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Thrombopoietin (TPO) and its receptor, MPL, are the primary regulators of platelet production and critical for hematopoietic stem cell (HSC) maintenance. Since TPO was first cloned in 1994, the physiological and pathological roles of TPO and MPL have been well characterized, culminating in the first MPL agonists being approved for the treatment of chronic immune thrombocytopenia in 2008. Dysregulation of the TPO-MPL signaling axis contributes to the pathogenesis of hematological disorders: decreased expression or function results in severe thrombocytopenia progressing to bone marrow failure, while hyperactivation of MPL signaling, either by mutations in the receptor or associated Janus kinase 2 (JAK2), results in pathological myeloproliferation. Despite its importance, it was only recently that the long-running debate over the mechanism by which TPO binding activates MPL has been resolved. This review will cover key aspects of TPO and MPL structure and function and their importance in receptor activation, discuss how these are altered in hematological disorders and consider how a greater understanding could lead to the development of better-targeted and more efficacious therapies.

Abstract Background The therapeutic use of [131I]meta-iodobenzylguanidine ([131I]MIBG) is often accompanied by hematological toxicity, primarily consisting of severe and persistent thrombocytopenia. We hypothesize that this is caused by selective uptake of MIBG via the serotonin transporter (SERT) located on platelets and megakaryocytes. In this study, we have investigated whether in vitro cultured human megakaryocytes are capable of selective plasma membrane transport of MIBG and whether pharmacological intervention with selective serotonin reuptake inhibitors (SSRIs) may prevent this radiotoxic MIBG uptake. Methods Peripheral blood CD34+ cells were differentiated to human megakaryocytic cells using a standardized culture protocol. Prior to [3H]serotonin and [125I]MIBG uptake experiments, the differentiation status of megakaryocyte cultures was assessed by flow cytometry. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to assess SERT and NET (norepinephrine transporter) mRNA expression. On day 10 of differentiation, [3H]serotonin and [125I]MIBG uptake assays were conducted. Part of the samples were co-incubated with the SSRI citalopram to assess SERT-specific uptake. HEK293 cells transfected with SERT, NET, and empty vector served as controls. Results In vitro cultured human megakaryocytes are capable of selective plasma membrane transport of MIBG. After 10 days of differentiation, megakaryocytic cell culture batches from three different hematopoietic stem and progenitor cell donors showed on average 9.2 ± 2.4 nmol of MIBG uptake per milligram protein per hour after incubation with 10–7 M MIBG (range: 6.6 ± 1.0 to 11.2 ± 1.0 nmol/mg/h). Co-incubation with the SSRI citalopram led to a significant reduction (30.1%—41.5%) in MIBG uptake, implying SERT-specific uptake of MIBG. A strong correlation between the number of mature megakaryocytes and SERT-specific MIBG uptake was observed. Conclusion Our study demonstrates that human megakaryocytes cultured in vitro are capable of MIBG uptake. Moreover, the SSRI citalopram selectively inhibits MIBG uptake via the serotonin transporter. The concomitant administration of citalopram to neuroblastoma patients during [131I]MIBG therapy might be a promising strategy to prevent the onset of thrombocytopenia.

Investigation of the bone marrow as the main compartment of hematopoiesis is critical in many research fields. Here, we adapted a centrifugation-based method for the isolation of murine bone marrow and compared it to the traditional flushing method. Analysis of primary hematopoietic stem cells, immune cells, and megakaryocytes revealed a comparable distribution of cellular (sub)populations. Furthermore, in vitro differentiated megakaryocytes displayed unaltered proplatelet formation. Strikingly, bone marrow isolation by centrifugation was considerably faster than the flushing method and significantly increased the cell yield. Thus, the centrifugation-based isolation method is highly suitable for the study of murine bone marrow cells.

Platelets are critical for haemostasis, thrombosis, and inflammatory responses, but the events that lead to mature platelet production remain incompletely understood. The bone marrow has been proposed to be a major site of platelet production, although there is indirect evidence that the lungs might also contribute to platelet biogenesis. Here, by directly imaging the lung microcirculation in mice, we show that a large number of megakaryocytes circulate through the lungs, where they dynamically release platelets. Megakaryocytes that release platelets in the lungs originate from extrapulmonary sites such as the bone marrow; we observed large megakaryocytes migrating out of the bone marrow space. The contribution of the lungs to platelet biogenesis is substantial, accounting for approximately 50% of total platelet production or 10 million platelets per hour. Furthermore, we identified populations of mature and immature megakaryocytes along with haematopoietic progenitors in the extravascular spaces of the lungs. Under conditions of thrombocytopenia and relative stem cell deficiency in the bone marrow, these progenitors can migrate out of the lungs, repopulate the bone marrow, completely reconstitute blood platelet counts, and contribute to multiple haematopoietic lineages. These results identify the lungs as a primary site of terminal platelet production and an organ with considerable haematopoietic potential.

Hematopoietic stem cells (HSCs) reside in a subzone of the bone marrow (BM) defined as the hematopoietic niche where, via the interplay of differentiation and self-renewal, they can give rise to immune and blood cells. Artificial hematopoietic niches were firstly developed in 2D in vitro cultures but the limited expansion potential and stemness maintenance induced the optimization of these systems to avoid the total loss of the natural tissue complexity. The next steps were adopted by engineering different materials such as hydrogels, fibrous structures with natural or synthetic polymers, ceramics, etc. to produce a 3D substrate better resembling that of BM. Cytokines, soluble factors, adhesion molecules, extracellular matrix (ECM) components, and the secretome of other niche-resident cells play a fundamental role in controlling and regulating HSC commitment. To provide biochemical cues, co-cultures, and feeder-layers, as well as natural or synthetic molecules were utilized. This review gathers key elements employed for the functionalization of a 3D scaffold that demonstrated to promote HSC growth and differentiation ranging from 1) biophysical cues, i.e., material, topography, stiffness, oxygen tension, and fluid shear stress to 2) biochemical hints favored by the presence of ECM elements, feeder cell layers, and redox scavengers. Particular focus is given to the 3D systems to recreate megakaryocyte products, to be applied for blood cell production, whereas HSC clinical application in such 3D constructs was limited so far to BM diseases testing.

Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types, including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatio-temporal regulation in megakaryocytes (MKs) is lacking. Optogenetics is a technique which allows spatio-temporal manipulation of molecular events in living cells or organisms. We took advantage of this method and expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide-triggered cGMP generation in BM MKs. In summary, we established for the first-time optogenetics in primary MKs and show that PDE5 is the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.

Rheumatoid arthritis (RA) is associated with an increased risk for cardiovascular events driven by abnormal platelet clotting effects. Platelets are produced by megakaryocytes, deriving from megakaryocyte erythrocyte progenitors (MEP) in the bone marrow. Increased megakaryocyte expansion across common autoimmune diseases was shown for RA, systemic lupus erythematosus (SLE) and primary Sjögren’s syndrome (pSS). In this context, we evaluated the role of the microbial-derived short chain fatty acid (SCFA) propionate on hematopoietic progenitors in the collagen induced inflammatory arthritis model (CIA) as we recently showed attenuating effects of preventive propionate treatment on CIA severity. In vivo, propionate treatment starting 21 days post immunization (dpi) reduced the frequency of MEPs in the bone marrow of CIA and naïve mice. Megakaryocytes numbers were reduced but increased the expression of the maturation marker CD61. Consistent with this, functional analysis of platelets showed an upregulated reactivity state following propionate-treatment. This was confirmed by elevated histone 3 acetylation and propionylation as well as by RNAseq analysis in Meg-01 cells. Taken together, we identified a novel nutritional axis that skews platelet formation and function.

Megakaryocytes (MKs) continuously produce platelets to support hemostasis and form a niche for hematopoietic stem cell maintenance in the bone marrow. MKs are also involved in inflammatory responses; however, the mechanism remains poorly understood. Using single-cell sequencing, we identified a CXCR4 highly expressed MK subpopulation, which exhibited both MK-specific and immune characteristics. CXCR4high MKs interacted with myeloid cells to promote their migration and stimulate the bacterial phagocytosis of macrophages and neutrophils by producing TNFα and IL-6. CXCR4high MKs were also capable of phagocytosis, processing, and presenting antigens to activate T cells. Furthermore, CXCR4high MKs also egressed circulation and infiltrated into the spleen, liver, and lung upon bacterial infection. Ablation of MKs suppressed the innate immune response and T cell activation to impair the anti-bacterial effects in mice under the Listeria monocytogenes challenge. Using hematopoietic stem/progenitor cell lineage-tracing mouse lines, we show that CXCR4high MKs were generated from infection-induced emergency megakaryopoiesis in response to bacterial infection. Overall, we identify the CXCR4high MKs, which regulate host-defense immune response against bacterial infection.

Within the bone marrow, megakaryocytes thrive in an environment rich in numerous growth factors that influences their development, maturation, and may play a role in signaling the start of platelet production. Up to now, experiments concerning localisation of various populations of cells in rat bone marrow showed their certain relationship to the blood vessels. There are strikingly few reliable sources of information available in the literature that confirm the relationship between the localisation of these cells in connection to nerve fibers. Therefore the purpose of the present study was to examine if megakaryocytes are located in a defined relation to sensory and sympathetic nerve fibers. The study used immature 6 weeks rats of the Wistar breed of both sexes. The animals were kept under constant lighting conditions (12 hours light-dark cycle) and temperature, and had unrestricted access to water and food (standard laboratory rodent feed).Double immunostaining method was applied with usage of secondary antibodies conjugated with fluorochromes (Cy3 and DTAF) to identify cells. The antibody against CD42d was used to immunolocalise megakaryocytes, while in order to identify the distribution of nerves, antibodies anti-NPY (for detection sympathetic nerve fibers) and anti-PGP 9.5 (sensory nerve fibers) were applied. These findings showed the presence of megakaryocyte and megakaryoblastic cells which were distributed with a close relationship to the blood vessels but some of them were located in parenchyma. It was shown that NPY-positive and PGP 9.5-positive cells were present in the vicinity of blood cells. Furthermore, some megakaryocytes were located near PGP 9.5-labelled cells. The relationship between sympathetic nerve fibers containing neuropeptide Y and megakaryocytes was also detected.

The immune system is comprised of two principal interconnected components called innate and adaptive immunity. While the innate immune system mounts a nonspecific response that provides protection against the spread of foreign pathogens, the adaptive immune system has developed to specifically recognize a given pathogen and lead to immunological memory. Platelets are small fragments produced from megakaryocytes in bone marrow and lungs. They circulate throughout the blood to monitor the integrity of the vasculature and to prevent bleeding. Given their large repertoire of immune receptors and inflammatory molecules, platelets and megakaryocytes can contribute to both innate and adaptive immunity. In adaptive immunity, platelets and megakaryocytes can process and present antigens to lymphocytes. Moreover, platelets, via FcγRIIA, rapidly respond to pathogens in an immune host when antibodies are present. This manuscript reviews the reported contributions of platelets and megakaryocytes with emphasis on antigen presentation and antibody response in adaptive immunity.

Understanding the role of bone marrow (BM) niches in determining hematopoietic stem cell (HSC) fate during acute inflammation is essential for developing new treatments. However, the complex molecular and cellular interactions and mechanisms involved remain largely unexplored. Here, we integrate in vitro live HSC signaling dynamics and fate quantification with in situ single-molecule detection in large BM volumes. Our results identify megakaryocytes (MEGs) as immune sensors and activators that respond to the inflammatory mediator lipopolysaccharide (LPS) by localized interleukin 1β (IL-1β) secretion. This in turn enhances p38 MAPK pathway activation only in neighboring HSCs, predisposing them to later granulocytic/monocytic differentiation. This effect can be blocked by IL-1β or p38 MAPK inhibition. Quantitative in situ single-molecule imaging in BM confirms that sparse MEGs respond to systemic LPS with a localized IL-1β secretion pulse, which sustains p38 MAPK signaling activation in Sca-1+ cells only within their close microenvironment. This research highlights the critical role of MEGs in guiding neighboring HSC fates during inflammation. It enhances our understanding of the BM microenvironment's role in directing stem cell lineage choice under inflammatory stress and identifies potential targets for modulating hematopoiesis and treating inflammatory blood disorders. [ABSTRACT FROM AUTHOR]

One of the breakthroughs in hematopoietic stem cell (HSC) field over the last decade is the discovery of two alternative differentiation routes from primitive HSCs to mature megakaryocytes: one through the stepwise hematopoietic hierarchy (stepwise route), and the other by direct differentiation (direct route). This raises a fundamental question of the physiological importance of two alternative differentiation routes for megakaryopoiesis. A major challenge in addressing this question is the lack of fate-mapping systems that distinguish the two differentiation routes. This work was initiated by designing genetic systems that distinguished the direct and stepwise differentiation routes for hematopoiesis. We found that Cd48-Dre specifically and constitutively marked all haematopoietic cells on the stepwise differentiation route. A combination of KitcreER, Cd48dre and Rosa26loxp-STOP-loxp-rox-loxp-ZsGreen-STOP-rox-tdTomato allowed inducible and simultaneous fate-mapping of haematopoietic stem and progenitor cells on the direct and stepwise differentiation routes. We mapped the turnover rates and differentiation kinetics of each branch of the hematopoietic hierarchy. We found that megakaryocytes were produced through the two routes with comparable kinetics and quantity under homeostasis. Single-cell RNA-sequencing of the fate-mapped megakaryocytes revealed that the direct and stepwise routes contributed to the niche-supporting and immune megakaryocytes respectively, but contributed to the platelet-producing megakaryocytes together. Consistent with this, megakaryocytes generated through different routes displayed different activities in vitro and in vivo. Chemotherapy preferentially enhanced megakaryopoiesis through the direct route, whereas inflammation preferentially enhanced megakaryopoiesis through the stepwise route. In summary, our work links the differentiation route to the cellular heterogeneity of adult megakaryocytes. Alternative differentiation routes result in variable combinations of functionally distinct megakaryocyte subpopulations poised for different physiological demands. [ABSTRACT FROM AUTHOR]

In contrast to most hematopoietic lineages, megakaryocytes (MKs) can derive rapidly and directly from hematopoietic stem cells (HSCs). The underlying mechanism is unclear, however. Here, we show that DNA damage induces MK markers in HSCs and that G2 arrest, an integral part of the DNA damage response, suffices for MK priming followed by irreversible MK differentiation in HSCs, but not in progenitors. We also show that replication stress causes DNA damage in HSCs and is at least in part due to uracil misincorporation in vitro and in vivo. Consistent with this notion, thymidine attenuated DNA damage, improved HSC maintenance, and reduced the generation of CD41+ MK-committed HSCs. Replication stress and concomitant MK differentiation is therefore one of the barriers to HSC maintenance. DNA damage-induced MK priming may allow rapid generation of a lineage essential to immediate organismal survival, while also removing damaged cells from the HSC pool. [Display omitted] • DNA damage induces megakaryocyte (MK) priming in hematopoietic stem cells (HSCs) • Pharmacological G2 arrest is sufficient for MK maturation from HSCs • Uracil misincorporation causes replication stress and MK priming in vitro • These effects are specific to HSCs In contrast to most blood cell lineages, megakaryocytes, which produce platelets essential for blood coagulation, can derive directly from hematopoietic stem cells. Garyn et al. show that arrest in the G2 phase of the cell cycle, which is induced by DNA damage, is sufficient to promote direct megakaryopoiesis. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

Megakaryocytes (MKs) are multifunctional hematopoietic cells that produce platelets, serve as components of bone marrow (BM) niches that support the development of hematopoietic stem and progenitor cell (HSPC) and provide inflammatory signals. MKs can dynamically change their activities during homeostasis and following stress, thereby regulating hematopoietic stem cell (HSC) function. Myelofibrosis (MF) is a progressive chronic myeloproliferative neoplasm (MPN) characterized by hyperactivation of JAK/STAT signaling and MK hyperplasia, which is associated with an aberrant inflammatory signature. Since JAK1/2 inhibitor alone is incapable of depleting the malignant HSC clones or reversing BM fibrosis, the identification of mechanisms that cooperate with MF JAK/STAT signaling to promote disease progression might help in developing combination therapies to modify disease outcomes. Chronic inflammation and MK hyperplasia result in an abnormal release of TGFβ1, which plays a critical role in the pathobiology of MF by contributing to the development of BM fibrosis. Dysregulated TGFβ signaling can also alter the hematopoietic microenvironment supporting the predominance of MF-HSCs and enhance the quiescence of the reservoir of wild-type HSCs. Upregulation of TGFβ1 levels is a relatively late event in MF, while during the early pre-fibrotic stage of MF the alarmin S100A8/S100A9 heterocomplex promotes pro-inflammatory responses and sustains the progression of MF-HSCs. In this review, we will discuss the recent advances in our understanding of the roles of abnormal megakaryopoiesis, and the altered microenvironment in MF progression and the development of novel combined targeted therapies to disrupt the aberrant interplay between MKs, the BM microenvironment and malignant HSCs which would potentially limit the expansion of MF-HSC clones. [ABSTRACT FROM AUTHOR]

Platelets are mainly known for their key role in hemostasis and thrombosis. However, studies over the last two decades have shown their strong implication in mechanisms associated with inflammation, thrombosis, and the immune system in various neoplastic, inflammatory, autoimmune, and infectious diseases. During sepsis, platelets amplify the recruitment and activation of innate immune cells at the site of infection and contribute to the elimination of pathogens. In certain conditions, these mechanisms can lead to thromboinflammation resulting in severe organ dysfunction. Here, we discuss the interactions of platelets with leukocytes, neutrophil extracellular traps (NETs), and endothelial cells during sepsis. The intrinsic properties of platelets that generate an inflammatory signal through the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome are discussed. As an example of immunothrombosis, the implication of platelets in vaccine-induced immune thrombotic thrombocytopenia is documented. Finally, we discuss the role of megakaryocytes (MKs) in thromboinflammation and their adaptive responses. [ABSTRACT FROM AUTHOR]

Pan‐proteasome inhibitors (pPIs) significantly improve outcomes in patients with multiple myeloma; however, their indiscriminate inhibition of multiple proteasome and immunoproteasome subunits causes diverse toxicities, including thrombocytopenia. We investigated the mechanisms underlying the platelet depletion induced by the pPIs bortezomib, carfilzomib, and ixazomib. An established thrombocytopenia model was adapted for each compound (bortezomib, ixazomib, and carfilzomib) to compare the following two pharmacodynamic mechanisms: a reversible inhibition of new progenitor cell formation (the myelosuppression model) and a reversible effect on the function of megakaryocytes to bud new platelets (platelet formation model). Bortezomib, ixazomib, and carfilzomib plasma concentration profiles and platelet counts were extracted from the literature. Pharmacokinetic (PK) and thrombocytopenia models were developed to predict the PK of these drugs and to describe their effects on proliferating cells and platelet budding. The PK models reproduced the exposure of the three compounds at steady state well compared with those reported in the literature. Both the platelet formation and myelosuppression models seemed able to describe the platelet depletion caused by bortezomib, ixazomib, and carfilzomib. Estimated structural parameters in the myelosuppression model were in the range of the values reported in the literature, whereas the mean transit time estimated with the platelet formation model was 3‐fold to 10‐fold higher than the highest reported value. The model of drug‐induced myelosuppression yielded estimates of structural parameters in the range of those previously reported. The platelet formation model captured the temporal variation reported in clinical studies. [ABSTRACT FROM AUTHOR]

Bone marrow megakaryocytes (MKs) undergo a maturation involving contacts with the microenvironment before extending proplatelets through sinusoids to deliver platelets in the bloodstream. We demonstrated that MKs assemble linear F-actin-enriched podosomes on collagen I fibers. Microscopy analysis evidenced an inverse correlation between the number of dot-like versus linear podosomes over time. Confocal videomicroscopy confirmed that they derived from each-other. This dynamics was dependent on myosin IIA. Importantly, MKs progenitors expressed the Tks4/5 adaptors, displayed a strong gelatinolytic ability and did not form linear podosomes. While maturing, MKs lost Tks expression together with digestive ability. However, those MKs were still able to remodel the matrix by exerting traction on collagen I fibers through a collaboration between GPVI, ß1 integrin and linear podosomes. Our data demonstrated that a change in structure and composition of podosomes accounted for the shift of function during megakaryopoiesis. These data highlight the fact that members of the invadosome family could correspond to different maturation status of the same entity, to adapt to functional responses required by differentiation stages of the cell that bears them. [ABSTRACT FROM AUTHOR]

Megakaryocytes are large hematopoietic cells present in the bone marrow cavity, comprising less than 0.1% of all bone marrow cells. Despite their small number, megakaryocytes play important roles in blood coagulation, inflammatory responses, and platelet production. However, little is known about changes in gene expression during megakaryocyte maturation. Here we identified the genes whose expression was changed during K562 leukemia cell differentiation into megakaryocytes using an Affymetrix GeneChip microarray to determine the multifunctionality of megakaryocytes. K562 cells were differentiated into mature megakaryocytes by treatment for 7 days with phorbol 12-myristate 13-acetate, and a microarray was performed using RNA obtained from both types of cells. The expression of 44,629 genes was compared between K562 cells and mature megakaryocytes, and 954 differentially expressed genes (DEGs) were selected based on a p-value < 0.05 and a fold change >2. The DEGs was further functionally classified using five major megakaryocyte function-associated clusters—inflammatory response, angiogenesis, cell migration, extracellular matrix, and secretion. Furthermore, interaction analysis based on the STRING database was used to generate interactions between the proteins translated from the DEGs. This study provides information on the bioinformatics of the DEGs in mature megakaryocytes after K562 cell differentiation. [ABSTRACT FROM AUTHOR]

In the clinic, the supply of platelets is frequently insufficient to meet transfusion needs. To address this issue, many scientists have established the derivation of functional platelets from CD34+ cells or human pluripotent stem cells (PSCs). However, the yield of platelets is still far below what is required. Here we found that the plant hormone abscisic acid (ABA) could increase the generation of megakaryocytes (MKs) and platelets from human induced PSCs (hiPSCs). During platelet derivation, ABA treatment promoted the generation of CD34+/CD45+ HPCs and CD41+ MKs on day 14 and then increased CD41+/CD42b+ MKs and platelets on day 19. Moreover, we found ABA-mediated activation of Akt and ERK1/2 signal pathway through receptors LANCL2 and GRP78 in a PKA-dependent manner on CD34+/CD45+ cells. In conclusion, our data suggest that ABA treatment can promote CD34+/CD45+ HPC proliferation and CD41+ MK differentiation. [ABSTRACT FROM AUTHOR]

Megakaryocytes (MKs) is an important component of the hematopoietic niche. Abnormal MK hyperplasia is a hallmark feature of myeloproliferative neoplasms (MPNs). The JAK2V617F mutation is present in hematopoietic cells in a majority of patients with MPNs. Using a murine model of MPN in which the human JAK2V617F gene is expressed in the MK lineage, we show that the JAK2V617F-bearing MKs promote hematopoietic stem cell (HSC) aging, manifesting as myeloid-skewed hematopoiesis with an expansion of CD41+ HSCs, a reduced engraftment and self-renewal capacity, and a reduced differentiation capacity. HSCs from 2-year-old mice with JAK2V617F-bearing MKs were more proliferative and less quiescent than HSCs from age-matched control mice. Examination of the marrow hematopoietic niche reveals that the JAK2V617F-bearing MKs not only have decreased direct interactions with hematopoietic stem/progenitor cells during aging but also suppress the vascular niche function during aging. Unbiased RNA expression profiling reveals that HSC aging has a profound effect on MK transcriptomic profiles, while targeted cytokine array shows that the JAK2V617F-bearing MKs can alter the hematopoietic niche through increased levels of pro-inflammatory and anti-angiogenic factors. Therefore, as a hematopoietic niche cell, MKs represent an important connection between the extrinsic and intrinsic mechanisms for HSC aging. [ABSTRACT FROM AUTHOR]

Background: Immune thrombocytopenia (ITP) is the most common etiology of acquired thrombocytopenia diseases in children. ITP is characterized by the immune-mediated decreased formation and excessive destruction of platelets. The pathogenesis and management of pediatric ITP are distinct from adult ITP. A disintegrin and metalloproteinase 17 (ADAM17) mediates the shedding of platelet receptor glycoprotein Ib α (GPIb α) in extracellular domain, functioning in the platelet activation and clearance. Our study aims to probe the roles and mechanisms of ADAM17 in pediatric ITP.Methods: The differently expressed ADAM17 in megakaryocytes was obtained from children with ITP through the next-generation RNA-Sequence. Hematoxylin-eosin and Giemsa staining were performed for cell morphology identification. Flow cytometry was applied to assess autoantibodies against platelets, subtypes of lymphocytes, the surface expression level of ADAM17 and polyploidization of megakaryocytes, as well as the full-length GP Ib α.Results: ADAM17 was significantly downregulated in megakaryocytes and platelets in children with ITP. Higher values of PDW and positive autoantibodies presence were observed in children with ITP. Loss of ADAM17 in mice led to defects in proplatelet formation and significantly elevated expression of phosphorylated myosin light chain (p-MLC) in megakaryocytes.Conclusions: Our study indicated that the downregulation of ADAM17 might be an innate cause of inefficient platelet production in pediatric ITP. [ABSTRACT FROM AUTHOR]

A major role for human (h)CXCL8 (interleukin-8) in the pathobiology of myelofibrosis (MF) has been suggested by observations indicating that MF megakaryocytes express increased levels of hCXCL8 and that plasma levels of this cytokine in MF patients are predictive of poor patient outcomes. Here, we demonstrate that, in addition to high levels of TGF-β, the megakaryocytes from the bone marrow of the Gata1 low mouse model of myelofibrosis express high levels of murine (m)CXCL1, the murine equivalent of hCXCL8, and its receptors CXCR1 and CXCR2. Treatment with the CXCR1/R2 inhibitor, Reparixin in aged-matched Gata1 low mice demonstrated reductions in bone marrow and splenic fibrosis. Of note, the levels of fibrosis detected using two independent methods (Gomori and reticulin staining) were inversely correlated with plasma levels of Reparixin. Immunostaining of marrow sections indicated that the bone marrow from the Reparixin-treated group expressed lower levels of TGF-β1 than those expressed by the bone marrow from vehicle-treated mice while the levels of mCXCL1, and expression of CXCR1 and CXCR2, were similar to that of vehicle-treated mice. Moreover, immunofluorescence analyses performed on bone marrow sections from Gata1 low mice indicated that treatment with Reparixin induced expression of GATA1 while reducing expression of collagen III in megakaryocytes. These data suggest that in Gata1low mice, Reparixin reduces fibrosis by reducing TGF-β1 and collagen III expression while increasing GATA1 in megakaryocytes. Our results provide a preclinical rationale for further evaluation of this drug alone and in combination with current JAK inhibitor therapy for the treatment of patients with myelofibrosis. [ABSTRACT FROM AUTHOR]

Platelets are small anucleate cells derived from the fragmentation of megakaryocytes and are involved in different biological processes especially hemostasis, thrombosis, and immune response. Despite their lack of nucleus, platelets contain a reservoir of megakaryocyte-derived RNAs and all the machinery useful for mRNA translation. Interestingly, platelet transcriptome was analyzed in health and diseases and led to the identification of disease-specific molecular signatures. Platelet contamination by leukocytes and erythrocytes during platelet purification is a major problem in transcriptomic analysis and the presence of few contaminants in platelet preparation could strongly alter transcriptome results. Since contaminant impacts on platelet transcriptome remains theoretical, we aimed to determine whether low leukocyte and erythrocyte contamination could cause great or only minor changes in platelet transcriptome. Using microarray technique, we compared the transcriptome of platelets from the same donor, purified by common centrifugation method or using magnetic microbeads to eliminate contaminating cells. We found that platelet transcriptome was greatly altered by contaminants, as the relative amount of 8274 transcripts was different between compared samples. We observed an increase of transcripts related to leukocytes and erythrocytes in platelet purified without microbeads, while platelet specific transcripts were falsely reduced. In conclusion, serious precautions should be taken during platelet purification process for transcriptomic analysis, in order to avoid platelets contamination and result alteration. [ABSTRACT FROM AUTHOR]

Amyloidosis was modeled in young mice by administration of aqueous solution of soy cream substitute and then, amyloidogenesis was corrected by oral administration of dry red wine. In mice with amyloidosis, the area of megakaryocytes decreased by 1.69 times, the relative content of oxyphilic forms of megakaryocytes increased by 4.33 times, and ploidy did not change in comparison with the corresponding parameters in intact mice. Administration of red dry grape wine against the background of formation of systemic amyloidosis changed the reaction of megakaryocytes: the content of polychromatophilic forms was comparable to the level of intact control, the content of oxyphilic forms decreased by 3.11 times, the RNA and DNA content in megakaryocyte nuclei decreased proportionally, and the ploidy decreased. Thus, red grape wine reduced the response of the megakaryocyte genome to the external supply of amyloidogenic substance. [ABSTRACT FROM AUTHOR]

Abstract Background Platelets are small anucleate cells that circulate in the blood in a resting state but can be activated by external cues. In case of need, platelets from blood donors can be transfused. As an alternative source, platelets can be produced from induced pluripotent stem cells (iPSCs); however, recovered numbers are low. Objectives To optimize megakaryocyte (MK) and platelet output from murine iPSCs, we investigated overexpression of the transcription factors GATA‐binding factor 1 (GATA1); nuclear factor, erythroid 2; and pre–B‐cell leukemia transcription factor 1 (Pbx1) and a hyperactive variant of the small guanosine triphosphatase RhoA (RhoAhc). Methods To avoid off‐target effects, we generated iPSCs carrying the reverse tetracycline‐responsive transactivator M2 (rtTA‐M2) in the Rosa26 locus and expressed the factors from Tet‐inducible gammaretroviral vectors. Differentiation of iPSCs was initiated by embryoid body (EB) formation. After EB dissociation, early hematopoietic progenitors were enriched and cocultivated on OP9 feeder cells with thrombopoietin and stem cell factor to induce megakaryocyte (MK) differentiation. Results Overexpression of GATA1 and Pbx1 increased MK output 2‐ to 2.5‐fold and allowed prolonged collection of MK. Cytologic and ultrastructural analyses identified typical MK with enlarged cells, multilobulated nuclei, granule structures, and an internal membrane system. However, GATA1 and Pbx1 expression did not improve MK maturation or platelet release, although in vitro–generated platelets were functional in spreading on fibrinogen or collagen‐related peptide. Conclusion We demonstrate that the use of rtTA‐M2 transgenic iPSCs transduced with Tet‐inducible retroviral vectors allowed for gene expression at later time points during differentiation. With this strategy we could identify factors that increased in vitro MK production.

Megakaryocytes (MKs) are multifunctional hematopoietic cells that produce platelets, serve as components of bone marrow (BM) niches that support the development of hematopoietic stem and progenitor cell (HSPC) and provide inflammatory signals. MKs can dynamically change their activities during homeostasis and following stress, thereby regulating hematopoietic stem cell (HSC) function. Myelofibrosis (MF) is a progressive chronic myeloproliferative neoplasm (MPN) characterized by hyperactivation of JAK/STAT signaling and MK hyperplasia, which is associated with an aberrant inflammatory signature. Since JAK1/2 inhibitor alone is incapable of depleting the malignant HSC clones or reversing BM fibrosis, the identification of mechanisms that cooperate with MF JAK/STAT signaling to promote disease progression might help in developing combination therapies to modify disease outcomes. Chronic inflammation and MK hyperplasia result in an abnormal release of TGFβ1, which plays a critical role in the pathobiology of MF by contributing to the development of BM fibrosis. Dysregulated TGFβ signaling can also alter the hematopoietic microenvironment supporting the predominance of MF-HSCs and enhance the quiescence of the reservoir of wild-type HSCs. Upregulation of TGFβ1 levels is a relatively late event in MF, while during the early pre-fibrotic stage of MF the alarmin S100A8/S100A9 heterocomplex promotes pro-inflammatory responses and sustains the progression of MF-HSCs. In this review, we will discuss the recent advances in our understanding of the roles of abnormal megakaryopoiesis, and the altered microenvironment in MF progression and the development of novel combined targeted therapies to disrupt the aberrant interplay between MKs, the BM microenvironment and malignant HSCs which would potentially limit the expansion of MF-HSC clones.

A major role for human (h)CXCL8 (interleukin-8) in the pathobiology of myelofibrosis (MF) has been suggested by observations indicating that MF megakaryocytes express increased levels of hCXCL8 and that plasma levels of this cytokine in MF patients are predictive of poor patient outcomes. Here, we demonstrate that, in addition to high levels of TGF-β, the megakaryocytes from the bone marrow of the Gata1low mouse model of myelofibrosis express high levels of murine (m)CXCL1, the murine equivalent of hCXCL8, and its receptors CXCR1 and CXCR2. Treatment with the CXCR1/R2 inhibitor, Reparixin in aged-matched Gata1low mice demonstrated reductions in bone marrow and splenic fibrosis. Of note, the levels of fibrosis detected using two independent methods (Gomori and reticulin staining) were inversely correlated with plasma levels of Reparixin. Immunostaining of marrow sections indicated that the bone marrow from the Reparixin-treated group expressed lower levels of TGF-β1 than those expressed by the bone marrow from vehicle-treated mice while the levels of mCXCL1, and expression of CXCR1 and CXCR2, were similar to that of vehicle-treated mice. Moreover, immunofluorescence analyses performed on bone marrow sections from Gata1low mice indicated that treatment with Reparixin induced expression of GATA1 while reducing expression of collagen III in megakaryocytes. These data suggest that in Gata1low mice, Reparixin reduces fibrosis by reducing TGF-β1 and collagen III expression while increasing GATA1 in megakaryocytes. Our results provide a preclinical rationale for further evaluation of this drug alone and in combination with current JAK inhibitor therapy for the treatment of patients with myelofibrosis.

Abstract Platelets are blood components that survive in circulation for 7 to 10 days in humans. Thus, platelet production by bone marrow (BM) megakaryocytes (MKs), and their removal from the blood circulation is precisely orchestrated to maintain an average platelet count. Abnormalities in both processes can result in thrombocytopenia (low platelet count) or thrombocytosis (high platelet count), often associated with the risk of bleeding or overt thrombus formation, respectively. Platelet glycans, particularly sialic acids, are indicators of platelet count. Loss of platelet sialic acids leads to platelet clearance. A State‐of‐the‐Art lecture titled “Platelet and Megakaryocyte Glycobiology” was presented at the ISTH virtual congress 2021 to discuss (i) the loss of O‐glycan sialic acid on BM MKs, revealing the Thomsen‐Friedenreich (TF) antigen as a new concept of thrombocytopenia; herein, impaired thrombopoiesis is attributed to activation of immune cells with a plasmacytoid dendritic cell signature; and (ii) upregulation of antibodies against the TF antigen in pediatric patients with immune thrombocytopenia (ITP), positing that glycan alterations such as MK asialylation can lead to immune cell responses. Here, we discuss our findings alongside new data presented at the 2020 and 2021 ISTH congresses on the role of sialic acids and glycans in regulating platelet count. Desialylation is a prominent feature in thrombocytopenia, notably in ITP presentation. We compare similarities between ITP mediated with shear‐stress and with storage‐related asialylation. We also discuss genes involved in sialic acid synthesis leading to thrombocytopenia. Increased awareness in gene‐regulating MK and platelet glycans is a giant leap to understanding the underpinning mechanisms of ITP and other forms of thrombocytopenia.

The lung is an important site of extramedullary platelet formation, and megakaryocytes in the lung participate in immune responses in addition to platelet production. In acute lung injury and chronic lung injury, megakaryocytes and platelets play a promoting or protective role through different mechanisms. The authors reviewed the role of megakaryocytes and platelets in common clinical lung injuries with different course of disease and different pathogenic factors in order to provide new thinking for the diagnosis and treatment of lung injuries.

Inflammatory insults affect platelet production, but it is yet unknown what mechanisms can drive rapid adaptations in thrombopoiesis. In this issue of Immunity , Petzold et al. (2022) propose that neutrophils "pluck" on megakaryocytes in the bone marrow to tune platelet release. [ABSTRACT FROM AUTHOR]

Protease-activated receptor-4 (PAR4) is a G protein-coupled receptor (GPCR) for thrombin and is proteolytically activated, similar to the prototypical PAR1. Due to the irreversible activation of PAR1, receptor trafficking is intimately linked to signal regulation. However, unlike PAR1, the mechanisms that control PAR4 trafficking are not known. Here, we sought to define the mechanisms that control PAR4 trafficking and signaling. In HeLa cells depleted of clathrin by siRNA, activated PAR4 failed to internalize. Consistent with clathrin-mediated endocytosis, expression of a dynamin dominant-negative K44A mutant also blocked activated PAR4 internalization. However, unlike most GPCRs, PAR4 internalization occurred independently of β-arrestins and the receptor's C-tail domain. Rather, we discovered a highly conserved tyrosine-based motif in the third intracellular loop of PAR4 and found that the clathrin adaptor protein complex-2 (AP-2) is important for internalization. Depletion of AP-2 inhibited PAR4 internalization induced by agonist. In addition, mutation of the critical residues of the tyrosine-based motif disrupted agonist-induced PAR4 internalization. Using Dami megakaryocytic cells, we confirmed that AP-2 is required for agonist-induced internalization of endogenous PAR4. Moreover, inhibition of activated PAR4 internalization enhanced ERK1/2 signaling, whereas Akt signaling was markedly diminished. These findings indicate that activated PAR4 internalization requires AP-2 and a tyrosine-based motif and occurs independent of β-arrestins, unlike most classical GPCRs. Moreover, these findings are the first to show that internalization of activated PAR4 is linked to proper ERK1/2 and Akt activation.

In a classical view of hematopoiesis, the various blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. We developed a cell-sorting scheme to resolve myeloid (My), erythroid (Er), and megakaryocytic (Mk) fates from single CD34(+) cells and then mapped the progenitor hierarchy across human development. Fetal liver contained large numbers of distinct oligopotent progenitors with intermingled My, Er, and Mk fates. However, few oligopotent progenitor intermediates were present in the adult bone marrow. Instead, only two progenitor classes predominate, multipotent and unipotent, with Er-Mk lineages emerging from multipotent cells. The developmental shift to an adult "two-tier" hierarchy challenges current dogma and provides a revised framework to understand normal and disease states of human hematopoiesis.

The article discusses a study on mapping megakaryocyte progenitors (MKPs) from pluripotent stem cells. It performs single-cell RNA sequencing in vitro differentiation and mapped it in vivo that identifies five surface markers, and culture optimization to increase MKP production. It concludes that in vitro in the MKP state mirrors the state in vivo thus preventing hemorrhage from platelet deficiency with platelet transfusions.

This study investigates the response of bone marrow (particularly megakaryocytes) in mice under the influence of diclofenac sodium for 10 days using intraperitoneal injection at various doses. A fundamentally new immunomagnetic separation method was applied during the experiment, which helped obtain pure lines of bone marrow cells, particularly megakaryocytes (MC), without admixtures of other cells or their particles. The resulting cells completely retain their structure and can be used in further research. The study determined that different doses of diclofenac sodium have different effects on different groups of diabetes mellitus cells CD34-megakaryocytes. The use of 1.0 mg/ml sharply negatively affects the state of early populations of megakaryocytes (decrease by 80%, p=0.05), a dose of 0.025 mg/ml had the least effect on this population of cells (22.8%, p=0.05). The greatest number of average forms of diabetes mellitus 34 was observed when using a dose of 0.95 mg/ml (22.8%, p=0.05), with a gradual decrease in the dose, the indicator of this group of cells decreased. A dose of 0.03 mg/ml did not affect the quantitative state of megakaryocytes, and a dose of 0.025 mg/ml caused a slight decrease (16.6%, p=0.05). Indicators of mature cells of megakaryocytes CD 34-decreased in all studied groups, however, their maximum value reached a maximum decrease by 0.25 mg/ml (55.2%, p=0.05), the dose of diclofenac sodium 0.03 mg/ml, lower (18.4%, p=0.05). Diclofenac sodium in different doses has different effects on the degree of differentiation of CD 34-. Its introduction positively affects the state of intermediate forms of megakaryocytes, except for minimal doses, while the effect on early and mature forms in all cases turned out to be negative. [ABSTRACT FROM AUTHOR]

Thrombocytopenia is defined as a platelet count of less than 1,50,000 per cubic millimeter. It is a hematological presentation which could have a multitude of causes. This study was conducted with an objective to identify the alterations in number and morphology of megakaryocytes in different hematological disorders causing thrombocytopenia. A prospective bone marrow aspiration study was conducted on 100 patients. Bone marrow aspiration was done, and smears were examined for quantitative as well as qualitative changes. Bone marrow aspirate films were studied by two pathologists separately and megakaryocytic alterations were documented and analyzed using Fischer's Exact Test. Out of a total of 100 cases of thrombocytopenia, 49% were megaloblastic anaemia, followed by ITP (13%), Dimorphic anaemia (11%), CML-blast crisis (7%) and AML (6%) among others. We observed an increase in the number of megakaryocytes in 28% (n=14) cases of megaloblastic anaemia, and 77% (n=10) cases of ITP. 67.35% (n=33) cases of megaloblastic anemia and 72.73% (n=8) cases of dimorphic anaemia showed hyperlobated megakaryocytes. 61.54% (n=8) cases of ITP showed hypolobated megakaryocytes. Cases of AML, Aplastic anaemia and hairy cell leukaemia showed normal number of nuclear lobes. 51.02 % (n=25) and 61.53 % (n=8) cases of megaloblastic anaemia and ITP respectively showed presence of dysplastic megakaryocytes. Detailed evaluation is key to establish the relationship between megakaryocytic alterations with the different causes of thrombocytopenia. [ABSTRACT FROM AUTHOR]

Objective: To investigate the application value of bone marrow megakaryocyte count, the proportion of megakaryocytes at each stage, and the platelet parameter in the clinical diagnosis of primary immune thrombocytopenia (ITP). Methods: The megakaryocyte and platelet parameter level in 62 ITP and 40 control group patients were compared and analyzed. Linear correlation analysis, Pearson correlation analysis, and ROC curves were performed for the correlation between megakaryocytes and platelet parameters. Results: Compared to the control group, the total number of megakaryocytes, the promegakaryocytes the granular megakaryocytes (GMeg), and naked megakaryocytes (NMeg), MPV, and P-LCR% in the ITP group increased. All differences were statistically significant (P <0.05). While the proportion of platelet-producing megakaryocytes (PMeg), PLT, and PCT decreased in the ITP group. These differences were statistically significant (P < 0.05). PLT was strongly positively correlated with PCT (r = 0.921, p<0.01). PCT was weakly positively with MPV (r = 0.309, p<0.05). MPV was positively correlated with P-LCR (r = 0.856, p<0.01). PDW was weakly positively correlated with P-LCR (r = 0.296, p<0.05) and Meg (r = 0.301, p<0.05), and negatively correlated with PMeg (r = -0.336, p<0.05). ROC curve analysis showed that PLT, PCT MPV and P-LCR% gave a high sensitivity（100.0%，81.0%，74.6%，90.5%，respectively.) and specificity (100.0%, 92.5%, 80.0%, 77.5%, respectively.) in diagnosis of ITP. Conclusion: The combined analysis of bone marrow megakaryocyte count, the proportion of megakaryocyte classification at each stage, and platelet parameters have an important reference value for auxiliary diagnosis of ITP. [ABSTRACT FROM AUTHOR]

Myeloproliferative neoplasms (MPNs) are characterized by upregulation of proinflammatory cytokines and immune dysregulation, which provide a reasonable basis for immunotherapy in patients. Megakaryocytes are crucial in the pathogenesis of primary myelofibrosis (PMF), the most clinically aggressive subtype of MPN. In this study, we aimed to explore PD‐L1 (programmed death‐ligand 1) expression in megakaryocytes and its clinical implications in PMF. We analyzed PD‐L1 expression on megakaryocytes in PMF patients by immunohistochemistry and correlated the results with clinicopathological features and molecular aberrations. We employed a two‐tier grading system considering both the proportion of cells positively stained and the intensity of staining. Among the 85 PMF patients, 41 (48%) showed positive PD‐L1 expression on megakaryocytes with the immune‐reactive score ranging from 1 to 12. PD‐L1 expression correlated closely with higher white blood cell count (p = 0.045), overt myelofibrosis (p = 0.010), JAK2V617F mutation (p = 0.011), and high‐molecular risk mutations (p = 0.045), leading to less favorable overall survival in these patients (hazard ratio 0.341, 95% CI 0.135–0.863, p = 0.023). Our study provides unique insights into the interaction between immunologic and molecular phenotypes in PMF patients. Future work to explore the translational potential of PD‐L1 in the clinical setting is needed. [ABSTRACT FROM AUTHOR]

Background: Thrombocytopenia is one of the most common hematological disease that can be life-threatening caused by bleeding complications. However, the treatment options for thrombocytopenia remain limited. Methods: In this study, giemsa staining, phalloidin staining, immunofluorescence and flow cytometry were used to identify the effects of 3,3ʹ-di-O-methylellagic acid 4ʹ-glucoside (DMAG), a natural ellagic acid derived from Sanguisorba officinalis L. (SOL) on megakaryocyte differentiation in HEL cells. Then, thrombocytopenia mice model was constructed by X-ray irradiation to evaluate the therapeutic action of DMAG on thrombocytopenia. Furthermore, the effects of DMAG on platelet function were evaluated by tail bleeding time, platelet aggregation and platelet adhesion assays. Next, network pharmacology approaches were carried out to identify the targets of DMAG. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to elucidate the underling mechanism of DMAG against thrombocytopenia. Finally, molecular docking simulation, molecular dynamics simulation and western blot analysis were used to explore the relationship between DAMG with its targets. Results: DMAG significantly promoted megakaryocyte differentiation of HEL cells. DMAG administration accelerated platelet recovery and megakaryopoiesis, shortened tail bleeding time, strengthened platelet aggregation and adhesion in thrombocytopenia mice. Network pharmacology revealed that ITGA2B, ITGB3, VWF, PLEK, TLR2, BCL2, BCL2L1 and TNF were the core targets of DMAG. GO and KEGG pathway enrichment analyses suggested that the core targets of DMAG were enriched in PI3K–Akt signaling pathway, hematopoietic cell lineage, ECM-receptor interaction and platelet activation. Molecular docking simulation and molecular dynamics simulation further indicated that ITGA2B, ITGB3, PLEK and TLR2 displayed strong binding ability with DMAG. Finally, western blot analysis evidenced that DMAG up-regulated the expression of ITGA2B, ITGB3, VWF, p-Akt and PLEK. Conclusion: DMAG plays a critical role in promoting megakaryocytes differentiation and platelets production and might be a promising medicine for the treatment of thrombocytopenia. [ABSTRACT FROM AUTHOR]

• Coronavirus disease 2019 (COVID-19)–associated cytokine storm may result in aberrant megakaryocyte formation. • Abnormal megakaryocyte behavior may cause severe COVID-19 clinical manifestations. • Elevated megakaryocytes in COVID-19 may lead to thrombosis and abnormal coagulopathy. • Patients with COVID-19 have large numbers of megakaryocytes in their cardiac and pulmonary systems. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly across the world and has resulted in more than 4.2 million global deaths as of July 30, 2021. For reasons that remain unknown, the coronavirus disease 2019 (COVID-19) clinically manifests itself in various levels of severity, with most patients positive for COVID-19 being asymptomatic or having only mild symptoms. However, clinical studies in severely ill patients have implicated that manifestations of this infection are due in part to abnormal megakaryocyte (MK) behavior. Additionally, COVID-19–associated cytokine storms have been found to induce aberrant MK formation, primarily through interleukin-6 and Janus kinase-signal transducer and activator of transcription signaling. Autopsy reports have revealed significantly higher rates of MKs in the pulmonary and cardiac systems, which may be responsible for the high rate of thrombotic complications and abnormal coagulopathies in patients with severe forms of COVID-19. This review examines MKs and their potential function in the clinical manifestations of SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID-19) is a global public health emergency with many clinical facets, and new knowledge about its pathogenetic mechanisms is deemed necessary; among these, there are certainly coagulation disorders. In the history of medicine, autopsies and tissue sampling have played a fundamental role in order to understand the pathogenesis of emerging diseases, including infectious ones; compared to the past, histopathology can be now expanded by innovative techniques and modern technologies. For the first time in worldwide literature, we provide a detailed postmortem and biopsy report on the marked increase, up to 1 order of magnitude, of naked megakaryocyte nuclei in the bone marrow and lungs from serious COVID-19 patients. Most likely related to high interleukin-6 serum levels stimulating megakaryocytopoiesis, this phenomenon concurs to explain well the pulmonary abnormal immunothrombosis in these critically ill patients, all without molecular or electron microscopy signs of megakaryocyte infection.

The first-line therapy for primary immune thrombocytopenia (ITP) is steroids, but about one-third of patients do not respond to steroids. Recent studies have shown megakaryocyte (MK) growth and development abnormalities and poorly compensated thrombopoiesis. Here, we attempted to determine the impact of MK morphological classification on steroid response. We enrolled 170 adult patients with primary ITP and divided them into steroid-sensitive ITP (109/170) and non-steroid-sensitive ITP (61/170) groups. In the univariate logistic model, female, reduced thrombocytogenic MK count (TMC), increased granular MK count to total MK count ratio (GMC/TM ratio), and elevated naked nucleus MK count to TM count ratio were significantly associated with steroid-sensitive ITP. In the multivariate logistic model, sex, reduced TMC, and increased GMC/TM ratio were independent predictors of steroid-sensitive ITP diagnosis. Based on the regression parameters, we established a predictive index with weighted risk score of 1 assigned each to sex, TMC, and GMC/TM ratio. A predictive index ≥2 points had the best area under the curve value (0.63) with 47.7% sensitivity and 78.7% specificity for predicting steroid sensitivity. These findings may help guide early treatment strategies in ITP.

Electron microscopy (EM) has a long history in megakaryocyte (MK) cellular biology. This chapter shows how the electron microscope, since its first appearance almost 90 years ago, has occupied center stage in the studies of MK morphology and function. It describes some of the more productive EM techniques that have shaped our understanding of the physiology of thrombopoiesis. These include the standard transmission and scanning EM techniques as well as the new imaging methods, correlative microscopy and volume EM which provide information on the 3D organization of MKs on different scales: single organelles, whole cells and tissues. For each technique, we list the advantages and limitations, the resolution that can be achieved, the technical difficulties and the applications in MK biology.

Recent advances in super-resolution (sub-diffraction limited) microscopy have yielded remarkable insights into the nanoscale architecture and behavior of cells. In addition to the capacity to provide sub 100 nm resolution, these technologies offer unique quantitative opportunities with particular relevance to platelet and megakaryocyte biology. In this review, we provide a short introduction to modern super-resolution microscopy, its applications in the field of platelet and megakaryocyte biology, and emerging quantitative approaches which will allow for unprecedented insights into the biology of these unique cell types.

The dynamics of platelet formation could only be investigated since the development of two-photon microscopy in combination with suitable fluorescent labeling strategies. In this review paper, we give an overview of recent advances in fluorescence imaging of the bone marrow that have contributed to our understanding of platelet biogenesis during the last decade. We make a brief survey through the perspectives and limitations of today’s intravital imaging, but also discuss complementary methods that may help to piece together the puzzle of megakaryopoiesis and platelet formation.