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Joseph E Italiano - One of the best experts on this subject based on the ideXlab platform.
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inducible gata1 suppression expands Megakaryocyte erythroid progenitors from embryonic stem cells
Journal of Clinical Investigation, 2015Co-Authors: Ji Yoo Noh, Shilpa Gandrebabbe, Yuhua Wang, Vince Hayes, Yu Yao, Paul Gadue, Spence K Sulliva, Stella T Chou, Kellie R Machlus, Joseph E ItalianoAbstract:Transfusion of donor-derived platelets is commonly used for thrombocytopenia, which results from a variety of clinical conditions and relies on a constant donor supply due to the limited shelf life of these cells. Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent a potential source of Megakaryocytes and platelets for transfusion therapies; however, the majority of current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny. In both mice and humans, mutations in the gene-encoding transcription factor GATA1 cause an accumulation of proliferating, developmentally arrested Megakaryocytes, suggesting that GATA1 suppression in ES and iPS cell–derived hematopoietic progenitors may enhance Megakaryocyte production. Here, we engineered ES cells from WT mice to express a doxycycline-regulated (dox-regulated) shRNA that targets Gata1 transcripts for degradation. Differentiation of these cells in the presence of dox and thrombopoietin (TPO) resulted in an exponential (at least 1013-fold) expansion of immature hematopoietic progenitors. Dox withdrawal in combination with multilineage cytokines restored GATA1 expression, resulting in differentiation into erythroblasts and Megakaryocytes. Following transfusion into recipient animals, these dox-deprived mature Megakaryocytes generated functional platelets. Our findings provide a readily reproducible strategy to exponentially expand ES cell–derived Megakaryocyte-erythroid progenitors that have the capacity to differentiate into functional platelet-producing Megakaryocytes.
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interpreting the developmental dance of the Megakaryocyte a review of the cellular and molecular processes mediating platelet formation
British Journal of Haematology, 2014Co-Authors: Joseph E Italiano, Jonatha N Tho, Kellie R MachlusAbstract:Summary Platelets are essential for haemostasis, and thrombocytopenia (platelet counts <150 × 109/l) is a major clinical problem encountered across a number of conditions, including immune thrombocytopenic purpura, myelodysplastic syndromes, chemotherapy, aplastic anaemia, human immunodeficiency virus infection, complications during pregnancy and delivery, and surgery. Circulating blood platelets are specialized cells that function to prevent bleeding and minimize blood vessel injury. Platelets circulate in their quiescent form, and upon stimulation, activate to release their granule contents and spread on the affected tissue to create a physical barrier that prevents blood loss. The current model of platelet formation states that large progenitor cells in the bone marrow, called Megakaryocytes, release platelets by extending long, branching processes, designated proplatelets, into sinusoidal blood vessels. This review will focus on different factors that impact Megakaryocyte development, proplatelet formation and platelet release. It will highlight recent studies on thrombopoeitin-dependent Megakaryocyte maturation, endomitosis and granule formation, cytoskeletal contributions to proplatelet formation, the role of apoptosis, and terminal platelet formation and release.
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mkl1 and mkl2 play redundant and crucial roles in Megakaryocyte maturation and platelet formation
Blood, 2012Co-Authors: Elenoe C Smith, Joseph E Italiano, Jonatha N Tho, Matthew T Devine, Vince P Schulz, Yanwe Guo, Stephanie A Massaro, Stephanie Halene, Patrick G GallagheAbstract:Serum response factor and its transcriptional cofactor MKL1 are critical for Megakaryocyte maturation and platelet formation. We show that MKL2, a homologue of MKL1, is expressed in Megakaryocytes and plays a role in Megakaryocyte maturation. Using a Megakaryocyte-specific Mkl2 knockout (KO) mouse on the conventional Mkl1 KO background to produce double KO (DKO) Megakaryocytes and platelets, a critical role for MKL2 is revealed. The decrease in Megakaryocyte ploidy and platelet counts of DKO mice is more severe than in Mkl1 KO mice. Platelet dysfunction in DKO mice is revealed by prolonged bleeding times and ineffective platelet activation in vitro in response to adenosine 5′-diphosphate. Electron microscopy and immunofluorescence of DKO Megakaryocytes and platelets indicate abnormal cytoskeletal and membrane organization with decreased granule complexity. Surprisingly, the DKO mice have a more extreme thrombocytopenia than mice lacking serum response factor (SRF) expression in the Megakaryocyte compartment. Comparison of gene expression reveals approximately 4400 genes whose expression is differentially affected in DKO compared with Megakaryocytes deficient in SRF, strongly suggesting that MKL1 and MKL2 have both SRF-dependent and SRF-independent activity in megakaryocytopoiesis.
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high content live cell imaging assay used to establish mechanism of trastuzumab emtansine t dm1 mediated inhibition of platelet production
Blood, 2012Co-Authors: Joseph E Italiano, Matthew T Devine, Jonathan N Thon, Antonija Jurak Begonja, Jay TibbittsAbstract:Proplatelet production represents a terminal stage of Megakaryocyte development during which long, branching processes composed of platelet-sized swellings are extended and released into the surrounding culture. Whereas the cytoskeletal mechanics driving these transformations have been the focus of many studies, significant limitations in our ability to quantify the rate and extent of proplatelet production have restricted the field to qualitative analyses of a limited number of cells over short intervals. A novel high-content, quantitative, live-cell imaging assay using the IncuCyte system (Essen BioScience) was therefore developed to measure the rate and extent of Megakaryocyte maturation and proplatelet production under live culture conditions for extended periods of time. As proof of concept, we used this system in the present study to establish a mechanism by which trastuzumab emtansine (T-DM1), an Ab-drug conjugate currently in clinical development for cancer, affects platelet production. High-content analysis of primary cell cultures revealed that T-DM1 is taken up by mouse Megakaryocytes, inhibits Megakaryocyte differentiation, and disrupts proplatelet formation by inducing abnormal tubulin organization and suppressing microtubule dynamic instability. Defining the pathways by which therapeutics such as T-DM1 affect Megakaryocyte differentiation and proplatelet production may yield strategies to manage drug-induced thrombocytopenias.
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visualization and manipulation of the platelet and Megakaryocyte cytoskeleton
Methods of Molecular Biology, 2012Co-Authors: Joseph E Italiano, Jonatha N ThoAbstract:Driven by the application of immunofluorescence (IF) microscopy and modern molecular biology approaches to cytoskeletal manipulation, the last 5 years have yielded considerable progress to our understanding of the molecular mechanisms governing Megakaryocyte development and platelet biogenesis. Such studies have visualized endomitotic spindle dynamics, characterized the maturation of the -demarcation membrane system, delineated the mechanics of organelle transport and microtubule assembly in living Megakaryocytes, described the process of platelet production in vivo, and revealed factors contributing to and the mechanisms driving proplatelet production and platelet release. Here, we describe methods to (1) culture Megakaryocytes from murine fetal livers, (2) manipulate the tubulin and actin cytoskeleton of both platelets and cultured Megakaryocytes, and (3) examine these by live-cell microscopy and fixed-cell immunofluorescence microscopy.
Ramesh A Shivdasani - One of the best experts on this subject based on the ideXlab platform.
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characterization of the Megakaryocyte demarcation membrane system and its role in thrombopoiesis
Blood, 2006Co-Authors: Harald Schulze, Manav Korpal, Jonatha Hurov, Sang We Kim, Jinghang Zhang, Lewis C Cantley, Thomas Graf, Ramesh A ShivdasaniAbstract:To produce blood platelets, Megakaryocytes elaborate proplatelets, accompanied by expansion of membrane surface area and dramatic cytoskeletal rearrangements. The invaginated demarcation membrane system (DMS), a hallmark of mature cells, has been proposed as the source of proplatelet membranes. By direct visualization of labeled DMS, we demonstrate that this is indeed the case. Late in Megakaryocyte ontogeny, the DMS gets loaded with PI-4,5-P2, a phospholipid that is confined to plasma membranes in other cells. Appearance of PI-4,5-P2 in the DMS occurs in proximity to PI-5-P-4-kinase α (PIP4Kα), and short hairpin (sh) RNA-mediated loss of PIP4Kα impairs both DMS development and expansion of Megakaryocyte size. Thus, PI-4,5-P2 is a marker and possibly essential component of internal membranes. PI-4,5-P2 is known to promote actin polymerization by activating Rho-like GTPases and Wiskott-Aldrich syndrome (WASp) family proteins. Indeed, PI-4,5-P2 in the Megakaryocyte DMS associates with filamentous actin. Expression of a dominant-negative N-WASp fragment or pharmacologic inhibition of actin polymerization causes similar arrests in proplatelet formation, acting at a step beyond expansion of the DMS and cell mass. These observations collectively suggest a signaling pathway wherein PI-4,5-P2 might facilitate DMS development and local assembly of actin fibers in preparation for platelet biogenesis.
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Megakaryocyte osteoblast interaction revealed in mice deficient in transcription factors gata 1 and nf e2
Journal of Bone and Mineral Research, 2003Co-Authors: Melissa A Kacena, Ramesh A Shivdasani, Kimberly M Wilso, Nancy Troiano, Ara Nazaria, Care M Gundberg, Mary L Ouxsei, Joseph A Lorenzo, Mark C HorowitzAbstract:Mice deficient in GATA-1 or NF-E2 have a 200–300% increase in bone volume and formation parameters. Osteoblasts and osteoclasts generated in vitro from mutant and control animals were similar in number and function. Osteoblast proliferation increased up to 6-fold when cultured with Megakaryocytes. A Megakaryocyte-osteoblast interaction plays a role in the increased bone formation in these mice. Introduction: GATA-1 and NF-E2 are transcription factors required for the differentiation of Megakaryocytes. Mice deficient in these factors have phenotypes characterized by markedly increased numbers of immature Megakaryocytes, a concomitant drastic reduction of platelets, and a striking increased bone mass. The similar bone phenotype in both animal models led us to explore the interaction between osteoblasts and Megakaryocytes. Materials and Methods: Histomorphometry, μCT, and serum and urine biochemistries were used to assess the bone phenotype in these mice. Wildtype and mutant osteoblasts were examined for differences in proliferation, alkaline phosphatase activity, and osteocalcin secretion. In vitro osteoclast numbers and resorption were measured. Because mutant osteoblasts and osteoclasts were similar to control cells, and because of the similar bone phenotype, we explored the interaction between cells of the osteoblast lineage and Megakaryocytes. Results: A marked 2- to 3-fold increase in trabecular bone volume and bone formation indices were observed in these mice. A 20- to 150-fold increase in trabecular bone volume was measured for the entire femoral medullary canal. The increased bone mass phenotype in these animals was not caused by osteoclast defects, because osteoclast number and function were not compromised in vitro or in vivo. In contrast, in vivo osteoblast number and bone formation parameters were significantly elevated. When wildtype or mutant osteoblasts were cultured with Megakaryocytes from GATA-1- or NF-E2-deficient mice, osteoblast proliferation increased over 3- to 6-fold by a mechanism that required cell-to-cell contact. Conclusions: These observations show an interaction between Megakaryocytes and osteoblasts, which results in osteoblast proliferation and increased bone mass, and may represent heretofore unrecognized anabolic pathways in bone.
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molecular and transcriptional regulation of Megakaryocyte differentiation
Stem Cells, 2001Co-Authors: Ramesh A ShivdasaniAbstract:Megakaryocytes, among the rarest of hematopoietic cells, serve the essential function of producing numerous platelets. Genetic studies have recently provided rich insights into the molecular and transcriptional regulation of Megakaryocyte differentiation and thrombopoiesis. Three transcription factors, GATA-1, FOG-1, and NF-E2, are essential regulators of distinct stages in Megakaryocyte differentiation, extending from the birth of early committed progenitors to the final step of platelet release; a fourth factor, Fli-1, likely also plays an important role. The putative transcriptional targets of these regulators, including the NF-E2-dependent hematopoietic-specific β-tubulin isoform β1, deepen our understanding of molecular mechanisms in platelet biogenesis. The study of rare syndromes of inherited thrombocytopenia in mice and man has also refined the emerging picture of Megakaryocyte maturation. Synthesis of platelet-specific organelles is mediated by a variety of regulators of intracellular vesicle membrane fusion, and platelet release is coordinated through extensive and dynamic reorganization of the actin and microtubule cytoskeletons. As in other aspects of hematopoiesis, characterization of recurrent chromosomal translocations in human leukemias provides an added dimension to the molecular underpinnings of Megakaryocyte differentiation. Long regarded as a mysterious cell, the Megakaryocyte is thus yielding many of its secrets, and mechanisms of thrombopoiesis are becoming clearer. Although this review focuses on transcriptional control mechanisms, it also discusses recent advances in broader consideration of the birth of platelets.
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blood platelets are assembled principally at the ends of proplatelet processes produced by differentiated Megakaryocytes
Journal of Cell Biology, 1999Co-Authors: Patrick Lecine, Ramesh A Shivdasani, Joseph E Italiano, John H. HartwigAbstract:Megakaryocytes release mature platelets in a complex process. Platelets are known to be released from intermediate structures, designated proplatelets, which are long, tubelike extensions of the Megakaryocyte cytoplasm. We have resolved the ultrastructure of the Megakaryocyte cytoskeleton at specific stages of proplatelet morphogenesis and correlated these structures with cytoplasmic remodeling events defined by video microscopy. Platelet production begins with the extension of large pseudopodia that use unique cortical bundles of microtubules to elongate and form thin proplatelet processes with bulbous ends; these contain a peripheral bundle of microtubules that loops upon itself and forms a teardrop-shaped structure. Contrary to prior observations and assumptions, time-lapse microscopy reveals proplatelet processes to be extremely dynamic structures that interconvert reversibly between spread and tubular forms. Microtubule coils similar to those observed in blood platelets are detected only at the ends of proplatelets and not within the platelet-sized beads found along the length of proplatelet extensions. Growth and extension of proplatelet processes is associated with repeated bending and bifurcation, which results in considerable amplification of free ends. These aspects are inhibited by cytochalasin B and, therefore, are dependent on actin. We propose that mature platelets are assembled de novo and released only at the ends of proplatelets, and that the complex bending and branching observed during proplatelet morphogenesis represents an elegant mechanism to increase the numbers of proplatelet ends.
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mice lacking transcription factor nf e2 provide in vivo validation of the proplatelet model of thrombocytopoiesis and show a platelet production defect that is intrinsic to Megakaryocytes
Blood, 1998Co-Authors: Patrick Lecine, Jean-luc Villeval, Bethany Swencki, Yuhui Xu, Paresh Vyas, Ramesh A ShivdasaniAbstract:Mechanisms of platelet production and release by mammalian Megakaryocytes are poorly understood. We used thrombocytopenic knockout mice to better understand these processes. Proplatelets are filamentous extensions of terminally differentiated Megakaryocytes that are thought to represent one mechanism of platelet release; however, these structures have largely been recognized in cultured cells and there has been no correlation between thrombocytopoiesis in vivo and proplatelet formation. Mice lacking transcription factor NF-E2 have a late arrest in Megakaryocyte maturation, resulting in profound thrombocytopenia. In contrast to normal Megakaryocytes, which generate abundant proplatelets, cells from these mice never produce proplatelets, even after prolonged stimulation with c-Mpl ligand. Similarly, Megakaryocytes from thrombocytopenic mice with lineage-selective loss of transcription factor GATA-1 produce proplatelets very rarely. These findings establish a significant correlation between thrombocytopoiesis and proplatelet formation and suggest that the latter represents a physiologic mechanism of platelet release. We further show that proplatelet formation by normal Megakaryocytes and its absence in cells lacking NF-E2 are independent of interactions with adherent (stromal) cells. Similarly, thrombocytopenia in NF-E2−/− mice reflects intrinsic defects in the Megakaryocyte lineage. These observations improve our understanding of platelet production and validate the study of proplatelets in probing the underlying mechanisms. © 1998 by The American Society of Hematology.
Najet Debili - One of the best experts on this subject based on the ideXlab platform.
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presence of a defect in karyokinesis during Megakaryocyte endomitosis
Cell Cycle, 2012Co-Authors: Larissa Lordier, Valeria Naim, Idinath Badirou, Philippe Rameau, Filippo Rosselli, Abdelali Jalil, William Vainchenker, Najet Debili, Jerome Larghero, Yunhua ChangAbstract:Megakaryocyte is the naturally polyploid cell that gives rise to platelets. Polyploidization occurs by endomitosis, a process corresponding to a late failure of cytokinesis with a backward movement of the daughter cells. Generally, a pure defect in cytokinesis produces a multinucleated cell, but Megakaryocytes are characterized by a single polylobulated nucleus with a 2N ploidy. Here, we show the existence of a defect in karyokinesis during the endomitotic process. From late telophase until the reversal of cytokinesis, some dipolar mitosis/endomitosis and most multipolar endomitosis present a thin DNA link between the segregated chromosomes surrounded by an incomplete nuclear membrane formation, which implies that sister chromatid separation is not complete. This observation may explain why polyploid Megakaryocytes display a single polylobulated nucleus along with an increase in ploidy.
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Megakaryocyte endomitosis is a failure of late cytokinesis related to defects in the contractile ring and rho rock signaling
Blood, 2008Co-Authors: Abdelali Jalil, Najet Debili, Jerome Larghero, Larissa Lordie, Frederic Aurade, Frederic LarbreAbstract:Megakaryocyte (MK) is the naturally polyploid cell that gives rise to platelets. Polyploidization occurs by endomitosis, which was a process considered to be an incomplete mitosis aborted in anaphase. Here, we used time-lapse confocal video microscopy to visualize the endomitotic process of primary human Megakaryocytes. Our results show that the switch from mitosis to endomitosis corresponds to a late failure of cytokinesis accompanied by a backward movement of the 2 daughter cells. No abnormality was observed in the central spindle of endomitotic MKs. A furrow formation was present, but the contractile ring was abnormal because accumulation of nonmuscle myosin IIA was lacking. In addition, a defect in cell elongation was observed in dipolar endomitotic MKs during telophase. RhoA and F-actin were partially concentrated at the site of furrowing. Inhibition of the Rho/Rock pathway caused the disappearance of F-actin at midzone and increased MK ploidy level. This inhibition was associated with a more pronounced defect in furrow formation as well as in spindle elongation. Our results suggest that the late failure of cytokinesis responsible for the endomitotic process is related to a partial defect in the Rho/Rock pathway activation.
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impaired megakaryocytopoiesis in type 2b von willebrand disease with severe thrombocytopenia
Blood, 2006Co-Authors: Paquita Nurden, William Vainchenker, Najet Debili, Regis Bobe, Raymonde Bredoux, Elisabeth Corvazier, Robert Combrie, Edith Fressinaud, D Meyer, Alan T NurdenAbstract:In type 2B von Willebrand disease, there is spontaneous binding of mutated von Willebrand factor (VWF) multimers to platelets. Here we report a family in which severe thrombocytopenia may also be linked to abnormal megakaryocytopoiesis. A heterozygous mutation in the VWF A1 domain gave a R1308P substitution in an interactive site for glycoprotein Ibα (GPIbα). Electron microscopy showed clusters of platelets in close contact. Binding of antibodies to the GPIbα N-terminal domain was decreased, whereas GPIX and GPV were normally detected. In Western blotting (WB), GPIbα, αIIb, and β3 were normally present. Proteins involved in Ca 2+ homeostasis were analyzed by quantitating platelet mRNA or by WB. Plasma membrane Ca 2+ ATPase (PMCA)-4b and type III inositol trisphosphate receptor (InsP 3 -R3) were selectively increased. The presence of degradation products of polyadenosine diphosphate (ADP)-ribose polymerase protein (PARP) suggested ongoing caspase-3 activity. These were findings typical of immature normal Megakaryocytes cultured from peripheral blood CD34 + cells with TPO. Significantly, Megakaryocytes from the patients in culture produced self-associated and interwoven proplatelets. Immunolocalization showed VWF not only associated with platelets, but already on the Megakaryocyte surface and within internal channels. In this family, type 2B VWD is clearly associated with abnormal platelet production.
Alan T Nurden - One of the best experts on this subject based on the ideXlab platform.
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impaired megakaryocytopoiesis in type 2b von willebrand disease with severe thrombocytopenia
Blood, 2006Co-Authors: Paquita Nurden, William Vainchenker, Najet Debili, Regis Bobe, Raymonde Bredoux, Elisabeth Corvazier, Robert Combrie, Edith Fressinaud, D Meyer, Alan T NurdenAbstract:In type 2B von Willebrand disease, there is spontaneous binding of mutated von Willebrand factor (VWF) multimers to platelets. Here we report a family in which severe thrombocytopenia may also be linked to abnormal megakaryocytopoiesis. A heterozygous mutation in the VWF A1 domain gave a R1308P substitution in an interactive site for glycoprotein Ibα (GPIbα). Electron microscopy showed clusters of platelets in close contact. Binding of antibodies to the GPIbα N-terminal domain was decreased, whereas GPIX and GPV were normally detected. In Western blotting (WB), GPIbα, αIIb, and β3 were normally present. Proteins involved in Ca 2+ homeostasis were analyzed by quantitating platelet mRNA or by WB. Plasma membrane Ca 2+ ATPase (PMCA)-4b and type III inositol trisphosphate receptor (InsP 3 -R3) were selectively increased. The presence of degradation products of polyadenosine diphosphate (ADP)-ribose polymerase protein (PARP) suggested ongoing caspase-3 activity. These were findings typical of immature normal Megakaryocytes cultured from peripheral blood CD34 + cells with TPO. Significantly, Megakaryocytes from the patients in culture produced self-associated and interwoven proplatelets. Immunolocalization showed VWF not only associated with platelets, but already on the Megakaryocyte surface and within internal channels. In this family, type 2B VWD is clearly associated with abnormal platelet production.
Alessandra Balduini - One of the best experts on this subject based on the ideXlab platform.
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Megakaryocytes contribute to the bone marrow matrix environment by expressing fibronectin type iv collagen and laminin
Stem Cells, 2014Co-Authors: Alessandro Malara, Manuela Currao, Cristian Gruppi, Giuseppe Celesti, Gianluca Viarengo, Chiara Buracchi, Luigi Laghi, David L Kaplan, Alessandra BalduiniAbstract:Megakaryocytes associate with the bone marrow vasculature where they convert their cytoplasm into proplatelets that protrude through the vascular endothelium into the lumen and release platelets. The extracellular matrix (ECM) microenvironment plays a critical role in regulating these processes. In this work we demonstrate that, among bone marrow ECM components, fibronectin, type IV collagen, and laminin are the most abundant around bone marrow sinusoids and constitute a pericellular matrix surrounding Megakaryocytes. Most importantly, we report, for the first time, that Megakaryocytes express components of the basement membrane and that these molecules contribute to the regulation of Megakaryocyte development and bone marrow ECM homeostasis both in vitro and in vivo. In vitro, fibronectin induced a threefold increase in the proliferation rate of mouse hematopoietic stem cells leading to higher Megakaryocyte output with respect to cells treated only with thrombopoietin or other matrices. However, Megakaryocyte ploidy level in fibronectin-treated cultures was significantly reduced. Stimulation with type IV collagen resulted in a 1.4-fold increase in Megakaryocyte output, while all tested matrices supported proplatelet formation to a similar extent in Megakaryocytes derived from fetal liver progenitor cells. In vivo, Megakaryocyte expression of fibronectin and basement membrane components was upregulated during bone marrow reconstitution upon 5-fluorouracil induced myelosuppression, while only type IV collagen resulted upregulated upon induced thrombocytopenia. In conclusion, this work demonstrates that ECM components impact Megakaryocyte behavior differently during their differentiation and highlights a new role for Megakaryocyte as ECM-producing cells for the establishment of cell niches during bone marrow regeneration. Stem Cells 2014;32:926–937
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high doses of romiplostim induce proliferation and reduce proplatelet formation by human Megakaryocytes
PLOS ONE, 2013Co-Authors: Manuela Currao, Carlo L. Balduini, Alessandra BalduiniAbstract:Background Romiplostim (AMG531) is a Thrombopoietin (TPO) receptor agonist with no homology with the endogenous TPO that has been used to treat patients affected by immune thrombocytopenia (ITP). Despite the use of TPO mimetics in the clinical practice, the mechanisms underlying their impact on Megakaryocyte function is still unknown. Methodology/Principal Findings In this project we took advantage of an in vitro human model, that we have established in our laboratory for long time to study Megakaryocyte development from human cord blood-derived progenitor cells, and we demonstrated that increasing doses of AMG531 (100 to 2000 ng/mL) determine a progressive increase of Megakaryocyte proliferation with a parallel decrease in Megakaryocyte ploidy and capacity of extending proplatelets. Most importantly, these differences in Megakaryocyte function seemed to be correlated to modulation of AKT phosphorylation. Conclusions/Significance Overall our results shed new light on the mechanisms and on the relevance of dosage related to AMG531 impact on Megakaryocyte function.
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Megakaryocytes of patients with myh9 related thrombocytopenia present an altered proplatelet formation
Thrombosis and Haemostasis, 2009Co-Authors: Alessandro Pecci, Valeria Bozzi, Stefania Badalucco, Mauro Torti, Carlo L. Balduini, Alessandro Malara, Alessandra BalduiniAbstract:MYH9-related disease (MYH9-RD) is an autosomal-dominant thrombocytopenia caused by mutations of MYH9, the gene for the heavy chain of myosin-IIA. Pathogenesis of thrombocytopenia of MYH9-RD is unknown. Recent studies in mice demonstrated that myosin-IIA is an inhibitor of proplatelet formation (PPF), and suggested that it could be involved in the suppression of PPF exerted by Megakaryocyte adhesion to type I collagen, which regulates the timing of platelet release within bone marrow. However, the consequences on PPF of the heterozygous mutations causative of the MYH9-RD have never been investigated. We studied the in-vitro PPF by Megakaryocytes obtained from four patients carrying the p.D1424N or the p.R1933X mutations. We demonstrated that MYH9-RD Megakaryocytes completely lose the physiologic suppression of proplatelet extension exerted by interaction with type I collagen, thus supporting the hypothesis that a premature platelet release within bone marrow contributes to pathogenesis of MYH9-related thrombocytopenia. Moreover, proplatelets extended by MYH9-RD Megakaryocytes presented a significant defect in branching in secondary processes (p=0.001) and formed a significantly lower number of proplatelet tips (p=0.005). Since platelets are assembled at the level of proplatelet tips, this defect could further contribute to pathogenesis of thrombocytopenia of MYH9-RD patients.
