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Becker IC, Barrachina MN, Lykins J, Camacho V, Stone AP, Chua BA, Signer RAJ, Machlus KR, Whiteheart SW, Roweth HG, Italiano JE. Inhibition of RhoA-mediated secretory autophagy in megakaryocytes mitigates myelofibrosis in mice. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.12.04.626665. [PMID: 39677616 PMCID: PMC11642871 DOI: 10.1101/2024.12.04.626665] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/17/2024]
Abstract
Megakaryocytes (MKs) are large, polyploid cells that contribute to bone marrow homeostasis through the secretion of cytokines such as transforming growth factor β1 (TGFβ1). During neoplastic transformation, immature MKs accumulate in the bone marrow where they induce fibrotic remodeling ultimately resulting in myelofibrosis. Current treatment strategies aim to prevent MK hyperproliferation, however, little is understood about the potential of targeting dysregulated cytokine secretion from neoplastic MKs as a novel therapeutic avenue. Unconventional secretion of TGFβ1 as well as interleukin 1β (IL1β) via secretory autophagy occurs in cells other than MKs, which prompted us to investigate whether similar mechanisms are utilized by MKs. Here, we identified that TGFβ1 strongly co-localized with the autophagy marker light chain 3B in native MKs. Disrupting secretory autophagy by inhibiting the small GTPase RhoA or its downstream effector Rho kinase (ROCK) markedly reduced TGFβ1 and IL1β secretion in vitro . In vivo , conditional deletion of the essential autophagy gene Atg5 from the hematopoietic system limited megakaryocytosis and aberrant cytokine secretion in an MPL W515L -driven transplant model. Similarly, mice with a selective deletion of Rhoa from the MK and platelet lineage were protected from progressive fibrosis. Finally, disease hallmarks in MPL W515L -transplanted mice were attenuated upon treatment with the autophagy inhibitor hydroxychloroquine or the ROCK inhibitor Y27632, either as monotherapy or in combination with the JAK2 inhibitor ruxolitinib. Overall, our data indicate that aberrant cytokine secretion is dependent on secretory autophagy downstream of RhoA, targeting of which represents a novel therapeutic avenue in the treatment of myelofibrosis. One Sentence Summary TGFβ1 is released from megakaryocytes via RhoA-mediated secretory autophagy, and targeting this process can alleviate fibrosis progression in a preclinical mouse model of myelofibrosis.
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Luis TC, Barkas N, Carrelha J, Giustacchini A, Mazzi S, Norfo R, Wu B, Aliouat A, Guerrero JA, Rodriguez-Meira A, Bouriez-Jones T, Macaulay IC, Jasztal M, Zhu G, Ni H, Robson MJ, Blakely RD, Mead AJ, Nerlov C, Ghevaert C, Jacobsen SEW. Perivascular niche cells sense thrombocytopenia and activate hematopoietic stem cells in an IL-1 dependent manner. Nat Commun 2023; 14:6062. [PMID: 37770432 PMCID: PMC10539537 DOI: 10.1038/s41467-023-41691-y] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2022] [Accepted: 09/11/2023] [Indexed: 09/30/2023] Open
Abstract
Hematopoietic stem cells (HSCs) residing in specialized niches in the bone marrow are responsible for the balanced output of multiple short-lived blood cell lineages in steady-state and in response to different challenges. However, feedback mechanisms by which HSCs, through their niches, sense acute losses of specific blood cell lineages remain to be established. While all HSCs replenish platelets, previous studies have shown that a large fraction of HSCs are molecularly primed for the megakaryocyte-platelet lineage and are rapidly recruited into proliferation upon platelet depletion. Platelets normally turnover in an activation-dependent manner, herein mimicked by antibodies inducing platelet activation and depletion. Antibody-mediated platelet activation upregulates expression of Interleukin-1 (IL-1) in platelets, and in bone marrow extracellular fluid in vivo. Genetic experiments demonstrate that rather than IL-1 directly activating HSCs, activation of bone marrow Lepr+ perivascular niche cells expressing IL-1 receptor is critical for the optimal activation of quiescent HSCs upon platelet activation and depletion. These findings identify a feedback mechanism by which activation-induced depletion of a mature blood cell lineage leads to a niche-dependent activation of HSCs to reinstate its homeostasis.
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Affiliation(s)
- Tiago C Luis
- Haematopoietic Stem Cell Biology Laboratory, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK.
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK.
- Centre for Inflammatory Disease, Department of Immunology and Inflammation, Imperial College London, W12 0NN, London, UK.
- Department of Life Sciences, Imperial College London, SW7 2AZ, London, UK.
| | - Nikolaos Barkas
- Haematopoietic Stem Cell Biology Laboratory, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
| | - Joana Carrelha
- Haematopoietic Stem Cell Biology Laboratory, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
| | - Alice Giustacchini
- Haematopoietic Stem Cell Biology Laboratory, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
- Molecular and Cellular Immunology Section, UCL Great Ormond Street Institute of Child Health, London, UK
- Human Technopole, Viale Rita Levi-Montalcini 1, 20157, Milan, Italy
| | - Stefania Mazzi
- Center for Hematology and Regenerative Medicine, Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, SE-141 86, Stockholm, Sweden
| | - Ruggiero Norfo
- Haematopoietic Stem Cell Biology Laboratory, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
| | - Bishan Wu
- Haematopoietic Stem Cell Biology Laboratory, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
| | - Affaf Aliouat
- Haematopoietic Stem Cell Biology Laboratory, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
| | - Jose A Guerrero
- Department of Haematology, University of Cambridge, Cambridge, UK
- National Health Service (NHS) Blood and Transplant, Cambridge Biomedical Campus, Cambridge, UK
| | - Alba Rodriguez-Meira
- Haematopoietic Stem Cell Biology Laboratory, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
| | - Tiphaine Bouriez-Jones
- Haematopoietic Stem Cell Biology Laboratory, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
| | - Iain C Macaulay
- Haematopoietic Stem Cell Biology Laboratory, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
- Earlham Institute, Norwich Research Park, NR4 7UZ, Norwich, UK
| | - Maria Jasztal
- Department of Haematology, University of Cambridge, Cambridge, UK
- National Health Service (NHS) Blood and Transplant, Cambridge Biomedical Campus, Cambridge, UK
| | - Guangheng Zhu
- Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto, ON, M5B 1W8, Canada
- CCOA Therapeutics Inc, Toronto, ON, M5B 1T8, Canada
| | - Heyu Ni
- Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto, ON, M5B 1W8, Canada
- CCOA Therapeutics Inc, Toronto, ON, M5B 1T8, Canada
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, M5S 1A1, Canada
- Canadian Blood Services Centre for Innovation, Toronto, ON, M5B 1W8, Canada
| | - Matthew J Robson
- Department of Biomedical Science, Charles E. Schmidt College of Medicine and Stiles-Nicholson Brain Institute, Florida Atlantic University, Jupiter, FL, 33458, USA
- Division of Pharmaceutical Sciences, James L. Winkle College of Pharmacy, University of Cincinnati, Cincinnati, OH, 45267, USA
| | - Randy D Blakely
- Department of Biomedical Science, Charles E. Schmidt College of Medicine and Stiles-Nicholson Brain Institute, Florida Atlantic University, Jupiter, FL, 33458, USA
| | - Adam J Mead
- Haematopoietic Stem Cell Biology Laboratory, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
| | - Claus Nerlov
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK
| | - Cedric Ghevaert
- Department of Haematology, University of Cambridge, Cambridge, UK
- National Health Service (NHS) Blood and Transplant, Cambridge Biomedical Campus, Cambridge, UK
| | - Sten Eirik W Jacobsen
- Haematopoietic Stem Cell Biology Laboratory, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK.
- MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, OX3 9DS, Oxford, UK.
- Center for Hematology and Regenerative Medicine, Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, SE-141 86, Stockholm, Sweden.
- Department of Cell and Molecular Biology, Karolinska Institutet, SE-171 77, Stockholm, Sweden.
- Department of Hematology, Karolinska University Hospital, Stockholm, Sweden.
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Kat M, van Moort I, Bürgisser PE, Kuijpers TW, Hofman M, Favier M, Favier R, Margadant C, Voorberg J, Bierings R. Mutations in Neurobeachin-like 2 do not impact Weibel-Palade body biogenesis and von Willebrand factor secretion in gray platelet syndrome Endothelial Colony Forming Cells. Res Pract Thromb Haemost 2023; 7:100086. [PMID: 36923710 PMCID: PMC10009729 DOI: 10.1016/j.rpth.2023.100086] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2022] [Revised: 12/13/2022] [Accepted: 01/25/2023] [Indexed: 02/16/2023] Open
Abstract
Background Patients with gray platelet syndrome (GPS) and Neurobeachin-like 2 (NBEAL2) deficiency produce platelets lacking alpha-granules (AGs) and present with lifelong bleeding symptoms. AGs are lysosome-related organelles and store the hemostatic protein von Willebrand factor (VWF) and the transmembrane protein P-selectin. Weibel-Palade bodies (WPBs) are lysosome-related organelles of endothelial cells and also store VWF and P-selectin. In megakaryocytes, NBEAL2 links P-selectin on AGs to the SNARE protein SEC22B on the endoplasmic reticulum, thereby preventing premature release of cargo from AG precursors. In endothelial cells, SEC22B drives VWF trafficking from the endoplasmic reticulum to Golgi and promotes the formation of elongated WPBs, but it is unclear whether this requires NBEAL2. Objectives To investigate a potential role for NBEAL2 in WPB biogenesis and VWF secretion using NBEAL2-deficient endothelial cells. Methods The interaction of SEC22B with NBEAL2 in endothelial cells was investigated by interatomic mass spectrometry and pull-down analysis. Endothelial colony forming cells were isolated from healthy controls and 3 unrelated patients with GPS and mutations in NBEAL2. Results We showed that SEC22B binds to NBEAL2 in ECs. Endothelial colony forming cells derived from a patient with GPS are deficient in NBEAL2 but reveal normal formation and maturation of WPBs and normal WPB cargo recruitment. Neither basal nor histamine-induced VWF secretion is altered in the absence of NBEAL2. Conclusions Although NBEAL2 deficiency causes the absence of AGs in patients with GPS, it does not impact WPB functionality in ECs. Our data highlight the differences in the regulatory mechanisms between these 2 hemostatic storage compartments.
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Affiliation(s)
- Marije Kat
- Molecular Hematology, Sanquin Research and Landsteiner Laboratory, Amsterdam University Medical Center, University of Amsterdam, the Netherlands
| | - Iris van Moort
- Hematology, Erasmus MC University Medical Center, Rotterdam, the Netherlands
| | - Petra E. Bürgisser
- Hematology, Erasmus MC University Medical Center, Rotterdam, the Netherlands
| | - Taco W. Kuijpers
- Molecular Hematology, Sanquin Research and Landsteiner Laboratory, Amsterdam University Medical Center, University of Amsterdam, the Netherlands
- Pediatric Immunology, Rheumatology and Infectious Diseases, Emma Children’s Hospital, Amsterdam University Medical Center, location AMC, Amsterdam, the Netherlands
| | - Menno Hofman
- Molecular Hematology, Sanquin Research and Landsteiner Laboratory, Amsterdam University Medical Center, University of Amsterdam, the Netherlands
| | - Marie Favier
- Assistance Publique-Hôpitaux de Paris, French Reference Center for Platelet Disorders, Armand Trousseau Children’s Hospital, Paris, France
| | - Rémi Favier
- Assistance Publique-Hôpitaux de Paris, French Reference Center for Platelet Disorders, Armand Trousseau Children’s Hospital, Paris, France
- INSERM, UMR 1287, Gustave Roussy Cancer Campus, Paris-Saclay University, Villejuif, France
| | - Coert Margadant
- Angiogenesis Laboratory, Cancer Center Amsterdam, Amsterdam University Medical Center, Amsterdam, the Netherlands
| | - Jan Voorberg
- Molecular Hematology, Sanquin Research and Landsteiner Laboratory, Amsterdam University Medical Center, University of Amsterdam, the Netherlands
- Experimental Vascular Medicine, Amsterdam University Medical Center, University of Amsterdam, the Netherlands
| | - Ruben Bierings
- Hematology, Erasmus MC University Medical Center, Rotterdam, the Netherlands
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Forstner D, Guettler J, Gauster M. Changes in Maternal Platelet Physiology during Gestation and Their Interaction with Trophoblasts. Int J Mol Sci 2021; 22:10732. [PMID: 34639070 PMCID: PMC8509324 DOI: 10.3390/ijms221910732] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2021] [Revised: 09/27/2021] [Accepted: 09/29/2021] [Indexed: 01/06/2023] Open
Abstract
Upon activation, maternal platelets provide a source of proinflammatory mediators in the intervillous space of the placenta. Therefore, platelet-derived factors may interfere with different trophoblast subtypes of the developing human placenta and might cause altered hormone secretion and placental dysfunction later on in pregnancy. Increased platelet activation, and the subsequent occurrence of placental fibrinoid deposition, are linked to placenta pathologies such as preeclampsia. The composition and release of platelet-derived factors change over gestation and provide a potential source of predicting biomarkers for the developing fetus and the mother. This review indicates possible mechanisms of platelet-trophoblast interactions and discusses the effect of increased platelet activation on placenta development.
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Affiliation(s)
- Désirée Forstner
- Division of Cell Biology, Histology and Embryology, Gottfried Schatz Research Center, Medical University of Graz, 8010 Graz, Austria; (J.G.); (M.G.)
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5
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The induction of a mesenchymal phenotype by platelet cloaking of cancer cells is a universal phenomenon. Transl Oncol 2021; 14:101229. [PMID: 34592589 PMCID: PMC8488306 DOI: 10.1016/j.tranon.2021.101229] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2021] [Accepted: 09/20/2021] [Indexed: 11/21/2022] Open
Abstract
Platelet cancer cell interactions are a key factor in driving the pro-metastatic phenotype. Platelet cancer cell interactions appear to be mediated by 5 key genes which have established roles in metastasis. Targeting these mediators of metastasis could improve outcomes for cancer patients. Tumour metastasis accounts for over 90% of cancer related deaths. The platelet is a key blood component, which facilitates efficient metastasis. This study aimed to understand the molecular mechanisms involved in tumour-platelet cell interactions. The interaction between cancer cells and platelets was examined in 15 epithelial cell lines, representing 7 cancer types. Gene expression analysis of EMT-associated and cancer stemness genes was performed by RT-PCR. Whole transcriptome analysis (WTA) was performed using Affymetrix 2.0ST arrays on a platelet co-cultured ovarian model. Platelet adhesion and activation occurred across all tumour types. WTA identified increases in cellular movement, migration, invasion, adhesion, development, differentiation and inflammation genes and decreases in processes associated with cell death and survival following platelet interaction. Increased invasive capacity was also observed in a subset of cell lines. A cross-comparison with a platelet co-cultured mouse model identified 5 common altered genes; PAI-1, PLEK2, CD73, TNC, and SDPR. Platelet cancer cell interactions are a key factor in driving the pro-metastatic phenotype and appear to be mediated by 5 key genes which have established roles in metastasis. Targeting these metastasis mediators could improve cancer patient outcomes.
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6
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Abstract
Upon activation, platelets release a plethora of factors which help to mediate their dynamic functions in hemostasis, inflammation, wound healing, tumor metastasis and angiogenesis. The majority of these bioactive molecules are released from α-granules, which are unique to platelets, and contain an incredibly diverse repertoire of cargo including; integral membrane proteins, pro-coagulant molecules, chemokines, mitogenic, growth and angiogenic factors, adhesion proteins, and microbicidal proteins. Clinically, activation of circulating platelets has increasingly been associated with various disease states. Biomarkers indicating the level of platelet activation in patients can therefore be useful tools to evaluate risk factors to predict future complications and determine treatment strategies or evaluate antiplatelet therapy. The irreversible nature of α-granule secretion makes it ideally suited as a marker of platelet activation. This review outlines the release and contents of platelet α-granules, as well as the membrane bound, and soluble α-granule cargo proteins that can be used as biomarkers of platelet activation.
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Affiliation(s)
- Christopher W Smith
- Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, B15 2TT, Birmingham, UK
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7
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Glembotsky AC, De Luca G, Heller PG. A Deep Dive into the Pathology of Gray Platelet Syndrome: New Insights on Immune Dysregulation. J Blood Med 2021; 12:719-732. [PMID: 34408521 PMCID: PMC8364843 DOI: 10.2147/jbm.s270018] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2021] [Accepted: 06/16/2021] [Indexed: 12/22/2022] Open
Abstract
The gray platelet syndrome (GPS) is a rare platelet disorder, characterized by impaired alpha-granule biogenesis in megakaryocytes and platelets due to NBEAL2 mutations. Typical clinical features include macrothrombocytopenia, bleeding and elevated vitamin B12 levels, while bone marrow fibrosis and splenomegaly may develop during disease progression. Recently, the involvement of other blood lineages has been highlighted, revealing the role of NBEAL2 outside the megakaryocyte-platelet axis. Low leukocyte counts, decreased neutrophil granulation and impaired neutrophil extracellular trap formation represent prominent findings in GPS patients, reflecting deranged innate immunity and associated with an increased susceptibility to infection. In addition, low numbers and impaired degranulation of NK cells have been demonstrated in animal models. Autoimmune diseases involving different organs and a spectrum of autoantibodies are present in a substantial proportion of GPS patients, expanding the syndromic spectrum of this disorder and pointing to dysregulation of the adaptive immune response. Low-grade inflammation, as evidenced by elevation of liver-derived acute-phase reactants, is another previously unrecognized feature of GPS which may contribute to disease manifestations. This review will focus on the mechanisms underlying the pathogenesis of blood cell abnormalities in human GPS patients and NBEAL2-null animal models, providing insight into the effects of NBEAL2 in hemostasis, inflammation and immunity.
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Affiliation(s)
- Ana C Glembotsky
- Departamento Hematología Investigación, Instituto de Investigaciones Médicas "Dr. A. Lanari", Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina.,Departamento Hematología Investigación, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad de Buenos Aires, Instituto de Investigaciones Médicas (IDIM), Buenos Aires, Argentina
| | - Geraldine De Luca
- Departamento Hematología Investigación, Instituto de Investigaciones Médicas "Dr. A. Lanari", Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina.,Departamento Hematología Investigación, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad de Buenos Aires, Instituto de Investigaciones Médicas (IDIM), Buenos Aires, Argentina
| | - Paula G Heller
- Departamento Hematología Investigación, Instituto de Investigaciones Médicas "Dr. A. Lanari", Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina.,Departamento Hematología Investigación, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad de Buenos Aires, Instituto de Investigaciones Médicas (IDIM), Buenos Aires, Argentina
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Braun A, Anders HJ, Gudermann T, Mammadova-Bach E. Platelet-Cancer Interplay: Molecular Mechanisms and New Therapeutic Avenues. Front Oncol 2021; 11:665534. [PMID: 34322381 PMCID: PMC8311658 DOI: 10.3389/fonc.2021.665534] [Citation(s) in RCA: 72] [Impact Index Per Article: 18.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2021] [Accepted: 06/17/2021] [Indexed: 12/18/2022] Open
Abstract
Although platelets are critically involved in thrombosis and hemostasis, experimental and clinical evidence indicate that platelets promote tumor progression and metastasis through a wide range of physical and functional interactions between platelets and cancer cells. Thrombotic and thromboembolic events are frequent complications in patients with solid tumors. Hence, cancer modulates platelet function by directly inducing platelet-tumor aggregates and triggering platelet granule release and altering platelet turnover. Also, platelets enhance tumor cell dissemination by activating endothelial cell function and recruiting immune cells to primary and metastatic tumor sites. In this review, we summarize current knowledge on the complex interactions between platelets and tumor cells and the host microenvironment. We also critically discuss the potential of anti-platelet agents for cancer prevention and treatment.
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Affiliation(s)
- Attila Braun
- Walther-Straub-Institute for Pharmacology and Toxicology, Ludwig-Maximilian-University, Member of the German Center for Lung Research (DZL), Munich, Germany
| | - Hans-Joachim Anders
- Division of Nephrology, Department of Medicine IV, Ludwig-Maximilians-University Hospital, Munich, Germany
| | - Thomas Gudermann
- Walther-Straub-Institute for Pharmacology and Toxicology, Ludwig-Maximilian-University, Member of the German Center for Lung Research (DZL), Munich, Germany
| | - Elmina Mammadova-Bach
- Walther-Straub-Institute for Pharmacology and Toxicology, Ludwig-Maximilian-University, Member of the German Center for Lung Research (DZL), Munich, Germany.,Division of Nephrology, Department of Medicine IV, Ludwig-Maximilians-University Hospital, Munich, Germany
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9
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Neutrophil specific granule and NETosis defects in gray platelet syndrome. Blood Adv 2021; 5:549-564. [PMID: 33496751 DOI: 10.1182/bloodadvances.2020002442] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2020] [Accepted: 12/06/2020] [Indexed: 12/15/2022] Open
Abstract
Gray platelet syndrome (GPS) is an autosomal recessive bleeding disorder characterized by a lack of α-granules in platelets and progressive myelofibrosis. Rare loss-of-function variants in neurobeachin-like 2 (NBEAL2), a member of the family of beige and Chédiak-Higashi (BEACH) genes, are causal of GPS. It is suggested that BEACH domain containing proteins are involved in fusion, fission, and trafficking of vesicles and granules. Studies in knockout mice suggest that NBEAL2 may control the formation and retention of granules in neutrophils. We found that neutrophils obtained from the peripheral blood from 13 patients with GPS have a normal distribution of azurophilic granules but show a deficiency of specific granules (SGs), as confirmed by immunoelectron microscopy and mass spectrometry proteomics analyses. CD34+ hematopoietic stem cells (HSCs) from patients with GPS differentiated into mature neutrophils also lacked NBEAL2 expression but showed similar SG protein expression as control cells. This is indicative of normal granulopoiesis in GPS and identifies NBEAL2 as a potentially important regulator of granule release. Patient neutrophil functions, including production of reactive oxygen species, chemotaxis, and killing of bacteria and fungi, were intact. NETosis was absent in circulating GPS neutrophils. Lack of NETosis is suggested to be independent of NBEAL2 expression but associated with SG defects instead, as indicated by comparison with HSC-derived neutrophils. Since patients with GPS do not excessively suffer from infections, the consequence of the reduced SG content and lack of NETosis for innate immunity remains to be explored.
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10
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Adipocyte Fatty Acid Transfer Supports Megakaryocyte Maturation. Cell Rep 2021; 32:107875. [PMID: 32640240 DOI: 10.1016/j.celrep.2020.107875] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2019] [Revised: 02/21/2020] [Accepted: 06/15/2020] [Indexed: 01/12/2023] Open
Abstract
Megakaryocytes (MKs) come from a complex process of hematopoietic progenitor maturation within the bone marrow that gives rise to de novo circulating platelets. Bone marrow microenvironment contains a large number of adipocytes with a still ill-defined role. This study aims to analyze the influence of adipocytes and increased medullar adiposity in megakaryopoiesis. An in vivo increased medullar adiposity in mice caused by high-fat-diet-induced obesity is associated to an enhanced MK maturation and proplatelet formation. In vitro co-culture of adipocytes with bone marrow hematopoietic progenitors shows that delipidation of adipocytes directly supports MK maturation by enhancing polyploidization, amplifying the demarcation membrane system, and accelerating proplatelet formation. This direct crosstalk between adipocytes and MKs occurs through adipocyte fatty acid transfer to MKs involving CD36 to reinforce megakaryocytic maturation. Thus, these findings unveil an influence of adiposity on MK homeostasis based on a dialogue between adipocytes and MKs.
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11
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Benlabiod C, Dagher T, Marty C, Villeval JL. Lessons from mouse models of MPN. INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY 2021; 366:125-185. [PMID: 35153003 DOI: 10.1016/bs.ircmb.2021.02.009] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
Over the past decades, a variety of MPN mouse models have been developed to express in HSC the main mutations identified in patients: JAK2V617F, CALRdel52 or ins5 and MPLW515L. These models mimic quite faithfully human PV or ET with their natural evolutions into MF and their hemostasis complications, demonstrating the driver function of these mutations in MPN. Here, we review these models and show how they have improved our general understanding of MPN regarding (1) the mechanisms of fibrosis, thrombosis/hemorrhages and disease initiation, (2) the roles of additional mutations and signaling pathways in disease progression and (3) the preclinical development of novel therapies. We also address controversial results between these models and remind how these models may differ from human MPN onset and also how basically mice are not humans, encouraging caution when one draw lessons from mice to humans. Furthermore, the contribution of germline genetic predisposition, HSC and niche aging, metabolic, oxidative, replicative or genotoxic stress, inflammation, immune escape and additional mutations need to be considered in further investigations to encompass the full complexity of human MPN in mice.
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Affiliation(s)
- Camelia Benlabiod
- INSERM, UMR 1287, Gustave Roussy, Villejuif, France; Université Paris-Saclay, UMR 1287, Gustave Roussy, Villejuif, France; Gustave Roussy, UMR 1287, Villejuif, France
| | - Tracy Dagher
- INSERM, UMR 1287, Gustave Roussy, Villejuif, France; Université Paris-Saclay, UMR 1287, Gustave Roussy, Villejuif, France; Gustave Roussy, UMR 1287, Villejuif, France
| | - Caroline Marty
- INSERM, UMR 1287, Gustave Roussy, Villejuif, France; Université Paris-Saclay, UMR 1287, Gustave Roussy, Villejuif, France; Gustave Roussy, UMR 1287, Villejuif, France.
| | - Jean-Luc Villeval
- INSERM, UMR 1287, Gustave Roussy, Villejuif, France; Université Paris-Saclay, UMR 1287, Gustave Roussy, Villejuif, France; Gustave Roussy, UMR 1287, Villejuif, France.
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12
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The endoplasmic reticulum protein SEC22B interacts with NBEAL2 and is required for megakaryocyte α-granule biogenesis. Blood 2021; 136:715-725. [PMID: 32384141 DOI: 10.1182/blood.2019004276] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2019] [Accepted: 04/20/2020] [Indexed: 12/14/2022] Open
Abstract
Studies of inherited platelet disorders have provided many insights into platelet development and function. Loss of function of neurobeachin-like 2 (NBEAL2) causes gray platelet syndrome (GPS), where the absence of platelet α-granules indicates NBEAL2 is required for their production by precursor megakaryocytes. The endoplasmic reticulum is a dynamic network that interacts with numerous intracellular vesicles and organelles and plays key roles in their development. The megakaryocyte endoplasmic reticulum is extensive, and in this study we investigated its role in the biogenesis of α-granules by focusing on the membrane-resident trafficking protein SEC22B. Coimmunoprecipitation (co-IP) experiments using tagged proteins expressed in human HEK293 and megakaryocytic immortalized megakaryocyte progenitor (imMKCL) cells established binding of NBEAL2 with SEC22B, and demonstrated that NBEAL2 can simultaneously bind SEC22B and P-selectin. NBEAL2-SEC22B binding was also observed for endogenous proteins in human megakaryocytes using co-IP, and immunofluorescence microscopy detected substantial overlap. SEC22B binding was localized to a region of NBEAL2 spanning amino acids 1798 to 1903, where 2 GPS-associated missense variants have been reported: E1833K and R1839C. NBEAL2 containing either variant did not bind SEC22B coexpressed in HEK293 cells. CRISPR/Cas9-mediated knockout of SEC22B in imMKCL cells resulted in decreased NBEAL2, but not vice versa. Loss of either SEC22B or NBEAL2 expression resulted in failure of α-granule production and reduced granule proteins in imMKCL cells. We conclude that SEC22B is required for α-granule biogenesis in megakaryocytes, and that interactions with SEC22B and P-selectin facilitate the essential role of NBEAL2 in granule development and cargo stability.
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Pluthero FG, Kahr WHA. Gray platelet syndrome: NBEAL2 mutations are associated with pathology beyond megakaryocyte and platelet function defects. J Thromb Haemost 2021; 19:318-322. [PMID: 33300270 DOI: 10.1111/jth.15177] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2020] [Accepted: 11/06/2020] [Indexed: 01/13/2023]
Affiliation(s)
- Fred G Pluthero
- Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada
| | - Walter H A Kahr
- Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada
- Division of Haematology/Oncology, Department of Paediatrics, University of Toronto and the Hospital for Sick Children, Toronto, ON, Canada
- Department of Biochemistry, University of Toronto, Toronto, ON, Canada
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14
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Mendoza-Almanza G, Burciaga-Hernández L, Maldonado V, Melendez-Zajgla J, Olmos J. Role of platelets and breast cancer stem cells in metastasis. World J Stem Cells 2020; 12:1237-1254. [PMID: 33312396 PMCID: PMC7705471 DOI: 10.4252/wjsc.v12.i11.1237] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/01/2020] [Revised: 08/23/2020] [Accepted: 09/22/2020] [Indexed: 02/06/2023] Open
Abstract
The high mortality rate of breast cancer is mainly caused by the metastatic ability of cancer cells, resistance to chemotherapy and radiotherapy, and tumor regression capacity. In recent years, it has been shown that the presence of breast cancer stem cells is closely associated with the migration and metastatic ability of cancer cells, as well as with their resistance to chemotherapy and radiotherapy. The tumor microenvironment is one of the main molecular factors involved in cancer and metastatic processes development, in this sense it is interesting to study the role of platelets, one of the main communicator cells in the human body which are activated by the signals they receive from the microenvironment and can generate more than one response. Platelets can ingest and release RNA, proteins, cytokines and growth factors. After the platelets interact with the tumor microenvironment, they are called "tumor-educated platelets." Tumor-educated platelets transport material from the tumor microenvironment to sites adjacent to the tumor, thus helping to create microenvironments conducive for the development of primary and metastatic tumors. It has been observed that the clone capable of carrying out the metastatic process is a cancer cell with stem cell characteristics. Cancer stem cells go through a series of processes, including epithelial-mesenchymal transition, intravasation into blood vessels, movement through blood vessels, extravasation at the site of the establishment of a metastatic focus, and site colonization. Tumor-educated platelets support all these processes.
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Affiliation(s)
| | | | - Vilma Maldonado
- Laboratorio de Epigenética, Instituto Nacional de Medicina Genómica, Ciudad de México 14610, Mexico
| | - Jorge Melendez-Zajgla
- Génómica funcional del cáncer, Instituto Nacional de Medicina Genómica, Ciudad de México 14610, Mexico
| | - Jorge Olmos
- Biotecnología Marina, Centro de Investigación Científica y de Estudios Superiores de Ensenada, Ensenada 22860, Mexico
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15
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Ido K, Nakane T, Tanizawa N, Makuuchi Y, Okamura H, Koh S, Nanno S, Nishimoto M, Hirose A, Nakamae M, Nakashima Y, Koh H, Hino M, Nakamae H. Acquired Gray Platelet Syndrome Associated with Primary Myelofibrosis. Intern Med 2020; 59:2751-2756. [PMID: 32641652 PMCID: PMC7691020 DOI: 10.2169/internalmedicine.4912-20] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
A 53-year-old man presented with uncontrolled bleeding caused by acquired platelet dysfunction accompanied by calreticulin-mutated primary myelofibrosis. Based on the detection of abnormal platelets, including large gray platelets, under light microscopy and the loss of the second wave of aggregation observed by light transmission aggregometry, the patient was diagnosed with platelet dysfunction accompanied by myeloproliferative neoplasms (MPNs). In addition, the absence of platelet α-granules was confirmed by electron microscopy. Therefore, this condition may be termed "acquired gray platelet syndrome." Acquired platelet dysfunction must be ruled out when abnormal platelets are observed in patients with MPNs.
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Affiliation(s)
- Kentaro Ido
- Hematology, Graduate School of Medicine, Osaka City University, Japan
| | - Takahiko Nakane
- Hematology, Graduate School of Medicine, Osaka City University, Japan
| | - Nao Tanizawa
- Hematology, Graduate School of Medicine, Osaka City University, Japan
| | - Yosuke Makuuchi
- Hematology, Graduate School of Medicine, Osaka City University, Japan
| | - Hiroshi Okamura
- Hematology, Graduate School of Medicine, Osaka City University, Japan
| | - Shiro Koh
- Hematology, Graduate School of Medicine, Osaka City University, Japan
| | - Satoru Nanno
- Hematology, Graduate School of Medicine, Osaka City University, Japan
| | | | - Asao Hirose
- Hematology, Graduate School of Medicine, Osaka City University, Japan
| | - Mika Nakamae
- Hematology, Graduate School of Medicine, Osaka City University, Japan
| | | | - Hideo Koh
- Hematology, Graduate School of Medicine, Osaka City University, Japan
| | - Masayuki Hino
- Hematology, Graduate School of Medicine, Osaka City University, Japan
| | - Hirohisa Nakamae
- Hematology, Graduate School of Medicine, Osaka City University, Japan
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16
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Sims MC, Mayer L, Collins JH, Bariana TK, Megy K, Lavenu-Bombled C, Seyres D, Kollipara L, Burden FS, Greene D, Lee D, Rodriguez-Romera A, Alessi MC, Astle WJ, Bahou WF, Bury L, Chalmers E, Da Silva R, De Candia E, Deevi SVV, Farrow S, Gomez K, Grassi L, Greinacher A, Gresele P, Hart D, Hurtaud MF, Kelly AM, Kerr R, Le Quellec S, Leblanc T, Leinøe EB, Mapeta R, McKinney H, Michelson AD, Morais S, Nugent D, Papadia S, Park SJ, Pasi J, Podda GM, Poon MC, Reed R, Sekhar M, Shalev H, Sivapalaratnam S, Steinberg-Shemer O, Stephens JC, Tait RC, Turro E, Wu JKM, Zieger B, Kuijpers TW, Whetton AD, Sickmann A, Freson K, Downes K, Erber WN, Frontini M, Nurden P, Ouwehand WH, Favier R, Guerrero JA. Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome. Blood 2020; 136:1956-1967. [PMID: 32693407 PMCID: PMC7582559 DOI: 10.1182/blood.2019004776] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2020] [Accepted: 07/02/2020] [Indexed: 12/12/2022] Open
Abstract
Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease.
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Affiliation(s)
- Matthew C Sims
- Department of Haematology, University of Cambridge, and
- National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
- Oxford Haemophilia and Thrombosis Centre, Oxford University Hospitals NHS Foundation Trust, NIHR Oxford Biomedical Research Centre, Oxford, United Kingdom
| | - Louisa Mayer
- Department of Haematology, University of Cambridge, and
- National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Janine H Collins
- Department of Haematology, University of Cambridge, and
- National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
- Department of Haematology, Barts Health NHS Trust, London, United Kingdom
| | - Tadbir K Bariana
- Department of Haematology, University of Cambridge, and
- Department of Haematology, Barts Health NHS Trust, London, United Kingdom
- Royal Free London NHS Foundation Trust, London, United Kingdom
| | - Karyn Megy
- Department of Haematology, University of Cambridge, and
- National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
- NIHR BioResource, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Cecile Lavenu-Bombled
- Assistance Publique-Hôpitaux de Paris, Centre de Reference des Pathologies Plaquettaires, Hôpitaux Armand Trousseau, Bicêtre, Robert Debré, Paris, France
| | - Denis Seyres
- Department of Haematology, University of Cambridge, and
- National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
- NIHR BioResource, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | | | - Frances S Burden
- Department of Haematology, University of Cambridge, and
- National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
- NIHR BioResource, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Daniel Greene
- Department of Haematology, University of Cambridge, and
- NIHR BioResource, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge, United Kingdom
- Medical Research Council Biostatistics Unit, Forvie Site, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Dave Lee
- Stoller Biomarker Discovery Centre, Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
| | - Antonio Rodriguez-Romera
- Department of Haematology, University of Cambridge, and
- National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Marie-Christine Alessi
- Centre for CardioVascular and Nutrition Research, INSERM 1263, INRAE 1260, Marseille, France
| | - William J Astle
- National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
- Medical Research Council Biostatistics Unit, Forvie Site, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Wadie F Bahou
- Department of Medicine, Stony Brook University, Stony Brook, NY
| | - Loredana Bury
- Department of Medicine, Section of Internal and Cardiovascular Medicine, University of Perugia, Perugia, Italy
| | | | - Rachael Da Silva
- Stoller Biomarker Discovery Centre, Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
| | - Erica De Candia
- Institute of Internal Medicine and Geriatrics, Catholic University School of Medicine, Rome, Italy
- Fondazione Policlinico Universitario Agostino Gemelli Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy
| | - Sri V V Deevi
- Department of Haematology, University of Cambridge, and
- NIHR BioResource, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Samantha Farrow
- Department of Haematology, University of Cambridge, and
- National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
- NIHR BioResource, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Keith Gomez
- Royal Free London NHS Foundation Trust, London, United Kingdom
| | - Luigi Grassi
- Department of Haematology, University of Cambridge, and
- National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
- NIHR BioResource, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Andreas Greinacher
- Institut für Immunologie und Transfusionsmedizin, Universitätsmedizin Greifswald, Greifswald, Germany
| | - Paolo Gresele
- Department of Medicine, Section of Internal and Cardiovascular Medicine, University of Perugia, Perugia, Italy
| | - Dan Hart
- The Royal London Hospital Haemophilia Centre, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom
| | - Marie-Françoise Hurtaud
- Assistance Publique-Hôpitaux de Paris, Centre de Reference des Pathologies Plaquettaires, Hôpitaux Armand Trousseau, Bicêtre, Robert Debré, Paris, France
| | - Anne M Kelly
- Department of Haematology, University of Cambridge, and
| | - Ron Kerr
- Department of Haematology, Ninewells Hospital and Medical School, Dundee, United Kingdom
| | - Sandra Le Quellec
- Service d'Hématologie Biologique, Hospices Civils de Lyon, Lyon, France
| | - Thierry Leblanc
- Assistance Publique-Hôpitaux de Paris, Centre de Reference des Pathologies Plaquettaires, Hôpitaux Armand Trousseau, Bicêtre, Robert Debré, Paris, France
| | - Eva B Leinøe
- Department of Haematology, Rigshospitalet, Copenhagen, Denmark
| | - Rutendo Mapeta
- Department of Haematology, University of Cambridge, and
- NIHR BioResource, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Harriet McKinney
- Department of Haematology, University of Cambridge, and
- National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
- NIHR BioResource, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Alan D Michelson
- Center for Platelet Research Studies, Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Harvard Medical School, Boston, MA
| | - Sara Morais
- Serviço de Hematologia Clínica, Hospital de Santo António, Centro Hospitalar Universitário do Porto, Porto, Portugal
- Unidade Multidisciplinar de Investigação Biomédica, Instituto de Ciências Biomédicas, Universidade do Porto, Porto, Portugal
| | - Diane Nugent
- Center for Inherited Bleeding Disorders, Children's Hospital of Orange County, Orange, CA
| | - Sofia Papadia
- Department of Haematology, University of Cambridge, and
- National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
- NIHR BioResource, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Soo J Park
- Division of Hematology and Oncology, Moores Cancer Center, University of California, San Diego, La Jolla, CA
| | - John Pasi
- The Royal London Hospital Haemophilia Centre, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom
| | - Gian Marco Podda
- Unità di Medicina 2, ASST Santi Paolo e Carlo, Dipartimento di Scienze della Salute, Università degli Studi di Milano, Milan, Italy
| | - Man-Chiu Poon
- University of Calgary Cumming School of Medicine and Southern Alberta Rare Blood and Bleeding Disorders Comprehensive Care Program, Calgary, AB, Canada
| | - Rachel Reed
- Stoller Biomarker Discovery Centre, Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
| | - Mallika Sekhar
- Department of Haematology, Royal Free London NHS Trust, London, United Kingdom
| | - Hanna Shalev
- Department of Pediatric Hematology/Oncology, Soroka Medical Center, Faculty of Medicine, Ben-Gurion University, Beer Sheva, Israel
| | - Suthesh Sivapalaratnam
- Department of Haematology, University of Cambridge, and
- Department of Haematology, Barts Health NHS Trust, London, United Kingdom
| | - Orna Steinberg-Shemer
- Department of Hematology-Oncology, Schneider Children's Medical Center of Israel, Petach Tikva, Israel
- Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
| | - Jonathan C Stephens
- Department of Haematology, University of Cambridge, and
- NIHR BioResource, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Robert C Tait
- Department of Haematology, Royal Infirmary, Glasgow, United Kingdom
| | - Ernest Turro
- Department of Haematology, University of Cambridge, and
- National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
- NIHR BioResource, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge, United Kingdom
- Medical Research Council Biostatistics Unit, Forvie Site, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - John K M Wu
- Division of Hematology-Oncology, University of British Columbia and BC Children's Hospital, Vancouver, BC, Canada
| | - Barbara Zieger
- Division of Pediatric Hematology and Oncology, Department of Pediatrics and Adolescent Medicine, Medical Center-Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Taco W Kuijpers
- Department of Pediatric Immunology, Rheumatology and Infectious Diseases, Emma Children's Hospital, Amsterdam University Medical Center, Amsterdam, The Netherlands
- Sanquin Research Institute, Department of Blood Cell Research, University of Amsterdam, Amsterdam, The Netherlands
| | - Anthony D Whetton
- Stoller Biomarker Discovery Centre, Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom
| | - Albert Sickmann
- Leibniz-Institut für Analytische Wissenschaften-ISAS-e. V., Dortmund, Germany
- Department of Chemistry, College of Physical Sciences, University of Aberdeen, Aberdeen, United Kingdom
- Medizinische Fakultät, Medizinisches Proteom Center, Ruhr-Universität Bochum, Bochum, Germany
| | - Kathleen Freson
- Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
| | - Kate Downes
- Department of Haematology, University of Cambridge, and
- NIHR BioResource, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Wendy N Erber
- Faculty of Health and Medical Sciences, The University of Western Australia, Crawley, Australia
- PathWest Laboratory Medicine, The University of Western Australia, Nedlands, Australia
| | - Mattia Frontini
- Department of Haematology, University of Cambridge, and
- National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
- British Heart Foundation, Cambridge Centre for Research Excellence, University of Cambridge, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Paquita Nurden
- Institut Hospitalo-Universitaire L'Institut de Rythmologie et Modélisation Cardiaque, Plateforme Technologique d'Innovation Biomédicale, Hôpital Xavier Arnozan, Pessac, France
| | - Willem H Ouwehand
- Department of Haematology, University of Cambridge, and
- National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
- NIHR BioResource, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge, United Kingdom
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom; and
| | - Remi Favier
- Assistance Publique-Hôpitaux de Paris, Centre de Reference des Pathologies Plaquettaires, Hôpitaux Armand Trousseau, Bicêtre, Robert Debré, Paris, France
- INSERM Unité Mixte de Recherche 1170, Gustave Roussy Cancer Campus, Universite Paris-Saclay, Villejuif, France
| | - Jose A Guerrero
- Department of Haematology, University of Cambridge, and
- National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
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17
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Beauchemin H, Shooshtharizadeh P, Pinder J, Dellaire G, Möröy T. Dominant negative Gfi1b mutations cause moderate thrombocytopenia and an impaired stress thrombopoiesis associated with mild erythropoietic abnormalities in mice. Haematologica 2020; 105:2457-2470. [PMID: 33054086 PMCID: PMC7556681 DOI: 10.3324/haematol.2019.222596] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2019] [Accepted: 11/21/2019] [Indexed: 11/30/2022] Open
Abstract
GFI1B-related thrombocytopenia (GFI1B-RT) is a rare bleeding disorder mainly caused by the presence of truncated GFI1B proteins with dominant-negative properties. The disease is characterized by low platelet counts, the presence of abnormal platelets, a megakaryocytic expansion and mild erythroid defects. However, no animal models faithfully reproducing the GFI1B-RT phenotype observed in patients exist. We had previously generated mice with floxed Gfi1b alleles that can be eliminated by Cre recombinase, but those animals developed a much more severe phenotype than GFI1B-RT patients and were of limited interest in assessing the disease. Using CRISPR/Cas9 technology, we have now established three independent mouse lines that carry mutated Gfi1b alleles producing proteins lacking DNA binding zinc fingers and thereby acting in a dominant negative (DN) manner. Mice heterozygous for these Gfi1b-DN alleles show reduced platelet counts and an expansion of megakaryocytes similar to features of human GFI1B-RT but lacking the distinctively large agranular platelets. In addition, Gfi1b-DN mice exhibit an expansion of erythroid precursors indicative of a mildly abnormal erythropoiesis but without noticeable red blood cell defects. When associated with megakaryocyte-specific ablation of the remaining allele, the Gfi1b-DN alleles triggered erythroid-specific deleterious defects. Gfi1b-DN mice also showed a delayed recovery from platelet depletion, indicating a defect in stress thrombopoiesis. However, injecting Gfi1b-DN mice with romiplostim, a thrombopoietin receptor super agonist, increased platelet numbers even beyond normal levels. Thus, our data support a causal link between DN mutations in GFI1B and thrombocytopenia and suggest that patients with GFI1B-RT could be treated successfully with thrombopoietin agonists.
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Affiliation(s)
- Hugues Beauchemin
- Institut de Recherches Cliniques de Montréal, IRCM, Montréal, Quebec
| | | | - Jordan Pinder
- Departments of Pathology and Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia
| | - Graham Dellaire
- Departments of Pathology and Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia
| | - Tarik Möröy
- Institut de Recherches Cliniques de Montréal, IRCM, Montréal, Quebec
- Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, Montréal, Quebec
- Division of Experimental Medicine, McGill University, Montréal, Quebec, Canada
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18
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A Genome-Wide Screen in Mice To Identify Cell-Extrinsic Regulators of Pulmonary Metastatic Colonisation. G3-GENES GENOMES GENETICS 2020; 10:1869-1877. [PMID: 32245826 PMCID: PMC7263671 DOI: 10.1534/g3.120.401128] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Metastatic colonization, whereby a disseminated tumor cell is able to survive and proliferate at a secondary site, involves both tumor cell-intrinsic and -extrinsic factors. To identify tumor cell-extrinsic (microenvironmental) factors that regulate the ability of metastatic tumor cells to effectively colonize a tissue, we performed a genome-wide screen utilizing the experimental metastasis assay on mutant mice. Mutant and wildtype (control) mice were tail vein-dosed with murine metastatic melanoma B16-F10 cells and 10 days later the number of pulmonary metastatic colonies were counted. Of the 1,300 genes/genetic locations (1,344 alleles) assessed in the screen 34 genes were determined to significantly regulate pulmonary metastatic colonization (15 increased and 19 decreased; P < 0.005 and genotype effect <-55 or >+55). While several of these genes have known roles in immune system regulation (Bach2, Cyba, Cybb, Cybc1, Id2, Igh-6, Irf1, Irf7, Ncf1, Ncf2, Ncf4 and Pik3cg) most are involved in a disparate range of biological processes, ranging from ubiquitination (Herc1) to diphthamide synthesis (Dph6) to Rho GTPase-activation (Arhgap30 and Fgd4), with no previous reports of a role in the regulation of metastasis. Thus, we have identified numerous novel regulators of pulmonary metastatic colonization, which may represent potential therapeutic targets.
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19
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Abstract
Introduction: Metastatic cancers are extremely difficult to treat, and account for the vast majority of cancer-related deaths. The dissemination of tumor cells to distant sites is highly dynamic, asynchronous, and involves both tumor and host intrinsic factors. Effective therapeutic targets to block metastasis will need to disrupt key pathways that are required for multiple stages of metastasis.Areas covered: This review discusses the heterogeneity of cancers and metastasis, with an emphasis on motility as a key driver trait of metastasis. Recent metastatic cancer studies that identified either host or cancer cell intrinsic factors important for metastasis, using single gene-deficient animal models or 3D intravital imaging of avian embryo models, are also discussed. Potential metastatic blocking targets are listed as they relate to metastatic cancer therapy.Expert opinion: The development of metastatic disease is a complex interplay of genetic and epigenetic factors from the host and cancer cells acting in a patient-specific manner. Inhibiting key driver traits of metastasis should yield survival benefit at any stage of the disease, and we look forward to the next generation of personalized medicines for cancer therapy that target cancer cell motility for increased therapeutic efficacy.
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Affiliation(s)
| | - Perrin H Beatty
- Department of Oncology, University of Alberta, Edmonton, Canada
| | - John D Lewis
- Department of Oncology, University of Alberta, Edmonton, Canada
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20
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Aspirin inhibits platelets from reprogramming breast tumor cells and promoting metastasis. Blood Adv 2020; 3:198-211. [PMID: 30670536 DOI: 10.1182/bloodadvances.2018026161] [Citation(s) in RCA: 33] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2018] [Accepted: 12/16/2018] [Indexed: 12/21/2022] Open
Abstract
It is now recognized that compounds released from tumor cells can activate platelets, causing the release of platelet-derived factors into the tumor microenvironment. Several of these factors have been shown to directly promote neovascularization and metastasis, yet how the feedback between platelet releasate and the tumor cell affects metastatic phenotype remains largely unstudied. Here, we identify that breast tumor cells secrete high levels of interleukin 8 (IL-8, CXCL8) in response to platelet releasate, which promotes their invasive capacity. Furthermore, we found that platelets activate the Akt pathway in breast tumor cells, and inhibition of this pathway eliminated IL-8 production. We therefore hypothesized inhibiting platelets with aspirin could reverse the prometastatic effects of platelets on tumor cell signaling. Platelets treated with aspirin did not activate the Akt pathway, resulting in reduced IL-8 secretion and impaired tumor cell invasion. Of note, patients with breast cancer receiving aspirin had lower circulating IL-8, and their platelets did not increase tumor cell invasion compared with patients not receiving aspirin. Our data suggest platelets support breast tumor metastasis by inducing tumor cells to secrete IL-8. Our data further support that aspirin acts as an anticancer agent by disrupting the communication between platelets and breast tumor cells.
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21
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Morodomi Y, Kanaji S, Won E, Kawamoto T, Kanaji T. Modified application of Kawamoto's film method for super-resolution imaging of megakaryocytes in undecalcified bone marrow. Res Pract Thromb Haemost 2020; 4:86-91. [PMID: 31989088 PMCID: PMC6971304 DOI: 10.1002/rth2.12276] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2019] [Revised: 09/25/2019] [Accepted: 09/27/2019] [Indexed: 12/11/2022] Open
Abstract
BACKGROUND Super-resolution microscopy has enabled high-resolution imaging of the actin cytoskeleton in megakaryocytes and platelets. These technologies have extended our knowledge of thrombopoiesis and platelet spreading using megakaryocytes and platelets cultured in vitro on matrix proteins. However, for better understanding of megakaryocytopoiesis and platelet production, high-resolution imaging of cells in an in vivo bone marrow microenvironment is required. Development of Kawamoto's film method greatly advanced the techniques of thin cryosectioning of hard tissues such as undecalcified bones. One obstacle that remains is the spherical aberration that occurs due to the difference in the refractive index for the light path, limiting the usage of Kawamoto's film method to lower magnification observation. OBJECTIVES To overcome the weakness of the conventional Kawamoto's film method for higher magnification observation of undecalcified bone marrow. METHODS We have modified the original method with a very simple method: flipping the film at the step of mounting the sections on the glass. RESULTS AND CONCLUSIONS This new method successfully led to the adjustment of the refractive index and enabled super-resolution imaging of megakaryocytes in undecalcified mouse femurs. Our modified method will expand the application of Kawamoto's film method and enable precise analysis of megakaryocytopoiesis and platelet production in the bone marrow microenvironment under pathophysiological conditions.
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Affiliation(s)
- Yosuke Morodomi
- Department of Molecular MedicineMERU‐Roon Research Center on Vascular BiologyThe Scripps Research InstituteLa JollaCalifornia
| | - Sachiko Kanaji
- Department of Molecular MedicineMERU‐Roon Research Center on Vascular BiologyThe Scripps Research InstituteLa JollaCalifornia
| | - Eric Won
- Department of Molecular MedicineMERU‐Roon Research Center on Vascular BiologyThe Scripps Research InstituteLa JollaCalifornia
- Department of Hematology & OncologyUniversity of CaliforniaSan DiegoCalifornia
- Rady Children’s HospitalSan DiegoCalifornia
| | - Tadafumi Kawamoto
- Department of BiochemistrySchool of Dental MedicineTsurumi UniversityTsurumi‐kuJapan
| | - Taisuke Kanaji
- Department of Molecular MedicineMERU‐Roon Research Center on Vascular BiologyThe Scripps Research InstituteLa JollaCalifornia
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22
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Abstract
Histology of bone marrow routinely identifies megakaryocytes that enclose neutrophils and other hematopoietic cells, a phenomenon termed emperipolesis. Preserved across mammalian species and enhanced with systemic inflammation and platelet demand, the nature and significance of emperipolesis remain largely unexplored. Recent advances demonstrate that emperipolesis is in fact a distinct form of cell-in-cell interaction. Following integrin-mediated attachment, megakaryocytes and neutrophils both actively drive entry via cytoskeletal rearrangement. Neutrophils enter a vacuole termed the emperisome which then releases them directly into the megakaryocyte cytoplasm. From this surprising location, neutrophils fuse with the demarcation membrane system to pass membrane to circulating platelets, enhancing the efficiency of thrombocytogenesis. Neutrophils then egress intact, carrying megakaryocyte membrane and potentially other cell components along with them. In this review, we summarize what is known about this intriguing cell-in-cell interaction and discuss potential roles for emperipolesis in megakaryocyte, platelet and neutrophil biology.
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Affiliation(s)
- Pierre Cunin
- Department of Medicine, Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School , Boston, MA, USA
| | - Peter A Nigrovic
- Department of Medicine, Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School , Boston, MA, USA.,Department of Medicine, Division of Immunology, Boston Children's Hospital, Harvard Medical School , Boston, MA, USA
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23
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Mammadova-Bach E, Braun A. Zinc Homeostasis in Platelet-Related Diseases. Int J Mol Sci 2019; 20:E5258. [PMID: 31652790 PMCID: PMC6861892 DOI: 10.3390/ijms20215258] [Citation(s) in RCA: 42] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2019] [Revised: 10/18/2019] [Accepted: 10/21/2019] [Indexed: 12/13/2022] Open
Abstract
Zn2+ deficiency in the human population is frequent in underdeveloped countries. Worldwide, approximatively 2 billion people consume Zn2+-deficient diets, accounting for 1-4% of deaths each year, mainly in infants with a compromised immune system. Depending on the severity of Zn2+ deficiency, clinical symptoms are associated with impaired wound healing, alopecia, diarrhea, poor growth, dysfunction of the immune and nervous system with congenital abnormalities and bleeding disorders. Poor nutritional Zn2+ status in patients with metastatic squamous cell carcinoma or with advanced non-Hodgkin lymphoma, was accompanied by cutaneous bleeding and platelet dysfunction. Forcing Zn2+ uptake in the gut using different nutritional supplementation of Zn2+ could ameliorate many of these pathological symptoms in humans. Feeding adult rodents with a low Zn2+ diet caused poor platelet aggregation and increased bleeding tendency, thereby attracting great scientific interest in investigating the role of Zn2+ in hemostasis. Storage protein metallothionein maintains or releases Zn2+ in the cytoplasm, and the dynamic change of this cytoplasmic Zn2+ pool is regulated by the redox status of the cell. An increase of labile Zn2+ pool can be toxic for the cells, and therefore cytoplasmic Zn2+ levels are tightly regulated by several Zn2+ transporters located on the cell surface and also on the intracellular membrane of Zn2+ storage organelles, such as secretory vesicles, endoplasmic reticulum or Golgi apparatus. Although Zn2+ is a critical cofactor for more than 2000 transcription factors and 300 enzymes, regulating cell differentiation, proliferation, and basic metabolic functions of the cells, the molecular mechanisms of Zn2+ transport and the physiological role of Zn2+ store in megakaryocyte and platelet function remain elusive. In this review, we summarize the contribution of extracellular or intracellular Zn2+ to megakaryocyte and platelet function and discuss the consequences of dysregulated Zn2+ homeostasis in platelet-related diseases by focusing on thrombosis, ischemic stroke and storage pool diseases.
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Affiliation(s)
- Elmina Mammadova-Bach
- University Hospital and Rudolf Virchow Center, University of Würzburg, 97080 Würzburg, Germany.
| | - Attila Braun
- Walther-Straub-Institute for Pharmacology and Toxicology, Ludwig-Maximilians University Munich, German Center for Lung Research, 80336 Munich, Germany.
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24
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Riley R, Khan A, Pai S, Warmke L, Winkler M, Gunning W. A Case of Chronic Thrombocytopenia in a 17-Year-Old Female. Lab Med 2019; 50:406-420. [PMID: 31228350 DOI: 10.1093/labmed/lmz013] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023] Open
Abstract
Storage pool deficiency (SPD) is a group of rare platelet disorders that result from deficiencies in α-granules, δ-granules, or both. One type of α-SPD is gray platelet syndrome (GPS), caused by mutations in the neurobeachin-like 2 (NBEAL2) gene that results in a bleeding diathesis, thrombocytopenia, splenomegaly, and progressive myelofibrosis. Due to the lack of α-granules, platelets have a gray and degranulated appearance by light microscopy. However, definitive diagnosis of GPS requires confirmation of α-granule deficiency by electron microscopy. Treatment is nonspecific, with the conservative utilization of platelet transfusions being the most important form of therapy. We present a case of a 17-year-old female with a past medical history of thrombocytopenia, first identified at the age of five. Her clinical symptomatology included chronic fatigue, gingival bleeding, bruising, menorrhagia, and leg pain. This report will discuss both the clinical and the pathophysiologic aspects of this rare platelet disorder.
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Affiliation(s)
- Roger Riley
- Departments of Pathology, Virginia Commonwealth University (VCU) School of Medicine, Richmond
| | - Asad Khan
- Departments of Pediatrics, Virginia Commonwealth University (VCU) School of Medicine, Richmond
| | - Shella Pai
- Departments of Pathology, Virginia Commonwealth University (VCU) School of Medicine, Richmond
| | - Laura Warmke
- Department of Pathology and Laboratory Medicine, University of Texas MD Anderson Cancer Center, Houston
| | | | - William Gunning
- Department of Pathology, University of Toledo College of Medicine, Toledo, Ohio
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25
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Guo BB, Linden MD, Fuller KA, Phillips M, Mirzai B, Wilson L, Chuah H, Liang J, Howman R, Grove CS, Malherbe JA, Leahy MF, Allcock RJ, Erber WN. Platelets in myeloproliferative neoplasms have a distinct transcript signature in the presence of marrow fibrosis. Br J Haematol 2019; 188:272-282. [PMID: 31426129 DOI: 10.1111/bjh.16152] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2019] [Accepted: 06/20/2019] [Indexed: 01/10/2023]
Abstract
Marrow fibrosis is a significant complication of myeloproliferative neoplasms (MPN) that affects up to 20% of patients and is associated with a poor prognosis. The pathological processes that lead to fibrotic progression are not well understood, but megakaryocytes have been implicated in the process. The aim of this study was to determine whether platelets, derived from megakaryocytes, have transcriptomic alterations associated with fibrosis. Platelets from MPN patients with and without fibrosis and non-malignant control individuals were assessed using next generation sequencing. Results from the initial training cohort showed discrete changes in platelet transcripts in the presence of marrow fibrosis. We identified more than 1000 differentially expressed transcripts from which a putative 3-gene fibrotic platelet signature (CCND1, H2AX [previously termed H2AFX] and CEP55) could be identified. This fibrosis-associated signature was assessed blinded on platelets from an independent test MPN patient cohort. The 3-gene signature was able to discriminate between patients with and without marrow fibrosis with a positive predictive value of 71% (93% specificity, 71% sensitivity). This demonstrates that assessment of dysregulated transcripts in platelets may be a useful monitoring tool in MPN to identify progression to marrow fibrosis. Further, sequential monitoring could have clinical applications for early prediction of progression to fibrosis.
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Affiliation(s)
- Belinda B Guo
- School of Biomedical Sciences, University of Western Australia, Crawley, WA, Australia
| | - Matthew D Linden
- School of Biomedical Sciences, University of Western Australia, Crawley, WA, Australia
| | - Kathryn A Fuller
- School of Biomedical Sciences, University of Western Australia, Crawley, WA, Australia.,PathWest Laboratory Medicine, Nedlands, WA, Australia
| | - Michael Phillips
- Centre for Medical Research, University of Western Australia, Crawley, WA, Australia
| | - Bob Mirzai
- School of Biomedical Sciences, University of Western Australia, Crawley, WA, Australia.,PathWest Laboratory Medicine, Nedlands, WA, Australia
| | - Lynne Wilson
- School of Biomedical Sciences, University of Western Australia, Crawley, WA, Australia.,PathWest Laboratory Medicine, Nedlands, WA, Australia
| | - Hun Chuah
- School of Biomedical Sciences, University of Western Australia, Crawley, WA, Australia.,Royal Perth Hospital, Department of Health Western Australia, Perth, WA, Australia
| | - James Liang
- School of Biomedical Sciences, University of Western Australia, Crawley, WA, Australia.,Sir Charles Gairdner Hospital, Department of Health Western Australia, Nedlands, WA, Australia
| | - Rebecca Howman
- Sir Charles Gairdner Hospital, Department of Health Western Australia, Nedlands, WA, Australia
| | - Carolyn S Grove
- School of Biomedical Sciences, University of Western Australia, Crawley, WA, Australia.,PathWest Laboratory Medicine, Nedlands, WA, Australia.,Sir Charles Gairdner Hospital, Department of Health Western Australia, Nedlands, WA, Australia
| | - Jacques A Malherbe
- School of Biomedical Sciences, University of Western Australia, Crawley, WA, Australia.,Medical School, University of Western Australia, Crawley, WA, Australia
| | - Michael F Leahy
- PathWest Laboratory Medicine, Nedlands, WA, Australia.,Royal Perth Hospital, Department of Health Western Australia, Perth, WA, Australia.,Medical School, University of Western Australia, Crawley, WA, Australia
| | - Richard J Allcock
- School of Biomedical Sciences, University of Western Australia, Crawley, WA, Australia.,PathWest Laboratory Medicine, Nedlands, WA, Australia
| | - Wendy N Erber
- School of Biomedical Sciences, University of Western Australia, Crawley, WA, Australia.,PathWest Laboratory Medicine, Nedlands, WA, Australia.,Medical School, University of Western Australia, Crawley, WA, Australia
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26
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Lo RW, Li L, Leung R, Pluthero FG, Kahr WHA. NBEAL2 (Neurobeachin-Like 2) Is Required for Retention of Cargo Proteins by α-Granules During Their Production by Megakaryocytes. Arterioscler Thromb Vasc Biol 2019; 38:2435-2447. [PMID: 30354215 DOI: 10.1161/atvbaha.118.311270] [Citation(s) in RCA: 39] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Objective- Human and mouse megakaryocytes lacking NBEAL2 (neurobeachin-like 2) produce platelets where α-granules lack protein cargo. This cargo is mostly megakaryocyte-synthesized, but some proteins, including FGN (fibrinogen), are endocytosed. In this study, we examined the trafficking of both types of cargo within primary megakaryocytes cultured from normal and NBEAL2-null mice, to determine the role of NBEAL2 in α-granule maturation. We also examined the interaction of NBEAL2 with the granule-associated protein P-selectin in human megakaryocytes and platelets. Approach and Results- Fluorescence microscopy was used to compare uptake of labeled FGN by normal and NBEAL2-null mouse megakaryocytes, which was similar in both. NBEAL2-null cells, however, showed decreased FGN retention, and studies with biotinylated protein showed rapid loss rather than increased degradation. Intracellular tracking via fluorescence microscopy revealed that in normal megakaryocytes, endocytosed FGN sequentially associated with compartments expressing RAB5 (Ras-related protein in brain 5), RAB7 (Ras-related protein in brain 7), and P-selectin, where it was retained. A similar initial pattern was observed in NBEAL2-null megakaryocytes, but then FGN passed from the P-selectin compartment to RAB11 (Ras-related protein in brain 11)-associated endosomes before release. Megakaryocyte-synthesized VWF (Von Willebrand factor) was observed to follow the same route out of NBEAL2-null cells. Immunofluorescence microscopy revealed intracellular colocalization of NBEAL2 with P-selectin in human megakaryocytes, proplatelets, and platelets. Native NBEAL2 and P-selectin were coimmunoprecipitated from platelets and megakaryocytes. Conclusions- NBEAL2 is not required for FGN uptake by megakaryocytes. NBEAL2 is required for the retention of both endocytosed and megakaryocyte-synthesized proteins by maturing α-granules, and possibly by platelet-borne granules. This function may involve interaction of NBEAL2 with P-selectin.
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Affiliation(s)
- Richard W Lo
- From the Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada (R.W.L., L.L., R.L., F.G.P., W.H.A.K.).,Department of Biochemistry, University of Toronto, ON, Canada (R.W.L., W.H.A.K.)
| | - Ling Li
- From the Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada (R.W.L., L.L., R.L., F.G.P., W.H.A.K.)
| | - Richard Leung
- From the Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada (R.W.L., L.L., R.L., F.G.P., W.H.A.K.)
| | - Fred G Pluthero
- From the Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada (R.W.L., L.L., R.L., F.G.P., W.H.A.K.)
| | - Walter H A Kahr
- From the Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada (R.W.L., L.L., R.L., F.G.P., W.H.A.K.).,Department of Biochemistry, University of Toronto, ON, Canada (R.W.L., W.H.A.K.).,Division of Haematology/Oncology, Department of Paediatrics, University of Toronto and The Hospital for Sick Children, ON, Canada (W.H.A.K.)
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27
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Balaphas A, Meyer J, Sadoul K, Fontana P, Morel P, Gonelle‐Gispert C, Bühler LH. Platelets and Platelet-Derived Extracellular Vesicles in Liver Physiology and Disease. Hepatol Commun 2019; 3:855-866. [PMID: 31304449 PMCID: PMC6601322 DOI: 10.1002/hep4.1358] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/20/2018] [Accepted: 03/12/2019] [Indexed: 02/06/2023] Open
Abstract
Beyond their role in hemostasis, platelets are proposed as key mediators of several physiological and pathophysiological processes of the liver, such as liver regeneration, toxic or viral acute liver injury, liver fibrosis, and carcinogenesis. The effects of platelets on the liver involve interactions with sinusoidal endothelial cells and the release of platelet-contained molecules following platelet activation. Platelets are the major source of circulating extracellular vesicles, which are suggested to play key roles in platelet interactions with endothelial cells in several clinical disorders. In the present review, we discuss the implications of platelet-derived extracellular vesicles in physiological and pathophysiological processes of the liver.
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Affiliation(s)
- Alexandre Balaphas
- Division of Digestive SurgeryGeneva University HospitalsGenevaSwitzerland
- Surgical Research UnitGeneva University HospitalsGenevaSwitzerland
- Geneva Medical SchoolUniversity of GenevaGenevaSwitzerland
| | - Jeremy Meyer
- Division of Digestive SurgeryGeneva University HospitalsGenevaSwitzerland
- Surgical Research UnitGeneva University HospitalsGenevaSwitzerland
- Geneva Medical SchoolUniversity of GenevaGenevaSwitzerland
| | - Karin Sadoul
- Regulation and Pharmacology of the Cytoskeleton, Institute for Advanced BiosciencesUniversité Grenoble AlpesGrenobleFrance
| | - Pierre Fontana
- Division of Angiology and HemostasisGeneva University HospitalsGenevaSwitzerland
- Geneva Platelet GroupUniversity of GenevaGenevaSwitzerland
| | - Philippe Morel
- Division of Digestive SurgeryGeneva University HospitalsGenevaSwitzerland
- Surgical Research UnitGeneva University HospitalsGenevaSwitzerland
- Geneva Medical SchoolUniversity of GenevaGenevaSwitzerland
| | - Carmen Gonelle‐Gispert
- Surgical Research UnitGeneva University HospitalsGenevaSwitzerland
- Geneva Medical SchoolUniversity of GenevaGenevaSwitzerland
| | - Leo H. Bühler
- Division of Digestive SurgeryGeneva University HospitalsGenevaSwitzerland
- Surgical Research UnitGeneva University HospitalsGenevaSwitzerland
- Geneva Medical SchoolUniversity of GenevaGenevaSwitzerland
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28
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Defective Zn 2+ homeostasis in mouse and human platelets with α- and δ-storage pool diseases. Sci Rep 2019; 9:8333. [PMID: 31171812 PMCID: PMC6554314 DOI: 10.1038/s41598-019-44751-w] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2018] [Accepted: 05/22/2019] [Indexed: 12/31/2022] Open
Abstract
Zinc (Zn2+) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn2+ storage and release. To visualize Zn2+ storage in human and mouse platelets, the Zn2+ specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d−/− mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn2+ release upon activation. Platelets from Nbeal2−/− mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn2+ levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn2+ homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2−/− and Unc13d−/− mice, and the impairment could be partially restored by extracellular Zn2+. Altogether, we conclude that the release of ionic Zn2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn2+ in platelet-dependent fibrin formation.
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29
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van Oorschot R, Hansen M, Koornneef JM, Marneth AE, Bergevoet SM, van Bergen MGJM, van Alphen FPJ, van der Zwaan C, Martens JHA, Vermeulen M, Jansen PWTC, Baltissen MPA, Gorkom BAPLV, Janssen H, Jansen JH, von Lindern M, Meijer AB, van den Akker E, van der Reijden BA. Molecular mechanisms of bleeding disorderassociated GFI1B Q287* mutation and its affected pathways in megakaryocytes and platelets. Haematologica 2019; 104:1460-1472. [PMID: 30655368 PMCID: PMC6601108 DOI: 10.3324/haematol.2018.194555] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2018] [Accepted: 01/09/2019] [Indexed: 12/13/2022] Open
Abstract
Dominant-negative mutations in the transcription factor Growth Factor Independence-1B (GFI1B), such as GFI1BQ287*, cause a bleeding disorder characterized by a plethora of megakaryocyte and platelet abnormalities. The deregulated molecular mechanisms and pathways are unknown. Here we show that both normal and Q287* mutant GFI1B interacted most strongly with the lysine specific demethylase-1 – REST corepressor - histone deacetylase (LSD1-RCOR-HDAC) complex in megakaryoblasts. Sequestration of this complex by GFI1BQ287* and chemical separation of GFI1B from LSD1 induced abnormalities in normal megakaryocytes comparable to those seen in patients. Megakaryocytes derived from GFI1BQ287*-induced pluripotent stem cells also phenocopied abnormalities seen in patients. Proteome studies on normal and mutant-induced pluripotent stem cell-derived megakaryocytes identified a multitude of deregulated pathways downstream of GFI1BQ287* including cell division and interferon signaling. Proteome studies on platelets from GFI1BQ287* patients showed reduced expression of proteins implicated in platelet function, and elevated expression of proteins normally downregulated during megakaryocyte differentiation. Thus, GFI1B and LSD1 regulate a broad developmental program during megakaryopoiesis, and GFI1BQ287* deregulates this program through LSD1-RCOR-HDAC sequestering.
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Affiliation(s)
- Rinske van Oorschot
- Department of Laboratory Medicine, Laboratory of Hematology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen
| | - Marten Hansen
- Department of Hematopoiesis, Sanquin Research-Academic Medical Center Landsteiner Laboratory, Amsterdam
| | | | - Anna E Marneth
- Department of Laboratory Medicine, Laboratory of Hematology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen
| | - Saskia M Bergevoet
- Department of Laboratory Medicine, Laboratory of Hematology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen
| | - Maaike G J M van Bergen
- Department of Laboratory Medicine, Laboratory of Hematology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen
| | | | | | - Joost H A Martens
- Department of Molecular Biology, Faculty of Science, Radboud University Nijmegen, Radboud Institute for Molecular Life Sciences, Nijmegen
| | - Michiel Vermeulen
- Department of Molecular Biology, Faculty of Science, Radboud University Nijmegen, Radboud Institute for Molecular Life Sciences, Nijmegen
| | - Pascal W T C Jansen
- Department of Molecular Biology, Faculty of Science, Radboud University Nijmegen, Radboud Institute for Molecular Life Sciences, Nijmegen
| | - Marijke P A Baltissen
- Department of Molecular Biology, Faculty of Science, Radboud University Nijmegen, Radboud Institute for Molecular Life Sciences, Nijmegen
| | | | - Hans Janssen
- Department of Biochemistry, the Netherlands Cancer Institute, Amsterdam, the Netherlands
| | - Joop H Jansen
- Department of Laboratory Medicine, Laboratory of Hematology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen
| | - Marieke von Lindern
- Department of Hematopoiesis, Sanquin Research-Academic Medical Center Landsteiner Laboratory, Amsterdam
| | | | - Emile van den Akker
- Department of Hematopoiesis, Sanquin Research-Academic Medical Center Landsteiner Laboratory, Amsterdam
| | - Bert A van der Reijden
- Department of Laboratory Medicine, Laboratory of Hematology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen
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30
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Shadur B, Asherie N, Newburger PE, Stepensky P. How we approach: Severe congenital neutropenia and myelofibrosis due to mutations in VPS45. Pediatr Blood Cancer 2019; 66:e27473. [PMID: 30294941 PMCID: PMC6249036 DOI: 10.1002/pbc.27473] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/30/2018] [Revised: 08/15/2018] [Accepted: 08/30/2018] [Indexed: 01/11/2023]
Abstract
Mutations in the VPS45 gene lead to a severe primary immune deficiency characterized by severe congenital neutropenia and primary myelofibrosis, leading to overwhelming infection and early death. This condition is exceedingly rare with only 16 patients previously reported, including four with successful hematopoietic stem cell transplantation. We review the pathophysiology underlying this condition and detail our approach to treatment, particularly vis-à-vis bone marrow transplantation and the challenges of transplanting into a diseased bone marrow niche. We provide an update on the progress of our three previously reported patients, and two additional patients transplanted at our center.
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Affiliation(s)
- Bella Shadur
- Bone Marrow Transplantation Department, Hadassah-Hebrew
University Medical Center, Jerusalem, Israel,Garvan Institute of Medical Research, Sydney,
Australia,University of New South Wales, Sydney, Australia
| | - Nathalie Asherie
- Bone Marrow Transplantation Department, Hadassah-Hebrew
University Medical Center, Jerusalem, Israel
| | - Peter E. Newburger
- Departments of Pediatrics & Molecular, Cell, and
Cancer Biology, University of Massachusetts Medical School, Worcester,
Massachusetts, USA
| | - Polina Stepensky
- Bone Marrow Transplantation Department, Hadassah-Hebrew
University Medical Center, Jerusalem, Israel
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31
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Cattaneo M. Inherited Disorders of Platelet Function. Platelets 2019. [DOI: 10.1016/b978-0-12-813456-6.00048-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
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32
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Tariket S, Guerrero JA, Garraud O, Ghevaert C, Cognasse F. Platelet α-granules modulate the inflammatory response under systemic lipopolysaccharide injection in mice. Transfusion 2018; 59:32-38. [PMID: 30394544 DOI: 10.1111/trf.14970] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2018] [Revised: 07/23/2018] [Accepted: 07/23/2018] [Indexed: 12/20/2022]
Abstract
BACKGROUND Beyond their role in hemostasis and thrombosis, platelets are also important mediators of inflammation by the release of hundreds of factors stored in their α-granules. Mutations in Nbeal2 cause gray platelet syndrome (GPS), characterized by the lack of platelet α-granules. This study aims to evaluate the immunological (proinflammatory) effects of platelet α-granules. STUDY DESIGN AND METHODS We performed an experiment using Nbeal2-/- mice, the mouse model of GPS. Systemic inflammation was induced by intravenous injection of lipopolysaccharide (LPS). Inflammatory response was assessed by quantification of inflammatory soluble factors and platelet biological response modifiers. RESULTS The lack of Nbeal2 (in Nbeal2 -/- mice, compared with controls) significantly reduced the recruitment of circulating neutrophils and monocytes. Moreover, after LPS injection, there was a significant increase in neutrophil and monocyte counts in control animals, compared with Nbeal2 -/- mice. The control of inflammation, evaluated by the production of anti-inflammatory cytokines, appeared to be greater in Nbeal2-/- mice compared with controls. Conversely, the production of certain inflammatory-soluble mediators known to characterize normal platelet secretion, such as soluble CD40 ligand (sCD40L), was decreased under experimental inflammation in Nbeal2 -/- mice. CONCLUSIONS These results show that α-granules play a direct role in platelet-mediated inflammation balance, confirming the need to further investigate platelet-associated inflammatory pathophysiology and inflammatory adverse events related to blood transfusion.
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Affiliation(s)
- Sofiane Tariket
- Université de Lyon, Groupe sur l'Immunité des Muqueuses et Agents Pathogènes (GIMAP)-EA3064, Saint-Etienne, France.,Établissement Français du Sang Auvergne-Rhône-Alpes, Saint-Etienne, France
| | - Jose A Guerrero
- Department of Haematology, University of Cambridge and National Health Service (NHS) Blood and Transplant, Cambridge, United Kingdom.,National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Olivier Garraud
- Université de Lyon, Groupe sur l'Immunité des Muqueuses et Agents Pathogènes (GIMAP)-EA3064, Saint-Etienne, France.,Institut National de la Transfusion Sanguine, Paris, France
| | - Cedric Ghevaert
- Department of Haematology, University of Cambridge and National Health Service (NHS) Blood and Transplant, Cambridge, United Kingdom.,National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Fabrice Cognasse
- Université de Lyon, Groupe sur l'Immunité des Muqueuses et Agents Pathogènes (GIMAP)-EA3064, Saint-Etienne, France.,Établissement Français du Sang Auvergne-Rhône-Alpes, Saint-Etienne, France
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33
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Megakaryocyte Contribution to Bone Marrow Fibrosis: many Arrows in the Quiver. Mediterr J Hematol Infect Dis 2018; 10:e2018068. [PMID: 30416700 PMCID: PMC6223581 DOI: 10.4084/mjhid.2018.068] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2018] [Accepted: 10/23/2018] [Indexed: 01/14/2023] Open
Abstract
In Primary Myelofibrosis (PMF), megakaryocyte dysplasia/hyperplasia determines the release of inflammatory cytokines that, in turn, stimulate stromal cells and induce bone marrow fibrosis. The pathogenic mechanism and the cells responsible for progression to bone marrow fibrosis in PMF are not completely understood. This review article aims to provide an overview of the crucial role of megakaryocytes in myelofibrosis by discussing the role and the altered secretion of megakaryocyte-derived soluble factors, enzymes and extracellular matrices that are known to induce bone marrow fibrosis.
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34
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Claushuis TAM, de Stoppelaar SF, de Vos AF, Grootemaat AE, van der Wel NN, Roelofs JJTH, Ware J, Van't Veer C, van der Poll T. Nbeal2 Deficiency Increases Organ Damage but Does Not Affect Host Defense During Gram-Negative Pneumonia-Derived Sepsis. Arterioscler Thromb Vasc Biol 2018; 38:1772-1784. [PMID: 29930006 DOI: 10.1161/atvbaha.118.311332] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Objective- Nbeal2-/- mice, a model of human gray platelet syndrome, have reduced neutrophil granularity and impaired host defense against systemic Staphylococcus aureus infection. We here aimed to study the role of Nbeal2 deficiency in both leukocytes and platelets during gram-negative pneumonia and sepsis. Approach and Results- We studied the role of Nbeal2 in platelets and leukocytes during murine pneumonia and sepsis by Klebsiella pneumoniae. Apart from platelet α-granule deficiency and reduced neutrophil granularity, also monocyte granularity was reduced in Nbeal2-/- mice, whereas plasma levels of MPO (myeloperoxidase), elastase, NGAL (neutrophil gelatinase-associated lipocalin), and MMP-9 (matrix metalloproteinase 9), and leukocyte CD11b expression were increased. Nbeal2-/- leukocytes showed unaltered in vitro antibacterial response and phagocytosis capacity against Klebsiella, and unchanged reactive nitrogen species and cytokine production. Also during Klebsiella pneumonia and sepsis, Nbeal2-/- mice had similar bacterial growth in lung and distant body sites, with enhanced leukocyte migration to the bronchoalveolar space. Despite similar infection-induced inflammation, organ damage was increased in Nbeal2-/- mice, which was also seen during endotoxemia. Platelet-specific Nbeal2 deficiency did not influence leukocyte functions, indicating that Nbeal2 directly modifies leukocytes. Transfusion of Nbeal2-/- but not of Nbeal2+/+ platelets into thrombocytopenic mice was associated with bleeding in the lung but similar host defense, pointing at a role for platelet α-granules in maintaining vascular integrity but not host defense during Klebsiella pneumosepsis. Conclusions- These data show that Nbeal2 deficiency-resulting in gray platelet syndrome-affects platelets, neutrophils, and monocytes, with intact host defense but increased organ damage during gram-negative pneumosepsis.
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Affiliation(s)
- Theodora A M Claushuis
- From the Center for Experimental and Molecular Medicine (T.A.M.C., S.F.d.S., A.F.d.V., C.v.V., T.v.d.P.)
| | - Sacha F de Stoppelaar
- From the Center for Experimental and Molecular Medicine (T.A.M.C., S.F.d.S., A.F.d.V., C.v.V., T.v.d.P.)
| | - Alex F de Vos
- From the Center for Experimental and Molecular Medicine (T.A.M.C., S.F.d.S., A.F.d.V., C.v.V., T.v.d.P.)
| | - Anita E Grootemaat
- Academic Medical Center, University of Amsterdam, The Netherlands; Electron Microscopy Center Amsterdam, Medical Biology, Academic Medical Center, The Netherlands (A.E.G., N.N.v.d.W.)
| | - Nicole N van der Wel
- Academic Medical Center, University of Amsterdam, The Netherlands; Electron Microscopy Center Amsterdam, Medical Biology, Academic Medical Center, The Netherlands (A.E.G., N.N.v.d.W.)
| | | | - Jerry Ware
- University of Arkansas for Medical Sciences, Little Rock (J.W.)
| | - Cornelis Van't Veer
- From the Center for Experimental and Molecular Medicine (T.A.M.C., S.F.d.S., A.F.d.V., C.v.V., T.v.d.P.)
| | - Tom van der Poll
- From the Center for Experimental and Molecular Medicine (T.A.M.C., S.F.d.S., A.F.d.V., C.v.V., T.v.d.P.)
- Division of Infectious Diseases, Academic Medical Center, University of Amsterdam, The Netherlands (T.v.d.P.)
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35
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Deppermann C, Kubes P. Start a fire, kill the bug: The role of platelets in inflammation and infection. Innate Immun 2018; 24:335-348. [PMID: 30049243 PMCID: PMC6830908 DOI: 10.1177/1753425918789255] [Citation(s) in RCA: 166] [Impact Index Per Article: 23.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2018] [Revised: 06/16/2018] [Accepted: 06/26/2018] [Indexed: 11/19/2022] Open
Abstract
Platelets are the main players in thrombosis and hemostasis; however they also play important roles during inflammation and infection. Through their surface receptors, platelets can directly interact with pathogens and immune cells. Platelets form complexes with neutrophils to modulate their capacities to produce reactive oxygen species or form neutrophil extracellular traps. Furthermore, they release microbicidal factors and cytokines that kill pathogens and influence the immune response, respectively. Platelets also maintain the vascular integrity during inflammation by a mechanism that is different from classical platelet activation. In this review we summarize the current knowledge about how platelets interact with the innate immune system during inflammation and infection and highlight recent advances in the field.
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Affiliation(s)
- Carsten Deppermann
- Calvin, Phoebe and Joan Snyder Institute for Chronic
Diseases, University of Calgary, Calgary, AB, Canada
| | - Paul Kubes
- Calvin, Phoebe and Joan Snyder Institute for Chronic
Diseases, University of Calgary, Calgary, AB, Canada
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36
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Parsons MEM, Szklanna PB, Guerrero JA, Wynne K, Dervin F, O'Connell K, Allen S, Egan K, Bennett C, McGuigan C, Gheveart C, Ní Áinle F, Maguire PB. Platelet Releasate Proteome Profiling Reveals a Core Set of Proteins with Low Variance between Healthy Adults. Proteomics 2018; 18:e1800219. [PMID: 29932309 DOI: 10.1002/pmic.201800219] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2018] [Revised: 06/09/2018] [Indexed: 12/18/2022]
Abstract
Upon activation, platelets release a powerful cocktail of soluble and vesicular signals, collectively termed the "platelet releasate" (PR). Although several studies have used qualitative/quantitative proteomic approaches to characterize PR; with debated content and significant inter-individual variability reported, confident, and reliable insights have been hindered. Using label-free quantitative (LFQ)-proteomics analysis, a reproducible, quantifiable investigation of the 1U mL-1 thrombin-induced PR from 32 healthy adults was conducted. MS proteomics data are available via ProteomeXchange, identifier PXD009310. Of the 894 proteins identified, 277 proteins were quantified across all donors and form a "core" PR. Bioinformatics and further LFQ-proteomic analysis revealed that the majority (84%) of "core" PR proteins overlapped with the protein composition of human platelet-derived exosomes. Vesicles in the exosomal-size range were confirmed in healthy-human PR and reduced numbers of similar-sized vesicles were observed in the PR of a mouse model of gray platelet syndrome, known to be deficient in platelet alpha-granules. Lastly, the variability of proteins in the PR was assessed, and reproducible secretion levels were found across all 32 healthy donors. Taken together, the PR contains valuable soluble and vesicular cargo and has low-population variance among healthy adults, rendering it a potentially useful platform for diagnostic fingerprinting of platelet-related disease.
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Affiliation(s)
- Martin E M Parsons
- SPHERE research group, Conway Institute, University College Dublin, Dublin 4, Ireland.,Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, Dublin 2, Ireland.,School of Biomolecular and Biomedical Science, University College Dublin, Dublin 4, Ireland
| | - Paulina B Szklanna
- SPHERE research group, Conway Institute, University College Dublin, Dublin 4, Ireland.,Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, Dublin 2, Ireland.,School of Biomolecular and Biomedical Science, University College Dublin, Dublin 4, Ireland
| | - Jose A Guerrero
- Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Camebridge, United Kingdom.,National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Kieran Wynne
- Proteomics Core, Conway Institute, University College Dublin, Dublin 4, Ireland
| | - Feidhlim Dervin
- School of Biomolecular and Biomedical Science, University College Dublin, Dublin 4, Ireland
| | - Karen O'Connell
- Department of Neurology, St Vincent's University Hospital, Dublin 4, Ireland.,School of Medicine, University College Dublin, Dublin 4, Ireland
| | - Seamus Allen
- SPHERE research group, Conway Institute, University College Dublin, Dublin 4, Ireland.,School of Biomolecular and Biomedical Science, University College Dublin, Dublin 4, Ireland.,School of Medicine, University College Dublin, Dublin 4, Ireland
| | - Karl Egan
- SPHERE research group, Conway Institute, University College Dublin, Dublin 4, Ireland.,Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, Dublin 2, Ireland.,School of Medicine, University College Dublin, Dublin 4, Ireland
| | - Cavan Bennett
- Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Camebridge, United Kingdom.,National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Christopher McGuigan
- Department of Neurology, St Vincent's University Hospital, Dublin 4, Ireland.,School of Medicine, University College Dublin, Dublin 4, Ireland
| | - Cedric Gheveart
- Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Camebridge, United Kingdom.,National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Fionnuala Ní Áinle
- SPHERE research group, Conway Institute, University College Dublin, Dublin 4, Ireland.,Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, Dublin 2, Ireland.,School of Medicine, University College Dublin, Dublin 4, Ireland.,Department of Haematology, Rotunda Hospital, Dublin 1, Ireland.,Department of Haematology, Mater Misericordiae University Hospital, Dublin 7, Ireland
| | - Patricia B Maguire
- SPHERE research group, Conway Institute, University College Dublin, Dublin 4, Ireland.,Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, Dublin 2, Ireland.,School of Biomolecular and Biomedical Science, University College Dublin, Dublin 4, Ireland.,UCD Institute for Discovery, O'Brien Centre for Science, University College Dublin, Dublin, Ireland
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37
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Abstract
Platelets patrol the vasculature and adhere at sites of vascular damage after trauma to limit blood loss. In recent years, however, it has become clear that platelets also contribute to pathophysiologic processes such as thrombosis, atherosclerosis, stroke, sepsis and many more. An exciting new role for them is in non-classical hemostasis to prevent bleeding in the inflamed vasculature. Recent studies suggest that GPVI, CLEC-2, integrin αIIbβ3 (GPIIb/IIIa), and the content of platelet α- and dense granules are important players in this process. This review summarizes the current knowledge about how platelets prevent vascular integrity during inflammation in the skin, lung, and the ischemic brain and their organ-specific role.
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Affiliation(s)
- Carsten Deppermann
- a Snyder Institute for Chronic Diseases , University of Calgary , Calgary , AB , Canada
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38
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Laffan M. Ein genomweiter Ansatz bei Thrombozyten-und Gerinnungsstörungen. Hamostaseologie 2017; 36:161-6. [DOI: 10.5482/hamo-14-11-0056] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2014] [Accepted: 01/13/2015] [Indexed: 11/05/2022] Open
Abstract
ZusammenfassungDie Sequenzierung von hunderttausenden menschlichen Exomen und Gesamtgenomen bietet einen immer genaueren und vollständigeren Katalog menschlicher Genvarianten. Die ersten Studien zum Verständnis von Thrombozytenstörungen anhand von genomweiten Daten wurden als genomweite Assoziationsstudien durchgeführt, in denen Loci identifiziert wurden, die mit Variationen der Blutzellparameter assoziiert sind. In diesen Studien wurden Norm-varianten genutzt, um die entsprechenden genetische Variation zu finden. Als nächstes wollten wir die genetische Grundlage von Gerinnungsstörungen untersuchen, die einen Schlüssel für neue Gene liefern könnte, welche Thrombozyten- und Gerinnungsfunktionen steuern. Das BRIDGE-Konsortium (www.bridgestudy. org) wird vom NIHR finanziert und bringt 13 Genforschungsprojekte zu seltenen Krankheiten zusammen. Ziel dieser Projekte ist die Erforschung bislang unterdiagnostizierter seltener Erbkrankheiten und die Identifizierung der zugrunde liegenden Mutationen. Wir verwendeten eine Cluster-Analyse, basierend auf der Human Phenotype Ontology, kombiniert mit Next-Generation Sequenzierungstechniken, um Patienten mit ähnlichen Phänotypen, die vermutlich aus den gleichen Gendefekten hervorgehen, leichter zu identifizieren. Vorläufige Ergebnisse bestätigen dieses Vorgehen in Clustern und ergaben auch eine Reihe neuer Gene, die für die normale und die pathologische Thrombozytenphysiologie wichtig sind.
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39
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Nbeal2 interacts with Dock7, Sec16a, and Vac14. Blood 2017; 131:1000-1011. [PMID: 29187380 DOI: 10.1182/blood-2017-08-800359] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2017] [Accepted: 11/21/2017] [Indexed: 12/11/2022] Open
Abstract
Mutations in NBEAL2, the gene encoding the scaffolding protein Nbeal2, are causal of gray platelet syndrome (GPS), a rare recessive bleeding disorder characterized by platelets lacking α-granules and progressive marrow fibrosis. We present here the interactome of Nbeal2 with additional validation by reverse immunoprecipitation of Dock7, Sec16a, and Vac14 as interactors of Nbeal2. We show that GPS-causing mutations in its BEACH domain have profound and possible effects on the interaction with Dock7 and Vac14, respectively. Proximity ligation assays show that these 2 proteins are physically proximal to Nbeal2 in human megakaryocytes. In addition, we demonstrate that Nbeal2 is primarily localized in the cytoplasm and Dock7 on the membrane of or in α-granules. Interestingly, platelets from GPS cases and Nbeal2-/- mice are almost devoid of Dock7, resulting in a profound dysregulation of its signaling pathway, leading to defective actin polymerization, platelet activation, and shape change. This study shows for the first time proteins interacting with Nbeal2 and points to the dysregulation of the canonical signaling pathway of Dock7 as a possible cause of the aberrant formation of platelets in GPS cases and Nbeal2-deficient mice.
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40
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Drube S, Grimlowski R, Deppermann C, Fröbel J, Kraft F, Andreas N, Stegner D, Dudeck J, Weber F, Rödiger M, Göpfert C, Drube J, Reich D, Nieswandt B, Dudeck A, Kamradt T. The Neurobeachin-like 2 Protein Regulates Mast Cell Homeostasis. THE JOURNAL OF IMMUNOLOGY 2017; 199:2948-2957. [PMID: 28887433 DOI: 10.4049/jimmunol.1700556] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/19/2017] [Accepted: 08/21/2017] [Indexed: 12/24/2022]
Abstract
The neurobeachin-like 2 protein (Nbeal2) belongs to the family of beige and Chediak-Higashi (BEACH) domain proteins. Loss-of-function mutations in the human NBEAL2 gene or Nbeal2 deficiency in mice cause gray platelet syndrome, a bleeding disorder characterized by macrothrombocytopenia, splenomegaly, and paucity of α-granules in megakaryocytes and platelets. We found that in mast cells, Nbeal2 regulates the activation of the Shp1-STAT5 signaling axis and the composition of the c-Kit/STAT signalosome. Furthermore, Nbeal2 mediates granule formation and restricts the expression of the transcription factors, IRF8, GATA2, and MITF as well as of the cell-cycle inhibitor p27, which are essential for mast cell differentiation, proliferation, and cytokine production. These data demonstrate the relevance of Nbeal2 in mast cells above and beyond granule biosynthesis.
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Affiliation(s)
- Sebastian Drube
- Institute of Immunology, Jena University Hospital, 07743 Jena, Germany;
| | - Randy Grimlowski
- Institute of Immunology, Jena University Hospital, 07743 Jena, Germany
| | - Carsten Deppermann
- Institute of Experimental Biomedicine, University Hospital and Rudolf Virchow Center for Experimental Biomedicine, University Würzburg, 97080 Würzburg, Germany
| | - Julia Fröbel
- Medical Faculty, Institute for Molecular and Clinical Immunology, 39120 Magdeburg, Germany; and
| | - Florian Kraft
- Institute of Immunology, Jena University Hospital, 07743 Jena, Germany
| | - Nico Andreas
- Institute of Immunology, Jena University Hospital, 07743 Jena, Germany
| | - David Stegner
- Institute of Experimental Biomedicine, University Hospital and Rudolf Virchow Center for Experimental Biomedicine, University Würzburg, 97080 Würzburg, Germany
| | - Jan Dudeck
- Medical Faculty, Institute for Molecular and Clinical Immunology, 39120 Magdeburg, Germany; and
| | - Franziska Weber
- Institute of Immunology, Jena University Hospital, 07743 Jena, Germany
| | - Mandy Rödiger
- Institute of Immunology, Jena University Hospital, 07743 Jena, Germany
| | | | - Julia Drube
- Center for Molecular Biomedicine, University Hospital Jena, 07745 Jena, Germany
| | - Daniela Reich
- Center for Molecular Biomedicine, University Hospital Jena, 07745 Jena, Germany
| | - Bernhard Nieswandt
- Institute of Experimental Biomedicine, University Hospital and Rudolf Virchow Center for Experimental Biomedicine, University Würzburg, 97080 Würzburg, Germany
| | - Anne Dudeck
- Medical Faculty, Institute for Molecular and Clinical Immunology, 39120 Magdeburg, Germany; and
| | - Thomas Kamradt
- Institute of Immunology, Jena University Hospital, 07743 Jena, Germany
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41
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Sowerby JM, Thomas DC, Clare S, Espéli M, Guerrero JA, Hoenderdos K, Harcourt K, Marsden M, Abdul-Karim J, Clement M, Antrobus R, Umrania Y, Barton PR, Flint SM, Juss JK, Condliffe AM, Lyons PA, Humphreys IR, Chilvers ER, Ouwehand WH, Dougan G, Smith KG. NBEAL2 is required for neutrophil and NK cell function and pathogen defense. J Clin Invest 2017; 127:3521-3526. [PMID: 28783043 PMCID: PMC5669559 DOI: 10.1172/jci91684] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2016] [Accepted: 06/23/2017] [Indexed: 12/02/2022] Open
Abstract
Mutations in the human NBEAL2 gene cause gray platelet syndrome (GPS), a bleeding diathesis characterized by a lack of α granules in platelets. The functions of the NBEAL2 protein have not been explored outside platelet biology, but there are reports of increased frequency of infection and abnormal neutrophil morphology in patients with GPS. We therefore investigated the role of NBEAL2 in immunity by analyzing the phenotype of Nbeal2-deficient mice. We found profound abnormalities in the Nbeal2-deficient immune system, particularly in the function of neutrophils and NK cells. Phenotyping of Nbeal2-deficient neutrophils showed a severe reduction in granule contents across all granule subsets. Despite this, Nbeal2-deficient neutrophils had an enhanced phagocyte respiratory burst relative to Nbeal2-expressing neutrophils. This respiratory burst was associated with increased expression of cytosolic components of the NADPH oxidase complex. Nbeal2-deficient NK cells were also dysfunctional and showed reduced degranulation. These abnormalities were associated with increased susceptibility to both bacterial (Staphylococcus aureus) and viral (murine CMV) infection in vivo. These results define an essential role for NBEAL2 in mammalian immunity.
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Affiliation(s)
- John M. Sowerby
- Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - David C. Thomas
- Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Simon Clare
- Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom
| | - Marion Espéli
- Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus, Cambridge, United Kingdom
- INSERM UMR-996, Inflammation, Chemokines and Immunopathology, Université Paris-Sud, Université Paris-Saclay, Clamart, France
| | - Jose A. Guerrero
- Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge, United Kingdom
- NHS Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Kim Hoenderdos
- Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Katherine Harcourt
- Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom
| | - Morgan Marsden
- Division of Infection and Immunity, Cardiff University, Cardiff, United Kingdom
| | - Juneid Abdul-Karim
- Division of Infection and Immunity, Cardiff University, Cardiff, United Kingdom
| | - Mathew Clement
- Division of Infection and Immunity, Cardiff University, Cardiff, United Kingdom
| | - Robin Antrobus
- Cambridge Institute for Medical Research, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Yagnesh Umrania
- Cambridge Institute for Medical Research, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Philippa R. Barton
- Cambridge Institute for Medical Research, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Shaun M. Flint
- Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Jatinder K. Juss
- Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Alison M. Condliffe
- Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Paul A. Lyons
- Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Ian R. Humphreys
- Division of Infection and Immunity, Cardiff University, Cardiff, United Kingdom
| | - Edwin R. Chilvers
- Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Willem H. Ouwehand
- Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom
- Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge, United Kingdom
- NHS Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
| | - Gordon Dougan
- Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus, Cambridge, United Kingdom
- Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom
| | - Kenneth G.C. Smith
- Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus, Cambridge, United Kingdom
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42
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Léon C, Dupuis A, Gachet C, Lanza F. The contribution of mouse models to the understanding of constitutional thrombocytopenia. Haematologica 2017; 101:896-908. [PMID: 27478199 DOI: 10.3324/haematol.2015.139394] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2016] [Accepted: 05/04/2016] [Indexed: 11/09/2022] Open
Abstract
Constitutional thrombocytopenias result from platelet production abnormalities of hereditary origin. Long misdiagnosed and poorly studied, knowledge about these rare diseases has increased considerably over the last twenty years due to improved technology for the identification of mutations, as well as an improvement in obtaining megakaryocyte culture from patient hematopoietic stem cells. Simultaneously, the manipulation of mouse genes (transgenesis, total or conditional inactivation, introduction of point mutations, random chemical mutagenesis) have helped to generate disease models that have contributed greatly to deciphering patient clinical and laboratory features. Most of the thrombocytopenias for which the mutated genes have been identified now have a murine model counterpart. This review focuses on the contribution that these mouse models have brought to the understanding of hereditary thrombocytopenias with respect to what was known in humans. Animal models have either i) provided novel information on the molecular and cellular pathways that were missing from the patient studies; ii) improved our understanding of the mechanisms of thrombocytopoiesis; iii) been instrumental in structure-function studies of the mutated gene products; and iv) been an invaluable tool as preclinical models to test new drugs or develop gene therapies. At present, the genetic determinants of thrombocytopenia remain unknown in almost half of all cases. Currently available high-speed sequencing techniques will identify new candidate genes, which will in turn allow the generation of murine models to confirm and further study the abnormal phenotype. In a complementary manner, programs of random mutagenesis in mice should also identify new candidate genes involved in thrombocytopenia.
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Affiliation(s)
- Catherine Léon
- UMR_S949, INSERM, Strasbourg, France Etablissement Français du Sang-Alsace (EFS-Alsace), Strasbourg, France Université de Strasbourg, France Fédération de Médecine Translationnelle de Strasbourg (FMTS), France
| | - Arnaud Dupuis
- UMR_S949, INSERM, Strasbourg, France Etablissement Français du Sang-Alsace (EFS-Alsace), Strasbourg, France Université de Strasbourg, France Fédération de Médecine Translationnelle de Strasbourg (FMTS), France
| | - Christian Gachet
- UMR_S949, INSERM, Strasbourg, France Etablissement Français du Sang-Alsace (EFS-Alsace), Strasbourg, France Université de Strasbourg, France Fédération de Médecine Translationnelle de Strasbourg (FMTS), France
| | - François Lanza
- UMR_S949, INSERM, Strasbourg, France Etablissement Français du Sang-Alsace (EFS-Alsace), Strasbourg, France Université de Strasbourg, France Fédération de Médecine Translationnelle de Strasbourg (FMTS), France
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Kurokawa T, Ohkohchi N. Platelets in liver disease, cancer and regeneration. World J Gastroenterol 2017; 23:3228-3239. [PMID: 28566882 PMCID: PMC5434428 DOI: 10.3748/wjg.v23.i18.3228] [Citation(s) in RCA: 75] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/08/2016] [Revised: 11/17/2016] [Accepted: 03/15/2017] [Indexed: 02/06/2023] Open
Abstract
Although viral hepatitis treatments have evolved over the years, the resultant liver cirrhosis still does not completely heal. Platelets contain proteins required for hemostasis, as well as many growth factors required for organ development, tissue regeneration and repair. Thrombocytopenia, which is frequently observed in patients with chronic liver disease (CLD) and cirrhosis, can manifest from decreased thrombopoietin production and accelerated platelet destruction caused by hypersplenism; however, the relationship between thrombocytopenia and hepatic pathogenesis, as well as the role of platelets in CLD, is poorly understood. In this paper, experimental evidence of platelets improving liver fibrosis and accelerating liver regeneration is summarized and addressed based on studies conducted in our laboratory and current progress reports from other investigators. In addition, we describe our current perspective based on the results of these studies. Platelets improve liver fibrosis by inactivating hepatic stellate cells, which decreases collagen production. The regenerative effect of platelets in the liver involves a direct effect on hepatocytes, a cooperative effect with liver sinusoidal endothelial cells, and a collaborative effect with Kupffer cells. Based on these observations, we ascertained the direct effect of platelet transfusion on improving several indicators of liver function in patients with CLD and liver cirrhosis. However, unlike the results of our previous clinical study, the smaller incremental changes in liver function in patients with CLD who received eltrombopag for 6 mo were due to patient selection from a heterogeneous population. We highlight the current knowledge concerning the role of platelets in CLD and cancer and anticipate a novel application of platelet-based clinical therapies to treat liver disease.
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Megakaryocytes in Myeloproliferative Neoplasms Have Unique Somatic Mutations. THE AMERICAN JOURNAL OF PATHOLOGY 2017; 187:1512-1522. [PMID: 28502479 DOI: 10.1016/j.ajpath.2017.03.009] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/10/2017] [Accepted: 03/23/2017] [Indexed: 02/06/2023]
Abstract
Myeloproliferative neoplasms (MPNs) are a group of related clonal hemopoietic stem cell disorders associated with hyperproliferation of myeloid cells. They are driven by mutations in the hemopoietic stem cell, most notably JAK2V617F, CALR, and MPL. Clinically, they have the propensity to progress to myelofibrosis and transform to acute myeloid leukemia. Megakaryocytic hyperplasia with abnormal features are characteristic, and it is thought that these cells stimulate and drive fibrotic progression. The biological defects underpinning this remain to be explained. In this study we examined the megakaryocyte genome in 12 patients with MPNs to determine whether there are somatic variants and whether there is any association with marrow fibrosis. We performed targeted next-generation sequencing for 120 genes associated with myeloid neoplasms on megakaryocytes isolated from aspirated bone marrow. Ten of the 12 patients had genomic defects in megakaryocytes that were not present in nonmegakaryocytic hemopoietic marrow cells from the same patient. The greatest allelic burden was in patients with increased reticulin deposition. The megakaryocyte-unique mutations were predominantly in genes that regulate chromatin remodeling, chromosome alignment, and stability. These findings show that genomic abnormalities are present in megakaryocytes in MPNs and that these appear to be associated with progression to bone marrow fibrosis.
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Chen CH, Lo RW, Urban D, Pluthero FG, Kahr WHA. α-granule biogenesis: from disease to discovery. Platelets 2017; 28:147-154. [DOI: 10.1080/09537104.2017.1280599] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Affiliation(s)
- Chang Hua Chen
- Department of Biochemistry, University of Toronto, Toronto, ON, Canada
| | - Richard W. Lo
- Department of Biochemistry, University of Toronto, Toronto, ON, Canada
| | - Denisa Urban
- Department of Biochemistry, University of Toronto, Toronto, ON, Canada
| | - Fred G. Pluthero
- Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada
| | - Walter H. A. Kahr
- Department of Biochemistry, University of Toronto, Toronto, ON, Canada
- Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada
- Division of Haematology/Oncology, Department of Paediatrics, University of Toronto and The Hospital for Sick Children, Toronto, ON, Canada
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van der Weyden L, Arends MJ, Campbell AD, Bald T, Wardle-Jones H, Griggs N, Velasco-Herrera MDC, Tüting T, Sansom OJ, Karp NA, Clare S, Gleeson D, Ryder E, Galli A, Tuck E, Cambridge EL, Voet T, Macaulay IC, Wong K, Spiegel S, Speak AO, Adams DJ. Genome-wide in vivo screen identifies novel host regulators of metastatic colonization. Nature 2017; 541:233-236. [PMID: 28052056 PMCID: PMC5603286 DOI: 10.1038/nature20792] [Citation(s) in RCA: 174] [Impact Index Per Article: 21.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2015] [Accepted: 11/15/2016] [Indexed: 12/17/2022]
Abstract
Metastasis is the leading cause of death for cancer patients. This multi-stage process requires tumour cells to survive in the circulation, extravasate at distant sites, then proliferate; it involves contributions from both the tumour cell and tumour microenvironment ('host', which includes stromal cells and the immune system). Studies suggest the early steps of the metastatic process are relatively efficient, with the post-extravasation regulation of tumour growth ('colonization') being critical in determining metastatic outcome. Here we show the results of screening 810 mutant mouse lines using an in vivo assay to identify microenvironmental regulators of metastatic colonization. We identify 23 genes that, when disrupted in mouse, modify the ability of tumour cells to establish metastatic foci, with 19 of these genes not previously demonstrated to play a role in host control of metastasis. The largest reduction in pulmonary metastasis was observed in sphingosine-1-phosphate (S1P) transporter spinster homologue 2 (Spns2)-deficient mice. We demonstrate a novel outcome of S1P-mediated regulation of lymphocyte trafficking, whereby deletion of Spns2, either globally or in a lymphatic endothelial-specific manner, creates a circulating lymphopenia and a higher percentage of effector T cells and natural killer (NK) cells present in the lung. This allows for potent tumour cell killing, and an overall decreased metastatic burden.
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Affiliation(s)
| | - Mark J Arends
- University of Edinburgh Division of Pathology, Edinburgh Cancer Research UK Cancer Centre, Institute of Genetics &Molecular Medicine, Edinburgh EH4 2XR, UK
| | | | - Tobias Bald
- Department of Dermatology, University Hospital Magdeburg, Magdeburg 39120, Germany
- Department of Immunology in Cancer and Infection Laboratory, QIMR Berghofer Medical Research Institute, Herston 4006, Australia
| | - Hannah Wardle-Jones
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK
| | - Nicola Griggs
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK
| | | | - Thomas Tüting
- Department of Dermatology, University Hospital Magdeburg, Magdeburg 39120, Germany
| | - Owen J Sansom
- Cancer Research UK Beatson Institute, Glasgow G61 1BD, UK
| | - Natasha A Karp
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK
| | - Simon Clare
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK
| | - Diane Gleeson
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK
| | - Edward Ryder
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK
| | - Antonella Galli
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK
| | - Elizabeth Tuck
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK
| | - Emma L Cambridge
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK
| | - Thierry Voet
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK
- Department of Human Genetics, University of Leuven (KU Leuven), Leuven, 3000, Belgium
| | - Iain C Macaulay
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK
| | - Kim Wong
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK
| | - Sarah Spiegel
- Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298-0614, USA
| | - Anneliese O Speak
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK
| | - David J Adams
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK
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47
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Songdej N, Rao AK. Inherited platelet dysfunction and hematopoietic transcription factor mutations. Platelets 2017; 28:20-26. [PMID: 27463948 PMCID: PMC5628047 DOI: 10.1080/09537104.2016.1203400] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2016] [Revised: 05/16/2016] [Accepted: 05/30/2016] [Indexed: 01/19/2023]
Abstract
Transcription factors (TFs) are proteins that bind to specific DNA sequences and regulate expression of genes. The molecular and genetic mechanisms in most patients with inherited platelet dysfunction are unknown. There is now increasing evidence that mutations in hematopoietic TFs are an important underlying cause for the defects in platelet production, morphology, and function. The hematopoietic TFs implicated in the patients with impaired platelet function include Runt related TF 1 (RUNX1), Fli-1 proto-oncogene, ETS TF (FLI1), GATA-binding protein 1 (GATA1), and growth factor independent 1B transcriptional repressor (GFI1B). These TFs act in a combinatorial manner to bind sequence-specific DNA within a promoter region to regulate lineage-specific gene expression, either as activators or as repressors. TF mutations induce rippling downstream effects by simultaneously altering the expression of multiple genes. Mutations involving these TFs affect diverse aspects of megakaryocyte biology and platelet production and function, culminating in thrombocytopenia, platelet dysfunction, and associated clinical features. Mutations in TFs may occur more frequently in the patients with inherited platelet dysfunction than generally appreciated. This review focuses on the alterations in hematopoietic TFs in the pathobiology of inherited platelet dysfunction.
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Affiliation(s)
- Natthapol Songdej
- a Sol Sherry Thrombosis Research Center, and Hematology Section, Department of Medicine , Lewis Katz School of Medicine at Temple University , Philadelphia , PA , USA
| | - A Koneti Rao
- a Sol Sherry Thrombosis Research Center, and Hematology Section, Department of Medicine , Lewis Katz School of Medicine at Temple University , Philadelphia , PA , USA
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48
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Bariana TK, Ouwehand WH, Guerrero JA, Gomez K. Dawning of the age of genomics for platelet granule disorders: improving insight, diagnosis and management. Br J Haematol 2016; 176:705-720. [PMID: 27984638 DOI: 10.1111/bjh.14471] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Inherited disorders of platelet granules are clinically heterogeneous and their prevalence is underestimated because most patients do not undergo a complete diagnostic work-up. The lack of a genetic diagnosis limits the ability to tailor management, screen family members, aid with family planning, predict clinical progression and detect serious consequences, such as myelofibrosis, lung fibrosis and malignancy, in a timely manner. This is set to change with the introduction of high throughput sequencing (HTS) as a routine clinical diagnostic test. HTS diagnostic tests are now available, affordable and allow parallel screening of DNA samples for variants in all of the 80 known bleeding, thrombotic and platelet genes. Increased genetic diagnosis and curation of variants is, in turn, improving our understanding of the pathobiology and clinical course of inherited platelet disorders. Our understanding of the genetic causes of platelet granule disorders and the regulation of granule biogenesis is a work in progress and has been significantly enhanced by recent genomic discoveries from high-powered genome-wide association studies and genome sequencing projects. In the era of whole genome and epigenome sequencing, new strategies are required to integrate multiple sources of big data in the search for elusive, novel genes underlying granule disorders.
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Affiliation(s)
- Tadbir K Bariana
- Katharine Dormandy Haemophilia Centre and Thrombosis Unit, Royal Free London NHS Foundation Trust, London, UK.,Department of Haematology, University College London Cancer Institute, London, UK.,Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge, UK
| | - Willem H Ouwehand
- Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge, UK.,NHS Blood and Transplant, Cambridge Biomedical Campus, Cambridge, UK.,NIHR BioResource, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge, UK.,Human Genetics, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK
| | - Jose A Guerrero
- Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge, UK.,NHS Blood and Transplant, Cambridge Biomedical Campus, Cambridge, UK
| | - Keith Gomez
- Katharine Dormandy Haemophilia Centre and Thrombosis Unit, Royal Free London NHS Foundation Trust, London, UK
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49
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Nurden AT, Nurden P. Should any genetic defect affecting α-granules in platelets be classified as gray platelet syndrome? Am J Hematol 2016; 91:714-8. [PMID: 26971401 DOI: 10.1002/ajh.24359] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2016] [Revised: 03/02/2016] [Accepted: 03/07/2016] [Indexed: 01/19/2023]
Abstract
There is much current interest in the role of the platelet storage pool of α-granule proteins both in hemostasis and non-hemostatic events. As well as in the arrest of bleeding, the secreted proteins participate in wound healing, inflammation, and innate immunity while in pathology they may be actors in arterial thrombosis and atherosclerosis as well as cancer and metastasis. For a long time, gray platelet syndrome (GPS) has been regarded as the classic inherited platelet disorder caused by an absence of α-granules and their contents. While NBEAL2 is the major source of mutations in GPS, other gene variants may give rise to significant α-granule deficiencies in platelets. These include GATA1, VPS33B, or VIPAS39 in the arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome and now GFI1B. Nevertheless, many phenotypic differences are associated with mutations in these genes. This critical review was aimed to assess genotype/phenotype variability in disorders of platelet α-granule biogenesis and to urge caution in grouping all genetic defects of α-granules as GPS. Am. J. Hematol. 91:714-718, 2016. © 2016 Wiley Periodicals, Inc.
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Affiliation(s)
- Alan T. Nurden
- Institut de Rhythmologie et de Modélisation Cardiaque, Plateforme Technologique d'Innovation Biomédicale, Hôpital Xavier Arnozan; Pessac France
| | - Paquita Nurden
- Institut de Rhythmologie et de Modélisation Cardiaque, Plateforme Technologique d'Innovation Biomédicale, Hôpital Xavier Arnozan; Pessac France
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50
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Johnson B, Fletcher SJ, Morgan NV. Inherited thrombocytopenia: novel insights into megakaryocyte maturation, proplatelet formation and platelet lifespan. Platelets 2016; 27:519-25. [PMID: 27025194 PMCID: PMC5000870 DOI: 10.3109/09537104.2016.1148806] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
The study of patients with inherited bleeding problems is a powerful approach in determining the function and regulation of important proteins in human platelets and their precursor, the megakaryocyte. The normal range of platelet counts in the bloodstream ranges from 150 000 to 400 000 platelets per microliter and is normally maintained within a narrow range for each individual. This requires a constant balance between thrombopoiesis, which is primarily controlled by the cytokine thrombopoietin (TPO), and platelet senescence and consumption. Thrombocytopenia can be defined as a platelet count of less than 150 000 per microliter and can be acquired or inherited. Heritable forms of thrombocytopenia are caused by mutations in genes involved in megakaryocyte differentiation, platelet production and platelet removal. In this review, we will discuss the main causative genes known for inherited thrombocytopenia and highlight their diverse functions and whether these give clues on the processes of platelet production, platelet function and platelet lifespan. Additionally, we will highlight the recent advances in novel genes identified for inherited thrombocytopenia and their suggested function.
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Affiliation(s)
- Ben Johnson
- a Institute of Cardiovascular Sciences, College of Medical and Dental Sciences , University of Birmingham , UK
| | - Sarah J Fletcher
- a Institute of Cardiovascular Sciences, College of Medical and Dental Sciences , University of Birmingham , UK
| | - Neil V Morgan
- a Institute of Cardiovascular Sciences, College of Medical and Dental Sciences , University of Birmingham , UK
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