelet reactivity to human alpha-thrombin in whole blood. Umbilical cord blood and day 1 peripheral blood were collected from 30 healthy term neonates and compared to peripheral blood from 20 normal adults. In whole blood samples without added agonist, there were no significant differences between neonates and adults in the platelet binding of monoclonal antibodies 6D1 (GPIb-specific) or 7E3 (GPIIb-IIIa complex-specific). As determined by S12 (a P-selectin-specific monoclonal antibody), neither neonates nor adults had circulating degranulated platelets. However, in both cord and peripheral whole blood samples, neonatal platelets were significantly less reactive than adult platelets to thrombin, ADP/epinephrine, and U46619, as determined by the extent of increase in the platelet surface expression of P-selectin and the GPIIb-IIIa complex, and the extent of decrease in the platelet surface expression of the GPIb-IX complex.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Neonatal platelets are less reactive than adult platelets to physiological agonists in whole blood. 774 Apr 70

Platelet stimulation by thrombin leads to the activation of phosphoinositide 3-kinase (PI 3K) and to the production of the D3 phosphoinositides, phosphatidylinositol 3,4-bisphosphate (PdtIns-3,4P2) and 3,4,5-trisphosphate (PdtIns-3,4,5-P3). Because changes in the levels of these phosphoinositides correlate with the kinetics of actin assembly, they have been proposed to mediate actin assembly, causing cell shape changes. Wortmannin and LY294002, two unrelated inhibitors of PI 3-K, were used to investigate the role of PI 3-K in platelet actin assembly and aggregation. Both PI 3-K inhibitors abrogated the production of PdtIns-3,4-P2 and PdtIns-3,4,5-P3 in thrombin receptor-activating peptide (TRAP)-stimulated cells. However, neither wortmannin nor LY294002 altered the kinetics of actin assembly or the exposure of nucleation sites in TRAP-stimulated cells. In contrast, PI 3-K inhibitors showed a specific inhibitory pattern of cell aggregation, characterized by a primary phase of aggregation followed by progressive disaggregation. Flow cytometry analysis with the PAC1 monoclonal antibody or with FITC-labeled fibrinogen indicated that wortmannin inhibited the maintenance of the platelet integrin GPIIb-IIIa in its active state. Wortmannin also inhibited, in a dose-dependent manner, platelet aggregation induced by the binding of the monoclonal antibodies P256 and LIBS-6 to GPIIb-IIIa. LIBS Fab-induced aggregation also led to the production of PdtIns-3,4-P2. Platelet secretion, as evidenced by the release of preloaded 14C-5-hydroxy-tryptamine secretion or P-selectin up-regulation, was not affected by PI 3-K inhibition. These results demonstrate that the generation of D3 phosphoinositides is not required for actin assembly in TRAP-activated platelets. However, PI 3-K stimulation is necessary for prolonged GPIIb-IIIa activation and irreversible platelet aggregation. PI 3-K stimulation downstream of GPIIb-IIIa engagement may provide positive feedback required to sustain active GPIIb-IIIa.
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PMID:Phosphoinositide 3-kinase inhibition spares actin assembly in activating platelets but reverses platelet aggregation. 774 73


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