A circle of life: platelet and megakaryocyte cytoskeleton dynamics in health and disease.

Platelets are blood cells derived from megakaryocytes that play a central role in regulating haemostasis and vascular integrity. The microtubule cytoskeleton of megakaryocytes undergoes a critical dynamic reorganization during cycles of endomitosis and platelet biogenesis. Quiescent platelets have a discoid shape maintained by a marginal band composed of microtubule bundles, which undergoes remarkable remodelling during platelet activation, driving shape change and platelet function. Disrupting or enhancing this process can cause platelet dysfunction such as bleeding disorders or thrombosis. However, little is known about the molecular mechanisms underlying the reorganization of the cytoskeleton in the platelet lineage. Recent studies indicate that the emergence of a unique platelet tubulin code and specific pathogenic tubulin mutations cause platelet defects and bleeding disorders. Frequently, these mutations exhibit dominant negative effects, offering valuable insights into both platelet disease mechanisms and the functioning of tubulins. This review will highlight our current understanding of the role of the microtubule cytoskeleton in the life and death of platelets, along with its relevance to platelet disorders.

SDF-1 promotes metastasis of NSCLC by enhancing chemoattraction of megakaryocytes through the PI3K/Akt signaling pathway.

Lung cancer (LC) is the leading cause of cancer-associated deaths worldwide, among which non-small-cell lung cancer (NSCLC) accounts for 80%. Stromal cell-derived factor-1 (SDF-1) inhibition results in a significant depletion of NSCLC metastasis. Additionally, SDF-1 is the only natural chemokine known to bind and activate the receptor CXCR4. Thus, we attempted to clarify the molecular mechanism of SDF-1 underlying NSCLC progression. Transwell migration, adhesion, and G-LISA assays were used to assess megakaryocytic chemotaxis in vitro and in vivo in terms of megakaryocytic migration, adherence, and RhoA activation, respectively. Western blotting was used to assess PI3K/Akt-associated protein abundances in MEG-01 cells and primary megakaryocytes under the indicated treatment. A hematology analyzer and flow cytometry were used to assess platelet counts in peripheral blood and newly formed platelet counts in Lewis LC mice under different treatments. Immunochemistry and flow cytometry were used to measure CD41+ megakaryocyte numbers in Lewis LC mouse tissue under different treatments. ELISA was used to measure serum TPO levels, and H&E staining was used to detect NSCLC metastasis.SDF-1 receptor knockdown suppressed megakaryocytic chemotaxis in Lewis LC mice. SDF-1 receptor inhibition suppressed megakaryocytic chemotaxis via the PI3K/Akt pathway. SDF-1 receptor knockdown suppressed CD41+ megakaryocyte numbers in vivo through PI3K/Akt signaling. SDF-1 receptor inhibition suppressed CD41+ megakaryocytes to hinder NSCLC metastasis. SDF-1 facilitates NSCLC metastasis by enhancing the chemoattraction of megakaryocytes via the PI3K/Akt signaling pathway, which may provide a potential new direction for seeking therapeutic plans for NSCLC.

The Role of TLR-2 in Lethal COVID-19 Disease Involving Medullary and Resident Lung Megakaryocyte Up-Regulation in the Microthrombosis Mechanism.

Patients with COVID-19 have coagulation and platelet disorders, with platelet alterations and thrombocytopenia representing negative prognostic parameters associated with severe forms of the disease and increased lethality. METHODS: The aim of this study was to study the expression of platelet glycoprotein IIIa (CD61), playing a critical role in platelet aggregation, together with TRL-2 as a marker of innate immune activation. RESULTS: A total of 25 patients were investigated, with the majority (24/25, 96%) having co-morbidities and dying from a fatal form of SARS-CoV-2(+) infection (COVID-19+), with 13 men and 12 females ranging in age from 45 to 80 years. When compared to a control group of SARS-CoV-2 (-) negative lungs (COVID-19-), TLR-2 expression was up-regulated in a subset of patients with deadly COVID-19 fatal lung illness. The proportion of Spike-1 (+) patients found by PCR and ISH correlates to the proportion of Spike-S1-positive cases as detected by digital pathology examination. Furthermore, CD61 expression was considerably higher in the lungs of deceased patients. In conclusion, we demonstrate that innate immune prolonged hyperactivation is related to platelet/megakaryocyte over-expression in the lung. CONCLUSIONS: Microthrombosis in deadly COVID-19+ lung disease is associated with an increase in the number of CD61+ platelets and megakaryocytes in the pulmonary interstitium, as well as their functional activation; this phenomenon is associated with increased expression of innate immunity TLR2+ cells, which binds the SARS-CoV-2 E protein, and significantly with the persistence of the Spike-S1 viral sequence.

Mapping genotypes to chromatin accessibility profiles in single cells.

In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

Decreasing circ_0014614 promotes the differentiation of bone marrow flineage cells into megakaryocytes in essential thrombocythemia via activiation of miR-138-5p/caspase3 axis.

BACKGROUND: Circular RNAs (circRNA) are pivotal in hematological diseases. Previous study showed that circ_0014614 (circDAP3) was significantly underexpressed in bone marrow-derived exosomes from essential thrombocythemia (ET) patients, affecting the differentiation of bone marrow lineage cells into megakaryocytes. METHODS: Fluorescence in situ hybridization (FISH) was used to display circ_0014614's primary cytoplasmic location in K562 cells. Cytoscape software was used to predict the circRNA-miRNA-mRNA networks, and their expression at the cellular level was detected by Quantitative reverse transcription-polymerase chain reaction (qRT-PCR). qRT-PCR was utilized to detect the expression levels of circ_0014614，miR-138-5p and caspase3 mRNA. Western blot was used to determine the protein levels of GATA-1, RUNX-1, NF-E2, CD41 and caspase3. The proliferation of K562 cells was assessed using the Cell Counting Kit-8 (CCK-8) Assay. Furthermore, the interplay between miR-138-5p and circ_0014614 or caspase3 was elucidated through a Dual-luciferase reporter assay. RESULTS: FISH assay indicated circ_0014614's primary cytoplasmic location in K562 cells. In ET bone marrow and K562 cells, circ_0014614 and caspase3 were down-regulated, whereas miR-138-5p saw a significant surge. Overexpressing circ_0014614 curtailed K562 cells' proliferation and differentiation. Further, circ_0014614 targeted miR-138-5p, with heightened miR-138-5p levels counteracting circ_0014614's inhibition. MiR-138-5p further targeted caspase3, and caspase3 silencing neutralized suppressed miR-138-5p's effects on K562 cell differentiation. CONCLUSION: Circ_0014614 was down-regulated in ET bone marrow and bone marrow lineage cells, and upregulating circ_0014614 can inhibit bone marrow lineage cells' proliferation and differentiation into megakaryocytes. Mechanistically, circ_0014614 functioned as ceRNA via sponging miR-138-5p and alleviated the inhibitory effect of miR-138-5p on its target caspase3, which potentially deters tumor activity in ET.

Daucosterol regulates JAK2-STAT3 signaling pathway to promote megakaryocyte differentiation.

Immune thrombocytopenia (ITP) is an autoimmune disease caused by the loss of immune tolerance to platelet autoantigens, resulting in reduced platelet production and increased platelet destruction. Impaired megakaryocyte differentiation and maturation is a key factor in the pathogenesis and treatment of ITP. Sarcandra glabra, a plant of the Chloranthaceae family, is commonly used in clinical practice to treat ITP, and daucosterol (Dau) is one of its active ingredients. However, whether Dau can treat ITP and the key mechanism of its effect are still unclear. In this study, we found that Dau could effectively promote the differentiation and maturation of megakaryocytes and the formation of polyploidy in the megakaryocyte differentiation disorder model constructed by co-culturing Dami and HS-5 cells. In vivo experiments showed that Dau could not only increase the number of polyploidized megakaryocytes in the ITP rat model, but also promote the recovery of platelet count. In addition, through network pharmacology analysis, we speculated that the JAK2-STAT3 signaling pathway might be involved in the process of Dau promoting megakaryocyte differentiation. Western blot results showed that Dau inhibited the expression of P-JAK2 and P-STAT3. In summary, these results provide a basis for further studying the pharmacological mechanism of Dau in treating ITP.

Megakaryocytic IGF1 coordinates activation and ferroptosis to safeguard hematopoietic stem cell regeneration after radiation injury.

BACKGROUND: Hematopoietic stem cell (HSC) regeneration underlies hematopoietic recovery from myelosuppression, which is a life-threatening side effect of cytotoxicity. HSC niche is profoundly disrupted after myelosuppressive injury, while if and how the niche is reshaped and regulates HSC regeneration are poorly understood. METHODS: A mouse model of radiation injury-induced myelosuppression was built by exposing mice to a sublethal dose of ionizing radiation. The dynamic changes in the number, distribution and functionality of HSCs and megakaryocytes were determined by flow cytometry, immunofluorescence, colony assay and bone marrow transplantation, in combination with transcriptomic analysis. The communication between HSCs and megakaryocytes was determined using a coculture system and adoptive transfer. The signaling mechanism was investigated both in vivo and in vitro, and was consolidated using megakaryocyte-specific knockout mice and transgenic mice. RESULTS: Megakaryocytes become a predominant component of HSC niche and localize closer to HSCs after radiation injury. Meanwhile, transient insulin-like growth factor 1 (IGF1) hypersecretion is predominantly provoked in megakaryocytes after radiation injury, whereas HSCs regenerate paralleling megakaryocytic IGF1 hypersecretion. Mechanistically, HSCs are particularly susceptible to megakaryocytic IGF1 hypersecretion, and mTOR downstream of IGF1 signaling not only promotes activation including proliferation and mitochondrial oxidative metabolism of HSCs, but also inhibits ferritinophagy to restrict HSC ferroptosis. Consequently, the delicate coordination between proliferation, mitochondrial oxidative metabolism and ferroptosis ensures functional HSC expansion after radiation injury. Importantly, punctual IGF1 administration simultaneously promotes HSC regeneration and hematopoietic recovery after radiation injury, representing a superior therapeutic approach for myelosuppression. CONCLUSIONS: Our study identifies megakaryocytes as a last line of defense against myelosuppressive injury and megakaryocytic IGF1 as a novel niche signal safeguarding HSC regeneration.

An intricate regulatory circuit between FLI1 and GATA1/GATA2/LDB1/ERG dictates erythroid vs. megakaryocytic differentiation.

During hematopoiesis, megakaryocytic erythroid progenitors (MEPs) differentiate into megakaryocytic or erythroid lineages in response to specific transcriptional factors, yet the regulatory mechanism remains to be elucidated. Using the MEP­like cell line HEL western blotting, RT­qPCR, lentivirus­mediated downregulation, flow cytometry as well as chromatin immunoprecipitation (ChIp) assay demonstrated that the E26 transformation­specific (ETS) transcription factor friend leukemia integration factor 1 (Fli­1) inhibits erythroid differentiation. The present study using these methods showed that while FLI1­mediated downregulation of GATA binding protein 1 (GATA1) suppresses erythropoiesis, its direct transcriptional induction of GATA2 promotes megakaryocytic differentiation. GATA1 is also involved in megakaryocytic differentiation through regulation of GATA2. By contrast to FLI1, the ETS member erythroblast transformation­specific­related gene (ERG) negatively controls GATA2 and its overexpression through exogenous transfection blocks megakaryocytic differentiation. In addition, FLI1 regulates expression of LIM Domain Binding 1 (LDB1) during erythroid and megakaryocytic commitment, whereas shRNA­mediated depletion of LDB1 downregulates FLI1 and GATA2 but increases GATA1 expression. In agreement, LDB1 ablation using shRNA lentivirus expression blocks megakaryocytic differentiation and modestly suppresses erythroid maturation. These results suggested that a certain threshold level of LDB1 expression enables FLI1 to block erythroid differentiation. Overall, FLI1 controlled the commitment of MEP to either erythroid or megakaryocytic lineage through an intricate regulation of GATA1/GATA2, LDB1 and ERG, exposing multiple targets for cell fate commitment and therapeutic intervention.

Alnustone promotes megakaryocyte differentiation and platelet production via the interleukin-17A/interleukin-17A receptor/Src/RAC1/MEK/ERK signaling pathway.

OBJECTIVES: Thrombocytopenia is a disease in which the number of platelets in the peripheral blood decreases. It can be caused by multiple genetic factors, and numerous challenges are associated with its treatment. In this study, the effects of alnustone on megakaryocytes and platelets were investigated, with the aim of developing a new therapeutic approach for thrombocytopenia. METHODS: Random forest algorithm was used to establish a drug screening model, and alnustone was identified as a natural active compound that could promote megakaryocyte differentiation. The effect of alnustone on megakaryocyte activity was determined using cell counting kit-8. The effect of alnustone on megakaryocyte differentiation was determined using flow cytometry, Giemsa staining, and phalloidin staining. A mouse model of thrombocytopenia was established by exposing mice to X-rays at 4 Gy and was used to test the bioactivity of alnustone in vivo. The effect of alnustone on platelet production was determined using zebrafish. Network pharmacology was used to predict targets and signaling pathways. Western blotting and immunofluorescence staining determined the expression levels of proteins. RESULTS: Alnustone promoted the differentiation and maturation of megakaryocytes in vitro and restored platelet production in thrombocytopenic mice and zebrafish. Network pharmacology and western blotting showed that alnustone promoted the expression of interleukin-17A and enhanced its interaction with its receptor, and thereby regulated downstream MEK/ERK signaling and promoted megakaryocyte differentiation. CONCLUSIONS: Alnustone can promote megakaryocyte differentiation and platelet production via the interleukin-17A/interleukin-17A receptor/Src/RAC1/MEK/ERK signaling pathway and thus provides a new therapeutic strategy for the treatment of thrombocytopenia.

Activating SRC/MAPK signaling via 5-HT1A receptor contributes to the effect of vilazodone on improving thrombocytopenia.

Thrombocytopenia caused by long-term radiotherapy and chemotherapy exists in cancer treatment. Previous research demonstrates that 5-Hydroxtrayptamine (5-HT) and its receptors induce the formation of megakaryocytes (MKs) and platelets. However, the relationships between 5-HT1A receptor (5-HTR1A) and MKs is unclear so far. We screened and investigated the mechanism of vilazodone as a 5-HTR1A partial agonist in promoting MK differentiation and evaluated its therapeutic effect in thrombocytopenia. We employed a drug screening model based on machine learning (ML) to screen the megakaryocytopoiesis activity of Vilazodone (VLZ). The effects of VLZ on megakaryocytopoiesis were verified in HEL and Meg-01 cells. Tg (itga2b: eGFP) zebrafish was performed to analyze the alterations in thrombopoiesis. Moreover, we established a thrombocytopenia mice model to investigate how VLZ administration accelerates platelet recovery and function. We carried out network pharmacology, Western blot, and immunofluorescence to demonstrate the potential targets and pathway of VLZ. VLZ has been predicted to have a potential biological action. Meanwhile, VLZ administration promotes MK differentiation and thrombopoiesis in cells and zebrafish models. Progressive experiments showed that VLZ has a potential therapeutic effect on radiation-induced thrombocytopenia in vivo. The network pharmacology and associated mechanism study indicated that SRC and MAPK signaling are both involved in the processes of megakaryopoiesis facilitated by VLZ. Furthermore, the expression of 5-HTR1A during megakaryocyte differentiation is closely related to the activation of SRC and MAPK. Our findings demonstrated that the expression of 5-HTR1A on MK, VLZ could bind to the 5-HTR1A receptor and further regulate the SRC/MAPK signaling pathway to facilitate megakaryocyte differentiation and platelet production, which provides new insights into the alternative therapeutic options for thrombocytopenia.

Thrombopoietin, the Primary Regulator of Platelet Production: From Mythos to Logos, a Thirty-Year Journey.

Thrombopoietin, the primary regulator of blood platelet production, was postulated to exist in 1958, but was only proven to exist when the cDNA for the hormone was cloned in 1994. Since its initial cloning and characterization, the hormone has revealed many surprises. For example, instead of acting as the postulated differentiation factor for platelet precursors, megakaryocytes, it is the most potent stimulator of megakaryocyte progenitor expansion known. Moreover, it also stimulates the survival, and in combination with stem cell factor leads to the expansion of hematopoietic stem cells. All of these growth-promoting activities have resulted in its clinical use in patients with thrombocytopenia and aplastic anemia, although the clinical development of the native molecule illustrated that "it's not wise to mess with mother nature", as a highly engineered version of the native hormone led to autoantibody formation and severe thrombocytopenia. Finally, another unexpected finding was the role of the thrombopoietin receptor in stem cell biology, including the development of myeloproliferative neoplasms, an important disorder of hematopoietic stem cells. Overall, the past 30 years of clinical and basic research has yielded many important insights, which are reviewed in this paper.

Long non-coding RNA NORAD regulates megakaryocyte differentiation and proplatelet formation via the DUSP6/ERK signaling pathway.

Megakaryopoiesis and platelet production is a complex process that is underpotential regulation at multiple stages. Many long non-coding RNAs (lncRNAs) are distributed in hematopoietic stem cells and platelets. lncRNAs may play important roles as key epigenetic regulators in megakaryocyte differentiation and proplatelet formation. lncRNA NORAD can affect cell ploidy by sequestering PUMILIO proteins, although its direct effect on megakaryocyte differentiation and thrombopoiesis is still unknown. In this study, we demonstrate NORAD RNA is highly expressed in the cytoplasm during megakaryocyte differentiation. Interestingly, we identified for the first time that NORAD has a strong inhibitory effect on megakaryocyte differentiation and proplatelet formation from cultured megakaryocytes. DUSP6/ERK1/2 pathway is activated in response to NORAD knockdown during megakaryocytopoiesis, which is achieved by sequestering PUM2 proteins. Finally, compared with the wild-type control mice, NORAD knockout mice show a faster platelet recovery after severe thrombocytopenia induced by 6 Gy total body irradiation. These findings demonstrate lncRNA NORAD has a key role in regulating megakaryocyte differentiation and thrombopoiesis, which provides a promising molecular target for the treatment of platelet-related diseases such as severe thrombocytopenia.

A subset of megakaryocytes regulates development of hematopoietic stem cell precursors.

Understanding the regulatory mechanisms facilitating hematopoietic stem cell (HSC) specification during embryogenesis is important for the generation of HSCs in vitro. Megakaryocyte emerged from the yolk sac and produce platelets, which are involved in multiple biological processes, such as preventing hemorrhage. However, whether megakaryocytes regulate HSC development in the embryonic aorta-gonad-mesonephros (AGM) region is unclear. Here, we use platelet factor 4 (PF4)-Cre;Rosa-tdTomato+ cells to report presence of megakaryocytes in the HSC developmental niche. Further, we use the PF4-Cre;Rosa-DTA (DTA) depletion model to reveal that megakaryocytes control HSC specification in the mouse embryos. Megakaryocyte deficiency blocks the generation and maturation of pre-HSCs and alters HSC activity at the AGM. Furthermore, megakaryocytes promote endothelial-to-hematopoietic transition in a OP9-DL1 coculture system. Single-cell RNA-sequencing identifies megakaryocytes positive for the cell surface marker CD226 as the subpopulation with highest potential in promoting the hemogenic fate of endothelial cells by secreting TNFSF14. In line, TNFSF14 treatment rescues hematopoietic cell function in megakaryocyte-depleted cocultures. Taken together, megakaryocytes promote production and maturation of pre-HSCs, acting as a critical microenvironmental control factor during embryonic hematopoiesis.

From Hematopoietic Stem Cells to Platelets: Unifying Differentiation Pathways Identified by Lineage Tracing Mouse Models.

Platelets are the terminal progeny of megakaryocytes, primarily produced in the bone marrow, and play critical roles in blood homeostasis, clotting, and wound healing. Traditionally, megakaryocytes and platelets are thought to arise from multipotent hematopoietic stem cells (HSCs) via multiple discrete progenitor populations with successive, lineage-restricting differentiation steps. However, this view has recently been challenged by studies suggesting that (1) some HSC clones are biased and/or restricted to the platelet lineage, (2) not all platelet generation follows the "canonical" megakaryocytic differentiation path of hematopoiesis, and (3) platelet output is the default program of steady-state hematopoiesis. Here, we specifically investigate the evidence that in vivo lineage tracing studies provide for the route(s) of platelet generation and investigate the involvement of various intermediate progenitor cell populations. We further identify the challenges that need to be overcome that are required to determine the presence, role, and kinetics of these possible alternate pathways.

Polyamine release and vesicular polyamine transporter expression in megakaryoblastic cells and platelets.

Polyamines not only play essential roles in cell growth and function of living organisms but are also released into the extracellular space and function as regulators of chemical transduction, although the cells from which they are released and their mode of release are not well understood. The vesicular polyamine transporter (VPAT), encoded by the SLC18B1 is responsible for the vesicular storage of spermine and spermidine, followed by their vesicular release from secretory cells. Focusing on VPAT will help identify polyamine-secreting cells and new polyamine functions. In this study, we investigated the possible involvement of VPAT in vesicular release of polyamines in MEG-01 clonal megakaryoblastic cells and platelets. RT-PCR, western blotting, and immunohistochemistry revealed VPAT expression in MEG-01 cells. MEG-01 cells secreted polyamines upon A23187 stimulation in the presence of Ca2+, which is temperature-dependent and sensitive to bafilomycin A1. A23187-induced polyamine secretion from MEG-01 cells was reduced by treatment with reserpine, VPAT inhibitors, or VPAT RNA interference. Platelets also expressed VPAT, displaying a punctate distribution, and released spermidine upon A23187 and thrombin stimulation. These findings have demonstrated VPAT-mediated vesicular polyamine release from MEG-01 cells, suggesting the presence of similar vesicular polyamine release mechanisms in platelets.

GFI1B and LSD1 repress myeloid traits during megakaryocyte differentiation.

The transcription factor Growth Factor Independence 1B (GFI1B) recruits Lysine Specific Demethylase 1 A (LSD1/KDM1A) to stimulate gene programs relevant for megakaryocyte and platelet biology. Inherited pathogenic GFI1B variants result in thrombocytopenia and bleeding propensities with varying intensity. Whether these affect similar gene programs is unknow. Here we studied transcriptomic effects of four patient-derived GFI1B variants (GFI1BT174N,H181Y,R184P,Q287*) in MEG01 megakaryoblasts. Compared to normal GFI1B, each variant affected different gene programs with GFI1BQ287* uniquely failing to repress myeloid traits. In line with this, single cell RNA-sequencing of induced pluripotent stem cell (iPSC)-derived megakaryocytes revealed a 4.5-fold decrease in the megakaryocyte/myeloid cell ratio in GFI1BQ287* versus normal conditions. Inhibiting the GFI1B-LSD1 interaction with small molecule GSK-LSD1 resulted in activation of myeloid genes in normal iPSC-derived megakaryocytes similar to what was observed for GFI1BQ287* iPSC-derived megakaryocytes. Thus, GFI1B and LSD1 facilitate gene programs relevant for megakaryopoiesis while simultaneously repressing programs that induce myeloid differentiation.

Osteomacs promote maintenance of murine hematopoiesis through megakaryocyte-induced upregulation of Embigin and CD166.

Maintenance of hematopoietic stem cell (HSC) function in the niche is an orchestrated event. Osteomacs (OM) are key cellular components of the niche. Previously, we documented that osteoblasts, OM, and megakaryocytes interact to promote hematopoiesis. Here, we further characterize OM and identify megakaryocyte-induced mediators that augment the role of OM in the niche. Single-cell mRNA-seq, mass spectrometry, and CyTOF examination of megakaryocyte-stimulated OM suggested that upregulation of CD166 and Embigin on OM augment their hematopoiesis maintenance function. CD166 knockout OM or shRNA-Embigin knockdown OM confirmed that the loss of these molecules significantly reduced the ability of OM to augment the osteoblast-mediated hematopoietic-enhancing activity. Recombinant CD166 and Embigin partially substituted for OM function, characterizing both proteins as critical mediators of OM hematopoietic function. Our data identify Embigin and CD166 as OM-regulated critical components of HSC function in the niche and potential participants in various in vitro manipulations of stem cells.

Quantitative and structural changes of blood platelet cytoskeleton proteins in multiple sclerosis (MS).

Epidemiological studies show that cardiovascular events related to platelet hyperactivity remain the leading causes of death among multiple sclerosis (MS) patients. Quantitative or structural changes of platelet cytoskeleton alter their morphology and function. Here, we demonstrated, for the first time, the structural changes in MS platelets that may be related to their hyperactivity. MS platelets were found to form large aggregates compared to control platelets. In contrast to the control, the images of overactivated, irregularly shaped MS platelets show changes in the cytoskeleton architecture, fragmented microtubule rings. Furthermore, MS platelets have long and numerous pseudopodia rich in actin filaments. We showed that MS platelets and megakaryocytes, overexpress ß1-tubulin and ß-actin mRNAs and proteins and have altered post-translational modification patterns. Moreover, we identified two previously undisclosed mutations in the gene encoding ß1-tubulin in MS. We propose that the demonstrated structural changes of platelet cytoskeleton enhance their ability to adhere, aggregate, and degranulate fueling the risk of adverse cardiovascular events in MS.

SARS-CoV-2 infection modifies the transcriptome of the megakaryocytes in the bone marrow.

ABSTRACT: Megakaryocytes (MKs), integral to platelet production, predominantly reside in the bone marrow (BM) and undergo regulated fragmentation within sinusoid vessels to release platelets into the bloodstream. Inflammatory states and infections influence MK transcription, potentially affecting platelet functionality. Notably, COVID-19 has been associated with altered platelet transcriptomes. In this study, we investigated the hypothesis that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection could affect the transcriptome of BM MKs. Using spatial transcriptomics to discriminate subpopulations of MKs based on proximity to BM sinusoids, we identified ∼19 000 genes in MKs. Machine learning techniques revealed that the transcriptome of healthy murine BM MKs exhibited minimal differences based on proximity to sinusoid vessels. Furthermore, at peak SARS-CoV-2 viremia, when the disease primarily affected the lungs, MKs were not significantly different from those from healthy mice. Conversely, a significant divergence in the MK transcriptome was observed during systemic inflammation, although SARS-CoV-2 RNA was never detected in the BM, and it was no longer detectable in the lungs. Under these conditions, the MK transcriptional landscape was enriched in pathways associated with histone modifications, MK differentiation, NETosis, and autoimmunity, which could not be explained by cell proximity to sinusoid vessels. Notably, the type I interferon signature and calprotectin (S100A8/A9) were not induced in MKs under any condition. However, inflammatory cytokines induced in the blood and lungs of COVID-19 mice were different from those found in the BM, suggesting a discriminating impact of inflammation on this specific subset of cells. Collectively, our data indicate that a new population of BM MKs may emerge through COVID-19-related pathogenesis.

The Y49H cytochrome c variant enhances megakaryocytic maturation of K-562 cells.

Five pathogenic variants in the gene encoding cytochrome c (CYCS) associated with mild autosomal dominant thrombocytopenia have been reported. Previous studies of peripheral blood CD34+ or CD45+ cells from subjects with the G42S CYCS variant showed an acceleration in megakaryopoiesis compared to wild-type (WT) cells. To determine whether this result reflects a common feature of the CYCS variants, the c.145T>C mutation (Y49H variant) was introduced into the endogenous CYCS locus in K-562 cells, which undergo megakaryocytic maturation in response to treatment with a phorbol ester. The c.145T>C (Y49H) variant enhanced the megakaryocyte maturation of the K-562 cells, and this effect was seen when the cells were cultured at both 18 % and 5 % oxygen. Thus, alteration of megakaryopoiesis is common to both the G42S and Y49H CYCS variants and may contribute to the low platelet phenotype. The Y49H CYCS variant has previously been reported to impair mitochondrial respiratory chain function in vitro, however using extracellular flux analysis the c.145T>C (Y49H) variant does not alter mitochondrial bioenergetics of the K-562 cells, consistent with the lack of a phenotype characteristic of mitochondrial diseases in CYCS variant families. The Y49H variant has also been reported to enhance the ability of cytochrome c to trigger caspase activation in the intrinsic apoptosis pathway. However, as seen in peripheral blood cells from G42S CYCS variant carriers, the presence of Y49H cytochrome c in K-562 cells did not significantly change their response to an apoptotic stimulus.