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)

Thrombopoietin (TPO) plays a crucial role in megakaryocyte differentiation and platelet production. c-Mpl, a receptor for TPO, is also expressed in terminally differentiated platelets. We investigated the effects of TPO on activation of p38 mitogen-activated protein kinase in human platelets. Thrombin, a thrombin receptor agonist peptide, a thromboxane A(2) analogue, collagen, crosslinking the glycoprotein VI, ADP, and epinephrine, but not phorbol 12, 13-dibutyrate activated p38. TPO did not activate p38 by itself, whereas TPO pretreatment potentiated the agonist-induced activation of p38. TPO did not promote phosphorylation of Hsp27 and cytosolic phospholipase A(2) by itself, but enhanced thrombin-induced phosphorylation of them. The specific p38 inhibitor SB203580 strongly inhibited such phosphorylation. Thus, TPO possesses the priming effect on p38 activation in human platelets and could affect platelet functions through the p38 pathway.
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PMID:Thrombopoietin potentiates agonist-stimulated activation of p38 mitogen-activated protein kinase in human platelets. 1040 23

Since megakaryocytes are the cellular precursors of platelets we have investigated whether they share responses to platelet agonists, in particular collagen. Although previous studies have reported responses to thrombin in non-human megakaryocytes, through studies of single cell calcium responses and protein tyrosine-phosphorylation we demonstrate for the first time that both isolated human megakaryocytes and CD41/61-positive megakaryocytes derived in culture from CD34+ cells share responses to the platelet agonists collagen, collagen-related peptide and thrombin. The responses to either collagen or CRP were seen only in the most mature megakaryocytes and not in megakaryocyte-like cell lines, suggesting that the response to collagen is a characteristic developed late during megakaryocyte differentiation. These primary cells offer the opportunity to use many molecular and cellular techniques to study and manipulate signalling events in response to platelet receptor agonists, which cannot be performed in the small, anucleate platelet itself.
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PMID:Collagen or collagen-related peptide cause (Ca2+)i elevation and increased tyrosine phosphorylation in human megakaryocytes. 1049 80

Glycoprotein V (GPV), a subunit of the platelet GPIb-V-IX receptor for von Willebrand factor and thrombin, is specifically found in platelets and mature megakaryocytes. Studies of the GPV gene can therefore provide insight into the mechanisms governing megakaryocyte differentiation. The human GPV promoter was isolated, and elements important for its tissue specific transcriptional activity were localized using systematic DNase I protection and reporter deletion assays. A -1413/+25 fragment inserted into a luciferase reporter construct displayed promoter activity in Dami and HEL but not in K562, HL60, or HeLa cells. Progressive 5' to 3' deletion showed a putative enhancer region in the -1413/-903 segment that contained closely spaced GATA and Ets sites protected from DNase I digestion in Dami extracts. Regions similar to a GPIIb gene repressor were found at -816 and -610, with the first exhibiting repressor activity in Dami and HEL cells and the second protected from DNAse I. Deletions from -362 to -103, an area containing protected sites for Sp1, STAT, and GATA, induced a progressive decrease in activity. The -103/+1 fragment, bearing a proximal Ets footprinted site and a GATA/Ets tandem footprint, displayed 75% activity relative to the full-length promoter and retained cell specificity. In summary, this work defines several regions of the GPV gene promoter important for its activity. It contains megakaryocyte-specific signals, including erythro-megakaryocytic GATA, and Ets cis-acting elements, GPIIb-like repressor domains, and binding sites for ubiquitous factors such as Sp1, ETF, and STAT.
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PMID:Functional characterization of the human platelet glycoprotein V gene promoter: A specific marker of late megakaryocytic differentiation. 1055 46

1. In human platelets, arachidonic acid is mainly metabolized by the two enzyme systems; cyclo-oxygenase and 12-lipoxygenase. Cyclo-oxygenase produces prostaglandin H(2) which is further converted to thromboxane B(2). 12-Lipoxygenase synthesizes 12(S)-hydroperoxyeicosatetraenoic acid which is reduced to 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). 2. An anti-platelet compound, OPC-29030, dose-dependently inhibited 12(S)-HETE production with an IC(50) of 0.06+/-0.01 microM, but not synthesis of thromboxane B(2) in human platelets. Although the compound suppressed 12(S)-HETE production in human platelets, cytosolic 12-lipoxygenase activity was not inhibited up to 10 microM. Essentially identical data were obtained with a 12-lipoxygenase of human erythroleukaemia cells which had megakaryocyte/platelet-like properties. 3. OPC-29030 also suppressed production of 5(S)-HETE, a 5-lipoxygenase product, in rat basophilic leukaemia cells without inhibiting enzyme activity. It has been shown that 5-lipoxygenase binds to membrane 5-lipoxygenase-activating protein (FLAP) to produce 5(S)-HETE, and thus FLAP inhibitor suppresses cellular 5(S)-HETE production. 4. A FLAP inhibitor, L-655,238, suppressed platelet 12(S)-HETE production, but had no effect on the 12-lipoxygenase activity. 5. Western blot analysis showed that platelet 12-lipoxygenase translocated from cytosol to membranes upon thrombin stimulation, and OPC-29030 suppressed this process in a dose-dependent manner. 6. These results suggest that the 12-lipoxygenase of human platelets binds to FLAP or a similar protein, and OPC-29030 suppresses 12(S)-HETE production by inhibiting a certain step of the 12-lipoxygenase translocation.
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PMID:An anti-platelet agent, OPC-29030, inhibits translocation of 12-lipoxygenase and 12-hydroxyeicosatetraenoic acid production in human platelets. 1058 25

Thrombin, a multifunctional protein, has been found to be involved in cellular mitogenesis, tumor growth, and metastasis, in addition to its well known effects on the initiation of platelet aggregation and secretion and the conversion of fibrinogen to fibrin to form blood clots. These properties of thrombin rely on its action as a serine protease, which cleaves the N-terminal region of a 7-transmembrane G protein receptor (protease-activated receptor, PAR-1), thus exposing a tethered end hexapeptide sequence capable of activating its receptor. Little is known about its effect on genes that regulate the cell cycle. This study was undertaken to investigate the possible mechanisms by which thrombin regulates tumor cell growth in several tumor cell lines: human CHRF megakaryocyte, DU145 prostate, MDAMB231 and MCF7 breast, U3A fibrosarcoma, and 2 murine fibroblast cell lines, MEFp53(-/-) and CD STAT(-/-). We have found that thrombin under the conditions of culture employed inhibits cell growth by both up-regulation of p21(waf/cip1) and induction of caspases via its PAR-1 receptor. The increased expression of p21(waf/cip1) by thrombin was p53 independent, STAT1 dependent, and protein synthesis independent. This was associated with tyrosine phosphorylation of JAK2 and STAT1, and nuclear translocation of STAT1. Induction of apoptosis is also PAR-1-specific, STAT1-dependent, and associated with up-regulation of caspases 1, 2, and 3. Our study establishes, for the first time, a link between PAR-1 receptor activation with the STAT signal pathway, which leads to cell cycle control and apoptosis. This observation broadens our understanding of the mechanism of PAR-1 activation and its effect on cell growth, and could possibly lead to therapeutic approaches for the treatment of cancer.
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PMID:Thrombin inhibits tumor cell growth in association with up-regulation of p21(waf/cip1) and caspases via a p53-independent, STAT-1-dependent pathway. 1069 50

Essential thrombocythemia (ET) is one of the less rare variants of the chronic myeloproliferative disorders (MPD). The present review questions the possible link between spontaneous megakaryocytopoiesis, platelet hyperreactivity, and the occurrence of platelet-mediated vascular manifestations in acquired and hereditary ET. In acquired ET, the role of thrombopoietin (TPO) is crucial to the observed hypermegakaryocytopoiesis, which is characterized by an increased proliferation of megakaryocyte (MK) progenitors, even in conditions of culture without addition of any known megakaryocyte colony-stimulating factors. An increased reactivity of megakaryocyte progenitors to TPO remains to be precisely delineated. A defective clearance of TPO by megakaryocytes and platelets because of a reduced number of TPO receptors is possible. TPO is able to enhance platelet aggregation induced by ADP, thrombin, and collagen. A point mutation in the TPO gene as the cause of increased TPO production in hereditary ET can readily explain both spontaneous megakaryocytopoiesis and platelet-mediated microvascular manifestations simulating the phenotype of acquired ET. Nevertheless, to date, no mutation of the TPO structural gene, as shown in two families with hereditary ET, and no mutations in the TPO receptor have been found in patients with acquired ET. There is good evidence that the microvascular circulation disturbances in ET are caused by intravascular activation and aggregation of hypersensitive platelets, with sludging or occlusion of the endarterial microvasculature. In this process, the generation of platelet-derived products, endothelial cell damage, fibromuscular intimal proliferation, and platelet thrombi are essential and can be inhibited by a platelet-specific regimen of aspirin, thus providing a rationale for using low-dose aspirin as an antithrombotic agent in thrombocythemia. In contrast, the generation of thrombin appears not to be essential for the formation of platelet thrombi, thereby explaining the inefficacy of coumadin derivatives and heparin in the treatment and prevention of microvascular circulation disturbances in hereditary and acquired ET.
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PMID:Spontaneous proliferative megakaryocytopoiesis and platelet hyperreactivity in essential thrombocythemia: is thrombopoietin the link? 1074 15

Megakaryocytopoiesis and thrombocytopoiesis result from the interactions between hematopoietic progenitor cells, humoral factors, and marrow stromal cells derived from mesenchymal stem cells (MSCs) or MSCs directly. MSCs are self-renewing marrow cells that provide progenitors for osteoblasts, adipocytes, chondrocytes, myocytes, and marrow stromal cells. MSCs are isolated from bone marrow aspirates and are expanded in adherent cell culture using an optimized media preparation. Culture-expanded human MSCs (hMSCs) express a variety of hematopoietic cytokines and growth factors and maintain long-term culture-initiating cells in long-term marrow culture with CD34(+) hematopoietic progenitor cells. Two lines of evidence suggest that hMSCs function in megakaryocyte development. First, hMSCs express messenger RNA for thrombopoietin, a primary regulator for megakaryocytopoiesis and thrombocytopoiesis. Second, adherent hMSC colonies in primary culture are often associated with hematopoietic cell clusters containing CD41(+) megakaryocytes. The physical association between hMSCs and megakaryocytes in marrow was confirmed by experiments in which hMSCs were copurified by immunoselection using an anti-CD41 antibody. To determine whether hMSCs can support megakaryocyte and platelet formation in vitro, we established a coculture system of hMSCs and CD34(+) cells in serum-free media without exogenous cytokines. These cocultures produced clusters of hematopoietic cells atop adherent MSCs. After 7 days, CD41(+) megakaryocyte clusters and pro-platelet networks were observed with pro-platelets increasing in the next 2 weeks. CD41(+) platelets were found in culture medium and expressed CD62P after thrombin treatment. These results suggest that MSCs residing within the megakaryocytic microenvironment in bone marrow provide key signals to stimulate megakaryocyte and platelet production from CD34(+) hematopoietic cells.
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PMID:Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34(+) hematopoietic progenitor cells. 1082 34

The mouse- and rat-platelet-specific hamster monoclonal antibody (MAb) 1C2, previously found to react with a thrombin-sensitive 74-kD glycoprotein, was now shown to recognize platelet glycoprotein V (GPV, CD42d). 1C2 reacted with NIH-3T3 cells in which recombinant mouse or rat GPV was expressed. Both 1C2 and 4A5, another mouse-platelet-specific rat MAb, immunoprecipitated GVP, although they recognized different epitopes. Side-by-side comparison confirmed that 1C2 as well as RPM.9, a MAb against rat GPV, recognized the same rat platelet molecule. In a mouse bone marrow culture, 1C2+ megakaryocytes emerged from CD41 (GPIIb)+1C2- megakaryocytes. Because 1C2+ megakaryocytes exhibited higher DNA ploidy distribution than CD41+ cells, GPV likely appears in the late stage of megakaryocyte maturation. This study established 1C2 as a MAb against mouse and rat GPV, namely CD42d, and as useful tool to study rodent megakaryopoiesis.
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PMID:Characterization of monoclonal antibodies against mouse and rat platelet glycoprotein V (CD42d). 1115 97

The study of megakaryocytopoiesis has been based largely on in vitro assays. We characterize an in vivo model of megakaryocyte and platelet development in which human peripheral blood stem cells (PBSCs) differentiate along megakaryocytic as well as myeloid/lymphoid lineages in sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD-SCID) mice. Human hematopoiesis preferentially occurs in the bone marrow of the murine recipients, and engraftment is independent of exogenous cytokines. Human colony-forming units-megakaryocyte (CFU-MK) develop predominantly in the bone marrow, and their presence correlates with the overall degree of human cell engraftment. Using a sensitive and specific flow cytometric assay, human platelets are detected in the peripheral blood from weeks 1 to 8 after transplantation. The number of circulating human platelets peaks at week 3 with a mean of 20 x 10(9)/L. These human platelets are functional as assessed by CD62P expression in response to thrombin stimulation in vitro. Exogenous cytokines have a detrimental effect on CFU-MK production after 2 weeks, and animals treated with these cytokines have no circulating platelets 8 weeks after transplantation. Although cytokine stimulation of human PBSCs ex vivo led to a significant increase in CFU-MK, CD34+/41+, and CD41+ cells, these ex vivo expanded cells provided only delayed and transient platelet production in vivo, and no CFU-MK developed in vivo after transplantation. In conclusion, xenogeneic transplantation of human PBSCs into NOD/SCID mice provides an excellent in vivo model to study human megakaryocytopoiesis and platelet production.
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PMID:Xenotransplantation of immunodeficient mice with mobilized human blood CD34+ cells provides an in vivo model for human megakaryocytopoiesis and platelet production. 1123 2

Store-regulated Ca(2+) entry (SOCE) is an important mechanism of elevating cytosolic [Ca(2+)]i in platelets, though the Ca(2+) influx channels involved are still unclear. We screened human platelets and their precursor cells (human stem cells and megakaryocytes) for the presence of candidate influx channels, i.e., isoforms of the Trp family of proteins. Primary stem cells were cultured with thrombopoietin to allow differentiation into megakaryocytes. The undifferentiated stem cells (CD34(+)) showed mRNA expression of only a spliced variant Trp1A. Immature (CD61(+)/CD42b(low)) and mature (CD61(+)/CD42b(high)) megakaryocytes as well as platelets expressed in addition unspliced Trp1 as well as Trp4 (less abundant) and Trp6 isoforms. This unspliced isoform appeared to be specific for cells of the megakaryocyte/platelet lineage, since immature (CD14(+)/CD61(-)/CD42b(-)) and mature monocytes expressed only the Trp1A isoform. This conclusion was confirmed by the presence of Trp1A, 3, 4 and 6 transcripts in the immature megakaryocytic Dami cell line, and of Trp1, 1A, 4 and 6 transcripts in the more mature CHRF-288 cell line. The up-regulation of Trp1, 4 and 6 in the lineage from primary stem cells to mature megakaryocytes and platelets was accompanied by increased influx of extracellular Ca(2+) after pretreatment of the cells with thapsigargin or thrombin. Expression of new Trp isoforms in the differentiated cells is thus accompanied by increased SOCE.
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PMID:Expression of transient receptor potential mRNA isoforms and Ca(2+) influx in differentiating human stem cells and platelets. 1142 Jan 22


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