Mature Megakaryocytes Research Articles

Xanthotoxin, a novel inducer of platelet formation, promotes thrombocytopoiesis via IL-1R1 and MEK/ERK signaling

BackgroundThrombocytopenia is a common hematological disease caused by many factors. It usually complicates critical diseases and increases morbidity and mortality. The treatment of thrombocytopenia remains a great challenge in clinical practice, however, its treatment options are limited. In this study, the active monomer xanthotoxin (XAT) was screened out to explore its medicinal value and provide novel therapeutic strategies for the clinical treatment of thrombocytopenia. MethodsThe effects of XAT on megakaryocyte differentiation and maturation were detected by flow cytometry, Giemsa and phalloidin staining. RNA-seq identified differentially expressed genes and enriched pathways. The signaling pathway and transcription factors were verified through WB and immunofluorescence staining. Tg (cd41: eGFP) transgenic zebrafish and mice with thrombocytopenia were used to evaluate the biological activity of XAT on platelet formation and the related hematopoietic organ index in vivo. ResultsXAT promoted the differentiation and maturation of Meg-01 cells in vitro. Meanwhile, XAT could stimulate platelet formation in transgenic zebrafish and recover platelet production and function in irradiation-induced thrombocytopenia mice. Further RNA-seq prediction and WB verification revealed that XAT activates the IL-1R1 target and MEK/ERK signaling pathway, and upregulates the expression of transcription factors related to the hematopoietic lineage to promote megakaryocyte differentiation and platelet formation. ConclusionXAT accelerates megakaryocyte differentiation and maturation to promote platelet production and recovery through triggering IL-1R1 and activating the MEK/ERK signaling pathway, providing a new pharmacotherapy strategy for thrombocytopenia.

Regulation of megakaryo/thrombopoiesis by endosomal toll-like receptor 7 and 8 activation of CD34+ cells in a viral infection model

BackgroundCD34+ cells, megakaryocytes (MKs), and platelets express toll-like receptors (TLRs) that enable these cells to amplify the host innate immune response. However, the role of TLR7/TLR8 activation in megakaryopoiesis has not yet been investigated. ObjectivesWe evaluated the effect of coxsackievirus B3 (CVB3) and synthetic TLR7/TLR8 agonists on the development of human MKs and production of platelets. MethodsCD34+ cells from human umbilical cord were inoculated with CVB3 or stimulated with synthetic TLR7/TLR8 agonists and then cultured in the presence of thrombopoietin. ResultsCD34+ cells, MK progenitor cells, and mature MKs expressed TLR7 and TLR8, and exposure to CVB3 resulted in productive infection, as determined by the presence of viral infectious particles in culture supernatants. Cell expansion, differentiation into MKs, MK maturation, and platelet biogenesis were significantly reduced in CD34+-infected cultures. The reduction in MK growth was not due to an alteration in cellular proliferation but was accompanied by an increase in cellular apoptosis and pyroptosis. Impairment of MK generation and maturation of viable cells were also associated with decreased expression of transcription factors involved in these processes. These effects were completely abrogated by TLR7 but not TLR8 antagonists and mimicked by TLR7 but not TLR8 agonists. CVB3 infection of CD34+ cells increased the immunophenotype of MKs characterized as CD148+/CD48+ or CD41+/CD53+ cells. ConclusionThese data suggest a novel role of TLR7 in megakaryo/thrombopoiesis that may contribute to a better understanding of the molecular basis underlying thrombocytopenia and the immunologic role of MKs in viral infection processes.

Isolation, In Vitro Differentiation, and Culture of Murine Megakaryocytes From Fetal Liver and Adult Bone Marrow

AbstractMegakaryocytes (MKs) are the source of circulating platelets and are readily recognized by their large size and distinctive morphology. Their poor representation in hematopoietic tissues often requires enrichment or considerable ex vivo expansion to generate cells for biochemical and cell biological studies. These experimental protocols describe the enrichment of primary MKs directly from the murine bone marrow as well as in vitro differentiation of fetal liver– or bone marrow–derived hematopoietic stem cells into MKs. Although in vitro–differentiated MKs are not synchronized in their maturation, they can be enriched over an albumin density gradient, and one‐third to one‐half of recovered cells will typically elaborate proplatelets. Support protocols describe methods for preparing fetal liver cells, identifying mature rodent MKs by staining for flow cytometry analysis, and immunofluorescence staining of fixed MKs for confocal laser scanning microscopy. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Isolation of mature bone marrow megakaryocytes by magnetic‐activated cell sortingBasic Protocol 2: Preparation of a megakaryocyte suspension culture from murine fetal livers or lineage‐depleted adult bone marrowSupport Protocol 1: Preparation of a single‐cell suspension from murine fetal livers for megakaryocyte cultureSupport Protocol 2: Megakaryocyte culture from lineage‐depleted murine bone marrowSupport Protocol 3: Quality control of megakaryocyte culture with flow cytometrySupport Protocol 4: Immunofluorescence staining of megakaryocytes for detection with confocal laser scanning microscopy

The intracellular and plasma membrane pools of phosphatidylinositol-4-monophosphate control megakaryocyte maturation and proplatelet formation

BackgroundMegakaryocytes (MKs) develop from hematopoietic stem cells after stimulation by the cytokine thrombopoietin. During megakaryopoiesis, MKs enlarge, undergo the process of endomitosis, and develop intracellular membranes (demarcation membrane system, DMS). During DMS formation, there is active transport of proteins, lipids, and membranes from the Golgi apparatus to the DMS. The most important phosphoinositide that controls anterograde transport from the Golgi apparatus to the plasma membrane (PM) is phosphatidylinositol-4-monophosphate (PI4P), whose levels are controlled by suppressor of actin mutations 1–like protein (Sac1) phosphatase at the Golgi and endoplasmic reticulum. ObjectivesHere we investigated the role of Sac1 and PI4P in megakaryopoiesis. MethodsWe analyzed Sac1 and PI4P localization in primary MKs derived from fetal liver or bone marrow and in the DAMI cell line by immunofluorescence. The intracellular and PM pools of PI4P in primary MKs were modulated by expression of Sac1 constructs from retroviral vector and inhibition of PI4 kinase IIIα, respectively. ResultsWe showed that in primary mouse MKs, PI4P is mostly found in the Golgi apparatus and the PM in immature MKs, while in mature MKs, it is found in the cell periphery and at the PM. The exogenous expression of wild-type but not C389S mutant (catalytically dead) Sac1 results in the perinuclear retention of the Golgi apparatus resembling immature MKs, with decreased ability to form proplatelets. The pharmacologic inhibition of PI4P production specifically at the PM also resulted in a significant decrease in MKs that form proplatelets. ConclusionThese results indicate that both intracellular and PM pools of PI4P mediate MK maturation and proplatelet formation.

Single-cell analysis of megakaryopoiesis in peripheral CD34+ cells: insights into ETV6-related thrombocytopenia

BackgroundGermline mutations in the ETV6 transcription factor gene are responsible for familial thrombocytopenia and leukemia predisposition syndrome. Although previous studies have shown that ETV6 plays an important role in megakaryocyte (MK) maturation and platelet formation, the mechanisms by which ETV6 dysfunction promotes thrombocytopenia remain unclear. ObjectivesTo decipher the transcriptional mechanisms and gene regulatory network linking ETV6 germline mutations and thrombocytopenia. MethodsPresuming that ETV6 mutations result in selective effects at a particular cell stage, we applied single-cell RNA sequencing to understand gene expression changes during megakaryopoiesis in peripheral CD34+ cells from healthy controls and patients with ETV6-related thrombocytopenia. ResultsAnalysis of gene expression and regulon activity revealed distinct clusters partitioned into 7 major cell stages: hematopoietic stem/progenitor cells, common-myeloid progenitors (CMPs), MK-primed CMPs, granulocyte-monocyte progenitors, MK-erythroid progenitors (MEPs), progenitor MKs/mature MKs, and platelet-like particles. We observed a differentiation trajectory in which MEPs developed directly from hematopoietic stem/progenitor cells and bypassed the CMP stage. ETV6 deficiency led to the development of aberrant cells as early as the MEP stage, which intensified at the progenitor MK/mature MK stage, with a highly deregulated core “ribosome biogenesis” pathway. Indeed, increased translation levels have been documented in patient CD34+-derived MKs with overexpression of ribosomal protein S6 and phosphorylated ribosomal protein S6 in both CD34+-derived MKs and platelets. Treatment of patient MKs with the ribosomal biogenesis inhibitor CX-5461 resulted in an increase in platelet-like particles. ConclusionThese findings provide novel insight into both megakaryopoiesis and the link among ETV6, translation, and platelet production.

Ribosome dysfunction underlies SLFN14-related thrombocytopenia

AbstractPathogenic missense variants in SLFN14, which encode an RNA endoribonuclease protein that regulates ribosomal RNA (rRNA) degradation, are known to cause inherited thrombocytopenia (TP) with impaired platelet aggregation and adenosine triphosphate secretion. Despite mild laboratory defects, the patients displayed an obvious bleeding phenotype. However, the function of SLFN14 in megakaryocyte (MK) and platelet biology remains unknown. This study aimed to model the disease in an immortalized MK cell line (imMKCL) and to characterize the platelet transcriptome in patients with the SLFN14 K219N variant. MK derived from heterozygous and homozygous SLFN14 K219N imMKCL and stem cells of blood from patients mainly presented with a defect in proplatelet formation and mitochondrial organization. SLFN14-defective platelets and mature MK showed signs of rRNA degradation; however, this was absent in undifferentiated imMKCL cells and granulocytes. Total platelet RNA was sequenced in 2 patients and 19 healthy controls. Differential gene expression analysis yielded 2999 and 2888 significantly (|log2 fold change| >1, false discovery rate <0.05) up- and downregulated genes, respectively. Remarkably, these downregulated genes were not enriched in any biological pathway, whereas upregulated genes were enriched in pathways involved in (mitochondrial) translation and transcription, with a significant upregulation of 134 ribosomal protein genes (RPGs). The upregulation of mitochondrial RPGs through increased mammalian target of rapamycin complex 1 (mTORC1) signaling in SLFN14 K219N MK seems to be a compensatory response to rRNA degradation. mTORC1 inhibition with rapamycin resulted in further enhanced rRNA degradation in SLFN14 K219N MK. Taken together, our study indicates dysregulation of mTORC1 coordinated ribosomal biogenesis is the disease mechanism for SLFN14-related TP.

Molecular analysis of phenotypic heterogeneity in JAK2V617F-positive myeloproliferative neoplasms reveals a potential target for therapy.

JAK2V617F is the most frequent mutation in BCR-ABL-negative myeloproliferative neoplasms (MPNs). It is an important but not the only determinant of MPN phenotype. We performed high-throughput sequencing on JAK2V617F+ essential thrombocythaemia (ET) and polycythaemia vera (PV) patient samples to unveil factors involved in phenotypic heterogeneity and to identify novel therapeutic targets for MPN. Two concurrent mutations that may affect phenotype were identified, including mutations in SH2B3, which is primarily prevalent in PV, and SF3B1, which is more commonly mutated in ET. Next, we conducted transcriptomic analysis at the haematopoietic stem cell (HSC) and megakaryocyte (MK)-erythroid progenitor (MEP) levels. Inflammatory signalling pathways were elevated in both ET HSCs and MEPs, unlike in PV HSCs and MEPs. Notably, Wnt/β-catenin signalling was uniquely upregulated during ET haematopoietic differentiation from HSC to MEP, and inhibiting Wnt/β-catenin signalling blocked MK differentiation in vitro. Consistently, Wnt/β-catenin inhibitor administration decreased platelet counts in JAK2V617F+ MPN mice by blocking MEPs and MK progenitors and by inhibiting maturation of MKs, while in wild-type mice, Wnt/β-catenin inhibitor did not significantly reduce platelet counts. In conclusion, our findings provide new insights into the mechanisms underlying phenotypic differentiation of JAK2V617F+ PV and ET and indicate Wnt/β-catenin signalling as a potential therapeutic target for MPN.

The role of CREG1 in megakaryocyte maturation and thrombocytopoiesis.

Abnormal megakaryocyte maturation and platelet production lead to platelet-related diseases and impact the dynamic balance between hemostasis and bleeding. Cellular repressor of E1A-stimulated gene 1 (CREG1) is a glycoprotein that promotes tissue differentiation. However, its role in megakaryocytes remains unclear. In this study, we found that CREG1 protein is expressed in platelets and megakaryocytes and was decreased in the platelets of patients with thrombocytopenia. A cytosine arabinoside-induced thrombocytopenia mouse model was established, and the mRNA and protein expression levels of CREG1 were found to be reduced in megakaryocytes. We established megakaryocyte/platelet conditional knockout (Creg1pf4-cre) and transgenic mice (tg-Creg1). Compared to Creg1fl/fl mice, Creg1pf4-cre mice exhibited thrombocytopenia, which was mainly caused by inefficient bone marrow (BM) thrombocytopoiesis, but not by apoptosis of circulating platelets. Cultured Creg1pf4-cre-megakaryocytes exhibited impairment of the actin cytoskeleton, with less filamentous actin, significantly fewer proplatelets, and lower ploidy. CREG1 directly interacts with MEK1/2 and promotes MEK1/2 phosphorylation. Thus, our study uncovered the role of CREG1 in the regulation of megakaryocyte maturation and thrombopoiesis, and it provides a possible theoretical basis for the prevention and treatment of thrombocytopenia.

Laboratory Markers of Platelet Production and Turnover

Platelets are formed from bone marrow megakaryocytes, circulate in blood for 7-10 days, and then are destroyed in the spleen and/or liver. Platelet production depends on the megakaryocyte population state in the bone marrow: number and size of the cells. The platelet turnover, i.e., the number of platelets passing through the bloodstream in a certain time, is determined by both the rate of their production and the rate of their destruction. The review considers laboratory markers, which are used to assess platelet production and turnover in the patients with hematologic and cardiovascular pathologies. These markers include some characteristics of platelets themselves: (i) content of reticulated ("young") forms in the blood detected by their staining with RNA dyes; (ii) indicators of the platelet size determined in hematology analyzers (mean volume, percentage of large forms) and in flow cytometers (light scattering level). Alterations of platelet production and turnover lead to the changes in blood plasma concentrations of such molecules as thrombopoietin (TPO, main mediator of megakaryocyte maturation and platelet formation in the bone marrow) and glycocalicin (soluble fragment of the membrane glycoprotein Ib detached from the surface of platelets during their destruction). Specific changes in the markers of platelet production and turnover have been observed in: (i) hypoproductive thrombocytopenias caused by suppression of megakaryocytes in the bone marrow; (ii) immune thrombocytopenias caused by accelerated clearance of the autoantibody-sensitized platelets; and (iii) thrombocytosis (both primary and reactive). The paper presents the data indicating that in patients with cardiovascular diseases an increased platelet turnover and changes in the corresponding markers (platelet size indexes and content of reticulated forms) are associated with the decreased efficacy of antiplatelet drugs and increased risk of thrombotic events, myocardial infarction, and unstable angina (acute coronary syndrome).

Dual specificity tyrosine phosphorylation regulated kinase 1B inhibition promotes megakaryocyte polyploidization and platelet production.

Platelets are produced from mature megakaryocytes which undergo polyploidization and proplatelet formation. Cell cycle regulation plays a crucial role in megakaryocyte terminal differentiation especially in polyploidization. Dual specificity tyrosine phosphorylation regulated kinase 1B (DYRK1B) controls cell cycle progression in cancer cells. The objective of this study was to determine DYRK1B function in megakaryocyte maturation and platelet production. A DYRK1B knock out mouse (DYKO) was generated with increased peripheral platelet count compared to the wild type (WT) mouse without affecting megakaryocyte numbers in bone marrow. Polyploidy and proplatelet formation were significantly enhanced when DYRK1B was depleted in vitro. DYRK1B inhibition promoted megakaryocyte maturation by simultaneously upregulating cyclin D1 and downregulating P27. Furthermore, there was platelet restoration in two mice disease models of transient thrombocytopenia. In summary, DYRK1B plays an important role in megakaryocyte maturation and platelet production by interacting with cyclin D1 and P27. DYRK1B inhibition has potential therapeutic value in transient thrombocytopenia treatment.

Novel chemical-structure TPOR agonist, TMEA, promotes megakaryocytes differentiation and thrombopoiesis via mTOR and ERK signalings

BackgroundNon-peptide thrombopoietin receptor (TPOR) agonists are promising therapies for the mitigation and treatment of thrombocytopenia. However, only few agents are available as safe and effective for stimulating platelet production for thrombocytopenic patients in the clinic. PurposeThis study aimed to develop a novel small molecule TPOR agonist and investigate its underlying regulation of function in megakaryocytes (MKs) differentiation and thrombopoiesis. MethodsA potential active compound that promotes MKs differentiation and thrombopoiesis was obtained by machine learning (ML). Meanwhile, the effect was verified in zebrafish model, HEL and Meg-01 cells. Next, the key regulatory target was identified by Drug Affinity Responsive Target Stabilization Assay (DARTS), Cellular Thermal Shift Assay (CETSA), and molecular simulation experiments. After that, RNA-sequencing (RNA-seq) was used to further confirm the associated pathways and evaluate the gene expression induced during MK differentiation. In vivo, irradiation (IR) mice, C57BL/6N-TPORem1cyagen (Tpor-/-) mice were constructed by CRISPR/Cas9 technology to examine the therapeutic effect of TMEA on thrombocytopenia. ResultsA natural chemical-structure small molecule TMEA was predicted to be a potential active compound based on ML. Obvious phenotypes of MKs differentiation were observed by TMEA induction in zebrafish model and TMEA could increase co-expression of CD41/CD42b, DNA content, and promote polyploidization and maturation of MKs in HEL and Meg-01 cells. Mechanically, TMEA could bind with TPOR protein and further regulate the PI3K/AKT/mTOR/P70S6K and MEK/ERK signal pathways. In vivo, TMEA evidently promoted platelet regeneration in mice with radiation-induced thrombocytopenia but had no effect on Tpor-/- and C57BL/6 (WT) mice. ConclusionTMEA could serve as a novel TPOR agonist to promote MKs differentiation and thrombopoiesis via mTOR and ERK signaling and could potentially be created as a promising new drug to treat thrombocytopenia.

Histone deacetylation-regulated cell surface Siglec-7 expression promoted megakaryocytic maturation and enhanced platelet-like particle release

BackgroundFunctioning as important hematologic cells for hemostasis, wound healing and immune defense platelets are produced before being released into the blood by cytoplasmic fragmentation at the end of the megakaryocyte (MK) differentiation, during which the involvement of both apoptosis and autophagy has been reported. Inhibitory sialic acid–binding immunoglobulin-like lectin-7 gene (Siglec-7) can be expressed on platelets and induce apoptosis on activation for uncharacterized function. ObjectiveWe aimed to investigate the regulatory mechanism for Siglec-7 activation along MK differentiation and its physiologic role during the MK maturation and platelet formation. MethodsBy using 2 well-established MK differentiation models (HEL and K562) and human primary CD34+ cell, we examined the upregulations of transcript and protein levels of Siglec-7 during MK differentiation, and the effect of Siglec-7 surface presence on MK differentiation and platelet-like particles (PLPs) release. ResultsWe show that both transcripts and surface Siglec-7 were elevated during MK differentiation, and the histone deacetylase 1 (HDAC1) acted as a negative regulator for Siglec-7 activation. By increasing Siglec-7 surface expression, we found that increased presence of Siglec-7 not only enhanced MK maturation but also the release of PLPs by activating caspase 3-dependent signaling, as evidenced in the observation of more CD41, polyploidy, and platelet factor 4 transcript formations. ConclusionIn this study, we demonstrated that Siglec-7 activation was subjected to epigenetic regulation, and the resulting induced expression of surface Siglec-7 played an important regulatory role in promoting MK differentiation, maturation, and PLP formation.

Akt-mediated mitochondrial metabolism regulates proplatelet formation and platelet shedding post vasopressin exposure

BackgroundPlatelet shedding from mature megakaryocytes (MKs) in thrombopoiesis is the critical step for elevating circulating platelets fast and efficiently, however, the underlying mechanism is still not well-illustrated, and the therapeutic targets and candidates are even less. ObjectivesIn order to investigate the mechanisms for platelet shedding after vasopressin treatment and find new therapeutic targets for thrombocytopenia. MethodsPlatelet production was evaluated both in vivo and in vitro after arginine vasopressin (AVP) administration. The underlying biological mechanism of AVP-triggered thrombopoiesis were then investigated by a series of molecular and bioinformatics techniques. Resultsit is observed that proplatelet formation and platelet shedding in the final stages of thrombopoiesis promoted by AVP, an endogenous hormone, can quickly increases peripheral platelets. This rapid elevation is thus able to speed up platelet recovery after radiation as expected. The mechanism analysis reveal that proplatelet formation and platelet release from mature MKs facilitated by AVP is mainly mediated by Akt-regulated mitochondrial metabolism. In particular, phosphorylated Akt regulates mitochondrial metabolism through driving the association of hexokinase-2 with mitochondrial voltage dependent anion channel-1 in AVP-mediated thrombopoiesis. Further studies suggest that this interaction is stabilized by IκBα, the expression of which is controlled by insulin-regulated membrane aminopeptidase. Conclusionthese data demonstrate that phosphorylated Akt-mediated mitochondrial metabolism regulates platelet shedding from MKs in response to AVP, which will provide new therapeutic targets and further drug discovery clues for thrombocytopenia treatment.

Reduced platelet glycoprotein Ibα shedding accelerates thrombopoiesis and COX-1 recovery: implications for aspirin dosing regimen.

Cardiovascular (CV) disease prevention with low-dose aspirin can be less effective in patients with a faster recovery of platelet (PLT) cyclooxygenase (COX)-1 activity during the 24-hour dosing interval. We previously showed that incomplete suppression of TXA2 over 24 hours can be rescued by a twice daily aspirin regimen. Here we show that reduced PLT glycoprotein (GP)Ibα shedding characterizes patients with accelerated COX-1 recovery and may contribute to higher thrombopoietin (TPO) production and higher rates of newly formed PLT, escaping aspirin inhibition over 24 hours. Two hundred aspirin-treated patients with high CV risk (100 with type 2 diabetes mellitus) were stratified according to the kinetics of PLT COX-1 activity recovery during the 10- to 24-hour dosing interval. Whole proteome analysis showed that PLT from patients with accelerated COX-1 recovery were enriched in proteins involved in cell survival, inhibition of apoptosis and cellular protrusion formation. In agreement, we documented increased plasma TPO, megakaryocyte maturation and proplatelet formation, and conversely increased PLT galactose and reduced caspase 3, phosphatidylserine exposure and ADAM17 activation, translating into diminished GPIbα cleavage and glycocalicin (GC) release. Treatment of HepG2 cells with recombinant GC led to a dose-dependent reduction of TPO mRNA in the liver, suggesting that reduced GPIbα ectodomain shedding may unleash thrombopoiesis. A cluster of clinical markers, including younger age, non-alcoholic fatty liver disease, visceral obesity and higher TPO/GC ratio, predicted with significant accuracy the likelihood of faster COX-1 recovery and suboptimal aspirin response. Circulating TPO/GC ratio, reflecting a dysregulation of PLT lifespan and production, may provide a simple tool to identify patients amenable to more frequent aspirin daily dosing.

Targeting TLR2/Rac1/cdc42/JNK Pathway to Reveal That Ruxolitinib Promotes Thrombocytopoiesis.

Thrombocytopenia has long been considered an important complication of chemotherapy and radiotherapy, which severely limits the effectiveness of cancer treatment and the overall survival of patients. However, clinical treatment options are extremely limited so far. Ruxolitinib is a potential candidate. The impact of ruxolitinib on the differentiation and maturation of K562 and Meg-01 cells megakaryocytes (MKs) was examined by flow cytometry, Giemsa and Phalloidin staining. A mouse model of radiation-injured thrombocytopenia (RIT) was employed to evaluate the action of ruxolitinib on thrombocytopoiesis. Network pharmacology, molecular docking, drug affinity responsive target stability assay (DARTS), RNA sequencing, protein blotting and immunofluorescence analysis were applied to explore the targets and mechanisms of action of ruxolitinib. Ruxolitinib can stimulate MK differentiation and maturation in a dose-dependent manner and accelerates recovery of MKs and thrombocytopoiesis in RIT mice. Biological targeting analysis showed that ruxolitinib binds directly to Toll Like Receptor 2 (TLR2) to activate Rac1/cdc42/JNK, and this action was shown to be blocked by C29, a specific inhibitor of TLR2. Ruxolitinib was first identified to facilitate MK differentiation and thrombocytopoiesis, which may alleviate RIT. The potential mechanism of ruxolitinib was to promote MK differentiation via activating the Rac1/cdc42/JNK pathway through binding to TLR2.

Mammalian STE20-like kinase-1/2 are activated in human platelets stimulated by collagen or thrombin and play a vital role in collagen-activated platelets

IntroductionMammalian ste20-like kinases-1/2 (MST1/2), the core kinases of the Hippo pathway, play critical roles in the biology of hematopoietic cells via noncanonical mechanisms and contributes to megakaryocyte differentiation, polyploidization, and maturation to produce platelets. However, the role of MST1/2 in platelet functions remains unclear. Materials and methodsIn this study, we investigated this topic by determining platelet aggregation and through flow cytometry, ATP release assay, clot retraction assay, and immunoblotting analysis. ResultsWe found that MST1/2 were rapidly phosphorylated and activated upon platelet stimulation by thrombin and collagen. XMU-MP-1, a specific inhibitor of MST1/2, blocks the activation of MST1/2 in platelets. Inhibitor-pretreated platelets showed impaired platelet aggregation and dense-granule secretion mediated by collagen, thrombin, and U46619, whereas ristocetin or ADP mediated platelet aggregation was unaffected by XMU-MP-1. Although platelet-mediated clot retraction was not affected by MST1/2 inhibitors, integrin αIIbβ3 activation was significantly attenuated in XMU-MP-1-treated platelets. Moreover, MST1/2 inhibition significantly attenuated the mobilization of platelet calcium ions and the secretion of α-granules induced by convulxin. ConclusionsThis study is the first to demonstrate that MST1/2 play vital roles in human platelets and contributes to collagen-induced platelet activation and aggregation.

Bifunctional effect of the inflammatory cytokine tumor necrosis factor α on megakaryopoiesis and platelet production

Background and ObjectivesPlatelets are affected by many factors, such as infectious or aseptic inflammation, and different inflammatory states may induce either thrombocytopenia or thrombocytosis. Tumor necrosis factor α (TNFα) is an important inflammatory cytokine that has been shown to affect the activity of hematopoietic stem cells. However, its role in megakaryocyte (MK) development and platelet production remains largely unknown. This study aimed to investigate the effects of TNFα on MK and platelet generation. Methods and ResultsThe ex vivo study with human CD34+ cells demonstrated that TNFα differentially modulated commitment toward the MK lineage. Specifically, a low concentration of 0.5 ng/ml TNFα promoted MK maturation, proplatelet formation, and platelet production, whereas a high concentration of 10 ng/ml or more TNFα exhibited a substantial inhibitory effect on MK and platelet production. The distinct effect of TNFα on MKs was mainly dependent on TNFα receptor 1. TNFα differentially regulated the MAPK–ERK1/2 signaling pathway and the cytoskeletal proteins cofilin and MLC2. The in vivo study with Balb/c mice indicated that low‐dose or high‐dose TNFα administration differentially affected short‐term platelet recovery after bone marrow transplantation. ConclusionsOur study revealed distinct roles for TNFα in megakaryopoiesis and thrombopoiesis and may provide new insights regarding the treatment for platelet disorders.

Insights into Regulatory Factors in Megakaryocyte Development and Function: Basic Mechanisms and Potential Targets.

Platelets are small, anucleate cellular fragments, which are produced by megakaryocytes, and play a key role in hemostasis and thrombus formation. The differentiation of megakaryocytes from hematopoietic stem cells in bone marrow and the development of megakaryocytes into platelets is a complex process. Various regulatory factorsin megakaryopoiesis including cytokines, growth factors, transcription factors, and gene expression, are all involved in the process of thrombocytopoiesis and play distinct roles in different stages of megakaryocytes development. In this review, we summarize the current state of knowledge ofmultiple regulatory factors including the TPO/Mpl signaling pathway, transcription factors, RasGTPases family, estrogen, and microRNAs. Altogether, we aimed to discuss more molecular mechanisms of megakaryocytes differentiation and maturation, and possess a better understanding of platelet formation.

Investigating ITP Plasma for Effect on Platelet Production Using the Autologous Peripheral Blood Based Megakaryopoiesis Assay

Investigating ITP plasma for effect on platelet production using the autologous peripheral blood based megakaryopoiesis assay Nikola Ivetic, Ishac Nazy, Rumi Claire, Angela Huynh John G. Kelton, Donald M. Arnold. Background: Studies focused on megakaryocyte number and function are needed to understand platelet underproduction as a potential mechanism of immune thrombocytopenia (ITP). Most experimental studies have relied on animal models, specialized cell lines, or exogenous and non-autologous sources of hematopoietic stem and progenitor cells (HSPCs) as a source of megakaryocytic cells and platelet production. We used HSPCs isolated from ITP patients themselves to examine the effects their plasma has megakaryocyte growth and platelet production. Study Design and Methods: Megakaryocytes were produced from peripheral blood CD34+ HSCs directly from patients with ITP and healthy controls. HSPC (CD45+ CD34+) were isolated, expanded for 4 days and then cultured with thrombopoietin (TPO) and stem cell factor (SCF) for 8 days. Megakaryocyte numbers were evaluated by flow cytometry using CD41a expression for megakaryopoiesis and CD42b expression for maturation. Platelet production was analyzed using CD41a expression and platelet forward and side scatter profile sizes. The addition of autologous ITP plasma and plasma depleted of IgG were also tested to determine the effects of antibodies on megakaryopoiesis and thrombopoiesis. Results: Peripheral blood derived megakaryocytes were cultured from HSPCs from ITP patients (N=10), and normal controls (N=9). Approximately 2,200±1,100 hematopoietic stem and progenitor cells were isolated per ml of peripheral blood and a 5-fold increase in stem cells was obtained after an expansion of the isolated ITP patient HSPC population. Following an additional 8 days of culture with TPO, 67±12% of the cultured cells were CD41a+ indicating they were undergoing megakaryopoiesis and 54±15% of the CD41a+ population expressed the CD42b marker for mature megakaryocytes. Of the 10 ITP cultures grown, four grew significantly more mature megakaryocytes in comparison to healthy controls. The addition of ITP plasma did not inhibit megakaryocyte growth or maturity, but platelet production was reduced with two samples demonstrating a 50% reduction in platelet levels in comparison to the . Relative to the number of mature megakaryocytes cultured there was a lower number of platelets detected in ITP cultures (3.34±1.9 platelets per mature megakaryocyte) in comparison to the healthy control group (8.1±1.4 platelets per mature megakaryocyte). Conclusions: The addition of patient plasma had no inhibiting effect on megakaryocyte counts in an autologous assay but a significant decrease in platelet number (thrombopoiesis) was detected. A subset of ITP HSPC generated more megakaryocytes when compared to healthy donors, indicating these cells are generating more megakaryocytes. This is to our knowledge the first autologous investigation of ITP plasma on patient HSPC undergoing megakaryopoiesis.

Sitagliptin Alleviates Excessive Autophagy in Macrophages in Immune Thrombocytopenia Patients to Promote Megakaryopoiesis Via the AMPK/mTOR Pathway

Introduction: The pathogenesis of ITP is complex and mainly manifests as autoantibody-mediated platelet destruction and impaired platelet production. Autophagy is a highly conserved cellular process that eliminates damaged organelles and promotes the degradation of misfolded proteins. Appropriate autophagy exerts a protective effect, while excessive autophagy may lead to cell death and increased inflammation. The specific role of bone marrow macrophages (BM MΦ) in regulating megakaryopoiesis in ITP patients has not been elucidated. Sitagliptin, a DPP-4 inhibitor, was reported to play a key role in the production and engraftment of hematopoietic cells by modulating SDF-1α and several colony-stimulating factors, thus enhancing hematopoietic stem and hematopoietic progenitor cell recovery. However, whether sitagliptin has the potential to promote megakaryocyte maturation and platelet production in ITP is unclear. Therefore, this article explores the effect of macrophage autophagy on megakaryopoiesis in ITP patients and investigates its underlying role in the pathogenesis of ITP in order to explore whether sitagliptin could be a potential treatment for ITP. Methods: BM MΦ and CD34+ cells were isolated from ITP patients and healthy control subjects. Excessive autophagy in bone marrow macrophages from ITP patients was confirmed by Western blotting and transmission electron microscopy. PCR and Western blotting were used to assess the expression of A20 and autophagy-related AMPK/mTOR/ULK1 pathway components in ITP-MΦ. Vectors carrying A20 cDNA/TNFAIP3-specific shRNA were transduced into the BM MΦ from either healthy control subjects or ITP patients. The different BM MΦ populations were separately cocultured with CD34+ cells. We compared the ability of BM MΦ to support megakaryocytopoiesis by determining the relative percentages of MKs at different stages using flow cytometry as well as CFU counts of megakaryocytes and platelets in culture. The mechanism by which sitagliptin normalized the impaired function of ITP-MΦ was evaluated in vitro. A prospective single-arm clinical cohort study was designed to explore the efficacy and safety of sitagliptin in the treatment of corticosteroid-resistant or relapsed ITP. Results: The expression of LC3-II/LC3-I and Beclin1 increased in ITP-MΦ, and the expression of P62 decreased. The number of autophagic vesicles also increased, indicating enhanced autophagy in ITP-MΦ. Transcriptome sequencing and untargeted metabolomics revealed that macrophage autophagy-related pathways were involved in the pathogenesis of ITP. Further studies confirmed that ITP-MΦ regulated megakaryopoiesis, which was dependent on the A20-mediated autophagy-related AMPK/mTOR/ULK1 pathway. Inhibition of A20 expression with A20-shRNA inhibited the AMPK/mTOR/ULK1 pathway and alleviated excessive autophagy in ITP-MΦ, thus increasing the number of megakaryocytes, the proportion of hyperploid megakaryocytes and the number of platelets produced in the ITP-MΦ coculture system. Inhibition of autophagy with the autophagy inhibitor 3-MA reversed the A20 overexpression-induced excessive autophagy in MΦ and normalized the differentiation and maturation of megakaryocytes. Inhibiting the AMPK/mTOR/ULK1 signaling pathway with the AMPK inhibitor Compound C reversed the A20 overexpression-induced increase in the expression of AMPK/mTOR/ULK1 autophagy pathway components in MΦ and normalized megakaryopoiesis. Sitagliptin was found to inhibit the expression of A20 and AMPK/mTOR/ULK1 autophagy pathway components in ITP-MΦ in vitro. Sitagliptin improved the ability of ITP-MΦ to promote the differentiation and development of megakaryocytes and platelet production. Sitagliptin was administered to a total of 56 patients with corticosteroid-resistant or relapsed ITP. Complete response and overall response were observed in 11 (19.6%) and 21 patients (37.5%), respectively. Conclusions: A20 mediated the AMPK/mTOR/ULK1 signaling pathway to regulate autophagy in ITP-MΦ and affected megakaryopoiesis and platelet formation. Sitagliptin could restore the proper function of the A20-mediated AMPK/mTOR/ULK1 signaling pathway in ITP-MΦ, thus promoting megakaryopoiesis. Sitagliptin resulted in a sustained response in patients with corticosteroid-resistant/recurrent ITP and exhibited good safety. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal