The role of AGK in thrombocytopoiesis and possible therapeutic strategies.

Abnormal megakaryocyte development and platelet production lead to thrombocytopenia or thrombocythemia and increase the risk of hemorrhage or thrombosis. Acylglycerol kinase (AGK) is a mitochondrial membrane kinase that catalyzes the formation of phosphatidic acid and lysophosphatidic acid. Mutation of AGK has been described as the major cause of Sengers syndrome, and the patients with Sengers syndrome have been reported to exhibit thrombocytopenia. In this study, we found that megakaryocyte/platelet-specific AGK-deficient mice developed thrombocytopenia and splenomegaly, mainly caused by inefficient bone marrow thrombocytopoiesis and excessive extramedullary hematopoiesis, but not by apoptosis of circulating platelets. It has been reported that the G126E mutation arrests the kinase activity of AGK. The AGK G126E mutation did not affect peripheral platelet counts or megakaryocyte differentiation, suggesting that the involvement of AGK in megakaryocyte development and platelet biogenesis was not dependent on its kinase activity. The Mpl/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (Stat3) pathway is the major signaling pathway regulating megakaryocyte development. Our study confirmed that AGK can bind to JAK2 in megakaryocytes/platelets. More interestingly, we found that the JAK2 V617F mutation dramatically enhanced the binding of AGK to JAK2 and greatly facilitated JAK2/Stat3 signaling in megakaryocytes/platelets in response to thrombopoietin. We also found that the JAK2 JAK homology 2 domain peptide YGVCF617CGDENI enhanced the binding of AGK to JAK2 and that cell-permeable peptides containing YGVCF617CGDENI sequences accelerated proplatelet formation. Therefore, our study reveals critical roles of AGK in megakaryocyte differentiation and platelet biogenesis and suggests that targeting the interaction between AGK and JAK2 may be a novel strategy for the treatment of thrombocytopenia or thrombocythemia.

RGS10 and RGS18 differentially limit platelet activation, promote platelet production, and prolong platelet survival.

G protein-coupled receptors are critical mediators of platelet activation whose signaling can be modulated by members of the regulator of G protein signaling (RGS) family. The 2 most abundant RGS proteins in human and mouse platelets are RGS10 and RGS18. While each has been studied individually, critical questions remain about the overall impact of this mode of regulation in platelets. Here, we report that mice missing both proteins show reduced platelet survival and a 40% decrease in platelet count that can be partially reversed with aspirin and a P2Y12 antagonist. Their platelets have increased basal (TREM)-like transcript-1 expression, a leftward shift in the dose/response for a thrombin receptor-activating peptide, an increased maximum response to adenosine 5'-diphosphate and TxA2, and a greatly exaggerated response to penetrating injuries in vivo. Neither of the individual knockouts displays this constellation of findings. RGS10-/- platelets have an enhanced response to agonists in vitro, but platelet count and survival are normal. RGS18-/- mice have a 15% reduction in platelet count that is not affected by antiplatelet agents, nearly normal responses to platelet agonists, and normal platelet survival. Megakaryocyte number and ploidy are normal in all 3 mouse lines, but platelet recovery from severe acute thrombocytopenia is slower in RGS18-/- and RGS10-/-18-/- mice. Collectively, these results show that RGS10 and RGS18 have complementary roles in platelets. Removing both at the same time discloses the extent to which this regulatory mechanism normally controls platelet reactivity in vivo, modulates the hemostatic response to injury, promotes platelet production, and prolongs platelet survival.

Platelet Heterogeneity in Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPNs) are a group of malignant disorders of the bone marrow where a dysregulated balance between proliferation and differentiation gives rise to abnormal numbers of mature blood cells. MPNs encompass a spectrum of disease entities with progressively more severe clinical features, including complications with thrombosis and hemostasis and an increased propensity for transformation to acute myeloid leukemia. There is an unmet clinical need for markers of disease progression. Our understanding of the precise mechanisms that influence pathogenesis and disease progression has been limited by access to disease-specific cells as biosources. Here, we review the landscape of MPN pathology and present blood platelets as potential candidates for disease-specific understanding. We conclude with our recent work discovering progressive platelet heterogeneity by subtype in a large clinical cohort of patients with MPN.

Roxadustat Does Not Affect Platelet Production, Activation, and Thrombosis Formation.

Objective: Roxadustat is a new medication for the treatment of renal anemia. EPO (erythropoietin)-the current treatment standard-has been reported to enhance platelet activation and production. However, to date, the effect of roxadustat on platelets is unclear. To address this deficiency, herein, we have evaluated the effect of roxadustat on platelet production and function. Approach and Results: We performed several mouse platelet functional assays in the presence/absence of in vitro and in vivo roxadustat treatment. Both healthy and 5/6 nephrectomized mice were utilized. The effect of roxadustat on platelet function of healthy volunteers and chronic kidney disease patients was also evaluated. For platelet production, megakaryocyte maturation and proplatelet formation were assayed in vitro. Peripheral platelet and bone marrow megakaryocyte counts were also determined. We found that roxadustat could not stimulate washed platelets directly, and platelet aggregation, spreading, clot retraction, and P-selectin/JON/A exposure were similar with or without in vitro or in vivo roxadustat treatment among both healthy and 5/6 nephrectomized mice. In vivo mouse thrombosis models were additionally performed, and no differences were detected between the vehicle and roxadustat treatment groups. EPO, which was considered a positive control in the present study, promoted platelet function and production as reported previously. Megakaryocyte maturation and proplatelet formation were also not significantly different between control mice and those treated with roxadustat. After receiving roxadustat for 14 days, no difference in the peripheral platelet count was observed in the mice. Conclusions: Administration of roxadustat has no significant impact on platelet production and function.

Bone marrow NLRP3 inflammasome-IL-1ß signal regulates post-myocardial infarction megakaryocyte development and platelet production.

Platelet plays an important role in the progression of atherosclerosis. Recently it has been reported that myocardial infarction (MI) triggers megakaryopoiesis and thrombopoiesis in the bone marrow and leads to increased circulating platelets, which might contribute to the aggravation of atherosclerosis. However, the underlying mechanisms remain unclear. Here, we analyzed post-MI bone marrow tissue and found that MI induced an upregulation of bone marrow NOD-like Receptor Protein 3 (NLRP3) and subsequent secretion of IL-1ß, an essential stimulator of megakaryopoiesis. Targeting NLRP3 using a specific inhibitor MCC950 reduced bone marrow IL-1ß expression. Using bone marrow whole-mount immunofluorescence staining combined with flow cytometry, we demonstrated that MCC950 reduced megakaryocyte cellularity and maturity, and effectively attenuated the excessive platelet production after MI. Importantly, mice subjected to MI treated with MCC950 showed a higher survival rate compared with the only MI group. Taken together, this study shows that bone marrow NLRP3-IL-1ß signal regulates megakaryocyte development and platelet production after myocardial infarction. It provides a new hint that pharmacological inhibition of NLRP3 might become a potential therapeutic approach for controlling excessive thrombopoiesis after MI.

In vitro effect of eltrombopag alone and in combination with azacitidine on megakaryopoiesis in patients with myelodysplastic syndrome.

Thrombocytopenia is a severe complication for patients with myelodysplastic syndrome (MDS). Eltrombopag increases platelet count in MDS patients but its combination with azacitidine elicited controversial results. We aimed to quantify the colony forming units of megakaryocytes (CFU-Mk) obtained from CD34+ bone marrow cells isolated from patients with MDS and from healthy donors that were cultured in vitro in the presence or absence of azacitidine and with or without the sequential addition of eltrombopag to the culture medium. CD34+ bone marrow cells from 6 MDS patients and 3 controls were expanded in vitro and cultured for 3 days with or without azacitidine. Subsequently, a CFU-Mk assay was performed in presence or absence of eltrombopag. The addition of eltrombopag in the CFU-Mk assay after mock treatment of CD34+ cells increased the number of CFU-Mk in both controls and patients. On the contrary, using azacitidine pretreated CD34+ cells, eltrombopag minimally increased CFU-Mk in controls and produced heterogeneous response in MDS patients with no change in two patients and CFU-Mk increase in four patients. In vitro CFU-Mk assay suggest that some MDS patients are likely to benefit from the sequential addition of eltrombopag after azacitidine treatment, in the context of a personalized medicine.

Histamine deficiency facilitates coronary microthrombosis after myocardial infarction by increasing neutrophil-platelet interactions.

Neutrophil-platelet interactions are responsible for thrombosis as well as inflammatory responses following acute myocardial infarction (AMI). While histamine has been shown to play a crucial role in many physiological and pathological processes, its effects on neutrophil-platelet interactions in thromboinflammatory complications of AMI remain elusive. In this study, we show a previously unknown mechanism by which neutrophil-derived histamine protects the infarcted heart from excessive neutrophil-platelet interactions and redundant arterial thrombosis. Using histamine-deficient (histidine decarboxylase knockout, HDC-/- ) and wild-type murine AMI models, we demonstrate that histamine deficiency increases the number of microthrombosis after AMI, in accordance with depressed cardiac function. Histamine-producing myeloid cells, mainly Ly6G+ neutrophils, directly participate in arteriole thrombosis. Histamine deficiency elevates platelet activation and aggregation by enhancing Akt phosphorylation and leads to dysfunctional characteristics in neutrophils which was confirmed by high levels of reactive oxygen species production and CD11b expression. Furthermore, HDC-/- platelets were shown to elicit neutrophil extracellular nucleosomes release, provoke neutrophil-platelet interactions and promote HDC-expressing neutrophils recruitment in arteriole thrombosis in vivo. In conclusion, we provide evidence that histamine deficiency promotes coronary microthrombosis and deteriorates cardiac function post-AMI, which is associated with the enhanced platelets/neutrophils function and neutrophil-platelet interactions.

USP7 inhibition inhibits proliferation and induces megakaryocytic differentiation in MDS cells by upregulating gelsolin.

Myelodysplastic syndrome (MDS), a largely incurable hematological malignancy, is driven by complex genetic and epigenetic alterations from an aberrant clone of hematopoietic stem/progenitor cells (HSPCs). Ubiquitin-specific protease 7 (USP7) has been demonstrated to have an important oncogenic role in the development of several cancer types, but its role in MDS is unknown. Here, we demonstrate that USP7 expression is elevated in MDS cell lines and patient samples. The USP7-selective small-molecule inhibitors P5091 and P22077 inhibited cell proliferation and induced megakaryocytic differentiation in both cell lines and primary cells. Furthermore, pharmacological inhibition of USP7 markedly suppressed the growth of MDS cell lines in xenograft mouse models. To explore the mechanisms underlying the observed phenotypic changes, we employed RNA-seq to compare the differences in genes after USP7 inhibitor treatment and found that gelsolin (GSN) expression was increased significantly after USP7 inhibitor treatment. Furthermore, knockdown of GSN attenuated the proliferation inhibition, apoptosis induction and megakaryocyte differentiation induced by USP7 inhibitors in MDS cells. Collectively, our findings identify previously unknown roles of USP7 and suggest that the USP7/GSN axis may be a potential therapeutic target in MDS.

Modeling individual time courses of thrombopoiesis during multi-cyclic chemotherapy.

BACKGROUND: Thrombocytopenia is a major side-effect of cytotoxic cancer therapies. The aim of precision medicine is to develop therapy modifications accounting for the individual's risk. METHODOLOGY/PRINCIPLE FINDINGS: To solve this task, we develop an individualized bio-mechanistic model of the dynamics of bone marrow thrombopoiesis, circulating platelets and therapy effects thereon. Comprehensive biological knowledge regarding cell differentiation, amplification, apoptosis rates, transition times and corresponding regulations are translated into ordinary differential equations. A model of osteoblast/osteoclast interactions was incorporated to mechanistically describe bone marrow support of quiescent cell stages. Thrombopoietin (TPO) as a major regulator is explicitly modelled including pharmacokinetics and-dynamics of TPO injections. Effects of cytotoxic drugs are modelled by transient depletions of proliferating cells. To calibrate the model, we used population data from the literature and close-meshed individual data of N = 135 high-grade non-Hodgkin's lymphoma patients treated with CHOP-like chemotherapies. To limit the number of free parameters, several parsimony assumptions were derived from biological data and tested via Likelihood methods. Heterogeneity of patients was explained by a few model parameters. The over-fitting issue of individual parameter estimation was successfully dealt with a virtual participation of each patient in population-based experiments. The model qualitatively and quantitatively explains a number of biological observations such as the role of osteoblasts in explaining long-term toxic effects, megakaryocyte-mediated feedback on stem cells, bi-phasic stimulation of thrombopoiesis by TPO, dynamics of megakaryocyte ploidies and non-exponential platelet degradation. Almost all individual time series could be described with high precision. We demonstrated how the model can be used to provide predictions regarding individual therapy adaptations. CONCLUSIONS: We propose a mechanistic thrombopoiesis model of unprecedented comprehensiveness in both, biological mechanisms considered and experimental data sets explained. Our innovative method of parameter estimation allows robust determinations of individual parameter settings facilitating the development of individual treatment adaptations during chemotherapy.

Melatonin enhances thrombopoiesis through ERK1/2 and Akt activation orchestrated by dual adaptor for phosphotyrosine and 3-phosphoinositides.

Melatonin (MT), endogenously secreted by the pineal gland, is closely related to multiple biological processes; however, its effect on thrombopoiesis is still not well illustrated. Here, we demonstrate that MT administration can elevate peripheral platelet levels. Analysis of different stages in thrombopoiesis reveals that MT has the capacity to promote the expansion of CD34+ and CD41+ cells, and accelerate proplatelet formation (PPF) and platelet production. Furthermore, in vivo experiments show that MT has a potential therapeutic effect on radiation-induced thrombocytopenia. The underlying mechanism suggests that both extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt signaling are involved in the processes of thrombopoiesis facilitated by MT. Interestingly, in addition to the direct regulation of Akt signaling by its upstream phosphoinositide 3-kinase (PI3K), ERK1/2 signaling is also regulated by PI3K via its effector, dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1), in megakaryocytes after MT treatment. Moreover, the expression level of DAPP1 during megakaryocyte differentiation is closely related to the activation of ERK1/2 and Akt at different stages of thrombopoiesis. In conclusion, our data suggest that MT treatment can promote thrombopoiesis, which is modulated by the DAPP1-orchestrated activation of ERK1/2 and Akt signaling.

Selinexor-induced thrombocytopenia results from inhibition of thrombopoietin signaling in early megakaryopoiesis.

Selinexor is the first oral selective inhibitor of nuclear export compound tested for cancer treatment. Selinexor has demonstrated a safety therapy profile with broad antitumor activity against solid and hematological malignancies in phases 2 and 3 clinical trials (#NCT03071276, #NCT02343042, #NCT02227251, #NCT03110562, and #NCT02606461). Although selinexor shows promising efficacy, its primary adverse effect is high-grade thrombocytopenia. Therefore, we aimed to identify the mechanism of selinexor-induced thrombocytopenia to relieve it and improve its clinical management. We determined that selinexor causes thrombocytopenia by blocking thrombopoietin (TPO) signaling and therefore differentiation of stem cells into megakaryocytes. We then used both in vitro and in vivo models and patient samples to show that selinexor-induced thrombocytopenia is indeed reversible when TPO agonists are administered in the absence of selinexor (drug holiday). In sum, these data reveal (1) the mechanism of selinexor-induced thrombocytopenia, (2) an effective way to reverse the dose-limiting thrombocytopenia, and (3) a novel role for XPO1 in megakaryopoiesis. The improved selinexor dosing regimen described herein is crucial to help reduce thrombocytopenia in selinexor patients, allowing them to continue their course of chemotherapy and have the best chance of survival. This trial was registered at www.clinicaltrials.gov as #NCT01607905.

Effects of Estrogens on Platelets and Megakaryocytes.

In women, oral menopausal hormonal therapy (MHT) is associated with adverse effects including an increased incidence of thromboembolic events, classically attributed to an increase in several liver-derived coagulation factors due to hepatic first pass. While platelets are central players in thrombus constitution, their implication in women treated with estrogens remains incompletely characterized. Platelets and their medullar progenitors, megakaryocytes, express estrogen receptors (ER) that may explain, at least in part, a sensitivity to hormonal changes. The purpose of this review is to summarize our current knowledge of estrogen actions on platelets and megakaryocytes in mice following in vivo administration and in women using MHT.

The Plant Hormone Abscisic Acid Is a Prosurvival Factor in Human and Murine Megakaryocytes.

Abscisic acid (ABA) is a phytohormone involved in pivotal physiological functions in higher plants. Recently, ABA has been proven to be also secreted and active in mammals, where it stimulates the activity of innate immune cells, mesenchymal and hematopoietic stem cells, and insulin-releasing pancreatic ß cells through a signaling pathway involving the second messenger cyclic ADP-ribose (cADPR). In addition to behaving like an animal hormone, ABA also holds promise as a nutraceutical plant-derived compound in humans. Many biological functions of ABA in mammals are mediated by its binding to the LANCL-2 receptor protein. A putative binding of ABA to GRP78, a key regulator of endoplasmic reticulum stress, has also been proposed. Here we investigated the role of exogenous ABA in modulating thrombopoiesis, the process of platelet generation. Our results demonstrate that expression of both LANCL-2 and GRP78 is up-regulated during hematopoietic stem cell differentiation into mature megakaryocytes (Mks). Functional ABA receptors exist in mature Mks because ABA induces an intracellular Ca2+ increase ([Ca2+] i ) through PKA activation and subsequent cADPR generation. In vitro exposure of human or murine hematopoietic progenitor cells to 10 µm ABA does not increase recombinant thrombopoietin (rTpo)-dependent Mk differentiation or platelet release. However, under conditions of cell stress induced by rTpo and serum deprivation, ABA stimulates, in a PKA- and cADPR-dependent fashion, the mitogen-activated kinase ERK 1/2, resulting in the modulation of lymphoma 2 (Bcl-2) family members, increased Mk survival, and higher rates of platelet production. In conclusion, we demonstrate that ABA is a prosurvival factor for Mks in a Tpo-independent manner.

Mesenchymal stem cell deficiency influences megakaryocytopoiesis through the TNFAIP3/NF-κB/SMAD pathway in patients with immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disease. Mesenchymal stem cells (MSCs) play important roles in the physiology and homeostasis of the haematopoietic system, including supporting megakaryocytic differentiation from CD34+ haematopoietic progenitor cells. Tumour necrosis factor alpha-induced protein 3 (TNFAIP3, also termed A20) plays a key role in terminating NF-κB signalling. Human genetic studies showed that the polymorphisms of the TNFAIP3 gene may contribute to ITP susceptibility. In this study, we showed a significant decrease in TNFAIP3 and increase in NF-κB/SMAD7 in ITP-MSCs. In co-cultures with CD34+ cells, NF-κB was overexpressed in MSCs from healthy controls (HC-MSCs) after transfection with NFKBIA (IκB)-specific short hairpin (sh)RNAs, resulting in MSC deficiency and a reduction in megakaryocytic differentiation and thrombopoiesis. Knockdown of TNFAIP3 expression using TNFAIP3-specific shRNAs in HC-MSCs affected megakaryocytopoiesis. However, IKBKB knockdown corrected megakaryocytopoiesis inhibition in the ITP-MSCs by decreasing NF-κB expression. Amplified TNFAIP3 expression in ITP-MSCs by TNFAIP3 cDNA can facilitate megakaryocyte differentiation. shRNA-mediated knockdown of SMAD7 expression rescued the impaired MSC function in ITP patients. Therefore, we demonstrate that a pathological reduction in TNFAIP3 levels induced NF-κB/SMAD7 pathway activation, causing a deficiency in MSCs in ITP patients. The ability of ITP-MSCs to support megakaryocytic differentiation and thrombopoiesis of CD34+ cells was impaired.

In vitro large scale production of megakaryocytes to functional platelets from human hematopoietic stem cells.

Megakaryocytopoiesis results in the formation of platelets, which are essential for hemostasis. Decreased production or increased destruction of platelets can cause thrombocytopenia, in which platelet transfusion is the mode of treatment. The present study is aimed in generation of megakaryocytes (MKs) and platelet from human hematopoietic stem cells (HSCs). The purity of HSCs was assessed through Flow cytometry and immunocytochemistry (ICC) studies. These pure HSCs were induced with thrombopoietin (TPO), similarly with Andrographis paniculata extract (APE) for 21 days to generate MKs. The APE is mainly composed of andrographolide which stimulates TPO from the liver, and this binds to CD110 present on the surface of HSCs and triggers the proliferation of HSCs and initiate higher MKs population subsequently, a large number of platelets. The results of the present study showed increased proliferation of HSCs grown in the presence of APE and revealed a high population of CD41a and CD42b positive MKs as enumerated by Flow cytometry compared with TPO induced MKs. These results also concurred with qRT-PCR and western blot analysis. The scanning electron microscopy (SEM) revealed the morphology of differentiated MKs and platelets were similar to human blood platelets. The differentiated MKs in APE exhibited polyploidy up to 32 N while TPO induced MKs showed polyploidy of 8 N, these results corroborated with colony forming unit assay. On thrombin stimulation, high expression of P-selectin (CD62p) and fibrinogen binding were detected in APE induced platelets. Autologous transplantation of platelets generated from APE may be a useful option in thrombocytopenia condition.

N-acetyl-L-cysteine improves bone marrow endothelial progenitor cells in prolonged isolated thrombocytopenia patients post allogeneic hematopoietic stem cell transplantation.

Prolonged isolated thrombocytopenia (PT) is a serious complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). According to murine studies, endothelial progenitor cells (EPCs) play a crucial role in the regulation of hematopoiesis and thrombopoiesis in the bone marrow (BM) microenvironment. We previously showed that the reduced frequency of BM EPCs was an independent risk factor for the occurrence of PT following allo-HSCT. However, the functional role of BM EPCs and methods to improve the impaired BM EPCs in PT patients are unknown. In the current case-control study, we investigated whether the BM EPCs in PT patients differed from those in good graft function patients. Moreover, we evaluated whether N-acetyl-L-cysteine (NAC, a reactive oxygen species [ROS] scavenger) could enhance BM EPCs from PT patients in vitro and in vivo. The PT patients exhibited dysfunctional BM EPCs characterized by high levels of ROS and apoptosis and decreased migration and angiogenesis capabilities. In vitro treatment with NAC improved the quantity and function of the BM EPCs cultivated from the PT patients by downregulating the p38 MAPK pathway and rescued the impaired BM EPCs to support megakaryocytopoiesis. Furthermore, according to the results of a preliminary clinical study, NAC is safe and effective in PT patients. In summary, these results suggested that the reduced and dysfunctional BM EPCs are involved in the occurrence of PT. The defective BM EPCs in the PT patients can be quantitatively and functionally improved by NAC, indicating that NAC is a promising therapeutic approach for PT patients following allo-HSCT.

Dopamine induces platelet production from megakaryocytes via oxidative stress-mediated signaling pathways.

Dopamine (DA), a catecholamine neurotransmitter, is known to for its diverse roles on hematopoiesis, yet its function in thrombopoiesis remains poorly understood. This study shows that DA stimulation can directly induce platelet production from megakaryocytes (MKs) in the final stages of thrombopoiesis via a reactive oxygen species (ROS)-dependent pathway. The mechanism was suggested by the results that DA treatment could significantly elevate the ROS levels in MKs, and time-dependently activate oxidative stress-mediated signaling, including p38 mitogen-activated protein kinase, c-Jun NH2-terminal kinase, and caspase-3 signaling pathways, while the antioxidants N-acetylcysteine and L-glutathione could effectively inhibit the activation of these signaling pathways, as well as the ROS increase and platelet production triggered by DA. Therefore, our data revealed that the direct role and mechanism of DA in thrombopoiesis, which provides new insights into the function recognition of DA in hematopoiesis.

Dual role of IL-21 in megakaryopoiesis and platelet homeostasis.

Gene profiling studies have indicated that in vitro differentiated human megakaryocytes express the receptor for IL-21 (IL-21R), an immunostimulatory cytokine associated with inflammatory disorders and currently under evaluation in cancer therapy. The aim of this study was to investigate whether IL-21 modulates megakaryopoiesis. We first checked the expression of IL-21 receptor on human bone marrow and in vitro differentiated megakaryocytes. We then investigated the effect of IL-21 on the in vitro differentiation of human blood CD34+ progenitors into megakaryocytes. Finally, we analyzed the consequences of hydrodynamic transfection-mediated transient expression of IL-21, on megakaryopoiesis and thrombopoiesis in mice. The IL-21Rα chain was expressed in human bone marrow megakaryocytes and was progressively induced during in vitro differentiation of human peripheral CD34+ progenitors, while the signal transducing γ chain was down-regulated. Consistently, the STAT3 phosphorylation induced by IL-21 diminished during the later stages of megakaryocytic differentiation. In vitro, IL-21 increased the number of colony-forming unit megakaryocytes generated from CD34+ cells and the number of megakaryocytes differentiated from CD34+ progenitors in a JAK3- and STAT3-dependent manner. Forced expression of IL-21 in mice increased the density of bi-potent megakaryocyte progenitors and bone marrow megakaryocytes, and the platelet generation, but increased platelet clearance with a consequent reduction in blood cell counts. Our work suggests that IL-21 regulates megakaryocyte development and platelet homeostasis. Thus, IL-21 may link immune responses to physiological or pathological platelet-dependent processes.

Hyaluronan based hydrogels provide an improved model to study megakaryocyte-matrix interactions.

Hyaluronan (HA) is a glycosamminoglican involved in cell biology as well as a relevant polymer for tissue engineering and regenerative medicine. Megakaryocytes (Mks) are immersed in a mesh of extracellular matrix (ECM) components that regulate their maturation in the bone marrow (BM) and the release of platelets into the bloodstream. While fibrous ECMs such as collagens and fibronectin have been demonstrated to differently regulate Mk function and platelet release, the role of HA, that fills the majority of the BM extracellular interstitial space, has not been investigated so far. Here we demonstrated that, although human Mks express HA receptors, they are not affected by HA in terms of in vitro differentiation, maturation and platelet formation. Importantly, chemical properties of HA were exploited to generate hydrogels with entrapped ECMs that represent a useful model to more closely mimic the tridimensional characteristics of the BM environment for studying Mk function. In conclusion, in this work we demonstrated that HA is an ideal candidate for a 3D ex vivo model of human BM ECM component environment.

Rapamycin and bafilomycin A1 alter autophagy and megakaryopoiesis.

Autophagy is an effective strategy for cell development by recycling cytoplasmic constituents. Genetic deletion of autophagy mediator Atg7 in hematopoietic stem cells (HSCs) can lead to failure of megakaryopoiesis and enhanced autophagy has been implicated in various hematological disorders such as immune thrombocytopenia and myelodysplastic syndrome. Here, we examined the hypothesis that optimal autophagy is essential for megakaryopoiesis and thrombopoiesis by altering autophagy using pharmacological approaches. When autophagy was induced by rapamycin or inhibited by bafilomycin A1 in fetal liver cells, we observed a significant decrease in high ploidy megakaryocytes, a reduction of CD41 and CD61 co-expressing cells, and less proplatelet or platelet formation. Additionally, reduced cell size was shown in megakaryocytes derived from rapamycin, but not bafilomycin A1-treated mouse fetal liver cells. However, when autophagy was altered in mature megakaryocytes, we observed no significant change in proplatelet formation, which was consistent with normal platelet counts, megakaryocyte numbers, and ploidy in Atg7flox/flox PF4-Cre mice with megakaryocyte- and platelet-specific deletion of autophagy-related gene Atg7. Therefore, our findings suggest that either induction or inhibition of autophagy in the early stage of megakaryopoiesis suppresses megakaryopoiesis and thrombopoiesis.