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Lok, S. et al. Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature 369, 565-568

University of Washington Seattle, Seattle, Washington, United States
Nature 06/1994; 369(6481):565-568. DOI: 10.1038/369565a0

ABSTRACT

THE major regulator of circulating platelet levels is believed to be a cytokine termed thrombopoietin1,2. It is thought to be a lineage-specific cytokine affecting the proliferation and maturation of committed cells resulting in the production of megakaryocytes and platelets. Despite considerable efforts by a number of laboratories, the unequivocal identification of thrombopoietin has proven elusive. Here we report the functional cloning of a murine complementary DNA encoding a ligand for the receptor encoded by the c-mpl proto-oncogene (c-Mpl) 3–5. The encoded polypeptide has a predicted molecular mass of 35,000 (M
r 35K). The protein has a novel two-domain structure with an amino-terminal domain homologous with erythropoietin and a carboxy-terminal domain rich in serine, threonine and proline residues and containing seven potential N-linked glycosylation sites. Intraperitoneal injections of mice with recombinant protein increase circulating platelet levels by greater than fourfold after 7 days. These results along with those presented in the accompanying report strongly suggest that the ligand for c-Mpl is thrombopoietin.

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    • "In support of this possibility, we found that the mean platelet volume , which is a measurement of the average size of the platelets and gives information about platelet production, was significantly decreased by rapamycin in PPVL rats (p < 0.01; Fig. 7C). Furthermore, the expression of thrombopoietin and its cell surface receptor c-Mpl, which are the most important physiological regulators of platelet formation and are expressed in the spleen [34], was significantly down-regulated by rapamycin in PPVL rats (p < 0.01; Fig. 7D). Therefore, it is possible that a deficient production of thrombopoietin secondary to rapamycin treatment may well contribute to the drop in blood platelet count observed in PPVL rats. "
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    ABSTRACT: Background & Aims: Splenomegaly is a frequent hallmark of portal hypertension that, in some cases, can be very prominent and cause symptoms like abdominal pain, splenic infarction, and cytopenia. This study characterizes the pathogenetic mechanisms leading to spleen enlargement in portal hypertensive rats and focuses on mTOR pathway as a potential modulator of splenomegaly in portal hypertension. Methods: Characterization of splenomegaly was performed by histological, hematological, immunohistochemical and Western blot analyses in rats with portal hypertension induced by portal vein ligation, and compared with sham-operated animals. The contribution of the mTOR signaling pathway to splenomegaly was determined in rats with fully developed portal hypertension and control rats by treatment with rapamycin or vehicle. Results: Our results illustrate that splenomegaly in portal hypertensive rats arises as a consequence of the interplay of several factors, including not only spleen congestion, as traditionally thought, but also enlargement and hyperactivation of the splenic lymphoid tissue, as well as increased angiogenesis and fibrogenesis. Since mTOR signaling plays a central role in immunological processes, angiogenesis and fibrogenesis, we next determined the involvement of mTOR in splenomegaly. Interestingly, mTOR signaling was overactivated in the spleen of portal hypertensive rats, and mTOR blockade by rapamycin profoundly ameliorated splenomegaly, causing a 44% decrease in spleen size. This effect was most likely accounted for the inhibitory action of rapamycin on lymphocyte proliferation, neovascularization and fibrosis. Conclusions: These findings shed light on the pathogenesis of splenomegaly in portal hypertension, and identify mTOR signaling as a potential target for therapeutic intervention in this disease. (c) 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
    Full-text · Dataset · Apr 2015
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    • "Prior to the identification of TPO, the receptor for TPO, called c-Mpl, was discovered as the product of the c-mpl gene, the wild-type homolog of the v-mpl oncogene (Vigon et al., 1992). The use of c-Mpl was instrumental in purifying thrombopoietin as its ligand (Bartley et al., 1994; de Sauvage et al., 1994; Lok et al., 1994; Sohma et al., 1994; Wendling et al., 1994). Although TPO-independent mechanisms exist in thrombopoiesis (Bunting et al., 1997), TPO/Mpl signaling is essential for proliferation and differentiation of megakaryocytes and platelet production. "
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    ABSTRACT: Thrombopoietin (TPO) is the principal regulator of thrombopoiesis and promotes the proliferation, differentiation and maturation of megakaryocytic progenitor cells in mammals. In this study we report on the molecular and functional characterization of goldfish TPO. Quantitative expression analysis of goldfish tpo revealed the highest mRNA levels in heart, followed by spleen, liver, brain, intestine and kidney tissues. Significant decrease of tpo and c-mpl expressions in goldfish primary kidney macrophage (PKM) cultures, as progenitor to macrophage development progressed, indicates that TPO is not involved in monopoiesis. Recombinant goldfish TPO (rgTPO) alone did not induce significant proliferation of progenitor cells, but TPO in cooperation with recombinant goldfish kit ligand A (rgKITLA) supported proliferation of progenitor cells in a dose-dependent manner. In response to rgTPO or a combination of rgTPO and rgKITLA, the mRNA levels of thrombopoietic markers cd41 and c-mpl as well as thrombo/erythropoietic transcription factors gata1 and lmo2 in sorted progenitor cells were up-regulated, while the mRNA levels of granulopoietic markers (cebpα and gcsfr) and the lymphoid transcription factor gata3 were down-regulated. Furthermore, rgTPO and rgKITLA synergistically stimulated thrombocytic colony-formation. Our results demonstrate that goldfish TPO has similar functions to mammalian TPO as a regulator of thrombopoiesis, and suggests a highly conserved molecular mechanism of thrombocyte development throughout evolution of vertebrates. Copyright © 2014. Published by Elsevier Ltd.
    Full-text · Article · Nov 2014 · Developmental & Comparative Immunology
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    • "times above normal value (Lok et al. 1994; Harker et al. 1996) and ameliorates chemotherapy/radiation-induced thrombocytopenia (Hokom et al. 1995; Ulich et al. 1995). Erythropoietin (EPO), a glycoprotein primarily produced by cells in the kidney and liver, where its production is upregulated by hypoxia, is best known as the principal factor regulating erythropoiesis in mammals and promotes survival, proliferation, and differentiation of erythroid progenitor/ precursor cells (Elliott et al. 2008; Paschos et al. 2008). "
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    ABSTRACT: Nuclear accidents or terrorist attacks could expose large numbers of people to ionizing radiation. Early biomarkers of radiation injury will be critical for triage, treatment, and follow-up of such individuals. The authors evaluated the utility of multiple blood biomarkers for early-response assessment of radiation exposure using a murine (CD2F1, males) total-body irradiation (TBI) model exposed to Co γ rays (0.6 Gy min) over a broad dose range (0-14 Gy) and timepoints (4 h-5 d). Results demonstrate: 1) dose-dependent changes in hematopoietic cytokines: Flt-3 ligand (Flt3L), interleukin 6 (IL-6), granulocyte colony stimulating factor (G-CSF), thrombopoietin (TPO), erythropoietin (EPO), and acute phase protein serum amyloid A (SAA); 2) dose-dependent changes in blood cell counts: lymphocytes, neutrophils, platelets, and ratio of neutrophils to lymphocytes; 3) protein results coupled with peripheral blood cell counts established very successful separation of groups irradiated to different doses; and 4) enhanced separation of dose was observed as the number of biomarkers increased. Results show that the dynamic changes in the levels of SAA, IL-6, G-CSF, and Flt3L reflect the time course and severity of acute radiation syndrome (ARS) and may function as prognostic indicators of ARS outcome. These results also demonstrate proof-in-concept that plasma proteins show promise as a complimentary approach to conventional biodosimetry for early assessment of radiation exposures and, coupled with peripheral blood cell counts, provide early diagnostic information to manage radiation casualty incidents effectively, closing a gap in capabilities to rapidly and effectively assess radiation exposure early, especially needed in case of a mass-casualty radiological incident.
    Full-text · Article · Jun 2014 · Health physics
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