Human Megakaryoblastic Cell Line Research Articles

ABCC4 impacts megakaryopoiesis and protects megakaryocytes against 6-mercaptopurine induced cytotoxicity

The role of ABCC4, an ATP-binding cassette transporter, in the process of platelet formation, megakaryopoiesis, is unknown. Here, we show that ABCC4 is highly expressed in megakaryocytes (MKs). Mining of public genomic data (ATAC-seq and genome wide chromatin interactions, Hi-C) revealed that key megakaryopoiesis transcription factors (TFs) interacted with ABCC4 regulatory elements and likely accounted for high ABCC4 expression in MKs. Importantly these genomic interactions for ABCC4 ranked higher than for genes with known roles in megakaryopoiesis suggesting a role for ABCC4 in megakaryopoiesis. We then demonstrate that ABCC4 is required for optimal platelet formation as in vitro differentiation of fetal liver derived MKs from Abcc4-/- mice exhibited impaired proplatelet formation and polyploidization, features required for optimal megakaryopoiesis. Likewise, a human megakaryoblastic cell line, MEG-01 showed that acute ABCC4 inhibition markedly suppressed key processes in megakaryopoiesis and that these effects were related to reduced cAMP export and enhanced dissociation of a negative regulator of megakaryopoiesis, protein kinase A (PKA) from ABCC4. PKA activity concomitantly increased after ABCC4 inhibition which was coupled with significantly reduced GATA-1 expression, a TF needed for optimal megakaryopoiesis. Further, ABCC4 protected MKs from 6-mercaptopurine (6-MP) as Abcc4-/- mice show a profound reduction in MKs after 6-MP treatment. In total, our studies show that ABCC4 not only protects the MKs but is also required for maximal platelet production from MKs, suggesting modulation of ABCC4 function might be a potential therapeutic strategy to regulate platelet production.

Megakaryocytes respond during sepsis and display innate immune cell behaviors.

Megakaryocytes (MKs) are precursors to platelets, the second most abundant cells in the peripheral circulation. However, while platelets are known to participate in immune responses and play significant functions during infections, the role of MKs within the immune system remains largely unexplored. Histological studies of sepsis patients identified increased nucleated CD61+ cells (MKs) in the lungs, and CD61+ staining (likely platelets within microthrombi) in the kidneys, which correlated with the development of organ dysfunction. Detailed imaging cytometry of peripheral blood from patients with sepsis found significantly higher MK counts, which we predict would likely be misclassified by automated hematology analyzers as leukocytes. Utilizing in vitro techniques, we show that both stem cell derived MKs (SC MKs) and cells from the human megakaryoblastic leukemia cell line, Meg-01, undergo chemotaxis, interact with bacteria, and are capable of releasing chromatin webs in response to various pathogenic stimuli. Together, our observations suggest that MK cells display some basic innate immune cell behaviors and may actively respond and play functional roles in the pathophysiology of sepsis.

SARS-CoV-2 Lysate Stimulation Impairs the Release of Platelet-like Particles and Megakaryopoiesis in the MEG-01 Cell Line.

SARS-CoV-2 infection causes a considerable inflammatory response coupled with impaired platelet reactivity, which can lead to platelet disorders recognized as negative prognostic factors in COVID-19 patients. The virus may cause thrombocytopenia or thrombocytosis during the different disease stages by destroying or activating platelets and influencing platelet production. While it is known that several viruses can impair megakaryopoiesis by generating an improper production and activation of platelets, the potential involvement of SARS-CoV-2 in affecting megakaryopoiesis is poorly understood. To this purpose, we explored, in vitro, the impact of SARS-CoV-2 stimulation in the MEG-01 cell line, a human megakaryoblastic leukemia cell line, considering its spontaneous capacity of releasing platelet-like particles (PLPs). We interrogated the effect of heat-inactivated SARS-CoV-2 lysate in the release of PLPs and activation from MEG-01, the signaling pathway influenced by SARS-CoV-2, and the functional effect on macrophagic skewing. The results highlight the potential influence of SARS-CoV-2 in the early stages of megakaryopoiesis by enhancing the production and activation of platelets, very likely due to the impairment of STATs signaling and AMPK activity. Overall, these findings provide new insight into the role of SARS-CoV-2 in affecting megakaryocyte-platelet compartment, possibly unlocking another avenue by which SARS-CoV-2 moves.

PAR4 Primarily Mediates Thrombin‐induced Morphological Changes in MEG‐01 Cells through PI3K/Akt and RhoA/ROCK Signaling Pathways

Platelet activation is critical in hemostasis and thrombosis. Thrombin most potently activates platelets via two protease‐activated receptors (PARs): PAR1 and PAR4. While PAR1 activation results in a rapid and transient signal required for the initiation phase of platelet aggregation, PAR4 activation induces a sustained signal associated with later phases responsible for stable thrombus formation. The present study focused on the differential signaling pathways by PAR1 and PAR4 involved in thrombin‐induced intracellular events in human megakaryoblastic leukemia cell line, MEG‐01. Interestingly, we found that thrombin triggers not only Gq/PLC‐mediated calcium signaling, but also G12/13/RhoA/ROCK‐mediated morphological changes in MEG‐01 cells via PAR1 and PAR4, which showed close similarity to those observed in platelets. We developed a new image‐based assay to quantify the morphological changes in living cells and extensively investigated on the underlying mechanism for thrombin‐induced morphological changes in MEG‐01 cells. Selective blockade of PAR1 and PAR4 by vorapaxar and BMS‐986120, respectively, indicated that PAR4 activation primarily mediates thrombin‐induced morphological changes in MEG‐01 cells. Moreover, treatment of a set of kinase inhibitors showed that the PAR4‐mediated morphological changes were predominantly mediated via PI3K/Akt and RhoA/ROCK signaling pathways. As in the platelets, PAR4‐mediated signaling pathways are significant in thrombin‐induced morphological changes in MEG‐01 cells. Based on the close similarity to thrombin‐induced signaling in platelets, MEG‐01 cell line is a useful complementary in vitro model that can be used in research on platelets and thrombosis.

PAR4-Mediated PI3K/Akt and RhoA/ROCK Signaling Pathways Are Essential for Thrombin-Induced Morphological Changes in MEG-01 Cells.

Thrombin stimulates platelets via a dual receptor system of protease-activated receptors (PARs): PAR1 and PAR4. PAR1 activation induces a rapid and transient signal associated with the initiation of platelet aggregation, whereas PAR4 activation results in a prolonged signal, required for later phases, that regulates the stable formation of thrombus. In this study, we observed differential signaling pathways for thrombin-induced PAR1 and PAR4 activation in a human megakaryoblastic leukemia cell line, MEG-01. Interestingly, thrombin induced both calcium signaling and morphological changes in MEG-01 cells via the activation of PAR1 and PAR4, and these intracellular events were very similar to those observed in platelets shown in previous studies. We developed a novel image-based assay to quantitatively measure the morphological changes in living cells, and observed the underlying mechanism for PAR1- and PAR4-mediated morphological changes in MEG-01 cells. Selective inhibition of PAR1 and PAR4 by vorapaxar and BMS-986120, respectively, showed that thrombin-induced morphological changes were primarily mediated by PAR4 activation. Treatment of a set of kinase inhibitors and 2-aminoethoxydiphenyl borate (2-APB) revealed that thrombin-mediated morphological changes were primarily regulated by calcium-independent pathways and PAR4 activation-induced PI3K/Akt and RhoA/ROCK signaling pathways in MEG-01 cells. These results indicate the importance of PAR4-mediated signaling pathways in thrombin-induced morphological changes in MEG-01 cells and provide a useful in vitro cellular model for platelet research.

Dengue virus infection impedes megakaryopoiesis in MEG-01 cells where the virus envelope protein interacts with the transcription factor TAL-1

Dengue virus (DENV) infection causes dengue fever in humans, which can lead to thrombocytopenia showing a marked reduction in platelet counts, and dengue hemorrhagic fever. The virus may cause thrombocytopenia either by destroying the platelets or by interfering with their generation via the process of megakaryopoiesis. MEG-01 is the human megakaryoblastic leukemia cell line that can be differentiated in vitro by phorbol-12-myristate-13-acetate (PMA) treatment to produce platelet-like-particles (PLPs). We have studied DENV infection of MEG-01 cells to understand its effect on megakaryopoiesis and the generation of PLPs. We observed that DENV could infect only naive MEG-01 cells, and differentiated cells were refractory to virus infection/replication. However, DENV-infected MEG-01 cells, when induced for differentiation with PMA, supported an enhanced viral replication. Following the virus infection, the MEG-01 cells showed a marked reduction in the surface expression of platelet markers (CD41, CD42a, and CD61), a decreased polyploidy, and significantly reduced PLP counts. DENV infection caused an enhanced Notch signaling in MEG-01 cells where the virus envelope protein was shown to interact with TAL-1, a host protein important for megakaryopoiesis. These observations provide new insight into the role of DENV in modulating the megakaryopoiesis and platelet production process.

Pro-apoptotic properties and mitochondrial functionality in platelet-like-particles generated from low Aspirin-incubated Meg-01 cells

Long-term therapy with low Aspirin (ASA) dose is basis to prevent thrombotic acute events. However, the anti-platelet mechanisms of ASA remain not completely known. The aim was to analyze if in vitro exposure of human megakaryocytes to low ASA concentration may alter the apoptotic features of the newly formed platelets. Cultured Meg-01 cells, a human megakaryoblastic cell line, were stimulated to form platelets with 10 nmol/L phorbol 12-myristate-13-acetate (PMA) in the presence and absence of ASA (0.33 mmol/L). Results revealed that platelet-like particles (PLPs) derived from ASA-exposed Meg-01 cells, showed higher content of pro-apoptotic proteins Bax and Bak than PLPs from non-ASA incubated Meg-01 cells. It was accompanied of reduced cytochrome C oxidase activity and higher mitochondrial content of PTEN-induced putative kinase-1 in PLPs from ASA-incubated Meg-01 cells. However, only after calcium ionophore A23187 stimulation, caspase-3 activity, the cytosolic cytochrome C content, and reduction of mitochondrial membrane potential were higher in PLPs from ASA-incubated megakaryocytes than in those from Meg-01 without ASA. Nitric oxide synthase 3 content was higher in PLPs from ASA-exposed Meg-01 cells than in PLPs from non-ASA incubated Meg-01 cells. The L-arginine antagonist, NG-Nitro-L-arginine Methyl Ester, reduced caspase-3 activity in A23187-stimulated PLPs generated from ASA-incubated Meg-01 cells. As conclusions exposure of megakaryocyte to ASA promotes that the newly generated PLPs have, under stimulating condition, higher sensitivity to go into apoptosis than those PLPs generated from Meg-01 cells without ASA. It could be associated with differences in mitochondrial functionality and NO formation.

Ibrutinib Suppresses Early Megakaryopoiesis but Enhances Proplatelet Formation.

Ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase, has a favorable safety profile in patients with B cell-related malignancies. A primary adverse effect of ibrutinib is thrombocytopenia in the early stages of treatment, but platelet counts increase or recover as treatment continues. Currently, the effects of ibrutinib on megakaryopoiesis remain unclear. In this study, we investigated the mechanism by which ibrutinib induces thrombocytopenia using cord blood CD34+ hematopoietic stem cells (HSCs), a human megakaryoblastic cell line (SET-2), and C57BL/6 mice. We show that treatment with ibrutinib can suppress CD34+ HSC differentiation into megakaryocytes (MKs) and decrease the number of colony-forming unit-MKs (CFU-MKs). The ibrutinib-dependent inhibition of early megakaryopoiesis seems to mainly involve impaired proliferation of progenitor cells without induction of apoptosis. The effects of ibrutinib on late-stage megakaryopoiesis, in contrast to early-stage megakaryopoiesis, include enhanced MK differentiation, ploidy, and proplatelet formation in CD34+ HSC-derived MKs and SET-2 cells. We also demonstrated that MK adhesion and spreading, but not migration, were inhibited by ibrutinib. Furthermore, we revealed that integrin αIIbβ3 outside-in signaling in MKs was inhibited by ibrutinib. Consistent with previous clinical observations, in C57BL/6 mice treated with ibrutinib, platelet counts decreased by days 2 to 7 and recovered to normal levels by day 15. Together, these results reveal the pathogenesis of ibrutinib-induced transient thrombocytopenia. In conclusion, ibrutinib suppresses early megakaryopoiesis, as evidenced by inhibition of MK progenitor cell proliferation and CFU-MK formation. Ibrutinib enhances MK differentiation, ploidy, and proplatelet formation, while it impairs integrin αIIbβ3 outside-in signaling.

Identification of Thrombospondin-1 As a Novel Substrate of Cereblon Responsible for IMiDs-Induced Venous Thromboembolism

Immunomodulatory drugs (IMiDs) such as lenalidomide and pomalidomide, are key drugs for the treatment of multiple myeloma (MM) and myelodysplastic syndrome(MDS) with chromosome 5q deletion[del(5q)].IMiDs modulate the ubiquitination of intracellular proteins by E3 ubiquitin ligase and alter the stability of target proteins via proteasomal degradation. IMiDs directly bind to cereblon (CRBN), a substrate receptor for the E3 ubiquitin ligase complex, and retarget the enzyme complex toward neo-substrates such as IKAROS family zinc finger 1/3 (IKZF1/3) and casein kinase 1α (CK1α) in MM and MDS with del(5q), respectively, which underlies their clinical efficacy in these disorders. Thus, the identification of cell specific neo-substrates should be a critical step to understand IMiDs-mediated pleiotropic effects. Clinically, IMiDs cause venous thromboembolism (VTE); however, the molecular machineries how IMiDs increase the risk of VTE still remains elusive. We, therefore, hypothesized that there is an unknown substrate for CRBN that induces thrombosis in megakaryocytes and platelets. To identify possible molecules that could interact with CRBN in human megakaryocytes, we performed screening analysis. Human CD34+hematopoietic stem/progenitor cells (HSPCs) from cord blood were cultured in a serum-free megakaryocytic condition for 7 days as previously reported. Most of the CD34+HSPCs were differentiated into CD41+CD42b+mature megakaryocytes, and we extracted human megakaryocytic protein. Cellular lysates from CD34+HSPCs-derived megakaryocytes and FLAG-tagged CRBN-overexpressing HEK293T cells were mixed with or without lenalidomide. CRBN-binding proteins were subsequently pulled down using anti-FLAG antibody, and subjected to mass spectrometry analysis. Mass spectrometry analysis revealed that THBS1 was coimmunoprecipitated with CRBN, and its interaction was decreased in the presence of lenalidomide. We therefore hypothesized that THBS1 could be a neo-substrate for CRBN. Consistent with the screening results, we found that IMiDs increased THBS1 protein in Meg-01 cells, a human megakaryoblastic leukemia cell line in a dose and time dependent manner, without affecting mRNA level. Proteasome inhibitor increased THBS1 level in Meg-01 cells, suggesting that THBS1 should be physiologically degraded via proteasome. The immunoprecipitation experiments showed CRBN and THBS1 conjugation, which was inhibited in the presence of Lenalidomide in vitro. Furthermore, we confirmed an increase in THBS1 protein level in the bone marrow and plasma of MM patients treated with IMiDs-containing regimen, suggesting that IMiDs actually induce the accumulation of THBS1 in vivo. These results collectively suggest that THBS1 is a neo-substrate for CRBN and IMiDs induces the accumulation of THBS1 through the inhibition of proteasomal THBS1 degradation in human. THBS1 is reported to competitively inhibit ADAMTS13 activity of von Willebrand Factor (vWF) multimer cleavage, leading to increased level of large vWF multimers. Since large vWF multimers promote thrombus formation and several studies showed increased plasma level of THBS1 correlates with the incidence of VTE, we tested whether the accumulation of THBS1 induced by IMiDs treatment could affect the size of vWF multimer in MM patients. vWF multimer analysis revealed the large vWF multimers increased in plasma of MM patients undergoing IMiDs treatment. These results collectively suggest that IMiDs-induced accumulation of THBS1 should be one of the molecular machineries for IMiDs-induced VTE in human. Disclosures Akashi: Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding; Sumitomo Dainippon, Kyowa Kirin: Consultancy.

Specific and Synergistic Targeting of JAK2V617F Cells By Interferon Alpha and Arsenic

N.M. and T.D. co-first authors, J.L.V. and Hd.T. co-last authorsIntroduction:We have shown in clinical trials and preclinical studies on Myeloproliferative Neoplasms (MPN) that interferon alpha (IFN) is able to specifically target JAK2V617F cells. However, achievement of complete molecular responses is rare and often requires long-term exposures to IFN, increasing the risk of toxicity. The mechanism implicated in the specific targeting of JAK2V617F cells remains elusive and its elucidation is crucial to improve efficiency of IFN therapy. PML is an IFN transcriptional target that drives the assembly of PML-nuclear bodies (NB), activating P53 or RB checkpoints and ultimately leading to cell cycle arrest or senescence. We explored the hypothesis that PML may contribute to IFN efficacy. This would then predict enhancement of IFN activity by arsenic (AS), a potent inducer of NB formation.Methods:To analyze IFN and/or AS effect on JAK2V617F-driven MPN, we developed chimeric animals transplanted with a mixture of bone marrow (BM) from wild type (WT) mice and conditional JAK2V617F KI/vav-Cre/Ubi-GFP mice monitoring allele burden by GFP expression. Animals, suffering from a polycythemia vera (PV)-like disease, were treated by IFN, AS or IFN/AS combination for 8 to 14 weeks. Blood was analyzed every 2 weeks. After treatment, animals were either (1) sacrificed to analyze BM, spleen and disease-initiating cells in secondary recipients or (2) kept alive to study disease recurrence. We also developed an UT-7 human megakaryoblastic cell line isogenic model by transducing JAK2WT or JAK2V617F. MPN patient samples were tested in clonogenic assays. Finally, the role of PML was assessed in the JAK2V617F-positive HEL cell line transduced by different shRNA for PML knock-down.Results:In vivo experiments demonstrated that IFN vs non-treated (NT) reduced leukocytosis (p<0.001), the platelet number (p<0.001), erythrocytosis (p=0.005) and the JAK2V617F allele burden in granulocytes (p<0.01), platelets (p=0.03) and RBC (p=0.03). More importantly, addition of AS improved the efficacy of IFN for reducing leukocytosis (p<0.03) and erythrocytosis (p=0.02) at 4-6 weeks, but not the platelet number. The effect of the IFN/AS combination was particularly striking in improving the reduction of allele burden compared to IFN alone in granulocytes (p=0.005), platelets (p=0.001) and RBC (p=0.001) (see figure). Improvement of IFN efficacy by addition of AS was also observed on splenomegaly and SLAM progenitor allele burden. The IFN/As combination efficiently decreased relapse rates (48%, p=0.007 vs NT) compared to IFN alone (73%, p=0.2 vs NT), as assessed on blood cells, allele burden and splenomegaly several weeks after treatment discontinuation. Moreover, none of the secondary mice transplanted with BM from IFN/AS-treated mice developed the disease, while 68% of the mice transplanted with BM from IFN alone-treated mice did. These data demonstrate the dramatic effect of the IFN/AS combination on disease initiating cells.We then look for underlying molecular mechanism. The number and size of PML-NB were increased in JAK2V617F vs. WT UT-7 cells at baseline (4.0 vs 6.6 dots/cell, p= 0.0003). As expected, IFN/AS significantly increased PML-NB in both cell types, but this increase was more important in JAK2V617F cells. Cell cycle arrest induced by IFN/AS was more prominent in UT-7 JAK2V617F cells compared to isogenic control, showing the specificity of the combination for mutant cells. IFN/AS had synergistic effects on JAK2V617F cells in terms of inhibition of proliferation and induction of senescence in contrast to single therapies. Critically, PML knock-down in the JAK2V617F-positive HEL cell line reduced the anti-proliferative effect and pro-senescent activity of the IFN/AS combination showing the key role of PML in these processes.Finally, comparison of JAK2V617F-positive CD34+ primary cells from PV patients to normal CD34+ cells demonstrated that IFN/AS specifically decreased proliferation and enhanced senescence as well as NB formation in JAK2V617F cells. Moreover, IFN/AS had a drastic effect on the clonogenic potential of BFU-E progenitors derived from 10 PV patients with a specific targeting of JAK2V617F colonies, which were reduced from 67% to 32%.Conclusion:Altogether, our results strongly suggest involvement of PML in IFN therapy of MPN and the potential utility of AS to greatly improve its efficacy. DisclosuresKiladjian:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees.

Stem cell factor and NSC87877 combine to enhance c-Kit mediated proliferation of human megakaryoblastic cells.

Enhancement of hematopoietic stem cells (HSCs) proliferation is a central aim in bone marrow transplantation (BMT). A stem cell factor (SCF) and c-Kit mediated extracellular signaling trigger proliferation of HSCs. This signaling is negatively regulated by protein tyrosine phosphatases (PTPs), SHP-1 and SHP-2. Although NSC87877 (N) is known to inhibit SHP-1/SHP-2, c-Kit-mediated HSCs proliferation by inhibiting SHP-1/SHP-2 has not been reported. This study investigated the combined effect of SCF (S) and N in c-Kit mediated proliferation and underlying mechanisms. The growth of human megakaryoblastic cell line, MO7e and HSCs, upon treatment with S and N alone, and in combination was assessed by PrestoBlue staining. The expression of c-Kit, phosphorylated c-Kit, SHP-1/SHP-2 and HePTP inhibition using S and N treatment were evaluated in the MO7e cells. Megakaryoblast cell proliferation was determined by quantification of Ki-67+, S-phase, BrdU+ and CFDA-SE+ cells using flow cytometry. The combination of S and N leads to enhanced cell growth compared with either S or N alone. Collectively, the results reveal a novel mechanism by which S in combination with N significantly enhances proliferation of human megakaryoblast cells. The pretreatment of N before S enhances proliferation of cells than S alone. This promising combination would likely play an essential role in enhancing the proliferation of cells.

Identification of eNOS‐based Megakaryocyte Subpopulations and Their Pharmacological Characterization by IFNγ and IL‐10

PurposeRecently, our laboratory identified in human blood two platelet subpopulations based on the presence or absence of a functional endothelial nitric oxide synthase (eNOS)‐signalling pathway and the ability to produce nitric oxide (NO). We have found that eNOS‐negative (eNOSneg) platelets, although less abundant are more reactive than eNOS‐positive (eNOSpos) platelets and initiate aggregate/thrombus formation, while eNOS‐positive platelets through their ability to generate NO limit aggregate size. As platelets derive from bone marrow megakaryocytes, we hypothesized that eNOSneg and eNOSpos subpopulations of megakaryocytes exist and give rise to their respective eNOS‐based platelet subpopulations. Additionally, we hypothesized that interferon‐γ (IFNγ) and interleukin‐10 (IL‐10), which are known to counter‐regulate eNOS expression and megakaryocyte differentiation, would determine in a concentration manner the ratio of eNOSneg to eNOSpos megakaryocytes.MethodsThe human megakaryoblastic cell line (Meg‐01) and megakaryocytes isolated from eNOS‐GFP transgenic mice were studied. RT‐PCR and DNA sequencing were performed to validate eNOS presence within Meg‐01. Flow cytometry was used to identify eNOSneg and eNOSpos megakaryocytes, and NO production was measured using DAF‐FM, a cell‐permeable fluorescent probe. Further, Meg‐01 cells were treated for 48 hours with IFNγ (0 – 100 ng/ml) and IL‐10 (0 – 100 ng/ml), and eNOS as well as inducible NOS (iNOS) were measured by flow cytometry and immunoblot, respectively.ResultsThe presence of eNOS was confirmed in Meg‐01 and eNOS‐GFP transgenic mice. Similar to human platelets, the majority of Meg‐01 cells were eNOSpos (91.5%±1.2 vs. 8.5%±1.2 eNOSneg, P&lt;0.05) and NO‐producing (92.0%±3.34 vs. 8.0%±6.68 non‐producing, P &lt; 0.05). Conversely, similar to mouse platelets, the majority of mouse megakaryocytes were eNOSneg (95.8%±0.7 vs. 4.2%±0.7 eNOSpos, P &lt; 0.05). The proinflammatory cytokine IFNγ decreased the percent eNOSpos Meg‐01 in a concentration‐dependent manner and also increased the level of iNOS within Meg‐01. IL‐10 in a concentration‐dependent manner (10–100ng/ml) reversed IFNγ‐induced iNOS expression, but not the decrease in percent eNOSpos Meg‐01. The changes in eNOS expression within Meg‐01 were further confirmed by qPCR within the total Meg‐01 population and demonstrated that IFNγ (10 ng/ml) caused a 6.4 fold decrease of eNOS mRNA but only 2.2 fold decrease in the presence IL‐10 (100ng/ml).ConclusionseNOS‐based megakaryocyte subpopulations exist within the Meg‐01 cell line and in eNOS‐GFP transgenic mice. Similar to previously shown for platelets, major species differences exist in eNOSpos to eNOSneg ratios between human and mouse megakaryocytes. IFNγ decreases eNOS expression and percent eNOSpos Meg‐01; while increasing iNOS expression. However, high concentrations of IL‐10 attenuated this effect. Our data show that pro‐inflammatory IFN‐γ may promote growth/differentiation of megakaryocytes with decreased levels/absence of eNOS, which may lead to formation of higher numbers of more reactive eNOSneg platelets potentially leading to higher risk of a pro‐thrombotic state. Further experiments are required to confirm whether eNOSpos and eNOSneg megakaryocytes give rise to their respective platelet subtypes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Promoter motifs required for c-mpl gene expression induced by thrombopoietin in CMK cells.

Thrombopoietin (TPO) and its receptor, c-Mpl, are the central regulators of megakaryocyte development and platelet production and are also crucial to regulate megakaryocytopoiesis. TPO remarkably elevated c-mpl promoter activity, while the protein kinase C (PKC) inhibitors, GF109203, H7 and Calphostin C, clearly reduced the steady level of its promoter activity. In the present study, motifs crucial for c-mpl promoter activity induced by TPO treatment have been analyzed using a human megakaryoblastic cell line, CMK. Destruction of the -107Sp1 and the -57Sp1 sites in the c-mpl promoter enhancer region resulted in decrease of the promoter activity by 53.1% and 64.4%, respectively, and destruction of -69Ets and -28Ets elements dramatically decreased the promoter activity by 96.4% and 87.8%, respectively, while mutation of -77GATA moderately reduced the activity by 31.4%. The result was in agreement with our previous report that showed the crucial motifs in the c-mpl promoter for the promoter activity induced by PMA-treatment. This indicates that TPO-induced activation of the c-mpl promoter activity is fully modulated by transcription through a PKC-dependent pathway and the two Sp1 and two Ets motifs are crucial for the activation of the c-mpl promoter activity rather than a GATA motif in the c-mpl promoter of CMK cells.

Platelet WDR1 suppresses platelet activity and is associated with cardiovascular disease

AbstractPlatelet activity plays a major role in hemostasis with increased platelet activity likely contributing to the pathogenesis of atherothrombosis. We sought to identify associations between platelet activity variability and platelet-related genes in healthy controls. Transcriptional profiling of platelets revealed that WD-40 repeat domain 1 (WDR1), an enhancer of actin-depolymerizing factor activity, is downregulated in platelet messenger RNA (mRNA) from subjects with a hyperreactive platelet phenotype. We used the human megakaryoblastic cell line MEG-01 as an in vitro model for human megakaryocytes and platelets. Stimulation of MEG-01 with thrombin reduced levels of WDR1 transcripts and protein. WDR1 knockdown (KD) in MEG-01 cells increased adhesion and spreading in both the basal and activated states, increased F-actin content, and increased the basal intracellular calcium concentration. Platelet-like particles (PLPs) produced by WDR1 KD cells were fewer in number but larger than PLPs produced from unmodified MEG-01 cells, and had significantly increased adhesion in the basal state and upon thrombin activation. In contrast, WDR1 overexpression reversed the WDR1 KD phenotype of megakaryocytes and PLPs. To translate the clinical significance of these findings, WDR1 expression was measured in platelet RNA from subjects with established cardiovascular disease (n = 27) and age- and sex-matched controls (n = 10). The WDR1 mRNA and protein level was significantly lower in subjects with cardiovascular disease. These data suggest that WDR1 plays an important role in suppressing platelet activity, where it alters the actin cytoskeleton dynamics, and downregulation of WDR1 may contribute to the platelet-mediated pathogenesis of cardiovascular disease.

Arachidonic Acid Metabolite 19(S)-HETE Induces Vasorelaxation and Platelet Inhibition by Activating Prostacyclin (IP) Receptor.

19(S)-hydroxy-eicosatetraenoic acid (19(S)-HETE) belongs to a family of arachidonic acid metabolites produced by cytochrome P450 enzymes, which play critical roles in the regulation of cardiovascular, renal and pulmonary functions. Although it has been known for a long time that 19(S)-HETE has vascular effects, its mechanism of action has remained unclear. In this study we show that 19(S)-HETE induces cAMP accumulation in the human megakaryoblastic leukemia cell line MEG-01. This effect was concentration-dependent with an EC50 of 520 nM, insensitive to pharmacological inhibition of COX-1/2 and required the expression of the G-protein Gs. Systematic siRNA-mediated knock-down of each G-protein coupled receptor (GPCR) expressed in MEG-01 followed by functional analysis identified the prostacyclin receptor (IP) as the mediator of the effects of 19(S)-HETE, and the heterologously expressed IP receptor was also activated by 19(S)-HETE in a concentration-dependent manner with an EC50 of 567 nM. Pretreatment of isolated murine platelets with 19(S)-HETE blocked thrombin-induced platelets aggregation, an effect not seen in platelets from mice lacking the IP receptor. Furthermore, 19(S)-HETE was able to relax mouse mesenteric artery- and thoracic aorta-derived vessel segments. While pharmacological inhibition of COX-1/2 enzymes had no effect on the vasodilatory activity of 19(S)-HETE these effects were not observed in vessels from mice lacking the IP receptor. These results identify a novel mechanism of action for the CYP450-dependent arachidonic acid metabolite 19(S)-HETE and point to the existence of a broader spectrum of naturally occurring prostanoid receptor agonists.

Proplatelet formation in megakaryocytes is associated with endoplasmic reticulum stress.

Although previous studies suggest that proplatelet formation in megakaryocytes involves caspase-3, the mechanism underlying the activation of caspase-3 is unknown. Here, we analyzed caspase activation in a human megakaryoblastic cell line, MEG-01, which forms proplatelets spontaneously. Specific activation of caspase-3 and caspase-4 was found in proplatelets. Consistent with previous observations of caspase-4 autoactivation in response to endoplasmic reticulum (ER) stress, several ER stress marker proteins were expressed during proplatelet formation. A pharmacological ER stressor enhanced platelet production via proplatelet formation, whereas inhibition of caspase-4 caused suppression. These results suggest that ER stress is a mechanism underlying the maturation of megakaryocytes.

The effect of amifostine on differentiation of the human megakaryoblastic Dami cell line.

Amifostine is a cytoprotective drug that was initially used to control and treat nuclear radiation injury and is currently used to provide organ protection in cancer patients receiving chemotherapy. Clinical studies have also found that amifostine has some efficacy in the treatment of cytopenia caused by conditions such as myelodysplastic syndrome and immune thrombocytopenia, both of which involve megakaryocyte maturation defects. We hypothesized that amifostine induced the differentiation of megakaryocytes and investigated this by exposing the human Dami megakaryocyte leukemia cell line to amifostine (1 mmol/L). After 12 days of amifostine exposure, optical microscopy showed that the proportion of Dami cells with diameters >20 μm had increased to 24.63%. Transmission electron microscopy identified the development of a platelet demarcation membrane system, while flow cytometry detected increased CD41a expression and decreased CD33 expression on the Dami cell surface. Ploidy analysis found that the number of polyploid cells with >4N DNA content increased to 27.96%. We did not detect any elevation in the mRNA or protein levels of megakaryocytic differentiation‐associated transcription factors GATA‐binding factor 1 (GATA‐1) and nuclear factor, erythroid 2 (NF‐E2), but nuclear import assay revealed an increased nuclear translocation of these proteins. These findings indicate that amifostine induced the differentiation of Dami cells into mature megakaryocytes via a mechanism involving increased nuclear translocation of the transcription factors, NF‐E2 and GATA‐1.

Thrombin Maybe Plays an Important Role in MK Differentiation into Platelets.

Objectives. After development and differentiation, megakaryocytes (MKs) can produce platelets. As is well known, thrombopoietin (TPO) can induce MKs to differentiate. The effect of thrombin on MKs differentiation is not clear. In this study, we used a human megakaryoblastic leukemia cell line (Meg-01) to assess the effect of thrombin on MKs differentiation. Methods. In order to interrogate the role of thrombin in Meg-01 cells differentiation, the changes of morphology, cellular function, and expression of diverse factors were analyzed. Results. The results show that thrombin suppresses Meg-01 cells proliferation and induces apoptosis and cell cycle arrest. Thrombin upregulates the expression of CD41b, which is one of the most important MK markers. Globin transcription factor 1 (GATA-1), an important transcriptional regulator, controls MK development and maturation. The expression of GATA-1 is also upregulated by thrombin in Meg-01 cells. The expression of B-cell lymphoma 2 (Bcl-2), an apoptosis-inhibitory protein, is downregulated by thrombin. Phosphorylated protein kinase B (p-AKT) and phosphorylated extracellular signal-regulated kinase (p-ERK) were upregulated by thrombin in Meg-01 cells. All the results are consistent with Meg-01 cells treated with TPO. Discussion and Conclusion. In conclusion, all these data indicate that thrombin maybe plays an important role in MK differentiation into platelets. However, whether the platelet-like particles are certainly platelets remains unknown.

DREAM, a Transcription Repressor, Is Critical for Calcium Signaling and Platelet Thrombus Formation Independently of Its Transcriptional Activity

DREAM (Downstream Regulatory Element Antagonistic Modulator) is a neuronal calcium sensor and acts as a transcriptional repressor for pain modulation. Recent studies showed that endothelial cell DREAM transcriptionally regulates NF-κB signaling during inflammation (Tiruppathi et al. Nat Immunol. 2014). However, it is unknown whether DREAM plays a role during thrombosis and hemostasis. Using real-time fluorescence intravital microscopy with DREAM knockout (KO) mice, we demonstrated that intravascular cell DREAM is required for platelet thrombus formation and fibrin generation at the site of laser-induced cremaster arteriolar injury. Studies of bone marrow chimeric mice further revealed that both hematopoietic and endothelial cell DREAM regulate thrombus formation following arteriolar injury. Using in vitro flow chamber assays, we found that DREAM is important for platelet thrombus formation on collagen-coated surfaces under shear conditions. Further, tail bleeding time and blood loss significantly increased in DREAM KO mice, compared with wild-type (WT) mice, suggesting the importance of DREAM for hemostasis as well as thrombus formation in vivo. We observed that DREAM KO platelets show markedly decreased aggregation, ATP secretion, P-selectin exposure, and αIIbβ3 integrin activation induced by thrombin, collagen-related peptide, ADP, and A23187 (calcium ionophore). Interestingly, DREAM deletion did not affect phorbol 12-myristate 13-acetate-induced platelet functions. Consistently, DREAM KO platelets exhibited decreased phosphorylation of phosphoinositide-3-kinase, AKT, and ERK, but not protein kinase C (PKC) isoforms during cell activation, suggesting that DREAM does not regulate protein kinase C signaling pathway. Electron microscopic analysis demonstrated that ultrastructural features of DREAM KO platelets are indistinguishable from those of WT platelets. Since DREAM is a transcription factor that binds to calcium in four EF-hand motifs, we investigated whether platelet DREAM regulates calcium signaling. Indeed, DREAM deletion in platelets resulted in significant reduction in both intracellular calcium release and store-operated calcium entry (SOCE) following agonist stimulation. When we transiently knocked down DREAM in a human megakaryoblastic leukemia cell line (MEG-01) using siRNA, we found that A23187-induced calcium release and SOCE are impaired by DREAM knockdown in MEG-01 cells without altering the expression of calcium signaling molecules, such as inositol trisphosphate receptor, stromal interaction molecule 1, and ORAI1. These defects were rescued by overexpression of siRNA-resistant WT but not EF-hand domain mutant DREAM. Therefore, our results provide novel evidence that platelet DREAM regulates calcium signaling independently of its transcriptional factor activity, thereby playing a critical role during thrombus formation and hemostasis in vivo. DisclosuresNo relevant conflicts of interest to declare.

Aspirin influences megakaryocytic gene expression leading to up-regulation of multidrug resistance protein-4 in human platelets.

AimThe aim of the study was to investigate whether human megakaryocytic cells have an adaptive response to aspirin treatment, leading to an enhancement of multidrug resistance protein-4 (MRP4) expression in circulating platelets responsible for a reduced aspirin action. We recently found that platelet MRP4 overexpression has a role in reducing aspirin action in patients after by-pass surgery. Aspirin enhances MRP4-mRNA levels in rat liver and drug administration transcriptionally regulates MRP4 gene expression through peroxisome proliferator-activated receptor-α (PPARα).MethodsThe effects induced by aspirin or PPARα agonist (WY14643) on MRP4 modulation were evaluated in vitro in a human megakaryoblastic DAMI cell line, in megakaryocytes (MKs) and in platelets obtained from human haematopoietic progenitor cell (HPC) cultures, and in vivo platelets obtained from aspirin treated healthy volunteers (HV).ResultsIn DAMI cells, aspirin and WY14643 treatment induced a significant increase in MRP4 and PPARα expression. In human MKs grown in the presence of either aspirin or WY14643, MRP4 and PPARα-mRNA were higher than in control cultures and derived platelets showed an enhancement in MRP4 protein expression. The ability of aspirin to modulate MRP4 expression in MKs and to transfer it to platelets was also confirmed in vivo. In fact, we found the highest MRP4 mRNA and protein expression in platelets obtained from HV after 15 days' aspirin treatment.ConclusionsThe present study provides evidence, for the first time, that aspirin treatment affects the platelet protein pattern through MK genomic modulation. This work represents an innovative and attractive approach, useful both to identify patients less sensitive to aspirin and to improve pharmacological treatment in cardiovascular high-risk patients.