Journal articles on the topic 'Megakaryocytes'
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1
Levene, R. B., J. M. Lamaziere, H. E. Broxmeyer, L. Lu, and E. M. Rabellino. "Human megakaryocytes. V. Changes in the phenotypic profile of differentiating megakaryocytes." Journal of Experimental Medicine 161, no. 3 (March 1, 1985): 457–74. http://dx.doi.org/10.1084/jem.161.3.457.
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Human megakaryocytes were studied for phenotypic changes occurring throughout differentiation using a panel of monoclonal antibodies raised against marrow megakaryocytes and blood platelets. 11 monoclonal antibody preparations were selected for restricted specificity against megakaryocytes and/or platelets after screening by immunofluorescence, complement-mediated cytolysis, and solid phase enzyme-linked immunosorbent assay. The expression of the cellular epitopes recognized by these reagents enabled the identification of three levels of megakaryocyte maturation characterized by distinct immunologic phenotypes. Based upon their reactivities against megakaryocytic cells at different ontogenetic levels, monoclonal antibodies were operationally categorized into three groups. Group A consisted of six different monoclonal antibodies that recognized antigens on the colony-forming unit-megakaryocyte (CFU-Mk), in vitro grown colony megakaryocytes, and early immature marrow megakaryocytes, only, and did not detect their respective epitopes on either mature megakaryocytes or platelets. A monoclonal antibody categorized in group B detected a cell antigen expressed by megakaryocytic cells at all maturational levels, but which is lost or suppressed during terminal differentiation and is not expressed on blood platelets. Group C included four different monoclonal antibodies raised against platelets that recognized antigenic determinants expressed on the CFU-Mk, colony megakaryocytes, early and mature megakaryocytes, and platelets. Three group C monoclonal antibodies (PC-1, PC-3, and PC-4) were specific for platelet glycoprotein IIb/IIIa. Additionally, group C monoclonal antibody PC-2 was unique in that it showed partial reactivity against the clonable progenitor for the erythroid series (BFU-E). Recognition of discrete phenotypic changes in differentiating megakaryocytes will enable multiparameter analyses of these cells as well as the study of factors regulating the dynamics of megakaryocytopoiesis in health and disease.
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Pokharel, S., P. Upadhyaya, S. Karki, P. Paudyal, B. Pradhan, and P. Poudel. "Megakaryocytic alterations in thrombocytopenia: A bone marrow aspiration study." Journal of Pathology of Nepal 6, no. 11 (March 17, 2016): 914–21. http://dx.doi.org/10.3126/jpn.v6i11.15673.
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Background: Megakaryocyte morphology plays an important role in thrombopoiesis. A defect in any stage of megakaryocytopoiesis can lead to dysmegakaryocytopoiesis and thrombocytopenia. This study was conducted to understand megakaryocytic alterations and their contribution in the diagnosis of cases of thrombocytopenia.Materials and Methods: This was a cross-sectional study was conducted on all consecutive cases of bone marrow aspirates of thrombocytopenia over a duration of one year in BPKIHS. Megakaryocyte morphology was studied with a 100X objective. Data were entered into Microsoft excel 10 and analysed with SPSS version 11.5. Descriptive statistics charted and Chi-square tests were done for inferential statistics to find any association at 95% Confidence Interval.Results: Among the 38 subjects, megakaryocytic thrombocytopenia (44.7%) was the most common cause of thrombocytopenia. Hypolobated megakaryocytes (63.2%), bare megakaryocytic nuclei (57.9%) were the common morphological changes in megakaryocytes. Odds of increased megakaryocyte count in megakaryocytic thrombocytopenia was found to be 12.5 times than for other causes of thrombocytopenia and the presence of bare megakaryocytic nuclei in MTP was statistically significant. (p –value<0.05)Conclusion: Many similarities were observed in megakaryocytic morphology among different hematological diseases. However, increased megakaryocyte count and presence of bare megakaryocytic nuclei, hypolobated forms were significant in megakaryocytic thrombocytopenia.
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Rivière, Christel, Frédéric Subra, Karine Cohen-Solal, Véronique Cordette-Lagarde, Remi Letestu, Christian Auclair, William Vainchenker, and Fawzia Louache. "Phenotypic and Functional Evidence for the Expression of CXCR4 Receptor During Megakaryocytopoiesis." Blood 93, no. 5 (March 1, 1999): 1511–23. http://dx.doi.org/10.1182/blood.v93.5.1511.
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Abstract The identification of stromal cell–derived factor (SDF)-1 as a chemoattractant for human progenitor cells suggests that this chemokine and its receptor might represent critical determinants for the homing, retention, and exit of precursor cells from hematopoietic organs. In this study, we investigated the expression profile of CXCR4 receptor and the biological activity of SDF-1 during megakaryocytopoiesis. CD34+ cells from bone marrow and cord blood were purified and induced to differentiate toward the megakaryocyte lineage by a combination of stem-cell factor (SCF) and recombinant human pegylated megakaryocyte growth and development factor (PEG-rhuMGDF). After 6 days of culture, a time where mature and immature megakaryocytes were present, CD41+ cells were immunopurified and CXCR4mRNA expression was studied. High transcript levels were detected by a RNase protection assay in cultured megakaryocytes derived from cord blood CD34+ cells as well as in peripheral blood platelets. The transcript levels were about equivalent to that found in activated T cells. By flow cytometry, a large fraction (ranging from 30% to 100%) of CD41+cells showed high levels of CXCR4 antigen on their surface, its expression increasing in parallel with the CD41 antigen during megakaryocytic differentiation. CXCR4 protein was also detected on peripheral blood platelets. SDF-1 acts on megakaryocytes by inducing intracellular calcium mobilization and actin polymerization. In addition, in in vitro transmigration experiments, a significant proportion of megakaryocytes was observed to respond to this chemokine. This cell migration was inhibited by pertussis toxin, indicating coupling of this signal to heterotrimeric guanine nucleotide binding proteins. Although a close correlation between CD41a and CXCR4 expession was observed, cell surface markers as well as morphological criteria indicate a preferential attraction of immature megakaryocytes (low level of CD41a and CD42a), suggesting that SDF-1 is a potent attractant for immature megakaryocytic cells but is less active on fully mature megakaryocytes. This hypothesis was further supported by the observation that SDF-1 induced the migration of colony forming unit–megakaryocyte progenitors (CFU-MK) and the expression of activation-dependent P-selectin (CD62P) surface antigen on early megakaryocytes, although no effect was observed on mature megakaryocytes and platelets. These results indicate that CXCR4 is expressed by human megakaryocytes and platelets. Furthermore, based on the lower responses of mature megakaryocytes and platelets to SDF-1 as compared with early precursors, these data suggest a role for this chemokine in the maintenance and homing during early stages of megakaryocyte development. Moreover, because megakaryocytes are also reported to express CD4, it becomes important to reevaluate the role of direct infection of these cells by the human immunodeficiency virus (HIV)-1 in HIV-1–related thrombocytopenia.
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Rivière, Christel, Frédéric Subra, Karine Cohen-Solal, Véronique Cordette-Lagarde, Remi Letestu, Christian Auclair, William Vainchenker, and Fawzia Louache. "Phenotypic and Functional Evidence for the Expression of CXCR4 Receptor During Megakaryocytopoiesis." Blood 93, no. 5 (March 1, 1999): 1511–23. http://dx.doi.org/10.1182/blood.v93.5.1511.405k02_1511_1523.
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The identification of stromal cell–derived factor (SDF)-1 as a chemoattractant for human progenitor cells suggests that this chemokine and its receptor might represent critical determinants for the homing, retention, and exit of precursor cells from hematopoietic organs. In this study, we investigated the expression profile of CXCR4 receptor and the biological activity of SDF-1 during megakaryocytopoiesis. CD34+ cells from bone marrow and cord blood were purified and induced to differentiate toward the megakaryocyte lineage by a combination of stem-cell factor (SCF) and recombinant human pegylated megakaryocyte growth and development factor (PEG-rhuMGDF). After 6 days of culture, a time where mature and immature megakaryocytes were present, CD41+ cells were immunopurified and CXCR4mRNA expression was studied. High transcript levels were detected by a RNase protection assay in cultured megakaryocytes derived from cord blood CD34+ cells as well as in peripheral blood platelets. The transcript levels were about equivalent to that found in activated T cells. By flow cytometry, a large fraction (ranging from 30% to 100%) of CD41+cells showed high levels of CXCR4 antigen on their surface, its expression increasing in parallel with the CD41 antigen during megakaryocytic differentiation. CXCR4 protein was also detected on peripheral blood platelets. SDF-1 acts on megakaryocytes by inducing intracellular calcium mobilization and actin polymerization. In addition, in in vitro transmigration experiments, a significant proportion of megakaryocytes was observed to respond to this chemokine. This cell migration was inhibited by pertussis toxin, indicating coupling of this signal to heterotrimeric guanine nucleotide binding proteins. Although a close correlation between CD41a and CXCR4 expession was observed, cell surface markers as well as morphological criteria indicate a preferential attraction of immature megakaryocytes (low level of CD41a and CD42a), suggesting that SDF-1 is a potent attractant for immature megakaryocytic cells but is less active on fully mature megakaryocytes. This hypothesis was further supported by the observation that SDF-1 induced the migration of colony forming unit–megakaryocyte progenitors (CFU-MK) and the expression of activation-dependent P-selectin (CD62P) surface antigen on early megakaryocytes, although no effect was observed on mature megakaryocytes and platelets. These results indicate that CXCR4 is expressed by human megakaryocytes and platelets. Furthermore, based on the lower responses of mature megakaryocytes and platelets to SDF-1 as compared with early precursors, these data suggest a role for this chemokine in the maintenance and homing during early stages of megakaryocyte development. Moreover, because megakaryocytes are also reported to express CD4, it becomes important to reevaluate the role of direct infection of these cells by the human immunodeficiency virus (HIV)-1 in HIV-1–related thrombocytopenia.
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Kaluzhny, Yulia, Guangyao Yu, Shishinn Sun, Paul A. Toselli, Bernhard Nieswandt, Carl W. Jackson, and Katya Ravid. "BclxL overexpression in megakaryocytes leads to impaired platelet fragmentation." Blood 100, no. 5 (September 1, 2002): 1670–78. http://dx.doi.org/10.1182/blood-2001-12-0263.
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Fragmentation of polyploid megakaryocytes into platelets has great relevance for blood homeostasis. Apoptotic cell death is a highly regulated genetic program, which has been observed in mature megakaryocytes fragmenting into platelets. The antiapoptotic protein BclxL has been reported as up-regulated during megakaryocytic differentiation in vitro, but absent during late megakaryopoiesis. Our study focused on examining BclxL levels in megakaryocytes in vivo and in assessing the effect of its overexpression in transgenic mice (via the platelet factor 4 [PF4] promoter) on megakaryocyte development and platelet fragmentation. Interestingly, in the wild-type and less in PF4-driven transgenic mice, BclxL was not detected in a fraction of the large mature megakaryocytes, suggesting a regulation on the protein level. BclxL overexpression was associated with a moderate increase in megakaryocyte number, with no significant change in ploidy level or platelet counts. When the mice were challenged by induction of immune thrombocytopenia, the rate of platelet recovery was significantly slower in the transgenic mice as compared with controls. Moreover, proplatelet formation in vitro by transgenic megakaryocytes was limited. Transgenic megakaryocytes displayed poorly developed platelet demarcation membranes and cell margin extensions. Our study indicates that regulated expression of BclxL in megakaryocytes is important for the development of cells with a high potential to fragment into platelets.
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Wen, Qiang Jeremy, Benjamin Goldenson, Sebastien Malinge, Priya Koppikar, Ross L. Levine, Ayalew Tefferi, and John Crispino. "Induction of Megakaryocyte Polyploidization in Combination with JAK Inhibition As a Novel Therapeutic Strategy for Myeloproliferative Neoplasms." Blood 118, no. 21 (November 18, 2011): 64. http://dx.doi.org/10.1182/blood.v118.21.64.64.
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Abstract Abstract 64 Megakaryocytes are one of the few cell types that undergo a modified form of the cell cycle termed endomitosis, in which cells skip the late stages of mitosis to become polyploid. Murine and human megakaryocytes commonly reach modal ploidy states of 32N and 16N, respectively, and can sometimes achieve DNA contents as high as 64N. Polyploidization is associated with upregulation of megakaryocyte lineage specific genes, proplatelet formation and expression of genes related to apoptosis. RNA expression array studies have shown that high ploidy states are strongly correlated with megakaryocyte differentiation and maturation. Importantly, the choice of a megakaryocyte to undergo polyploidization and differentiation is inextricably linked to exit from the proliferative cell cycle. Given that megakaryocytes in patients with essential thrombocythemia are hyperproliferative and that those in primary myelofibrosis fail to undergo normal differentiation or polyploidization, we hypothesized that small molecule inducers of polyploidization would drive these cells to exit the proliferative cell cycle and undergo terminal differentiation or death. In collaboration with the Broad Institute, we performed a high throughput screen and identified small molecules that induce polyploidization and proliferative arrest of malignant megakaryocytes, including those that express MPLW515L and JAK2 V617F. We have shown that these compounds, including the Rho kinase inhibitor dimethylfasudil (diMF), selectively increase polyploidization, expression of megakaryocyte cell surface markers, and apoptosis of murine and human megakaryocytic cell lines and primary cells. Furthermore, diMF blocked the growth of primary human AMKL blasts both in vitro and in vivo. With respect to MPNs, diMF showed potent activity against megakaryocytic cell lines and primary cells expressing either JAK2 V617F or MPL W515L alleles commonly associated with these disorders. diMF inhibited proliferation, induced polyploidization and upregulation of lineage specific markers CD41 and CD42, and increased apoptosis of megakaryocytes transduced with JAK2 V617F or MPLW515L. diMF also significantly reduced megakaryocyte colony forming units (CFU-MK), and induced polyploidization and differentiation of bone marrow and fetal liver megakaryocytes from Gata1 mutant mice, which develop a PMF-like disease. Given that diMF induces growth arrest, polyploidization and apoptosis of cells that express activated mutants of JAK2 and MPL, we predicted that it, as well as other small molecule inducers of polyploidy, would be efficient at restraining aberrant megakaryocyte proliferation in both PMF and ET. To assay the effectiveness of diMF in these disorders, we treated peripheral blood mononuclear cells from patients with either PMF or ET with diMF and monitored growth and maturation of megakaryocytes. We discovered that diMF induced polyploidization and subsequent apoptosis of both types of MPN primary samples. diMF also reduced CFU-MK of these MPN patient samples. Next, we assessed the activity of diMF in a model of MPN in which congenic recipients of MPLW515L transduced Balb/C bone marrow cells develop a rapid MPN characterized by leukocytosis, thrombocytosis, bone marrow fibrosis, and death. diMF led to a significant decrease of fibrosis in the bone marrow, diminished infiltration of megakaryocytes and granulocytes in the liver, and a profound reduction in the numbers of megakaryocytes within the spleen of a mouse model of PMF. diMF also led to a significant reduction in the platelet count and a trend towards decreased white cell count, with no effect on hematocrit. Overall, diMF results were comparable to intermediate doses of INCB16562. These encouraging results strongly suggest that diMF induces a decline in megakaryocyte lineage, which leads to reduction in platelet count, and support pre-clinical development of diMF for megakaryocytic subtypes of MPNs. Of note, diMF did not inhibit the phosphorylation of Stat5 or Stat3, suggesting that it acts through a mechanism distinct from JAK2 inhibitors. Interestingly, combination of diMF with a selective JAK2 inhibitor greatly enhanced the efficacy of diMF to inhibit proliferation and induce apoptosis in MPLW515L transduced megakaryocytic cell line. These data support combining JAK inhibition and induction of megakaryocyte polyploidy as a new therapeutic strategy for MPNs. Disclosures: No relevant conflicts of interest to declare.
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Noh, Ji-Yoon, Shilpa Gandre-Babbe, Yuhuan Wang, Vincent Hayes, Yu Yao, Paul Gadue, Spencer Sullivan, et al. "Inducible Gata1 Suppression As a Novel Strategy to Expand Physiologic Megakaryocyte Production from Embryonic Stem Cells." Blood 124, no. 21 (December 6, 2014): 3846. http://dx.doi.org/10.1182/blood.v124.21.3846.3846.
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Abstract Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent potential sources of megakaryocytes and platelets for transfusion therapy. However, most current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny, including platelet-releasing megakaryocytes. Mutations in the mouse and human genes encoding transcription factor GATA1 cause accumulation of proliferating, developmentally arrested megakaryocytes. Previously, we reported that in vitro differentiation of Gata1-null murine ES cells generated self-renewing hematopoietic progenitors termed G1ME cells that differentiated into erythroblasts and megakaryocytes upon restoration of Gata1 cDNA by retroviral transfer. However, terminal maturation of Gata1-rescued megakaryocytes was aberrant with immature morphology and no proplatelet formation, presumably due to non-physiological expression of GATA1. We now engineered wild type (WT) murine ES cells that express doxycycline (dox)-regulated Gata1 short hairpin (sh) RNAs to develop a strategy for Gata1-blockade that upon its release, restores physiologic GATA1 expression during megakaryopoiesis. In vitro hematopoietic differentiation of control scramble shRNA-expressing ES cells with dox and thrombopoietin (TPO) produced megakaryocytes that underwent senescence after 7 days. Under similar differentiation conditions, Gata1 shRNA-expressing ES cells produced immature hematopoietic progenitors, termed G1ME2 cells, which replicated continuously for more than 40 days, resulting in ~1013-fold expansion (N=4 separate experiments). Upon dox withdrawal with multi-lineage cytokines present (EPO, TPO, SCF, GMCSF and IL3), endogenous GATA1 expression was restored to G1ME2 cells followed by differentiation into erythroblasts and megakaryocytes, but no myeloid cells. In clonal methylcellulose assays, dox-deprived G1ME2 cells produced a mixture of erythroid, megakaryocytic and erythro-megakaryocytic colonies. In liquid culture with TPO alone, dox-deprived G1ME2 cells formed mature megakaryocytes in 5-6 days, as determined by morphology, ultrastructure, acetylcholinesterase staining, upregulated megakaryocytic gene expression (Vwf, Pf4, Gp1ba, Selp, Ppbp), CD42b surface expression, increased DNA ploidy and proplatelet production. Compared to G1ME cells rescued with Gata1 cDNA retrovirus, dox-deprived G1ME2 cells exhibited more robust megakaryocytic maturation, similar to that of megakaryocytes produced from cultured fetal liver. Importantly, G1ME2 cell-derived megakaryocytes generated proplatelets in vitro and functional platelets in vivo (~40 platelets/megakaryocyte with a circulating half life of 5-6 hours). These platelets were actively incorporated into growing arteriolar thrombi at sites of laser injury and subsequently expressed the platelet activation marker p-selectin (N=3-4 separate experiments). Our findings indicate that precise timing and magnitude of a transcription factor is required for proper terminal hematopoiesis. We illustrate this principle using a novel, readily reproducible strategy to expand ES cell-derived megakaryocyte-erythroid progenitors and direct their differentiation into megakaryocytes and then into functional platelets in clinically relevant numbers. Disclosures No relevant conflicts of interest to declare.
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Ghai, Shuchi, and Sharada Rai. "Megakaryocytic morphology in Janus kinase 2 V617F positive myeloproliferative neoplasm." South Asian Journal of Cancer 06, no. 02 (April 2017): 075–78. http://dx.doi.org/10.4103/2278-330x.208854.
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Abstract Context: Alterations in megakaryocyte morphology are the hallmark of myeloproliferative neoplasms (MPNs). These neoplasm are also associated with Janus kinase 2 (JAK2) V617F mutation in nearly 95% patients with polycythemia vera (PV), 40% patients of essential thrombocythemia (ET) and 50% patients of myelofibrosis (MF). The utility of megakaryocyte morphology in these disorders in correlation with JAK2 V617F remains unresolved. Aims: The aim of the study was to assess the morphology of megakaryocytes in bone marrow aspirates (BMAs) and bone marrow biopsies of patients of BCR-ABL negative MPNs with JAK2 V617F mutation. Settings and Design: This study was a retrospective and prospective, hospital-based study undertaken for a period ranging from January 2011 to April 2015. Subjects and Methods: Assessment of morphological features of megakaryocytes in 15 BMAs and their respective biopsies which included seven cases of PV, three cases of ET, and five cases of MF with JAK2 V617F mutation. Statistical Analysis Used: Chi-square test and Fisher exact test were used to compare the different features of megakaryocytes. Software version SPSS 13.0 was used. Results: Megakaryocytes in ET were found to have characteristically large size with staghorn multinucleated nuclei and exhibiting large amount of cytoplasm. MF showed dense clustering of megakaryocytes with staghorn nucleus along with sinusoidal dilatation and intrasinusoidal hematopoiesis. PV showed loose and dense clustering of megakaryocytes with a predominance of cloud-like nuclei. Few of the megakaryocytic morphologic features showed overlap between MF and PV and between ET and early MF. Conclusions: Megakaryocytic morphology can aid in the accurate diagnosis of the different subcategories of MPNs. This would help in categorization of clinically suspicious patients of JAK2 V617F negative patients.
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Ghai, Shuchi, and Sharada Rai. "Megakaryocytic morphology in Janus kinase 2 V617F positive myeloproliferative neoplasm." South Asian Journal of Cancer 06, no. 02 (April 2017): 075–78. http://dx.doi.org/10.4103/2278-330x.208854.
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Abstract Context: Alterations in megakaryocyte morphology are the hallmark of myeloproliferative neoplasms (MPNs). These neoplasm are also associated with Janus kinase 2 (JAK2) V617F mutation in nearly 95% patients with polycythemia vera (PV), 40% patients of essential thrombocythemia (ET) and 50% patients of myelofibrosis (MF). The utility of megakaryocyte morphology in these disorders in correlation with JAK2 V617F remains unresolved. Aims: The aim of the study was to assess the morphology of megakaryocytes in bone marrow aspirates (BMAs) and bone marrow biopsies of patients of BCR-ABL negative MPNs with JAK2 V617F mutation. Settings and Design: This study was a retrospective and prospective, hospital-based study undertaken for a period ranging from January 2011 to April 2015. Subjects and Methods: Assessment of morphological features of megakaryocytes in 15 BMAs and their respective biopsies which included seven cases of PV, three cases of ET, and five cases of MF with JAK2 V617F mutation. Statistical Analysis Used: Chi-square test and Fisher exact test were used to compare the different features of megakaryocytes. Software version SPSS 13.0 was used. Results: Megakaryocytes in ET were found to have characteristically large size with staghorn multinucleated nuclei and exhibiting large amount of cytoplasm. MF showed dense clustering of megakaryocytes with staghorn nucleus along with sinusoidal dilatation and intrasinusoidal hematopoiesis. PV showed loose and dense clustering of megakaryocytes with a predominance of cloud-like nuclei. Few of the megakaryocytic morphologic features showed overlap between MF and PV and between ET and early MF. Conclusions: Megakaryocytic morphology can aid in the accurate diagnosis of the different subcategories of MPNs. This would help in categorization of clinically suspicious patients of JAK2 V617F negative patients.
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James, Chloé, Emma C. Josefsson, Michael J. White, Katya J. Henley, and Benjamin T. Kile. "Deletion of Bcl-X in the Megakaryocyte Compartment Results in Profound Thrombocytopenia Caused by Impaired Platelet Production and Survival." Blood 114, no. 22 (November 20, 2009): 1495. http://dx.doi.org/10.1182/blood.v114.22.1495.1495.
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Abstract Abstract 1495 Poster Board I-518 Recent studies have suggested a role for the intrinsic apoptosis pathway in megakaryocytic differentiation and platelet shedding. The intrinsic pathway is regulated by the Bcl-2 family of pro- and anti- cell death proteins. We recently demonstrated that platelet life span is controlled by an intrinsic cell death pathway, whereby the anti-apoptotic protein Bcl-xL constrains the pro-apoptotic activity of Bak to maintain platelet survival. As Bcl-xL is expressed in megakaryocytes, we investigated whether this protein is required for megakaryocyte survival, differentiation and/or platelet shedding. We specifically deleted the Bcl-x gene in the megakaryocyte lineage by crossing mice carrying a floxed allele of Bcl-x with mice carrying a platelet factor 4-regulated Cre transgene. Bcl-xfl/flCre+ mice were profoundly thrombocytopenic (26 ± 5 × 103/μl, n=14) compared with Bcl-xfl/flCre− animals (1157 ± 202 × 103/μl, n=13). Platelet life span in these mice was reduced to only 5 hours, as compared to 5 days in wild type littermates. This result confirmed that Bcl-xL is absolutely required for platelet survival. To determine whether Bcl-x deletion has an impact on platelet production, we analyzed the megakaryocyte compartment in Bcl-xfl/flCre+ and Bcl-xfl/flCre− mice. We observed that the number of megakaryocyte progenitors, and number of megakaryocytes in the bone marrow were increased in Bcl-xfl/flCre+ mice (23 ± 9 megakaryocyte progenitors vs 11 ± 5, and 51 ± 9 megakaryocytes vs 12 ± 1). This result suggested that Bcl-xL is not required for the survival of megakaryocytes or their progenitors. To determine whether Bcl-xL is required for the last step of megakaryocyte differentiation, i.e. platelet shedding, we cultured fetal liver cells with thrombopoietin. Large megakaryocytes were isolated after 3 days of differentiation on discontinuous bovine serum albumin gradient. They were cultured for 3 more days in the same media and the percentage of megakaryocytes displaying proplatelets was determined each day. Interestingly, Bcl-xfl/flCre+ megakaryocytes died much more quickly than Bcl-xfl/flCre− megakaryocytes, and almost none of those that survived were able to form proplatelets. Our study indicates that Bcl-xL is not only essential for platelet survival, but it is also required for the survival of mature megakaryocytes at the stage of platelet shedding. Disclosures: No relevant conflicts of interest to declare.
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Bhandari, Anuja, Anil Dev Pant, Anjan Shrestha, and Jiwan Thapa. "Megakaryocytic alterations in bone marrow aspiration smears in thrombocytopenia." Journal of Pathology of Nepal 9, no. 2 (September 29, 2019): 1523–29. http://dx.doi.org/10.3126/jpn.v9i2.25408.
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Background: Thrombocytopenia can be seen in any patients irrespective of age and the causes are diverse. This study was done to determine the frequency of various conditions associated with thrombocytopenia and to assess the number and morphology of megakaryocytes in various cases of thrombocytopenia. Megakaryocytic alterations in myelodysplastic versus non-myelodysplastic conditions were also evaluated in this study.
Materials and methods: This was a prospective study conducted on 132 cases of bone marrow aspirates of thrombocytopenia over a duration of one year from 14th April 2017 to 13th April 2018 in Tribhuvan University Teaching Hospital, Kathmandu. All the statistical evaluation was done by using SPSS version 24.
Results: In this study of 132 cases, megakaryocytic thrombocytopenia was the most common cause of thrombocytopenia for which bone marrow aspiration was done. The most common dysplastic change observed in megakaryocytes was micro megakaryocyte which was more commonly seen in MDS. Nondysplastic changes frequently observed were immature forms followed by bare nuclei and were more frequently seen in megakaryocytic thrombocytopenia followed by in myelodysplastic syndromes.
Conclusions: Many similar morphological changes were observed in megakaryocytes among different hematological diseases. So, the presence of dysplastic megakaryocyte should not prompt an interpretation of myelodysplastic syndromes and should always be correlated with patient’s clinical and hematological parameter.
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Sakaguchi, M., T. Sato, and JE Groopman. "Human immunodeficiency virus infection of megakaryocytic cells." Blood 77, no. 3 (February 1, 1991): 481–85. http://dx.doi.org/10.1182/blood.v77.3.481.481.
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Abstract The human immunodeficiency virus (HIV) is capable of infecting certain cells of hematopoietic lineage, particularly monocyte-macrophages and T lymphocytes. Recently, the possibility that cells of megakaryocytic lineage are susceptible to HIV infection has been raised. We have characterized infection of the permanent megakaryocytic cell line CMK by HIV in vitro. CMK cells were easily infected by HIV type 2 (HIV-2), producing significant amounts of virus in culture. Infection appeared to be mediated by the CD4 surface antigen on CMK cells. Three different strains of HIV-1 were able to minimally infect CMK cells, suggesting there may be isolates of HIV tropic for megakaryocytes. Infection of CMK cells led to downregulation of the CD4 surface antigen but no discernable change in expression of megakaryocyte-associated proteins glycoprotein Ib and glycoprotein IIb/IIIa. These observations support the likelihood that megakaryocytes are susceptible to HIV infection, and cell lines of megakaryocytic origin may provide a useful model to study effects of the retrovirus on megakaryocyte function.
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Sakaguchi, M., T. Sato, and JE Groopman. "Human immunodeficiency virus infection of megakaryocytic cells." Blood 77, no. 3 (February 1, 1991): 481–85. http://dx.doi.org/10.1182/blood.v77.3.481.bloodjournal773481.
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The human immunodeficiency virus (HIV) is capable of infecting certain cells of hematopoietic lineage, particularly monocyte-macrophages and T lymphocytes. Recently, the possibility that cells of megakaryocytic lineage are susceptible to HIV infection has been raised. We have characterized infection of the permanent megakaryocytic cell line CMK by HIV in vitro. CMK cells were easily infected by HIV type 2 (HIV-2), producing significant amounts of virus in culture. Infection appeared to be mediated by the CD4 surface antigen on CMK cells. Three different strains of HIV-1 were able to minimally infect CMK cells, suggesting there may be isolates of HIV tropic for megakaryocytes. Infection of CMK cells led to downregulation of the CD4 surface antigen but no discernable change in expression of megakaryocyte-associated proteins glycoprotein Ib and glycoprotein IIb/IIIa. These observations support the likelihood that megakaryocytes are susceptible to HIV infection, and cell lines of megakaryocytic origin may provide a useful model to study effects of the retrovirus on megakaryocyte function.
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Zauli, Giorgio, Marco Vitale, Elisabetta Falcieri, Davide Gibellini, Alessandra Bassini, Claudio Celeghini, Marta Columbaro, and Silvano Capitani. "In Vitro Senescence and Apoptotic Cell Death of Human Megakaryocytes." Blood 90, no. 6 (September 15, 1997): 2234–43. http://dx.doi.org/10.1182/blood.v90.6.2234.
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Abstract To investigate the fate of human megakaryocytes, CD34+ hematopoietic progenitor cells were purified from the peripheral blood or bone marrow of healthy donors and seeded in serum-free chemically defined suspension cultures. In the presence of thrombopoietin (TPO; 100 ng/mL), CD34-derived cells showed an eightfold numerical expansion and a progressive maturation along the megakaryocytic lineage. Megakaryocyte maturation was characterized ultrastructurally by the presence of a demarcation membrane system and phenotypically by a high surface expression of αIIbβ3 integrin. The number of mature megakaryocytes peaked at days 12 to 15 of culture. On the other hand, the number of platelets released in the culture supernatant by CD34-derived megakaryocytes peaked at days 18 to 21, when a high percentage of megakaryocytes showed the characteristic features of apoptosis, as evaluated by electron microscopy, terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end-labeling technique (TUNEL) and uptake of propidium iodide. In other experiments, primary αIIbβ3+ megakaryocytic cells were directly purified from the bone marrow aspirates of normal donors and seeded in serum-free suspension cultures. In the absence of cytokines, αIIbβ3+ megakaryocytes progressively underwent apoptotic cell death. The addition of TPO but not interleukin-3 or erythropoietin showed some protection of αIIbβ3+ cells from apoptosis at early culture times (days 2 to 4), but it did not show any significant effect at later time points. These findings suggest that the terminal phase of the megakaryocyte life span is characterized by the onset of apoptosis, which can be modulated only to a certain extent by TPO.
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Zauli, Giorgio, Marco Vitale, Elisabetta Falcieri, Davide Gibellini, Alessandra Bassini, Claudio Celeghini, Marta Columbaro, and Silvano Capitani. "In Vitro Senescence and Apoptotic Cell Death of Human Megakaryocytes." Blood 90, no. 6 (September 15, 1997): 2234–43. http://dx.doi.org/10.1182/blood.v90.6.2234.2234_2234_2243.
Full textAbstract:
To investigate the fate of human megakaryocytes, CD34+ hematopoietic progenitor cells were purified from the peripheral blood or bone marrow of healthy donors and seeded in serum-free chemically defined suspension cultures. In the presence of thrombopoietin (TPO; 100 ng/mL), CD34-derived cells showed an eightfold numerical expansion and a progressive maturation along the megakaryocytic lineage. Megakaryocyte maturation was characterized ultrastructurally by the presence of a demarcation membrane system and phenotypically by a high surface expression of αIIbβ3 integrin. The number of mature megakaryocytes peaked at days 12 to 15 of culture. On the other hand, the number of platelets released in the culture supernatant by CD34-derived megakaryocytes peaked at days 18 to 21, when a high percentage of megakaryocytes showed the characteristic features of apoptosis, as evaluated by electron microscopy, terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end-labeling technique (TUNEL) and uptake of propidium iodide. In other experiments, primary αIIbβ3+ megakaryocytic cells were directly purified from the bone marrow aspirates of normal donors and seeded in serum-free suspension cultures. In the absence of cytokines, αIIbβ3+ megakaryocytes progressively underwent apoptotic cell death. The addition of TPO but not interleukin-3 or erythropoietin showed some protection of αIIbβ3+ cells from apoptosis at early culture times (days 2 to 4), but it did not show any significant effect at later time points. These findings suggest that the terminal phase of the megakaryocyte life span is characterized by the onset of apoptosis, which can be modulated only to a certain extent by TPO.
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Hitchcock, Ian S., Norma E. Fox, Nicolas Prévost, Katherine Sear, Sanford J. Shattil, and Kenneth Kaushansky. "Roles of focal adhesion kinase (FAK) in megakaryopoiesis and platelet function: studies using a megakaryocyte lineage–specific FAK knockout." Blood 111, no. 2 (January 15, 2008): 596–604. http://dx.doi.org/10.1182/blood-2007-05-089680.
Full textAbstract:
Focal adhesion kinase (FAK) plays a key role in mediating signaling downstream of integrins and growth factor receptors. In this study, we determined the roles of FAK in vivo by generating a megakaryocyte lineage–specific FAK-null mouse (Pf4-Cre/FAK-floxed). Megakaryocyte and platelet FAK expression was ablated in Pf4-Cre/FAK-floxed mice without affecting expression of the FAK homologue PYK2, although PYK2 phosphorylation was increased in FAK−/− megakaryocytes in response to fibrinogen. Megakaryopoiesis is greatly enhanced in Pf4-Cre/FAK-floxed mice, with significant increases in megakaryocytic progenitors (CFU-MK), mature megakaryocytes, megakaryocyte ploidy, and moderate increases in resting platelet number and platelet recovery following a thrombocytopenic stress. Thrombopoietin (Tpo)–mediated activation of Lyn kinase, a negative regulator of megakaryopoiesis, is severely attenuated in FAK-null megakaryocytes compared with wild-type controls. In contrast, Tpo-mediated activation of positive megakaryopoiesis regulators such as ERK1/2 and AKT is increased in FAK-null megakaryocytes, providing a plausible explanation for the observed increases in megakaryopoiesis in these mice. In Pf4-Cre/FAK-floxed mice, rebleeding times are significantly increased, and FAK-null platelets exhibit diminished spreading on immobilized fibrinogen. These studies establish clear roles for FAK in megakaryocyte growth and platelet function, setting the stage for manipulation of this component of the Tpo signaling apparatus for therapeutic benefit.
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Frontelo, Pilar, Deepa Manwani, Mariann Galdass, Holger Karsunky, Felix Lohmann, Patrick G. Gallagher, and James J. Bieker. "Novel role for EKLF in megakaryocyte lineage commitment." Blood 110, no. 12 (December 1, 2007): 3871–80. http://dx.doi.org/10.1182/blood-2007-03-082065.
Full textAbstract:
Abstract Megakaryocytes and erythroid cells are thought to derive from a common progenitor during hematopoietic differentiation. Although a number of transcriptional regulators are important for this process, they do not explain the bipotential result. We now show by gain- and loss-of-function studies that erythroid Krüppel-like factor (EKLF), a transcription factor whose role in erythroid gene regulation is well established, plays an unexpected directive role in the megakaryocyte lineage. EKLF inhibits the formation of megakaryocytes while at the same time stimulating erythroid differentiation. Quantitative examination of expression during hematopoiesis shows that, unlike genes whose presence is required for establishment of both lineages, EKLF is uniquely down-regulated in megakaryocytes after formation of the megakaryocyte-erythroid progenitor. Expression profiling and molecular analyses support these observations and suggest that megakaryocytic inhibition is achieved, at least in part, by EKLF repression of Fli-1 message levels.
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Flaumenhaft, Robert, James R. Dilks, Jennifer Richardson, Eva Alden, Sunita R. Patel-Hett, Elisabeth Battinelli, Giannoula L. Klement, Martha Sola-Visner, and Joseph E. Italiano. "Megakaryocyte-derived microparticles: direct visualization and distinction from platelet-derived microparticles." Blood 113, no. 5 (January 29, 2009): 1112–21. http://dx.doi.org/10.1182/blood-2008-06-163832.
Full textAbstract:
Abstract Platelet microparticles are a normal constituent of circulating blood. Several studies have demonstrated positive correlations between thrombotic states and platelet microparticle levels. Yet little is known about the processes by which platelet microparticles are generated in vivo. We now characterize microparticles derived directly from megakaryocytes. Video microscopy of live mouse megakaryocytes demonstrated that microparticles form as submicron beads along the lengths of slender, unbranched micropodia. These microparticles are CD41+, CD42b+, and express surface phosphatidylserine. Megakaryocyte microparticle generation is resistant to inhibition of microtubule assembly, which is critical to platelet formation, and augmented by inhibition of actin polymerization. To determine whether circulating microparticles are derived primarily from activated platelets or megakaryocytes, we identified markers that distinguish between these 2 populations. CD62P and LAMP-1 were found only on mouse microparticles from activated platelets. In contrast, full-length filamin A was found in megakaryocyte-derived microparticles, but not microparticles from activated platelets. Circulating microparticles isolated from mice were CD62P−, LAMP-1− and expressed full-length filamin A, indicating a megakaryocytic origin. Similarly, circulating microparticles isolated from healthy volunteers were CD62P− and expressed full-length filamin A. Cultured human megakaryocytes elaborated microparticles that were CD41+, CD42b+, and express surface phosphatidylserine. These results indicate that direct production by megakaryocytes represents a physiologic means to generate circulating platelet microparticles.
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Vyas, Paresh, Kenneth Ault, Carl W. Jackson, Stuart H. Orkin, and Ramesh A. Shivdasani. "Consequences of GATA-1 Deficiency in Megakaryocytes and Platelets." Blood 93, no. 9 (May 1, 1999): 2867–75. http://dx.doi.org/10.1182/blood.v93.9.2867.
Full textAbstract:
Abstract In the absence of the hematopoietic transcription factor GATA-1, mice develop thrombocytopenia and an increased number of megakaryocytes characterized by marked ultrastructural abnormalities. These observations establish a critical role for GATA-1 in megakaryopoiesis and raise the question as to how GATA-1 influences megakaryocyte maturation and platelet production. To begin to address this, we have performed a more detailed examination of the megakaryocytes and platelets produced in mice that lack GATA-1 in this lineage. Our analysis demonstrates that compared with their normal counterparts, GATA-1–deficient primary megakaryocytes exhibit significant hyperproliferation in liquid culture, suggesting that the megakaryocytosis seen in animals is nonreactive. Morphologically, these mutant megakaryocytes are small and show evidence of retarded nuclear and cytoplasmic development. A significant proportion of these cells do not undergo endomitosis and express markedly lower levels of mRNA of all megakaryocyte-associated genes tested, including GPIb, GPIbβ, platelet factor 4 (PF4), c-mpl, and p45 NF-E2. These results are consistent with regulation of a program of megakaryocytic differentiation by GATA-1. Bleeding times are significantly prolonged in mutant animals. GATA-1–deficient platelets show abnormal ultrastructure, reminiscent of the megakaryocytes from which they are derived, and exhibit modest but selective defects in platelet activation in response to thrombin or to the combination of adenosine diphosphate (ADP) and epinephrine. Our findings indicate that GATA-1 serves multiple functions in megakaryocyte development, influencing both cellular growth and maturation.
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Vyas, Paresh, Kenneth Ault, Carl W. Jackson, Stuart H. Orkin, and Ramesh A. Shivdasani. "Consequences of GATA-1 Deficiency in Megakaryocytes and Platelets." Blood 93, no. 9 (May 1, 1999): 2867–75. http://dx.doi.org/10.1182/blood.v93.9.2867.409k24_2867_2875.
Full textAbstract:
In the absence of the hematopoietic transcription factor GATA-1, mice develop thrombocytopenia and an increased number of megakaryocytes characterized by marked ultrastructural abnormalities. These observations establish a critical role for GATA-1 in megakaryopoiesis and raise the question as to how GATA-1 influences megakaryocyte maturation and platelet production. To begin to address this, we have performed a more detailed examination of the megakaryocytes and platelets produced in mice that lack GATA-1 in this lineage. Our analysis demonstrates that compared with their normal counterparts, GATA-1–deficient primary megakaryocytes exhibit significant hyperproliferation in liquid culture, suggesting that the megakaryocytosis seen in animals is nonreactive. Morphologically, these mutant megakaryocytes are small and show evidence of retarded nuclear and cytoplasmic development. A significant proportion of these cells do not undergo endomitosis and express markedly lower levels of mRNA of all megakaryocyte-associated genes tested, including GPIb, GPIbβ, platelet factor 4 (PF4), c-mpl, and p45 NF-E2. These results are consistent with regulation of a program of megakaryocytic differentiation by GATA-1. Bleeding times are significantly prolonged in mutant animals. GATA-1–deficient platelets show abnormal ultrastructure, reminiscent of the megakaryocytes from which they are derived, and exhibit modest but selective defects in platelet activation in response to thrombin or to the combination of adenosine diphosphate (ADP) and epinephrine. Our findings indicate that GATA-1 serves multiple functions in megakaryocyte development, influencing both cellular growth and maturation.
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Giraudier, Stéphane, Hédia Chagraoui, Emiko Komura, Stéphane Barnache, Benoit Blanchet, Jean Pierre LeCouedic, David F. Smith, et al. "Overexpression of FKBP51 in idiopathic myelofibrosis regulates the growth factor independence of megakaryocyte progenitors." Blood 100, no. 8 (October 15, 2002): 2932–40. http://dx.doi.org/10.1182/blood-2002-02-0485.
Full textAbstract:
Idiopathic myelofibrosis (IMF) is a chronic myeloproliferative disorder characterized by megakaryocyte hyperplasia and bone marrow fibrosis. Biologically, an autonomous megakaryocyte growth and differentiation is noticed, which contributes to the megakaryocyte accumulation. To better understand the molecular mechanisms involved in this spontaneous growth, we searched for genes differentially expressed between normal megakaryocytes requiring cytokines to grow and IMF spontaneously proliferating megakaryocytes. Using a differential display technique, we found that the immunophilin FKBP51 was 2 to 8 times overexpressed in megakaryocytes derived from patients' CD34+ cells in comparison to normal megakaryocytes. Overexpression was moderate and confirmed in 8 of 10 patients, both at the mRNA and protein levels. Overexpression of FKBP51 in a UT-7/Mpl cell line and in normal CD34+ cells induced a resistance to apoptosis mediated by cytokine deprivation with no effect on proliferation. FKBP51 interacts with both calcineurin and heat shock protein (HSP)70/HSP90. However, a mutant FKBP51 deleted in the HSP70/HSP90 binding site kept the antiapoptotic effect, suggesting that the calcineurin pathway was responsible for the FKBP51 effect. Overexpression of FKBP51 in UT-7/Mpl cells induced a marked inhibition of calcineurin activity. Pharmacologic inhibition of calcineurin by cyclosporin A mimicked the effect of FKBP51. The data support the conclusion that FKBP51 inhibits apoptosis through a calcineurin-dependent pathway. In conclusion, FKBP51 is overexpressed in IMF megakaryocytes and this overexpression could be, in part, responsible for the megakaryocytic accumulation observed in this disorder by regulating their apoptotic program.
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Guo, Yinshi, Chao Niu, Peter Breslin, Minghui Tang, Shubin Zhang, Wei Wei, Ameet R. Kini, et al. "c-Myc–mediated control of cell fate in megakaryocyte-erythrocyte progenitors." Blood 114, no. 10 (September 3, 2009): 2097–106. http://dx.doi.org/10.1182/blood-2009-01-197947.
Full textAbstract:
Abstract It has been found that c-Myc protein plays a critical role in controlling self-renewal versus differentiation in hematopoietic stem cells. We report that c-Myc also controls the fate of megakaryocyte-erythrocyte progenitors through regulating the differentiation of erythroid and megakaryocytic progenitors. In addition to the significant reduction of granulocytes/macrophages and B and T lymphocytes because of the reduction of their corresponding progenitors, we found significantly increased numbers of megakaryocytic progenitors and mature megakaryocytes in bone marrow and spleens of c-Myc-knockout (c-Myc−/−) mice. Differentiation of erythrocytes was blocked at the erythroid progenitor stage. This increased megakaryocytopoiesis is a cell-intrinsic defect of c-Myc-mutant hematopoietic stem cells, as shown by transplantation studies. Furthermore, we found that c-Myc is required for polyploidy formation but not for cytoplasmic maturation of megakaryocytes. Megakaryocytes from c-Myc−/− mice are significantly smaller in size and lower in ploidy than those of control mice; however, because of the dramatic increase in megakaryocyte number, although fewer platelets are produced by each megakaryocyte, a greater than 3-fold increase in platelet number was consistently observed in c-Myc−/− mice. Thus, c-Myc−/− mice develop a syndrome of severe thrombocytosis-anemia-leukopenia because of significant increases in megakaryocytopoiesis and concomitant blockage of erythrocyte differentiation and reductions in myelolymphopoiesis.
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Takafuta, Toshiro, Kingo Fujirmura, Hironori Kawano, Masaaki Noda, Tetsuro Fujimoto, Kenji Oda, Takeshi Shimomura, and Atsushi Kuramoto. "Expression of Platelet Membrane Glycoprotein V in Human Megakaryocytes and Megakaryocytic Cell Lines: A Study Using a Novel Monoclonal Antibody against GPV." Thrombosis and Haemostasis 72, no. 05 (1994): 762–69. http://dx.doi.org/10.1055/s-0038-1648955.
Full textAbstract:
SummaryGlycoprotein V (GPV) is a platelet membrane protein with a molecular weight of 82 kD, and one of the leucine rich glycoproteins (LRG). By reverse transcription-polymerase chain reaction (RT-PCR), GPV cDNA was amplified from mRNA of platelets and megakaryocytic cell lines. However, since there are few reports indicating whether GPV protein is expressed in megakaryocytes as a lineage and maturation specific protein, we studied the GPV expression at the protein level by using a novel monoclonal antibody (1D9) recognizing GPV. Flow cytometric and immunohistochemical analysis indicated that GPV was detected on the surface and in the cytoplasm of only the megakaryocytes in bone marrow aspirates. In a megakaryocytic cell line UT-7, GPV antigen increased after treatment with phorbol-12-myri-state-13-acetate (PMA). These data indicate that only megakaryocytes specifically express the GPV protein among hematopoietic cells and that the expression of GPV increases with differentiation of the megakaryocyte as GPIb-IX complex.
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Banu, N., D. J. Price, R. London, B. Deng, M. Mark, P. J. Godowski, and H. Avraham. "Modulation of megakaryocytopoiesis by human macrophage-stimulating protein, the ligand for the RON receptor." Journal of Immunology 156, no. 8 (April 15, 1996): 2933–40. http://dx.doi.org/10.4049/jimmunol.156.8.2933.
Full textAbstract:
Abstract We observed that human megakaryocytes expressed the heterodimeric tyrosine kinase RON, which serves as a receptor for macrophage-stimulating protein (MSP). MSP appears to be structurally related to hepatocyte growth factor (HGF), which is a pleiotropic growth factor for a broad spectrum of tissues and cell types. The effects of human rMSP and rHGF on permanent human megakaryocytic cell lines as well as on human and murine primary marrow megakaryocytes were studied. MSP enhanced the maturation of the primary bone marrow megakaryocytes and human megakaryocytic cell lines, CMK and DAMI, as assessed by an increase in ploidy content. The increase in ploidy was blocked by specific Abs for MSP and by anti-IL-6 Abs. MSP treatment of primary human marrow megakaryocytes, DAMI cells, or CMK cells resulted in enhanced secretion of IL-6. The addition of MSP to cultures of immature murine megakaryoblasts showed a significant growth response, similar to that of exogenous IL-6. This increased growth of immature murine megakaryoblasts in response to MSP was abrogated either by Abs against MSP or by neutralizing mAbs to IL-6. HGF, over a range of concentrations (10 to 100 ng/ml) alone or in combination with IL-3, granulocyte-macrophage-CSF, or IL-6, had no effect on differentiation of human or murine marrow megakaryocytes. These results indicate that megakaryocytes express a novel tyrosine kinase receptor (RON), and that its ligand, MSP, appears capable of regulating megakaryocyte maturation, possibly via an autocrine mechanism mediated by induction of the cytokine IL-6.
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Davizon-Castillo, Pavel, Kenneth L. Jones, George D. Trahan, and Jorge Di Paola. "Single-Cell RNA-Seq Analysis of Native Murine Megakaryocytes from Young and Old Mice Reveals Significant Metabolic and Mitochondrial Differences throughout Megakaryocyte Development." Blood 132, Supplement 1 (November 29, 2018): 1286. http://dx.doi.org/10.1182/blood-2018-99-120006.
Full textAbstract:
Abstract Traditional transcriptome analysis of megakaryocytes relies on ex-vivo culture and expansion of hematopoietic cells in thrombopoietin-rich media for several days prior to RNA isolation for bulk RNA-seq analysis. This approach has been widely used, however, it is not clear as to what extend this ex vivo expansion of megakaryocytes represents the transcriptome landscape of megakaryocytes at the time of harvest from the bone marrow. In order to get a more detailed megakaryocytic transcriptome landscape immediately after bone marrow harvest, we have optimized a method to perform single cell RNA-seq analysis of native freshly isolated bone marrow megakaryocytes. This was accomplished throughout a series of steps to enrich murine bone marrow for megakaryocytes followed by cell capture using the 10X Genomics platform. In order to assess the transcriptome of native murine megakaryocytes and the effect of age on transcriptional signatures we performed single cell RNA seq analysis of 3 young mice (age 2-3 months old) and 3 old mice (>18 months old). Bioinformatics analyses identified seven transcriptionally different clusters of cells that represent the megakaryocyte ploidy status (Figures 1 and 2). Within these seven regions, 3 of them appear to represent late states of maturation (regions 5, 6 and 7) and possibly, the pro-platelet forming groups of cells due to the elevated expression of megakaryocyte transcripts such as Vwf, Pf4, Itga2b, Itgb3, Gata-1 and Mpl. Ingenuity pathway analysis (IPA) shows that the top three differentially regulated pathways between megakaryocytes from young and old mice are: a) protein ubiquitination; b) mitochondrial dysfunction and; c) oxidative phosphorylation. Furthermore, we validated the RNA-seq analysis at the protein level by measuring ALDHA1 from platelet lysates, the top differentially expressed transcript between groups. Finally, platelets from old mice have a distinctive mitochondrial phenotype characterized by elevated mitochondrial mass and significantly elevated oxygen consumption upon activation by thrombin, features that might be directly contributing to platelet hyperreactivity of aging. In summary, we present a novel methodology to study the transcriptional profile of native megakaryocytes. This initial approach highlights that metabolic and mitochondrial pathways appear to be important modulators of megakaryocyte and platelet development and function. Disclosures No relevant conflicts of interest to declare.
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Mei, RL, and SA Burstein. "Megakaryocytic maturation in murine long-term bone marrow culture: role of interleukin-6." Blood 78, no. 6 (September 15, 1991): 1438–47. http://dx.doi.org/10.1182/blood.v78.6.1438.1438.
Full textAbstract:
Abstract Megakaryocytic maturation was analyzed in long-term bone marrow cultures in the absence of added growth factors. Megakaryocytes could be observed for periods of up to 13 weeks in both the supernatant and stromal layer of these cultures. Using acetylcholinesterase staining for enumeration and sizing of megakaryocytes, and a novel rat antimurine platelet monoclonal antibody (MoAb) that detects only megakaryocytes in bone marrow, the number, volume, and ploidy of these cells were assessed microscopically and by flow cytometry. Correlation of these measurements with ambient interleukin-6 (IL-6) levels showed no relationship between IL-6 bioactivity and megakaryocyte number. Conversely, the relatively high IL-6 bioactivity present during the first 2 weeks of culture was correlated with increased megakaryocytic size and ploidy, while the relatively lower IL-6 bioactivity present after week 3 corresponded to decreased megakaryocytic size and ploidy. Addition of neutralizing anti-IL-6 MoAb decreased megakaryocytic size and ploidy at times when ambient IL-6 levels were relatively high, while the addition of exogenous IL-6 increased size and ploidy at times when endogenous IL-6 concentrations were low. The data show that long- term bone marrow cultures can be used as a means to evaluate megakaryocytic maturation in vitro, and suggest that, to some extent, IL-6 plays a role in the maturation process in this system.
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Mei, RL, and SA Burstein. "Megakaryocytic maturation in murine long-term bone marrow culture: role of interleukin-6." Blood 78, no. 6 (September 15, 1991): 1438–47. http://dx.doi.org/10.1182/blood.v78.6.1438.bloodjournal7861438.
Full textAbstract:
Megakaryocytic maturation was analyzed in long-term bone marrow cultures in the absence of added growth factors. Megakaryocytes could be observed for periods of up to 13 weeks in both the supernatant and stromal layer of these cultures. Using acetylcholinesterase staining for enumeration and sizing of megakaryocytes, and a novel rat antimurine platelet monoclonal antibody (MoAb) that detects only megakaryocytes in bone marrow, the number, volume, and ploidy of these cells were assessed microscopically and by flow cytometry. Correlation of these measurements with ambient interleukin-6 (IL-6) levels showed no relationship between IL-6 bioactivity and megakaryocyte number. Conversely, the relatively high IL-6 bioactivity present during the first 2 weeks of culture was correlated with increased megakaryocytic size and ploidy, while the relatively lower IL-6 bioactivity present after week 3 corresponded to decreased megakaryocytic size and ploidy. Addition of neutralizing anti-IL-6 MoAb decreased megakaryocytic size and ploidy at times when ambient IL-6 levels were relatively high, while the addition of exogenous IL-6 increased size and ploidy at times when endogenous IL-6 concentrations were low. The data show that long- term bone marrow cultures can be used as a means to evaluate megakaryocytic maturation in vitro, and suggest that, to some extent, IL-6 plays a role in the maturation process in this system.
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Jackson, CW, SA Steward, NK Hutson, and RA Ashmun. "Interaction of ristocetin and bovine plasma with guinea pig megakaryocytes: a means to enrich megakaryocytes based on membrane rather than physical characteristics." Blood 69, no. 1 (January 1, 1987): 173–79. http://dx.doi.org/10.1182/blood.v69.1.173.173.
Full textAbstract:
Abstract We have investigated whether megakaryocytes can be aggregated by ristocetin and bovine plasma and whether such aggregation can be used as a step in the purification of megakaryocytes from marrow cell suspensions. Guinea pig marrow cell suspensions were first enriched for megakaryocytes by density equilibrium centrifugation in continuous Percoll density gradients. The megakaryocyte-enriched marrow was stirred in a platelet aggregometer to which ristocetin or bovine plasma was added. Megakaryocytes were aggregated by both ristocetin and bovine plasma with the proportion aggregated being related to the concentration of ristocetin or bovine plasma. Maximal aggregation (greater than 90% of megakaryocytes) was achieved with 2.0 mg/mL ristocetin or 5% bovine plasma and required five minutes. All maturation stages of morphologically recognizable megakaryocytes were aggregated. The megakaryocyte aggregates were separated from the marrow suspension by sedimentation at 1 g and the megakaryocytes disaggregated by dilution with media (ristocetin aggregated) or addition of dextran sulfate (bovine plasma aggregated). Megakaryocyte purity and recovery were higher with bovine plasma than with ristocetin. A mean of 92% of the megakaryocytes in the bovine plasma aggregated cell suspensions were recovered with megakaryocytes constituting an average of 76% of the final cell suspensions. The viability as well as the diameters and DNA content distribution of these megakaryocytes were similar to those of the starting population. We conclude that guinea pig megakaryocytes behave like platelets in that they can be aggregated with ristocetin or bovine plasma and that megakaryocyte aggregation induced by ristocetin or bovine plasma provides a means to enrich these cells based on membrane rather than physical characteristics. This approach yields purified megakaryocyte populations that are representative of those in unfractionated marrow.
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Jackson, CW, SA Steward, NK Hutson, and RA Ashmun. "Interaction of ristocetin and bovine plasma with guinea pig megakaryocytes: a means to enrich megakaryocytes based on membrane rather than physical characteristics." Blood 69, no. 1 (January 1, 1987): 173–79. http://dx.doi.org/10.1182/blood.v69.1.173.bloodjournal691173.
Full textAbstract:
We have investigated whether megakaryocytes can be aggregated by ristocetin and bovine plasma and whether such aggregation can be used as a step in the purification of megakaryocytes from marrow cell suspensions. Guinea pig marrow cell suspensions were first enriched for megakaryocytes by density equilibrium centrifugation in continuous Percoll density gradients. The megakaryocyte-enriched marrow was stirred in a platelet aggregometer to which ristocetin or bovine plasma was added. Megakaryocytes were aggregated by both ristocetin and bovine plasma with the proportion aggregated being related to the concentration of ristocetin or bovine plasma. Maximal aggregation (greater than 90% of megakaryocytes) was achieved with 2.0 mg/mL ristocetin or 5% bovine plasma and required five minutes. All maturation stages of morphologically recognizable megakaryocytes were aggregated. The megakaryocyte aggregates were separated from the marrow suspension by sedimentation at 1 g and the megakaryocytes disaggregated by dilution with media (ristocetin aggregated) or addition of dextran sulfate (bovine plasma aggregated). Megakaryocyte purity and recovery were higher with bovine plasma than with ristocetin. A mean of 92% of the megakaryocytes in the bovine plasma aggregated cell suspensions were recovered with megakaryocytes constituting an average of 76% of the final cell suspensions. The viability as well as the diameters and DNA content distribution of these megakaryocytes were similar to those of the starting population. We conclude that guinea pig megakaryocytes behave like platelets in that they can be aggregated with ristocetin or bovine plasma and that megakaryocyte aggregation induced by ristocetin or bovine plasma provides a means to enrich these cells based on membrane rather than physical characteristics. This approach yields purified megakaryocyte populations that are representative of those in unfractionated marrow.
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Deng, Bijia, Naheed Banu, Beth Malloy, Philip Hass, Jian Feng Wang, Lisa Cavacini, Dan Eaton, and Hava Avraham. "An Agonist Murine Monoclonal Antibody to the Human c-Mpl Receptor Stimulates Megakaryocytopoiesis." Blood 92, no. 6 (September 15, 1998): 1981–88. http://dx.doi.org/10.1182/blood.v92.6.1981.
Full textAbstract:
Abstract Thrombopoietin (TPO) is a hematopoietic growth factor that stimulates megakaryocytopoiesis and platelet production in vivo and promotes the development of identifiable megakaryocytes in vitro. We have developed a murine monoclonal antibody, BAH-1, raised against human megakaryocytic cells, which specifically recognizes the c-Mpl receptor and shows agonist activity by stimulating megakaryocytopoiesis in vitro. BAH-1 antibody specifically binds to platelets and to recombinant c-Mpl with high affinity. Similar to TPO, BAH-1 alone supported the formation of colony-forming unit-megakaryocyte (CFU-MK) colonies. The combination of BAH-1 plus interleukin-3 or of BAH-1 plus human TPO significantly increased the number of human CFU-MK colonies. In addition, BAH-1 monoclonal antibody stimulated the proliferation and maturation of primary bone marrow megakaryocytes in a dynamic heterogeneous liquid culture system. Individual large megakaryocytes as well as small megakaryocytic cells were observed in cultures of CD34+ CD41+cells in the presence of BAH-1 antibodies. Similar to TPO, BAH-1 antibody induced a significant response of murine immature megakaryocytes as observed by an increase in the detectable numbers of acetylcholinesterase-positive megakaryocytes. No effects of BAH-1 antibody were observed on colony-forming unit–granulocyte-macrophage, burst-forming unit-erythroid, or colony-forming unit-erythroid colonies. In vivo studies showed that BAH-1, alone or in combination with TPO, expands the numbers of megakaryocytic progenitor cells in myelosuppressed mice. This antibody should prove useful in understanding the structure-function aspects of the c-Mpl receptor as well as in evaluating the effects of the sustained activation of this receptor in preclinical models of severe thrombocytopenia. © 1998 by The American Society of Hematology.
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Deng, Bijia, Naheed Banu, Beth Malloy, Philip Hass, Jian Feng Wang, Lisa Cavacini, Dan Eaton, and Hava Avraham. "An Agonist Murine Monoclonal Antibody to the Human c-Mpl Receptor Stimulates Megakaryocytopoiesis." Blood 92, no. 6 (September 15, 1998): 1981–88. http://dx.doi.org/10.1182/blood.v92.6.1981.418k15_1981_1988.
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Thrombopoietin (TPO) is a hematopoietic growth factor that stimulates megakaryocytopoiesis and platelet production in vivo and promotes the development of identifiable megakaryocytes in vitro. We have developed a murine monoclonal antibody, BAH-1, raised against human megakaryocytic cells, which specifically recognizes the c-Mpl receptor and shows agonist activity by stimulating megakaryocytopoiesis in vitro. BAH-1 antibody specifically binds to platelets and to recombinant c-Mpl with high affinity. Similar to TPO, BAH-1 alone supported the formation of colony-forming unit-megakaryocyte (CFU-MK) colonies. The combination of BAH-1 plus interleukin-3 or of BAH-1 plus human TPO significantly increased the number of human CFU-MK colonies. In addition, BAH-1 monoclonal antibody stimulated the proliferation and maturation of primary bone marrow megakaryocytes in a dynamic heterogeneous liquid culture system. Individual large megakaryocytes as well as small megakaryocytic cells were observed in cultures of CD34+ CD41+cells in the presence of BAH-1 antibodies. Similar to TPO, BAH-1 antibody induced a significant response of murine immature megakaryocytes as observed by an increase in the detectable numbers of acetylcholinesterase-positive megakaryocytes. No effects of BAH-1 antibody were observed on colony-forming unit–granulocyte-macrophage, burst-forming unit-erythroid, or colony-forming unit-erythroid colonies. In vivo studies showed that BAH-1, alone or in combination with TPO, expands the numbers of megakaryocytic progenitor cells in myelosuppressed mice. This antibody should prove useful in understanding the structure-function aspects of the c-Mpl receptor as well as in evaluating the effects of the sustained activation of this receptor in preclinical models of severe thrombocytopenia. © 1998 by The American Society of Hematology.
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Wang, Xiaofen, Ying Wang, Chao Qi, Sai Qiao, Suwen Yang, Rongrong Wang, Hong Jin, and Jun Zhang. "The Application of Morphogo in the Detection of Megakaryocytes from Bone Marrow Digital Images with Convolutional Neural Networks." Technology in Cancer Research & Treatment 22 (January 2023): 153303382211500. http://dx.doi.org/10.1177/15330338221150069.
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The evaluation of megakaryocytes is an important part of the work up on bone marrow smear examination. It has significance in the differential diagnosis, therapeutic efficacy assessment, and predication of prognosis of many hematologic diseases. The process of manual identification of megakaryocytes are tedious and lack of reproducibility; therefore, a reliable method of automated megakaryocytic identification is urgently needed. Three hundred and thirty-three bone marrow aspirate smears were digitized by Morphogo system. Pathologists annotated megakaryocytes on the digital images of marrow smears are applied to construct a large dataset for testing the system's predictive performance. Subsequently, we obtained megakaryocyte count and classification for each sample by different methods (system-automated analysis, system-assisted analysis, and microscopic examination) to study the correlation between different counting and classification methods. Morphogo system localized cells likely to be megakaryocytes on digital smears, which were later annotated by pathologists and the system, respectively. The system showed outstanding performance in identifying megakaryocytes in bone marrow smears with high sensitivity (96.57%) and specificity (89.71%). The overall correlation between the different methods was confirmed the high consistency ( r ≥ 0.7218, R2 ≥ 0.5211) with microscopic examination in classifying megakaryocytes. Morphogo system was proved as a reliable screen tool for analyzing megakaryocytes. The application of Morphogo system shows promises to advance the automation and standardization of bone marrow smear examination.
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Kuhl, Christiane, Ann Atzberger, Francisco Iborra, Bernhard Nieswandt, Catherine Porcher, and Paresh Vyas. "GATA1-Mediated Megakaryocyte Differentiation and Growth Control Can Be Uncoupled and Mapped to Different Domains in GATA1." Molecular and Cellular Biology 25, no. 19 (October 1, 2005): 8592–606. http://dx.doi.org/10.1128/mcb.25.19.8592-8606.2005.
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ABSTRACT The DNA-binding hemopoietic zinc finger transcription factor GATA1 promotes terminal megakaryocyte differentiation and restrains abnormal immature megakaryocyte expansion. How GATA1 coordinates these fundamental processes is unclear. Previous studies of synthetic and naturally occurring mutant GATA1 molecules demonstrate that DNA-binding and interaction with the essential GATA1 cofactor FOG-1 (via the N-terminal finger) are required for gene expression in terminally differentiating megakaryocytes and for platelet production. Moreover, acquired mutations deleting the N-terminal 84 amino acids are specifically detected in megakaryocytic leukemia in human Down syndrome patients. In this study, we have systematically dissected GATA1 domains required for platelet release and control of megakaryocyte growth by ectopically expressing modified GATA1 molecules in primary GATA1-deficient fetal megakaryocyte progenitors. In addition to DNA binding, distinct N-terminal regions, including residues in the first 84 amino acids, promote platelet release and restrict megakaryocyte growth. In contrast, abrogation of GATA1-FOG-1 interaction leads to loss of differentiation, but growth of blocked immature megakaryocytes is controlled. Thus, distinct GATA1 domains regulate terminal megakaryocyte gene expression leading to platelet release and restrain megakaryocyte growth, and these processes can be uncoupled.
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Khetawat, Gopal, Nauder Faraday, Michele L. Nealen, K. Vinod Vijayan, Everlie Bolton, Stephen J. Noga, and Paul F. Bray. "Human megakaryocytes and platelets contain the estrogen receptor β and androgen receptor (AR): testosterone regulates AR expression." Blood 95, no. 7 (April 1, 2000): 2289–96. http://dx.doi.org/10.1182/blood.v95.7.2289.
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Abstract Gender differences in vascular thromboses are well known, and there is evidence that platelets may be involved in these differences and that sex hormones affect platelet function. We characterized the expression of the estrogen receptor (ER ), estrogen receptor β (ER β), progesterone receptor (PR), and androgen receptor (AR) in the megakaryocyte lineage. Megakaryocytes generated ex vivo from normal human CD34+ stem cells contained RNA for ER β and AR, which increased with cell differentiation. Platelets and human erythroleukemia (HEL) cells also contained ER β and AR transcripts. No ER or PR messenger RNA or protein was detected in the megakaryocyte lineage. Immunofluorescence microscopy showed that ER β protein was present in glycoprotein (GP) IIb+ megakaryocytes and the HEL megakaryocytic cell line in a predominantly cytoplasmic location. AR showed a cytoplasmic and nuclear distribution in GPIIb+ and GPIIb− cells derived from CD34+ cells and in HEL cells. Western immunoblotting confirmed the presence of ER β and AR in platelets. Megakaryocyte and HEL AR expression was up-regulated by 1, 5, and 10 nmol/L testosterone, but down-regulated by 100 nmol/L testosterone. These findings indicate a regulated ability of megakaryocytes to respond to testosterone and suggest a potential mechanism through which sex hormones may mediate gender differences in platelet function and thrombotic diseases.
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Khetawat, Gopal, Nauder Faraday, Michele L. Nealen, K. Vinod Vijayan, Everlie Bolton, Stephen J. Noga, and Paul F. Bray. "Human megakaryocytes and platelets contain the estrogen receptor β and androgen receptor (AR): testosterone regulates AR expression." Blood 95, no. 7 (April 1, 2000): 2289–96. http://dx.doi.org/10.1182/blood.v95.7.2289.007k03_2289_2296.
Full textAbstract:
Gender differences in vascular thromboses are well known, and there is evidence that platelets may be involved in these differences and that sex hormones affect platelet function. We characterized the expression of the estrogen receptor (ER ), estrogen receptor β (ER β), progesterone receptor (PR), and androgen receptor (AR) in the megakaryocyte lineage. Megakaryocytes generated ex vivo from normal human CD34+ stem cells contained RNA for ER β and AR, which increased with cell differentiation. Platelets and human erythroleukemia (HEL) cells also contained ER β and AR transcripts. No ER or PR messenger RNA or protein was detected in the megakaryocyte lineage. Immunofluorescence microscopy showed that ER β protein was present in glycoprotein (GP) IIb+ megakaryocytes and the HEL megakaryocytic cell line in a predominantly cytoplasmic location. AR showed a cytoplasmic and nuclear distribution in GPIIb+ and GPIIb− cells derived from CD34+ cells and in HEL cells. Western immunoblotting confirmed the presence of ER β and AR in platelets. Megakaryocyte and HEL AR expression was up-regulated by 1, 5, and 10 nmol/L testosterone, but down-regulated by 100 nmol/L testosterone. These findings indicate a regulated ability of megakaryocytes to respond to testosterone and suggest a potential mechanism through which sex hormones may mediate gender differences in platelet function and thrombotic diseases.
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Halene, Stephanie, Yuan Gao, Katherine Hahn, Stephanie Massaro, Joseph E. Italiano, Vincent Schulz, Sharon Lin, Gary M. Kupfer, and Diane S. Krause. "Serum response factor is an essential transcription factor in megakaryocytic maturation." Blood 116, no. 11 (September 16, 2010): 1942–50. http://dx.doi.org/10.1182/blood-2010-01-261743.
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Abstract Serum response factor (Srf) is a MADS–box transcription factor that is critical for muscle differentiation. Its function in hematopoiesis has not yet been revealed. Mkl1, a cofactor of Srf, is part of the t(1;22) translocation in acute megakaryoblastic leukemia, and plays a critical role in megakaryopoiesis. To test the role of Srf in megakaryocyte development, we crossed Pf4-Cre mice, which express Cre recombinase in cells committed to the megakaryocytic lineage, to SrfF/F mice in which functional Srf is no longer expressed after Cre-mediated excision. Pf4-Cre/SrfF/F knockout (KO) mice are born with normal Mendelian frequency, but have significant macrothrombocytopenia with approximately 50% reduction in platelet count. In contrast, the BM has increased number and percentage of CD41+ megakaryocytes (WT: 0.41% ± 0.06%; KO: 1.92% ± 0.12%) with significantly reduced ploidy. KO mice show significantly increased megakaryocyte progenitors in the BM by FACS analysis and CFU-Mk. Megakaryocytes lacking Srf have abnormal stress fiber and demarcation membrane formation, and platelets lacking Srf have abnormal actin distribution. In vitro and in vivo assays reveal platelet function defects in KO mice. Critical actin cytoskeletal genes are down-regulated in KO megakaryocytes. Thus, Srf is required for normal megakaryocyte maturation and platelet production partly because of regulation of cytoskeletal genes.
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Heazlewood, Shen Y., Tanveer Ahmad, Monika Mohenska, Belinda B. Guo, Pradnya Gangatirkar, Emma C. Josefsson, Sarah L. Ellis, et al. "The RNA-binding protein SRSF3 has an essential role in megakaryocyte maturation and platelet production." Blood 139, no. 9 (March 3, 2022): 1359–73. http://dx.doi.org/10.1182/blood.2021013826.
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Abstract RNA processing is increasingly recognized as a critical control point in the regulation of different hematopoietic lineages including megakaryocytes responsible for the production of platelets. Platelets are anucleate cytoplasts that contain a rich repertoire of RNAs encoding proteins with essential platelet functions derived from the parent megakaryocyte. It is largely unknown how RNA binding proteins contribute to the development and functions of megakaryocytes and platelets. We show that serine-arginine–rich splicing factor 3 (SRSF3) is essential for megakaryocyte maturation and generation of functional platelets. Megakaryocyte-specific deletion of Srsf3 in mice led to macrothrombocytopenia characterized by megakaryocyte maturation arrest, dramatically reduced platelet counts, and abnormally large functionally compromised platelets. SRSF3 deficient megakaryocytes failed to reprogram their transcriptome during maturation and to load platelets with RNAs required for normal platelet function. SRSF3 depletion led to nuclear accumulation of megakaryocyte mRNAs, demonstrating that SRSF3 deploys similar RNA regulatory mechanisms in megakaryocytes as in other cell types. Our study further suggests that SRSF3 plays a role in sorting cytoplasmic megakaryocyte RNAs into platelets and demonstrates how SRSF3-mediated RNA processing forms a central part of megakaryocyte gene regulation. Understanding SRSF3 functions in megakaryocytes and platelets provides key insights into normal thrombopoiesis and platelet pathologies as SRSF3 RNA targets in megakaryocytes are associated with platelet diseases.
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Yang, Zhong-Fa, Timothy M. Chlon, John Crispino, and Alan G. Rosmarin. "Gabp Transcription Factor Is Required for the Development of Mouse Megakaryocytes." Blood 116, no. 21 (November 19, 2010): 2604. http://dx.doi.org/10.1182/blood.v116.21.2604.2604.
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Abstract Abstract 2604 GABP transcription factor has been implicated in the regulation of genes that are required for normal megakaryocytic differentiation. Megakaryocytes express several related ets factors, including Fli-1, ets2, and GABP, and it has been unclear if any single ets factor plays a non-redundant role in these cells. The tetrameric GABP transcription factor complex contains two molecules of GABPα, which binds DNA, and two molecules of GABPβ, which encodes the transcription activation domain. We created mice with loxP recombination sites which flank exons that encode Gabpa ets-related DNA-binding domains (floxed Gabpa, or Gabpa fl/fl), and bred them to mice that carry Mx1-Cre. In response to injection with the synthetic polynucleotide, pIC, these mice express Cre recombinase and efficiently delete Gabpa; these animals are referred to as knock-out (KO) mice. Control mice, which carry floxed Gabpa but lack Mx1-Cre, were treated identically with pIC. Platelet counts of KO mice declined to less than 50,000 within nine days, while platelet counts in control mice were unaffected. One half of KO mice died within two weeks of Gabpa deletion due to widespread visceral hemorrhage. Histologic examination of the bone marrow and spleen reveals a loss of megakaryocytes in KO mice, compared to control animals. Residual megakaryocytes in KO mice exhibit increased expression of platelet-specific antigens, CD41 and CD42, and a significant increase of DNA ploidy. Because Gabpa KO mice died with a striking loss of megakaryocytes and platelets, yet megakaryocytic differentiation appeared to be unimpaired, we sought to better define the nature of this defect. Bone marrow from Gabpa fl/fl mice was infected with a retrovirus that expresses Cre and green fluorescent protein (GFP), or control virus that expresses only GFP; grown for three days in liquid culture conditions that foster megakaryocytic differentiation; and analyzed for CD41 and CD42 expression, ploidy, and apoptosis. Gabpa was efficiently deleted by the Cre-bearing virus, and Gabpa deletion was associated with increased expression of CD41 and CD42, and increased DNA ploidy. However, Gabpa deletion was also associated with increased megakaryocytic-associated apoptosis, and in vitro megakaryocyte colony formation was dramatically reduced in Gabpα null cells. In summary, deletion of Gabpa in mice is associated with plummeting platelet counts, widespread visceral hemorrhage, and a loss of splenic and bone marrow megakaryocytes. In vitro analysis demonstrates intact megakaryocytic differentiation and a profound loss of megakaryocytic progenitor cells. The increased expression of megakaryocytic antigens and DNA ploidy may indicate that Gabpa deletion enhances megakaryocytic differentiation or, alternatively, it may represent selective loss of more immature megakaryocytic cells following Gabpa disruption. Data that directly test these alternative hypotheses will be presented. In summary, we demonstrate that GABP plays a non-redundant role in megakaryocyte development, that GABP is required for the proliferation of committed megakaryocytic progenitors, but that GABP is not required for the later stages of megakaryocytic maturation. Disclosures: No relevant conflicts of interest to declare.
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Geddis, Amy E. "Ontology of Megakaryopoesis." Blood 114, no. 22 (November 20, 2009): SCI—8—SCI—8. http://dx.doi.org/10.1182/blood.v114.22.sci-8.sci-8.
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Abstract Abstract SCI-8 Compared to red cells, whose passage from embryonic to adult stages is marked by the expression of distinct forms of hemoglobin, the development of megakaryocytes during embryogenesis is less well understood. However, certain shared characteristics between megakaryocytes, endothelial cells, hematopoietic stem cells and erythrocytes infer developmental relationships between these lineages. Recent data support the model that hematopoietic stem cells derive from the hemangioblast, and that megakaryocytes and erythrocytes develop from a common precursor both in primitive and adult hematopoiesis. Evidence of these common origins can be found in the genetic programs that are activated during hematopoiesis, in that many of the cell surface markers and transcriptions factors that are characteristic of megakaryocytes can also be found in endothelial cells, stem cells and erythrocytes. In this session I will review current views on developmental thrombopoiesis, key megakaryocytic transcription factors and the experimental and clinical phenotypes associated with their disruption, and current controversies in lineage choice during megakaryocyte differentiation. Disclosures Geddis: Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding.
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Baccini, Véronique, Lydia Roy, Natacha Vitrat, Hédia Chagraoui, Siham Sabri, Jean-Pierre Le Couedic, Najet Debili, Françoise Wendling, and William Vainchenker. "Role of p21Cip1/Waf1 in cell-cycle exit of endomitotic megakaryocytes." Blood 98, no. 12 (December 1, 2001): 3274–82. http://dx.doi.org/10.1182/blood.v98.12.3274.
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Abstract The cyclin-dependent kinase inhibitor p21Waf-1/Cip-1 is expressed at high level during megakaryocyte differentiation, but its precise function remains unknown. In this study, it is confirmed that p21 was expressed at a high level in hypoploid (2N and 4N) and polyploid (at least 8N) human megakaryocytes derived from CD34+ cells. A high expression of p27Kip1, p16, cyclin E, and cyclin D3 was also found in both populations associated with a hypophosphorylated form of retinoblastoma protein, suggesting that the majority of hypoploid and polyploid megakaryocytes are G1-arrested cells. As human megakaryocytes grown in vitro present a defect in their polyploidization, the study switched to the murine model. The modal ploidy of megakaryocytes derived from lineage-negative cells was 32N, and an elevated expression of p21 was found in high-ploidy megakaryocytes. In addition, p21 and p27 were coexpressed in the majority of mature polyploid megakaryocytes. The p21 was detected by immunofluorescence in megakaryocytes derived from p53−/− mice, demonstrating a p53-independent regulation during megakaryocyte differentiation. Megakaryocytopoiesis of p21−/− mice was subsequently studied. No marked abnormality in the ploidy of primary or cultured megakaryocytes was detected. Overexpression of p21 in p21−/− or normal murine megakaryocytes and in human megakaryocytes showed in all these cases a marked inhibition in megakaryocyte polyploidization. In conclusion, while a reciprocal relation is observed between p21 levels in megakaryocytes and the cycling state of the cells, p21 is not essential for the determination of the ploidy profile in normal megakaryocytes in vivo. However, high levels of its expression in cultured megakaryocytes arrest the endomitotic cell cycle.
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Ishida, Yoji, Toshiharu Ito, and Shin-ichiro Kuriya. "Effects of C-Mpl Ligand on Cytoplasmic Maturation of Murine Megakaryocytes and on Platelet Production." Journal of Histochemistry & Cytochemistry 46, no. 1 (January 1998): 49–57. http://dx.doi.org/10.1177/002215549804600107.
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To test the hypothesis that the c-mpl ligand is not a primary factor in thromb-ocytopoiesis, we investigated the biological effects of recombinant human (rh) c-mpl ligand on differentiation of murine progenitor cells and on maturation of the cultured murine megakaryocytes under serum-free conditions on the basis of ploidy distribution, megakaryocyte/platelet-specific surface antigen CD 61 [glycoprotein (GP) IIIa], and cytoplasmic acetylcholinesterase (AchE) expression in vitro. In addition, we studied the effect of c-mpl ligand on proplatelet formation (PPF) by murine mature megakaryocytes. AchE was less strongly expressed in cultured megakaryocytic cells stimulated by c-mpl ligand than in those stimulated by recombinant murine (rm) IL-3 + rh IL-6 during the differentiation of progenitor cells. Less CD 61 was expressed by c-mpl ligand during both the differentiation of progenitor cells and the maturation of megakaryocytes compared with that by rm IL-3 + rh IL-6. Endomitosis, however, was more stimulated by c-mpl ligand than by rm IL-3 + rh IL-6 under both conditions. Furthermore, PPF of mature megakaryocytes was not stimulated by c-mpl ligand. These results indicate that c-mpl ligand stimulates the nuclear development of megakaryocytic cells but that it does not stimulate cytoplasmic maturation and PPF as much as IL-6. These data strongly suggest that c-mpl ligand is not a primary factor in platelet production.
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Berthebaud, Magali, Christel Rivière, Peggy Jarrier, Adlen Foudi, Yanyan Zhang, Daniel Compagno, Anne Galy, William Vainchenker, and Fawzia Louache. "RGS16 is a negative regulator of SDF-1–CXCR4 signaling in megakaryocytes." Blood 106, no. 9 (November 1, 2005): 2962–68. http://dx.doi.org/10.1182/blood-2005-02-0526.
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AbstractRegulators of G-protein signaling (RGS) constitute a family of proteins involved in the negative regulation of signaling through heterotrimeric G protein–coupled receptors (GPCRs). Several RGS proteins have been implicated in the down-regulation of chemokine signaling in hematopoietic cells. The chemokine stromal-cell–derived factor 1 (SDF-1) activates migration of hematopoietic progenitors cells but fails to activate mature megakaryocytes despite high levels of CXC chemokine receptor 4 (CXCR4) receptor expression in these cells. This prompted us to analyze RGS expression and function during megakaryocyte differentiation. We found that RGS16 and RGS18 mRNA expression was up-regulated during this process. Overexpressing RGS16 mRNA in the megakaryocytic MO7e cell line inhibited SDF-1–induced migration, mitogen-activated protein kinase (MAPK) and protein kinase B (AKT) activation, whereas RGS18 overexpression had no effect on CXCR4 signaling. Knocking down RGS16 mRNA via lentiviral-mediated RNA interference increased CXCR4 signaling in MO7e cells and in primary megakaryocytes. Thus, our data reveal that RGS16 is a negative regulator of CXCR4 signaling in megakaryocytes. We postulate that RGS16 regulation is a mechanism that controls megakaryocyte maturation by regulating signals from the microenvironment.
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Tanaka, R., K. Koike, T. Imai, M. Shiohara, T. Kubo, Y. Amano, A. Komiyama, and T. Nakahata. "Stem cell factor enhances proliferation, but not maturation, of murine megakaryocytic progenitors in serum-free culture." Blood 80, no. 7 (October 1, 1992): 1743–49. http://dx.doi.org/10.1182/blood.v80.7.1743.1743.
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Abstract The effects of recombinant rat stem cell factor (SCF/c-kit ligand) on murine megakaryocytopoiesis were studied using partially purified bone marrow cells derived from normal and 5-fluorouracil (5-FU)-treated mice in a serum-free culture system. SCF alone did not support the formation of megakaryocyte (M) and granulocyte-macrophage-megakaryocyte (GMM) colonies. However, the addition of SCF to cultures containing interleukin-3 (IL-3) resulted in a significant increase in the number of M and GMM colonies formed by bone marrow cells from normal mice, whereas IL-6 augmented only M colony growth. The stimulatory effect of SCF was approximately three to four times as high as that of IL-6 on the primitive progenitors capable of megakaryocytic-lineage expression derived from 5-FU-treated mice. In addition, SCF, but not IL-6, significantly increased the number of constituent cells in the individual M colonies supported by IL-3. On the other hand, SCF did not exert any effect on the size and DNA content of megakaryocytes in IL-3- dependent M and GMM colonies, whereas IL-6 enhanced the maturation of megakaryocytes. These results suggest that SCF stimulates the proliferative process in megakaryocytic progenitors and that the main activity of IL-6 is the promotion of megakaryocyte maturation.
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Tanaka, R., K. Koike, T. Imai, M. Shiohara, T. Kubo, Y. Amano, A. Komiyama, and T. Nakahata. "Stem cell factor enhances proliferation, but not maturation, of murine megakaryocytic progenitors in serum-free culture." Blood 80, no. 7 (October 1, 1992): 1743–49. http://dx.doi.org/10.1182/blood.v80.7.1743.bloodjournal8071743.
Full textAbstract:
The effects of recombinant rat stem cell factor (SCF/c-kit ligand) on murine megakaryocytopoiesis were studied using partially purified bone marrow cells derived from normal and 5-fluorouracil (5-FU)-treated mice in a serum-free culture system. SCF alone did not support the formation of megakaryocyte (M) and granulocyte-macrophage-megakaryocyte (GMM) colonies. However, the addition of SCF to cultures containing interleukin-3 (IL-3) resulted in a significant increase in the number of M and GMM colonies formed by bone marrow cells from normal mice, whereas IL-6 augmented only M colony growth. The stimulatory effect of SCF was approximately three to four times as high as that of IL-6 on the primitive progenitors capable of megakaryocytic-lineage expression derived from 5-FU-treated mice. In addition, SCF, but not IL-6, significantly increased the number of constituent cells in the individual M colonies supported by IL-3. On the other hand, SCF did not exert any effect on the size and DNA content of megakaryocytes in IL-3- dependent M and GMM colonies, whereas IL-6 enhanced the maturation of megakaryocytes. These results suggest that SCF stimulates the proliferative process in megakaryocytic progenitors and that the main activity of IL-6 is the promotion of megakaryocyte maturation.
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Iacovino, Michelina, Mitsujiro Osawa, Zhaohui Xu, Melissa G. Prather, and Michael Kyba. "HoxA2 Regulates Proliferation of an Embryonic Megakaryocyte Progenitor, Which Can Effectively Produce Platelets In Vitro." Blood 110, no. 11 (November 16, 2007): 1266. http://dx.doi.org/10.1182/blood.v110.11.1266.1266.
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Abstract Megakaryocytes are rare cells in the bone marrow, and are solely responsible for thrombopoiesis throughout the life of the organism. Various transcription factors regulating megakaryopoiesis have been identified, including GATA1, Fli1 and NF-E2, which are important for the maturation and differentiation of megakaryocytes and SCL and FOG1, which play important roles in the specification and proliferation of the early megakaryocyte progenitor but no single factor capable of sustaining long-term proliferation of the megakaryocyte progenitor has been identified. The rarity of this progenitor, and the difficulty of proliferating megakaryocytes in vitro have seriously hampered the effort to produce large quantities platelets in vitro, and to date the only source of platelets for transplantation remains human donors. In this study, through a gain of function screen of Hox genes in which each paralog group was tested, we identified HoxA2 as an important transcription factor for the proliferation of early embryonic megakaryocytic progenitors. HoxA2 promoted the outgrowth of undifferentiated megakaryocyte progenitors when transduced into precirculation yolk sac stem and progenitor cells, however it had no effect on megakaryocytic progenitors derived from definitive HSCs of adult bone marrow. To study this unique embryonic regulatory circuit further, we generated an inducible mouse embryonic stem cell line, in which the expression of murine HoxA2 is regulated by doxycycline. Embryoid body differentiation of these cells, followed by sorting and reculture of the Kit+/CD41+ hematopoietic progenitor fraction at day 6 in the presence of continual HoxA2 expression allowed undifferentiated c-Kit+/CD41+/CD45+ megakaryocyte progenitors to proliferate for upwards of 2 months. When doxycycline is removed, numerous gene expression changes take plase, and these progenitors differentiate into mature c-Kitneg/CD41high/CD45neg megakaryocytes, which undergo proplatelet formation, releasing large quantities of platelets (∼107/ml) into the medium. As this rate of production can be sustained for weeks, a highly efficient platelet bioreactor is enabled. The platelets express the integrin αIIb-β3 (GPIIb-IIIa complex), and upon stimulation they bind fibrinogen, demonstrating functionality. We are currently investigating the transplantation potential of these cells.Our data suggest that adult and embryonic megakaryopoiesis have distinct regulatory circuits, and demonstrate a novel and uniquely embryonic role for HoxA2, which opens up the possibility of producing industrial quantities of platelets in vitro for therapeutic purposes.
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Arnold, Julie T., Najat C. Daw, Paula E. Stenberg, Deepthi Jayawardene, Deo Kumar Srivastava, and Carl W. Jackson. "A Single Injection of Pegylated Murine Megakaryocyte Growth and Development Factor (MGDF ) Into Mice Is Sufficient to Produce a Profound Stimulation of Megakaryocyte Frequency, Size, and Ploidization." Blood 89, no. 3 (February 1, 1997): 823–33. http://dx.doi.org/10.1182/blood.v89.3.823.
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Abstract Despite numerous studies investigating the action of c-mpl ligand, no reports have defined the in vivo changes in megakaryocytopoiesis in response to a single injection of this cytokine. Here we compare the kinetics of the megakaryocytopoietic response in C57Bl/6J mice administered 25 μg/kg or 250 μg/kg of pegylated (PEG) murine megakaryocyte growth and development factor (MGDF ) as a single intravenous injection. Megakaryocytes of mice treated with MGDF had normal ultrastructure, showing a typical distribution of the demarcation membrane system, α-granules, and other cytoplasmic organelles. Megakaryocyte ploidy, size, and frequency were markedly increased with both MGDF doses. Megakaryocyte ploidy was maximally increased from a modal value of 16N to 64N on day 3, with both doses of MGDF. Similarly, a comparable increase in megakaryocyte size occurred in the two MGDF groups. Increased megakaryocyte size was coupled to the increase in megakaryocyte ploidy, and no evidence for independent regulation of megakaryocyte size within individual ploidy classes was apparent. In contrast to megakaryocyte ploidy and size, the increase in megakaryocyte frequency was markedly different with the two doses of MGDF. The proportion of 2N and 4N cells was increased from a baseline of 0.035% to 0.430% by day 4 in mice treated with the higher dose of MGDF, but only to 0.175% in mice administered 25 μg/kg of MGDF. The marked increase in the pool of these immature megakaryocytes translated to a sustained elevation in the frequency of polyploid megakaryocytes (8N cells and greater). In contrast to the sustained increase in the frequency of polyploid cells, the level of polyploidization was downregulated on days 6 to 10, but normalized by day 14. We conclude that a single injection of MGDF is able to expand the megakaryocytic pool in a dose-dependent manner, which, with subsequent maturation, should lead to an increased rate of platelet production.
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Suehiro, Youko, Dragoslava Kika Veljkovic, Nola Fuller, Yasuaki Motomura, Jean Marc Massé, Elisabeth M. Cramer, and Catherine P. M. Hayward. "Endocytosis and storage of plasma factor V by human megakaryocytes." Thrombosis and Haemostasis 94, no. 09 (2005): 585–92. http://dx.doi.org/10.1160/th05-02-0141.
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SummaryFactor V is an essential coagulation cofactor that circulates in plasma and platelet α-granules where it is stored complexed to multimerin 1 (MMRN1). To gain insights into the origin and processing of human platelet factor V, and factor V-MMRN1 complexes, we studied factor V in cultured megakaryocytes. Factor V mRNA was detected in all megakaryocyte cultures. However, like albumin, IgG and fibrinogen, factorV protein was detectable only in megakaryocytes cultured with exogenous protein. The amount of factor V associated with megakaryocytes was influenced by the exogenous factorV concentration. Similar to platelet factor V, megakaryocyte factor V was proteolyzed and complexed with megakaryocyte-synthesized MMRN1. With secretagogues, megakaryocytes released factorV, IgG, fibrinogen and MMRN1. Immunofluorescent and electron microscopy confirmed factorV uptake by endocytosis and its trafficking to megakaryocyte α-granules. These data provide direct evidence that human megakaryocytes process plasma-derived factor V into α-granules and generate factor V-MMRN1 complexes from endogenously and exogenously synthesized proteins.
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Stenberg, PE, JH Beckstead, RP McEver, and J. Levin. "Immunohistochemical localization of membrane and alpha-granule proteins in plastic-embedded mouse bone marrow megakaryocytes and murine megakaryocyte colonies." Blood 68, no. 3 (September 1, 1986): 696–702. http://dx.doi.org/10.1182/blood.v68.3.696.696.
Full textAbstract:
Abstract Using an immunoperoxidase technique that permits optimal antigen localization at the light microscope level, we have detected two platelet alpha-granule constituents and three platelet membrane glycoproteins in mouse bone marrow megakaryocytes and in murine megakaryocyte colonies grown in soft agar culture for three to seven days. Using polyclonal antibodies prepared against human platelet proteins, we have demonstrated labeling for von Willebrand factor, fibrinogen, and the membrane glycoproteins IIIa and GMP-140 in both bone marrow megakaryocytes and megakaryocyte colonies after seven days of culture. Using monoclonal antibodies to membrane glycoproteins IIb and GMP-140, we have demonstrated label in mouse bone marrow megakaryocytes. Granulocyte and macrophage colonies were negative for each of these markers. Murine bone marrow megakaryocytes and megakaryocyte colonies demonstrated a similar enzyme histochemical pattern: weakly positive for alpha-naphthyl acetate esterase and negative for chloroacetate esterase. These data indicate that megakaryocytes grown in soft agar culture express many of the same glycoproteins as bone marrow megakaryocytes. Furthermore, the ability of antibodies directed against human platelet membrane glycoproteins to identify murine megakaryocyte glycoproteins indicates that these constituents have been highly conserved during evolution.
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Stenberg, PE, JH Beckstead, RP McEver, and J. Levin. "Immunohistochemical localization of membrane and alpha-granule proteins in plastic-embedded mouse bone marrow megakaryocytes and murine megakaryocyte colonies." Blood 68, no. 3 (September 1, 1986): 696–702. http://dx.doi.org/10.1182/blood.v68.3.696.bloodjournal683696.
Full textAbstract:
Using an immunoperoxidase technique that permits optimal antigen localization at the light microscope level, we have detected two platelet alpha-granule constituents and three platelet membrane glycoproteins in mouse bone marrow megakaryocytes and in murine megakaryocyte colonies grown in soft agar culture for three to seven days. Using polyclonal antibodies prepared against human platelet proteins, we have demonstrated labeling for von Willebrand factor, fibrinogen, and the membrane glycoproteins IIIa and GMP-140 in both bone marrow megakaryocytes and megakaryocyte colonies after seven days of culture. Using monoclonal antibodies to membrane glycoproteins IIb and GMP-140, we have demonstrated label in mouse bone marrow megakaryocytes. Granulocyte and macrophage colonies were negative for each of these markers. Murine bone marrow megakaryocytes and megakaryocyte colonies demonstrated a similar enzyme histochemical pattern: weakly positive for alpha-naphthyl acetate esterase and negative for chloroacetate esterase. These data indicate that megakaryocytes grown in soft agar culture express many of the same glycoproteins as bone marrow megakaryocytes. Furthermore, the ability of antibodies directed against human platelet membrane glycoproteins to identify murine megakaryocyte glycoproteins indicates that these constituents have been highly conserved during evolution.
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Kozuma, Yukinori, Hiroshi Kojima, Satoshi Yuki, Hidenori Suzuki, and Toshiro Nagasawa. "GATA-1, Transcriptionally, and Thrombopoietin-Mediated Akt Activation, Post-Translationally, Regulate Continuous Expression of Bcl-xL Protein during Megakaryopoiesis." Blood 108, no. 11 (November 16, 2006): 1140. http://dx.doi.org/10.1182/blood.v108.11.1140.1140.
Full textAbstract:
Abstract Thrombopoietin (TPO) plays a relevant role for megakaryocyte differentiation from stem cells. One of the important biological activities of TPO is to prevent the apoptosis of megakaryocytic cells. As an anti-apoptotic protein Bcl-xL, which has been proved to be indispensable for erythroid differentiation, is also abundantly expressed in megakaryocytes, it is assumed that Bcl-xL plays an important role for megakaryopoiesis. We thus investigated the expression of Bcl-xL during megakaryopoiesis and the underlying regulatory mechanism. In stem cell-derived megakaryocytes, expression of Bcl-xL increased in the early- and mid-stages of the differentiation. Both in vitro in stem cell-derived megakaryocyteic cell culture and in vivo in an animal model injected with anti-platelet antibody, expression of Bcl-xL protein was maintained until platelet-producing stage of the megakaryopoiesis. TPO-depletion caused significant decrease in Bcl-xL protein level without affecting its mRNA in both stem cell-derived megakaryocytes and TPO-dependent megakaryocytic UT7/TPO cells. As a 12-kD fragment of Bcl-xL appeared by the withdrawal of TPO, we considered that Bcl-xL was cleaved upon TPO-depletion. This cleavage was blocked by a caspase-3-specific inhibitor, suggesting that caspase cleaves Bcl-xL in TPO-depleted megakaryocytes. Furthermore, pretreatment of UT7/TPO cells with a phosphatidylinositol 3-kinase (PI3K) inhibitor resulted in the cleavage of Bcl-xL even in the presence of TPO. We thus hypothesized that PI3K or its downstream signaling molecule inhibits the activation of caspase-3 and consequent cleavage of Bcl-xL. To prove this possibility, we prepared UT7/TPO cells transfected with constitutively active Akt-1. When TPO was depleted, the transfectant was significantly less liable to caspase-3 activation and Bcl-xL cleavage. Concerning transcriptional regulation of Bcl-xL, suppression of GATA-1 in UT7/TPO using siRNA caused decreased expression of both c-Mpl and Bcl-xL. Taken together, we conclude that GATA-1 regulates the expression of both c-Mpl and Bcl-xL, and once Bcl-xL is expressed, its protein level is maintained by the TPO-mediated Akt activation.