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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.

Summary Neonatal and adult megakaryocytes differ in proliferative capacity and ploidy levels, and neonatal and adult platelets differ in function, gene expression, and protein content. The mechanisms underlying these differences are incompletely understood. CDK8 and CDK19 are transcriptional kinases part of the CDK‐mediator complex, which regulates gene transcription in a cell‐specific manner. We discovered that cortistatin A, a potent highly selective inhibitor of CDK8/CDK19, significantly reduced cell expansion and increased ploidy in cord blood‐derived megakaryocytes. These phenotypic changes were associated with gene expression changes that partially overlapped developmentally regulated genes. These findings might have relevance for the management of developmental megakaryocyte disorders. [ABSTRACT FROM AUTHOR]

Blood platelets are produced by megakaryocytes (MKs), their parent cells, which are in the bone marrow. Once mature, MK pierces through the sinusoid vessel, and the initial protrusion further elongates as proplatelet or buds to release platelets. The mechanisms controlling the decision to initiate proplatelet and platelet formation are unknown. Here, we show that the mechanical properties of the microenvironment prevent proplatelet and platelet release in the marrow stroma while allowing this process in the bloodstream. Loss of marrow confinement following myelosuppression led to inappropriate proplatelet and platelet release into the extravascular space. We further used an inert viscoelastic hydrogel to evaluate the impact of compressive stress. Transcriptional analysis showed that culture in three-dimensional gel induced upregulation of genes related to the Rho-GTPase pathway. We found higher Rho-GTPase activation, myosin light chain phosphorylation and F-actin under mechanical constraints while proplatelet formation was inhibited. The use of latrunculin-A to decrease F-actin promoted microtubule-dependent budding and proplatelet extension inside the gel. Additionally, ex vivo exposure of intact bone marrow to latrunculin-A triggered proplatelet extensions in the interstitial space. In vivo, this confinement-mediated high intracellular tension is responsible for the formation of the peripheral zone, a unique actin-rich structure. Cytoskeleton reorganization induces the disappearance of the peripheral zone upon reaching a liquid milieu to facilitate proplatelet and platelet formation. Hence, our data provide insight into the mechanisms preventing ectopic platelet release in the marrow stroma. Identifying such pathways is especially important for understanding pathologies altering marrow mechanics such as chemotherapy or myelofibrosis. [ABSTRACT FROM AUTHOR]

From haematopoietic stem cells to megakaryocytes (Mks), cells undergo various mechanical forces that affect Mk differentiation, maturation and proplatelet formation. The mechanotransductor PIEZO1 appears to be a natural candidate for sensing these mechanical forces and regulating megakaryopoiesis and thrombopoiesis. Gain‐of‐function mutations of PIEZO1 cause hereditary xerocytosis, a haemolytic anaemia associated with thrombotic events. If some functions of PIEZO1 have been reported in platelets, few data exist on PIEZO1 role in megakaryopoiesis. To address this subject, we used an in vitro model of Mk differentiation from CD34+ cells and studied step‐by‐step the effects of PIEZO1 activation by the chemical activator YODA1 during Mk differentiation and maturation. We report that PIEZO1 activation by 4 μM YODA1 at early stages of culture induced cytosolic calcium ion influx and reduced cell maturation. Indeed, CD41+CD42+ numbers were reduced by around 1.5‐fold, with no effects on proliferation. At later stages of Mk differentiation, PIEZO1 activation promoted endomitosis and proplatelet formation that was reversed by PIEZO1 gene invalidation with a shRNA‐PIEZO1. Same observations on endomitosis were reproduced in HEL cells induced into Mks by PMA and treated with YODA1. We provide for the first time results suggesting a dual role of PIEZO1 mechanotransductor during megakaryopoiesis. [ABSTRACT FROM AUTHOR]

Background Allogeneic hematopoietic stem cell transplant for myeloid neoplasms with increased fibrosis is uncommon; morphologic features posttransplant can be concerning for persistent disease. Methods In this retrospective study, we identified 22 patients transplanted for myeloproliferative neoplasms or chronic myelomonocytic leukemia with fibrosis at our institution, and reviewed slides from pretransplant and posttransplant bone marrow biopsies. Clinical features and results of molecular, chimerism, and cytogenetic studies were retrieved from the medical record. Results Pretransplant bone marrow biopsies commonly exhibited hypercellularity, atypical megakaryocytes, and reticulin fibrosis. At day 100, 36% of biopsies had reticulin grade >MF1 and 33% of those tested had positive molecular studies, with no significant associations between day 100 marrow characteristics and molecular profile or peripheral count recovery times. In the 1 year posttransplant biopsies (n = 12), 7 of 9 had negative molecular studies; of these, none had reticulin grade >MF1, 1 had trichrome 1+, 2 had atypical megakaryocytes, and 1 was hypercellular. Conclusions Supporting recent literature, our study indicates that persistent day 100 reticulin fibrosis/collagen deposition does not show an association with day 100 molecular status. Our study additionally provides data for 12 patients with 1 year posttransplant marrow biopsies, with the majority of those lacking either increased fibrosis or molecular evidence of persistent disease. [ABSTRACT FROM AUTHOR]

Emperipolesis is a cell-within-cell phenomenon distinct from phagocytosis more often described in Rosai-Dorfman disease, where usually lymphocytes or other bone marrow cells (plasma cells, erythroblasts or neutrophils) are entirely surrounded but not engulfed by macrophages as the host cell, but occasionally megakaryocytes and neoplastic could be. Mesothelial cell has been described in a couple of cases of lymphomas affecting serous membranes, but never described in pleuritis. In the present work, the first case of emperipolesis by mesothelial cells in a patient with self-limited pleural effusion was demonstrated by immunohistochemistry and Electron Microscopy studies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Studies conducted on animal models have identified several therapeutic targets for myelofibrosis, the most severe of the myeloproliferative neoplasms. Unfortunately, many of the drugs which were effective in pre-clinical settings had modest efficacy when tested in the clinic. This discrepancy suggests that treatment for this disease requires combination therapies. To rationalize possible combinations, the efficacy in the Gata1low model of drugs currently used for these patients (the JAK1/2 inhibitor Ruxolitinib) was compared with that of drugs targeting other abnormalities, such as p27kip1 (Aplidin), TGF-β (SB431542, inhibiting ALK5 downstream to transforming growth factor beta (TGF-β) signaling and TGF-β trap AVID200), P-selectin (RB40.34), and CXCL1 (Reparixin, inhibiting the CXCL1 receptors CXCR1/2). The comparison was carried out by expressing the endpoints, which had either already been published or had been retrospectively obtained for this study, as the fold change of the values in the corresponding vehicles. In this model, only Ruxolitinib was found to decrease spleen size, only Aplidin and SB431542/AVID200 increased platelet counts, and with the exception of AVID200, all the inhibitors reduced fibrosis and microvessel density. The greatest effects were exerted by Reparixin, which also reduced TGF-β content. None of the drugs reduced osteopetrosis. These results suggest that future therapies for myelofibrosis should consider combining JAK1/2 inhibitors with drugs targeting hematopoietic stem cells (p27Kip1) or the pro-inflammatory milieu (TGF-β or CXCL1). [ABSTRACT FROM AUTHOR]

A key step in platelet production is the migration of megakaryocytes to the vascular sinusoids within the bone marrow. This homing is mediated by the chemokine CXCL12 and its receptor CXCR4. CXCR4 is also a positive regulator of platelet activation and thrombosis. Pim-1 kinase has been shown to regulate CXCR4 signalling in other cell types, and we have previously described how Pim kinase inhibitors attenuate platelet aggregation to CXCL12. However, the mechanism by which Pim-1 regulates CXCR4 signalling in platelets and megakaryocytes has yet to be elucidated. Using human platelets, murine bone marrow-derived megakaryocytes, and the megakaryocyte cell line MEG-01, we demonstrate that pharmacological Pim kinase inhibition leads to reduced megakaryocyte and platelet function responses to CXCL12, including reduced megakaryocyte migration and platelet granule secretion. Attenuation of CXCL12 signalling was found to be attributed to the reduced surface expression of CXCR4. The decrease in CXCR4 surface levels was found to be mediated by rapid receptor internalisation, in the absence of agonist stimulation. We demonstrate that pharmacological Pim kinase inhibition disrupts megakaryocyte and platelet function by reducing constitutive CXCR4 surface expression, decreasing the number of receptors available for agonist stimulation and signalling. These findings have implications for the development and use of Pim kinase inhibitors for the treatment of conditions associated with elevated circulating levels of CXCL12/SDF1α and increased thrombotic risk. [ABSTRACT FROM AUTHOR]

\nIntroduction: Serologic characterization of antihuman platelet antigen (HPA) alloantibodies is crucial in fetal neonatal alloimmune thrombocytopenia. The gold standard MAIPA assay requires fresh platelets from HPA-genotyped donors, which is challenging for some laboratories. Megakaryocytes express HPA epitopes and offer an alternative source for detecting anti-HPA antibodies. The objective of this study was to assess the efficacy of a novel assay called monoclonal antibody immobilization of megakaryocyte antigens (MAIMA) for detecting anti-HPA antibodies. Methods: CD34+ cells from buffy coats were differentiated into megakaryocytes in vitro. The performance of the MAIMA assay was evaluated using WHO reference reagents for HPA-1a, HPA-3a, and HPA-5b, along with sera samples from patients who had well-characterized anti-HPA antibodies. Results: The WHO anti-HPA-1a reference reagent showed similar binding to megakaryocytes and platelets in MAIMA and MAIPA, respectively. On the other hand, optical density (OD) values for the WHO anti-HPA-3a reference reagent were lower in MAIMA than in MAIPA. Anti-HPA-5b antibodies were not detectable in MAIMA. Patients’ sera containing anti-HPA-1a antibodies were successfully detected in MAIMA in all clinical samples. Moreover, OD values in MAIPA and MAIMA showed high correlation (r = 0.96, p < 0.001). MAIMA was reactive for samples with anti-HPA-3a as well as anti-HPA-3b; however, OD values were lower compared to MAIPA. Interestingly, all patient samples with anti-HPA-5b antibodies were tested negative in MAIMA. Conclusion: In vitro generated megakaryocytes can be used to detect anti-HPA-1a alloantibodies. However, despite this potential, they may be less suitable for the detection of alloantibodies against other HPAs such as HPA-5b. The identification of antibodies to human platelet antigens (HPA) is crucial. The standard test (MAIPA assay) is difficult because it requires fresh platelets from specific donors. This study investigated a new method, monoclonal antibody immobilization of megakaryocyte antigens (MAIMA), using in vitro generated megakaryocytes from CD34+ cells. The assay was tested against WHO reference reagents and patient samples with known anti-HPA antibodies. Results showed that MAIMA successfully detected anti-HPA-1a antibodies and correlated well with MAIPA. However, MAIMA had lower sensitivity for anti-HPA-3a and anti-HPA-5b antibodies. Notably, anti-HPA-5b antibodies were not detectable by MAIMA. In conclusion, in vitro generated megakaryocytes can effectively detect anti-HPA-1a antibodies. However, MAIMA may be less suitable for antibodies against other HPAs, such as HPA-5b. This study highlights the potential of MAIMA as an alternative assay and provides insight into its strengths and limitations in detecting specific anti-HPA antibodies. [ABSTRACT FROM AUTHOR]

Background: Developments in the field of platelet biology led to attention given to their origins and constituents. Objectives: we aimed to highlight the important factors involved in megakaryocytosis and platelet origin. Methods: We searched websites including PupMed, Medline, Google scholars, and Cochrane library for megakaryocytosis. Results: Platelets are a blood component. Indeed, they are not true cells but the cytoplasmic fragmentation of mature megakaryocytes. Their production process (megakaryocytopoiesis) occurs in bone marrow under the regulation of thrombopoietin, starting with the commitment and differentiation of HSCs and ending with the formation of polyploid megakaryocytes and the release of platelets in the blood stream as proplatelets. Recent research has focused on the implications of platelets not only in their primary role in hemostasis but also in other physiological, pathophysiological, and regenerative processes. The biological importance of platelets originates from their bioactive contents in the three intracellular storage granules, particularly alpha granules, as they have a wide range of growth factors. Conclusion: this resulted in the use of platelets at the target site in concentrations above the baseline, named platelet concentrates (PRP, PRF, and CGF), which are rich in platelets and growth factors. [ABSTRACT FROM AUTHOR]

The study of the physiological and pathophysiological processes under extreme conditions facilitates a better understanding of the state of a healthy organism and can also shed light on the pathogenesis of diseases. In recent years, it has become evident that gravitational stress affects both the whole organism and individual cells. We have previously demonstrated that simulated microgravity inhibits proliferation, induces apoptosis, changes morphology, and alters the surface marker expression of megakaryoblast cell line MEG-01. In the present work, we investigate the expression of cell cycle cyclins in MEG-01 cells. We performed several experiments for 24 h, 72 h, 96 h and 168 h. Flow cytometry and Western blot analysis demonstrated that the main change in the levels of cyclins expression occurs under conditions of simulated microgravity after 96 h. Thus, the level of cyclin A expression showed an increase in the RPM group during the first 4 days, followed by a decrease, which, together with the peak of cyclin D, may indicate inhibition of the cell cycle in the G2 phase, before mitosis. In addition, based on the data obtained by PCR analysis, we were also able to see that both cyclin A and cyclin B expression showed a peak at 72 h, followed by a gradual decrease at 96 h. STED microscopy data also confirmed that the main change in cyclin expression of MEG-01 cells occurs at 96 h, under simulated microgravity conditions, compared to static control. These results suggested that the cell cycle disruption induced by RPM-simulated microgravity in MEG-01 cells may be associated with the altered expression of the main regulators of the cell cycle. Thus, these data implicate the development of cellular stress in MEG-01 cells, which may be important for proliferating human cells exposed to microgravity in real space. [ABSTRACT FROM AUTHOR]

Objectives VEXAS syndrome is an adult-onset autoinflammatory disease caused by a somatic pathogenic mutation in the UBA1 (ubiquitin-like modifier activating enzyme 1) gene. Patients present with rheumatologic manifestations and cytopenias and may have an increased predisposition to myelodysplastic syndrome (MDS) and plasma cell neoplasms. Prior studies have reported on the peripheral blood and bone marrow findings in patients with VEXAS syndrome. Due to the protean clinical presentation and lack of specificity of morphologic features (eg, vacuoles in early erythroid and granulocytic precursors), an optimal screening methodology to identify these patients in a timely fashion is desirable. Methods To further evaluate and describe the salient diagnostic morphologic features in VEXAS syndrome, we carried out a comprehensive study of the largest single-institution cohort to date. Diagnostic and follow-up bone marrow biopsy specimens from 52 male patients with molecularly identified VEXAS syndrome underwent central review. Results Cytopenias were common in all cases, primarily macrocytic anemia, monocytopenia, and thrombocytopenia. Bone marrow aspirate and biopsy were often hypercellular, with an increased myeloid/erythroid ratio, granulocytic hyperplasia with left shift, erythroid left shift, and megakaryocyte hyperplasia, which exhibited a range of striking morphologic findings. Distinctly vacuolated myeloid and erythroid precursors were seen in more than 95% of cases. Conclusions Our data reveal potential novel diagnostic features, such as a high incidence of monocytopenia and distinct patterns of atypical megakaryopoiesis, that appear different from dysmegakaryopoiesis typically associated with MDS. In our experience, those findings are suggestive of VEXAS, in the appropriate clinical context. [ABSTRACT FROM AUTHOR]

Native and engineered extracellular vesicles generated from human megakaryocytes (huMkEVs) or from the human megakaryocytic cell line CHRF (CHEVs) interact with tropism delivering their cargo to both human and murine hematopoietic stem and progenitor cells (HSPCs). To develop non-viral delivery vectors to HSPCs based on MkEVs, we first confirmed, using NOD-scid IL2Rγnull (NSG™) mice, the targeting potential of the large EVs, enriched in microparticles (huMkMPs), chosen for their large cargo capacity. 24 h post intravenous infusion into NSG mice, huMkEVs induced a nearly 50% increase in murine platelet counts. PKH26-labeled huMkEVs or CHEVs localized to the HSPC-rich bone marrow preferentially interacting with murine HSPCs, thus confirming their receptor-mediated tropism for NSG HSPCs, and their potential to treat thromobocytopenias. We explored this tropism to functionally deliver synthetic cargo, notably plasmid DNA coding for a fluorescent reporter, to NSG HSPCs both in vitro and in vivo. We loaded huMkEVs with plasmid DNA either through electroporation or by generating hybrid particles with preloaded liposomes. Both methods facilitated successful functional targeted delivery of pDNA, as tissue weight-normalized fluorescence intensity of the expressed fluorescent reporter was significantly higher in bone marrow than other tissues. Furthermore, the fraction of fluorescent CD117+ HSPCs was nearly 19-fold higher than other cell types within the bone marrow 72-h following administration of the hybrid particles, further supporting that HSPC tropism is retained when using hybrid particles. These data demonstrate the potential of these EVs as a non-viral, HSPC-specific cargo vehicle for gene therapy applications to treat hematological diseases.

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. [ABSTRACT FROM AUTHOR]

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. Synopsis: The contribution of immune cells to the emergence of hematopoietic stem cells (HSCs) during embryonic development remains unclear. Here, combined genetic and single-cell sequencing analyses reveal megakaryocytes as a critical niche component in the mouse embryonic aorta-gonad-mesonephros (AGM) region regulating HSC precursor generation. Megakaryocyte depletion reduces HSC activity in the AGM region. Megakaryocyte depletion decreases pre-HSC formation and maturation. CD226+ megakaryocytes most potently enhance HSC specification and promote endothelial-to-hematopoietic transition by secreting TNFSF14. TNFSF14 treatment rescues formation of hematopoietic cells in the megakaryocyte-deficient cocultures. CD226-positive megakaryocytes control hematopoietic stem cell specification in the developing mouse embryo. [ABSTRACT FROM AUTHOR]

Background: Clonal cytopenia of undetermined significance (CCUS) is defined as somatic mutations of myeloid malignancy‐associated genes in the blood or bone marrow with one or more persistent unexplained cytopenias that do not meet diagnostic criteria for a defined myeloid neoplasm. CCUS with isolated thrombocytopenia (CCUS‐IT) is rare. Methods: This is a retrospective case series of patients with prolonged isolated thrombocytopenia, a pathogenic mutation on a myeloid molecular panel, and a bone marrow biopsy with morphologic atypia below the WHO‐defined diagnostic threshold for dysplasia. Results: Five male patients were identified with a median age at CCUS‐IT diagnosis of 61 years (56–74). Median duration of thrombocytopenia prior to CCUS‐IT diagnosis was 4 years (3–12), and median platelet count at CCUS‐IT diagnosis was 41 × 103/μL (26–80). All patients had megakaryocytic hyperplasia and megakaryocytes with hyperchromasia and high nuclear‐cytoplasmic ratio. Pathogenic SRSF2 mutations were identified in all 5 patients with median variant allele frequency of 36% (28%–50%). Three patients were treated with IVIg and/or steroids with no response; one of three responded to thrombopoietin receptor agonists. Three patients progressed to MDS and one to AML. Discussion: We describe the clinicopathological features of CCUS‐IT which can mimic immune thrombocytopenia. [ABSTRACT FROM AUTHOR]

We recently achieved the first-in-human transfusion of induced pluripotent stem cell-derived platelets (iPSC-PLTs) as an alternative to standard transfusions, which are dependent on donors and therefore variable in supply. However, heterogeneity characterized by thrombopoiesis-biased or immune-biased megakaryocytes (MKs) continues to pose a bottleneck against the standardization of iPSC-PLT manufacturing. To address this problem, here we employ microRNA (miRNA) switch biotechnology to distinguish subpopulations of imMKCLs, the MK cell lines producing iPSC-PLTs. Upon miRNA switch-based screening, we find imMKCLs with lower let-7 activity exhibit an immune-skewed transcriptional signature. Notably, the low activity of let-7a-5p results in the upregulation of RAS like proto-oncogene B (RALB) expression, which is crucial for the lineage determination of immune-biased imMKCL subpopulations and leads to the activation of interferon-dependent signaling. The dysregulation of immune properties/subpopulations, along with the secretion of inflammatory cytokines, contributes to a decline in the quality of the whole imMKCL population. The authors pioneered the iPSC-derived platelet transfusion in human. Here they employ miRNA switches, identifying RALB as a determinant of immune megakaryocytes and a marker for quality control, advancing standardization of iPSC-platelet production. [ABSTRACT FROM AUTHOR]

Mechanosensation is a fundamental function through which cells sense mechanical stimuli by initiating intracellular ion currents. Ion channels play a pivotal role in this process by orchestrating a cascade of events leading to the activation of downstream signaling pathways in response to particular stimuli. Piezo1 is a cation channel that reacts with Ca2+ influx in response to pressure sensation evoked by tension on the cell lipid membrane, originating from cell–cell, cell–matrix, or hydrostatic pressure forces, such as laminar flow and shear stress. The application of such forces takes place in normal physiological processes of the cell, but also in the context of different diseases, where microenvironment stiffness or excessive/irregular hydrostatic pressure dysregulates the normal expression and/or activation of Piezo1. Since Piezo1 is expressed in several blood cell lineages and mutations of the channel have been associated with blood cell disorders, studies have focused on its role in the development and function of blood cells. Here, we review the function of Piezo1 in different blood cell lineages and related diseases, with a focus on megakaryocytes and platelets. [ABSTRACT FROM AUTHOR]

Coagulation disorders are described in COVID-19 and long COVID patients. In particular, SARS-CoV-2 infection in megakaryocytes, which are precursors of platelets involved in thrombotic events in COVID-19, long COVID and, in rare cases, in vaccinated individuals, requires further investigation, particularly with the emergence of new SARS-CoV-2 variants. CD147, involved in the regulation of inflammation and required to fight virus infection, can facilitate SARS-CoV-2 entry into megakaryocytes. MCT4, a co-binding protein of CD147 and a key player in the glycolytic metabolism, could also play a role in SARS-CoV-2 infection. Here, we investigated the susceptibility of megakaryocytes to SARS-CoV-2 infection via CD147 and MCT4. We performed infection of Dami cells and human CD34+ hematopoietic progenitor cells induced to megakaryocytic differentiation with SARS-CoV-2 pseudovirus in the presence of AC-73 and syrosingopine, respective inhibitors of CD147 and MCT4 and inducers of autophagy, a process essential in megakaryocyte differentiation. Both AC-73 and syrosingopine enhance autophagy during differentiation but only AC-73 enhances megakaryocytic maturation. Importantly, we found that AC-73 or syrosingopine significantly inhibits SARS-CoV-2 infection of megakaryocytes. Altogether, our data indicate AC-73 and syrosingopine as inhibitors of SARS-CoV-2 infection via CD147 and MCT4 that can be used to prevent SARS-CoV-2 binding and entry into megakaryocytes, which are precursors of platelets involved in COVID-19-associated coagulopathy. [ABSTRACT FROM AUTHOR]

Interleukins, a diverse family of cytokines produced by various cells, play crucial roles in immune responses, immunoregulation, and a wide range of physiological and pathological processes. In the context of megakaryopoiesis, thrombopoiesis, and platelet function, interleukins have emerged as key regulators, exerting significant influence on the development, maturation, and activity of megakaryocytes (MKs) and platelets. While the therapeutic potential of interleukins in platelet-related diseases has been recognized for decades, their clinical application has been hindered by limitations in basic research and challenges in drug development. Recent advancements in understanding the molecular mechanisms of interleukins and their interactions with MKs and platelets, coupled with breakthroughs in cytokine engineering, have revitalized the field of interleukin-based therapeutics. These breakthroughs have paved the way for the development of more effective and specific interleukin-based therapies for the treatment of platelet disorders. This review provides a comprehensive overview of the effects of interleukins on megakaryopoiesis, thrombopoiesis, and platelet function. It highlights the potential clinical applications of interleukins in regulating megakaryopoiesis and platelet function and discusses the latest bioengineering technologies that could improve the pharmacokinetic properties of interleukins. By synthesizing the current knowledge in this field, this review aims to provide valuable insights for future research into the clinical application of interleukins in platelet-related diseases. [ABSTRACT FROM AUTHOR]

Functional bone marrow studies have focused primarily on hematopoietic progenitors, leaving limited knowledge about other fragile populations, such as bone marrow adipocytes (BMAds) and megakaryocytes. The isolation of these cells is challenging due to rupture susceptibility and large size. We introduce here a label-free cytometry microsystem, MarrowCellDLD, based on deterministic lateral displacement. MarrowCellDLD enables the isolation of large, fragile BM-derived cells based on intrinsic size properties while preserving their viability and functionality. Bone marrow adipocytes, obtained from mouse and human stromal line differentiation, as well as megakaryocytes, from primary human CD34+ hematopoietic stem and progenitor cells, were used for validation. Precise micrometer-range separation cutoffs were adapted for each cell type. Cells were sorted directly in culture media, without pre-labeling steps, and with real-time imaging for quality control. At least 106 cells were retrieved intact per sorting round. Our method outperformed two FACS instruments in purity and yield, particularly for large cell size fractions. MarrowCellDLD represents a non-destructive sorting tool for large, fragile BM-derived cells, facilitating the separation of pure populations of BMAds and megakaryocytes to further investigate their physiological and pathological roles. [ABSTRACT FROM AUTHOR]

BACKGROUND: Platelets play vital roles in repair of the minute vascular damages in day to day life, wound repair, the innate immune response and metastatic tumour cell biology. AIMS AND OBJECTIVES: The aim of our study was to examine bone marrow aspiration smears for changes in megakaryocytes in patients with thrombocytopenia. MATERIALS AND METHODS: It was a prospective study carried out from June 2022 to June 2023. A series of 53 bone marrow aspirates were examined in patients with thrombocytopenia to look for changes in megakaryocytes. RESULTS: Out of the 53 cases megakaryocytes were suppressed in 20 cases (37%), inadequate in 9 cases (17%), hypoactive in 11 cases (21%), absent in 6 cases (11.5%) and adequate in 7 cases (13.5%) of thrombocytopenia. In our study nutritional deficiency was the most common cause of thrombocytopenia. CONCLUSION: Peripheral blood and bone marrow examination is crucial for patients with thrombocytopenia. Understanding the morphological changes of megakaryocytes in bone marrow aspirates can improve the diagnostic accuracy. [ABSTRACT FROM AUTHOR]

Background: The essential roles of platelets in thrombosis have been well recognized. Unexpectedly, thrombosis is prevalent during thrombocytopenia induced by cytotoxicity of biological, physical and chemical origins, which could be suffered by military personnel and civilians during chemical, biological, radioactive, and nuclear events. Especially, thrombosis is considered a major cause of mortality from radiation injury-induced thrombocytopenia, while the underlying pathogenic mechanism remains elusive. Methods: A mouse model of radiation injury-induced thrombocytopenia was built by exposing mice to a sublethal dose of ionizing radiation (IR). The phenotypic and functional changes of platelets and megakaryocytes (MKs) were determined by a comprehensive set of in vitro and in vivo assays, including flow cytometry, flow chamber, histopathology, Western blotting, and chromatin immunoprecipitation, in combination with transcriptomic analysis. The molecular mechanism was investigated both in vitro and in vivo, and was consolidated using MK-specific knockout mice. The translational potential was evaluated using a human MK cell line and several pharmacological inhibitors. Results: In contrast to primitive MKs, mature MKs (mMKs) are intrinsically programmed to be apoptosis-resistant through reprogramming the Bcl-xL-BAX/BAK axis. Interestingly, mMKs undergo minority mitochondrial outer membrane permeabilization (MOMP) post IR, resulting in the activation of the cyclic GMP-AMP synthase-stimulator of IFN genes (cGAS-STING) pathway via the release of mitochondrial DNA. The subsequent interferon-β (IFN-β) response in mMKs upregulates a GTPase guanylate-binding protein 2 (GBP2) to produce large and hyperreactive platelets that favor thrombosis. Further, we unmask that autophagy restrains minority MOMP in mMKs post IR. Conclusions: Our study identifies that megakaryocytic mitochondria-cGAS/STING-IFN-β-GBP2 axis serves as a fundamental checkpoint that instructs the size and function of platelets upon radiation injury and can be harnessed to treat platelet pathologies. [ABSTRACT FROM AUTHOR]

In this study, we reported that novel single-chain fusion proteins linking thromboxane A2 (TXA2) receptor (TP) to a selected G-protein α-subunit q (SC-TP-Gαq) or to α-subunit s (SC-TP-Gαs) could be stably expressed in megakaryocytes (MKs). We tested the MK-released platelet-linked particles (PLPs) to be used as a vehicle to deliver the overexpressed SC-TP-Gαq or the SC-TP-Gαs to regulate human platelet function. To understand how the single-chain TP-Gα fusion proteins could regulate opposite platelet activities by an identical ligand TXA2, we tested their dual functions—binding to ligands and directly linking to different signaling pathways within a single polypeptide chain—using a 3D structural model. The immature MKs were cultured and transfected with cDNAs constructed from structural models of the individual SC-TP-Gαq and SC-TP-Gαs, respectively. After transient expression was identified, the immature MKs stably expressing SC-TP-Gαq or SC-TP-Gαs (stable cell lines) were selected. The stable cell lines were induced into mature MKs which released PLPs. Western blot analysis confirmed that the released PLPs were carrying the recombinant SC-TP-Gαq or SC-TP-Gαs. Flow cytometry analysis showed that the PLPs carrying SC-TP-Gαq were able to perform the activity by promoting platelet aggregation. In contrast, PLPs carrying SC-TP-Gαs reversed Gq to Gs signaling to inhibit platelet aggregation. This is the first time demonstrating that SC-TP-Gαq and SC-TP-Gαs were successfully overexpressed in MK cells and released as PLPs with proper folding and programmed biological activities. This bio-engineering led to the formation of two sets of biologically active PLP forms mediating calcium and cAMP signaling, respectively. As a result, these PLPs are able to bind to identical endogenous TXA2 with opposite activities, inhibiting and promoting platelet aggregation as reprogrammed for therapeutic process. Results also demonstrated that the nucleus-free PLPs could be used to deliver recombinant membrane-bound GPCRs to regulate cellular activity in general. [ABSTRACT FROM AUTHOR]

Human embryonic stem cells (hESCs) have become an ideal cell source for the ex vivo generation of megakaryocyte (MK) and platelet products for clinical applications. However, an ongoing challenge is to establish scalable culture systems to maximize the yield of stem cell‐derived MKs that release platelets. We defined a specific dynamic 3D manufacturing system in a baffled‐flow manner that could remarkably facilitate megakaryopoiesis and increase the yield of platelet‐producing MKs from hESCs within a 12‐day induction period. Additionally, an increased number of >16N ploidy MKs, proplatelets, and platelets were generated from induced cells harvested on Day 12 using the specific dynamic culture method. The specific dynamic culture method significantly enhanced endothelium‐to‐haematopoietic transition and early haematopoiesis. More importantly, MK fate was significantly facilitated in a specific dynamic manner during early haematopoiesis. Mechanistically, this dynamic culture significantly enhanced mitochondrial function via the oxidative phosphorylation pathway and caused differentiation skewing of hESCs toward megakaryopoiesis. This study can aid in the automatic and scalable production of MKs from stem cells using baffled‐flow bioreactors and assist in the manufacturing of hESC‐derived MK and platelet products. [ABSTRACT FROM AUTHOR]

Megakaryocytes (MKs), the largest and rarest cells of the hematopoietic system, differentiate by increasing their size, DNA and cytoplasmic contents during maturation in order to release high numbers of blood platelets into the circulation. The gold-standard to study these complex cells is the isolation of primary MKs from the native bone marrow (BM). This is typically achieved by using fluorescence- or magnetic-activated cell sorting. However, both methods are timeconsuming and require a trained experimenter who is able to operate highly priced special equipment. Here, we demonstrate a simple and rapid alternative method to enrich mature MKs (=16 N) from murine adult BM by size exclusion. The purity of the MK fraction reached 70-80% after isolation (100- to 250-fold enrichment). Reanalysis of isolated MKs by confocal microscopy revealed the expected expression of lineage-defining MK- and platelet-specific surface receptors, including CD42a/b/d and CD41/CD61. In addition, we detected a clear enrichment of MKspecific proteins/transcripts like ß1-tubulin, ß3-integrin, GPVI and GPIba, whereas the neutrophil marker Ly6G was only detectable in the BM sample. Taken together, we demonstrate that the protocol proposed in this Technical Report is a compatible addition to established isolation methods. [ABSTRACT FROM AUTHOR]

The field of proteomics and its application to platelet biology, is rapidly and promisingly developing. Platelets (and megakaryocytes) are postulated as biosensors of health and disease, and their proteome poses as a tool to identify the specific health-disease hallmarks. Furthermore, the clinical management of certain pathologies where platelets are active players demands the development of alternative treatments, such is the case in patients where the balance thrombosis-bleeding is compromised, and a proteomics approach might aid at the identification of novel targets. Hereby, the mouse and human platelet proteomes and secretomes from public databases are compared, which shows that human and mouse platelets share a highly conserved proteome, considering identified proteins, and most importantly, their relative abundance. These supports, also interspecies wise, the use of the proteomics tool in the field, substantiated by a growing number of clinically relevant studies in humans or preclinical models. While the study of platelets through proteomics seems accessible and direct (i.e. noninvasive blood sampling, enucleated), there are some points of concern regarding the quality control of samples for such proteomics studies. Importantly, the quality of the generated data is improving over the years, which will allow cross-study comparisons. In parallel, the application of proteomics to the megakaryocyte compartment has a promising but long journey ahead. We foresee and encourage the application of platelet proteomics for diagnostic/prognostic purposes even beyond hematopoiesis and transfusion medicine, and as a tool that will procure the improvement of current therapies and the development of alternative treatment options. [ABSTRACT FROM AUTHOR]

The anucleate human platelets contain a broad pattern of mRNAs and other RNA transcripts. The high quantitative similarity of mRNAs in megakaryocytes and platelets from different sources points to a common origin, and suggests a random redistribution of mRNA species upon proplatelet formation. A comparison of the classified platelet transcriptome (17.6k transcripts) with the identified platelet proteome (5.2k proteins) indicates an underrepresentation of: (i) nuclear but not of other organellar proteins; (ii) membrane receptors and channels with low transcript levels; (iii) transcription/translation proteins; and (iv) so far uncharacterized proteins. In this review, we discuss the technical, normalization and databasedependent possibilities and challenges to come to a complete, genome-wide platelet transcriptome and proteome. Such a reference transcriptome and proteome can serve to further elucidate intra-subject and inter-subject differences in platelets in health and disease. Applications may also lay in the aid of genetic diagnostics. [ABSTRACT FROM AUTHOR]

The bone marrow (BM) hematopoietic system (HS) gives rise to blood cells originating from hematopoietic stem cells (HSCs), including megakaryocytes (MKs) and red blood cells (erythrocytes; RBCs). Many steps of the cell-fate decision remain to be elucidated, being important for cancer treatment. To explore the role of Wnt/β-catenin for MK and RBC differentiation, we activated β-catenin signaling in platelet-derived growth factor b (Pdgfb)-expressing cells of the HS using a Cre-lox approach (Ctnnb1BM-GOF). FACS analysis revealed that Pdgfb is mainly expressed by megakaryocytic progenitors (MKPs), MKs and platelets. Recombination resulted in a lethal phenotype in mutants (Ctnnb1BM-GOFwt/fl, Ctnnb1BM-GOFfl/fl) 3 weeks after tamoxifen injection, showing an increase in MKs in the BM and spleen, but no pronounced anemia despite reduced erythrocyte counts. BM transplantation (BMT) of Ctnnb1BM-GOF BM into lethally irradiated wildtype recipients (BMT-Ctnnb1BM-GOF) confirmed the megakaryocytic, but not the lethal phenotype. CFU-MK assays in vitro with BM cells of Ctnnb1BM-GOF mice supported MK skewing at the expense of erythroid colonies. Molecularly, the runt-related transcription factor 1 (RUNX1) mRNA, known to suppress erythropoiesis, was upregulated in Ctnnb1BM-GOF BM cells. In conclusion, β-catenin activation plays a key role in cell-fate decision favoring MK development at the expense of erythroid production. [ABSTRACT FROM AUTHOR]

Influenza viruses pose a serious threat to public health, with severe cases often characterized by lung damage and inflammation. However, the underlying mechanisms of these processes remain poorly understood. This study aimed to investigate the essential role of megakaryocytes (MKs) and platelets (PLTs) in influenza A virus (IAV) infections. Conducted at the Department of Rare Respiratory Diseases, Cystic Fibrosis, and Pulmonology, Nord University Hospital, Marseille, France, the study collected seventy blood samples between October 2018 and March 2019. Samples were obtained from healthy individuals and patients diagnosed with IAV. Messenger RNA was extracted from isolated PLTs and subjected to quantitative real-time-polymerase chain reaction using sets of primers targeting immune marker genes. Western blot analysis was also performed for confirmation, focusing on Fas Ligand (FasL). Results showed that PLTs from IAV-infected individuals expressed the FasL, tumor necrosis factor-related apoptosis-inducing ligand, and Granulysin (GNLY) receptors when activated. Furthermore, an in vitro assay revealed the presence of FasL receptors on infected CMK cell lines. In vivo investigations demonstrated that activated MKs and PLTs in mice also expressed FasL. Interestingly, none of the immune receptors under investigation were found in both MKs and PLTs in mouse model studies. In conclusion, MKs and PLTs play a significant role in influencing immune responses that may help prevent viral spread during infection. However, further examination of their mechanisms of action is warranted. Understanding the involvement of these cells in influenza pathogenesis could offer valuable insights for developing potential therapeutic strategies. [ABSTRACT FROM AUTHOR]

Platelets have a wide range of functions including critical roles in hemostasis, thrombosis, and immunity. We hypothesized that during acute inflammation, such as in life-threatening sepsis, there are fundamental changes in the sites of platelet production and phenotypes of resultant platelets. Here, we showed during sepsis that the spleen was a major site of megakaryopoiesis and platelet production. Sepsis provoked an adrenergic-dependent mobilization of megakaryocyte-erythrocyte progenitors (MEPs) from the bone marrow to the spleen, where IL-3 induced their differentiation into megakaryocytes (MKs). In the spleen, immune-skewed MKs produced a CD40 ligandhi platelet population with potent immunomodulatory functions. Transfusions of post-sepsis platelets enriched from splenic production enhanced immune responses and reduced overall mortality in sepsis-challenged animals. These findings identify a spleen-derived protective platelet population that may be broadly immunomodulatory in acute inflammatory states such as sepsis.

Abstract Bone marrow fibrosis represents an important structural change in the marrow that interferes with some of its normal functions. The aetiopathogenesis of fibrosis is not well established except in its primary form. The present review consolidates current understanding of marrow fibrosis. We searched PubMed without time restriction using key words: bone marrow and fibrosis as the main stem against the terms: growth factors, cytokines and chemokines, morphology, megakaryocytes and platelets, myeloproliferative disorders, myelodysplastic syndrome, collagen biosynthesis, mesenchymal stem cells, vitamins and minerals and hormones, and mechanism of tissue fibrosis. Tissue marrow fibrosis-related papers were short listed and analysed for the review. It emerged that bone marrow fibrosis is the outcome of complex interactions between growth factors, cytokines, chemokines and hormones together with their facilitators and inhibitors. Fibrogenesis is initiated by mobilisation of special immunophenotypic subsets of mesenchymal stem cells in the marrow that transform into fibroblasts. Fibrogenic stimuli may arise from neoplastic haemopoietic or non-hematopoietic cells, as well as immune cells involved in infections and inflammatory conditions. Autoimmunity is involved in a small subset of patients with marrow fibrosis. Megakaryocytes and platelets are either directly involved or are important intermediaries in stimulating mesenchymal stem cells. MMPs, TIMPs, TGF-β, PDGRF, and basic FGF and CRCXL4 chemokines are involved in these processes. Genetic and epigenetic changes underlie many of these conditions.

Mitochondrial DNA copy number (mtDNA-CN) measured from blood specimens is a minimally invasive marker of mitochondrial function that exhibits both inter-individual and intercellular variation. To identify genes involved in regulating mitochondrial function, we performed a genome-wide association study (GWAS) in 465,809 White individuals from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium and the UK Biobank (UKB). We identified 133 SNPs with statistically significant, independent effects associated with mtDNA-CN across 100 loci. A combination of fine-mapping, variant annotation, and co-localization analyses was used to prioritize genes within each of the 133 independent sites. Putative causal genes were enriched for known mitochondrial DNA depletion syndromes (p = 3.09 × 10-15) and the gene ontology (GO) terms for mtDNA metabolism (p = 1.43 × 10-8) and mtDNA replication (p = 1.2 × 10-7). A clustering approach leveraged pleiotropy between mtDNA-CN associated SNPs and 41 mtDNA-CN associated phenotypes to identify functional domains, revealing three distinct groups, including platelet activation, megakaryocyte proliferation, and mtDNA metabolism. Finally, using mitochondrial SNPs, we establish causal relationships between mitochondrial function and a variety of blood cell-related traits, kidney function, liver function and overall (p = 0.044) and non-cancer mortality (p = 6.56 × 10-4).

Studies conducted on animal models have identified several therapeutic targets for myelofibrosis, the most severe of the myeloproliferative neoplasms. Unfortunately, many of the drugs which were effective in pre-clinical settings had modest efficacy when tested in the clinic. This discrepancy suggests that treatment for this disease requires combination therapies. To rationalize possible combinations, the efficacy in the Gata1low model of drugs currently used for these patients (the JAK1/2 inhibitor Ruxolitinib) was compared with that of drugs targeting other abnormalities, such as p27kip1 (Aplidin), TGF-β (SB431542, inhibiting ALK5 downstream to transforming growth factor beta (TGF-β) signaling and TGF-β trap AVID200), P-selectin (RB40.34), and CXCL1 (Reparixin, inhibiting the CXCL1 receptors CXCR1/2). The comparison was carried out by expressing the endpoints, which had either already been published or had been retrospectively obtained for this study, as the fold change of the values in the corresponding vehicles. In this model, only Ruxolitinib was found to decrease spleen size, only Aplidin and SB431542/AVID200 increased platelet counts, and with the exception of AVID200, all the inhibitors reduced fibrosis and microvessel density. The greatest effects were exerted by Reparixin, which also reduced TGF-β content. None of the drugs reduced osteopetrosis. These results suggest that future therapies for myelofibrosis should consider combining JAK1/2 inhibitors with drugs targeting hematopoietic stem cells (p27Kip1) or the pro-inflammatory milieu (TGF-β or CXCL1).

The study of the physiological and pathophysiological processes under extreme conditions facilitates a better understanding of the state of a healthy organism and can also shed light on the pathogenesis of diseases. In recent years, it has become evident that gravitational stress affects both the whole organism and individual cells. We have previously demonstrated that simulated microgravity inhibits proliferation, induces apoptosis, changes morphology, and alters the surface marker expression of megakaryoblast cell line MEG-01. In the present work, we investigate the expression of cell cycle cyclins in MEG-01 cells. We performed several experiments for 24 h, 72 h, 96 h and 168 h. Flow cytometry and Western blot analysis demonstrated that the main change in the levels of cyclins expression occurs under conditions of simulated microgravity after 96 h. Thus, the level of cyclin A expression showed an increase in the RPM group during the first 4 days, followed by a decrease, which, together with the peak of cyclin D, may indicate inhibition of the cell cycle in the G2 phase, before mitosis. In addition, based on the data obtained by PCR analysis, we were also able to see that both cyclin A and cyclin B expression showed a peak at 72 h, followed by a gradual decrease at 96 h. STED microscopy data also confirmed that the main change in cyclin expression of MEG-01 cells occurs at 96 h, under simulated microgravity conditions, compared to static control. These results suggested that the cell cycle disruption induced by RPM-simulated microgravity in MEG-01 cells may be associated with the altered expression of the main regulators of the cell cycle. Thus, these data implicate the development of cellular stress in MEG-01 cells, which may be important for proliferating human cells exposed to microgravity in real space.

Backgrounds: The Qing-Fei-Pai-Du Decoction (QFPDD) was reported to ameliorate the inflammatory coagulopathy and thrombocytopenia in COVID-19 patients. This work investigated the effects of QFPDD in endotoxaemia rats and then further studied its influence on platelet production using cultured megakaryocytes. Methods: The coagulation indexes and platelet aggregation reactivity of LPS-induced endotoxemia rats with or without QFPDD treatment were analyzed. The platelet parameters, the expression of thrombopoietin (TPO) in the liver and the kidney, number of the megakaryocytes in bone marrow smear of rats were checked. The maturation and platelet production of human megakaryoblastic MEG-01 cells treated with QFPDD or isochlorogenic acid A (ISA), an active component of QFPDD, were observed. Results: QFPDD improved the coagulation indexes of LPS-induced endotoxaemia rats and decreased the hyper-reactivity of platelets. QFPDD ameliorated LPS-induced decrease in platelet number, partially alleviated increase in liver TPO expression and exhibited no influence on increase in the number of bone marrow megakaryocytes. QFPDD promoted maturation and production of platelet-like particles of MEG-01 megakaryocytes. The effects of QFPDD on MEG-01 megakaryocytes might be related to activation of AKT pathway and up-regulation of related transcription factors. ISA also could induce the maturation of MEG-01 cells. Conclusions: QFPDD effectively ameliorated coagulopathy and thrombocytopenia in rats under inflammatory state. The mechanisms of QFPDD in ameliorating thrombocytopenia might be based on both decreasing effects on platelet depletion in coagulation and inducing effects on maturation and platelet production of megakaryocytes. ISA might be one of the components which contribute to the effects of QFPDD on megakaryocytes.

The field of proteomics and its application to platelet biology, is rapidly and promisingly developing. Platelets (and megakaryocytes) are postulated as biosensors of health and disease, and their proteome poses as a tool to identify the specific health-disease hallmarks. Furthermore, the clinical management of certain pathologies where platelets are active players demands the development of alternative treatments, such is the case in patients where the balance thrombosis-bleeding is compromised, and a proteomics approach might aid at the identification of novel targets. Hereby, the mouse and human platelet proteomes and secretomes from public databases are compared, which shows that human and mouse platelets share a highly conserved proteome, considering identified proteins, and most importantly, their relative abundance. These supports, also interspecies wise, the use of the proteomics tool in the field, substantiated by a growing number of clinically relevant studies in humans or preclinical models. While the study of platelets through proteomics seems accessible and direct (i.e. noninvasive blood sampling, enucleated), there are some points of concern regarding the quality control of samples for such proteomics studies. Importantly, the quality of the generated data is improving over the years, which will allow cross-study comparisons. In parallel, the application of proteomics to the megakaryocyte compartment has a promising but long journey ahead. We foresee and encourage the application of platelet proteomics for diagnostic/prognostic purposes even beyond hematopoiesis and transfusion medicine, and as a tool that will procure the improvement of current therapies and the development of alternative treatment options.

Summary: The diagnosis of myeloproliferative neoplasms (MPN) requires the integration of clinical, morphological, genetic and immunophenotypic findings. Recently, there has been a transformation in our understanding of the cellular and molecular mechanisms underlying disease initiation and progression in MPN. This has been accompanied by the widespread application of high‐resolution quantitative molecular techniques. By contrast, microscopic interpretation of bone marrow biopsies by haematologists/haematopathologists remains subjective and qualitative. However, advances in tissue image analysis and artificial intelligence (AI) promise to transform haematopathology. Pioneering studies in bone marrow image analysis offer to refine our understanding of the boundaries between reactive samples and MPN subtypes and better capture the morphological correlates of high‐risk disease. They also demonstrate potential to improve the evaluation of current and novel therapeutics for MPN and other blood cancers. With increased therapeutic targeting of diverse molecular, cellular and extra‐cellular components of the marrow, these approaches can address the unmet need for improved objective and quantitative measures of disease modification in the context of clinical trials. This review focuses on the state‐of‐the‐art in image analysis/AI of bone marrow tissue, with an emphasis on its potential to complement and inform future clinical studies and research in MPN. [ABSTRACT FROM AUTHOR]

Obesity is a major health issue that can increase the risk of cardiovascular morbidity and mortality. Obesity is characterized by an elevated prothrombotic state in which dysregulated platelet activation is a major component due to accumulation of adipose depots, insulin resistance, endothelial dysfunction, low-grade chronic inflammation and oxidative stress. Obesity also alters the homeostasis of the bone marrow, the site where megakaryopoiesis and platelet production take place, which include bone marrow adipose tissue as an important constituent of this microenvironment. Here, we provide an up-to-date review focusing on bone marrow adipose tissue and the influence of obesity on megakaryocytes and platelets that increases the cardiovascular risk. [ABSTRACT FROM AUTHOR]

Platelets are anucleated cells derived from megakaryocytes that are primarily responsible for hemostasis. However, in recent years, these cytoplasts have become increasingly recognized as immune cells, able to detect, interact with, and kill pathogens. As platelets are involved in both immunity and coagulation, they have a central role in immunothrombosis, a physiological process in which immune cells induce the formation of microthrombi to both prevent the spread of pathogens, and to help facilitate clearance. In this review, we will highlight the role of platelets as key players in the inflammatory and innate immune response against bacterial and viral infection, including direct and indirect interactions with pathogens and other immune cells. • Platelets function as immune cells, directly linking coagulation and immunity. • Platelets respond to pathogens by direct recognition or in response to inflammatory stimuli from other immune cells. • Platelets represent a key regulator between protective hemostasis and pathogenic immunothrombosis. [ABSTRACT FROM AUTHOR]

Autophagy, the continuous recycling of intracellular building blocks, molecules, and organelles is necessary to preserve cellular function and homeostasis. In this context, it was demonstrated that autophagy plays an important role in megakaryopoiesis, the development and differentiation of hematopoietic progenitor cells into megakaryocytes. Furthermore, in recent years, autophagic proteins were detected in platelets, anucleate cells generated by megakaryocytes, responsible for hemostasis, thrombosis, and a key cell in inflammation and host immune responses. In the last decade studies have indicated the occurrence of autophagy in platelets. Moreover, autophagy in platelets was subsequently demonstrated to be involved in platelet aggregation, adhesion, and thrombus formation. Here, we review the current knowledge about autophagy in platelets, its function, and clinical implications. However, at the advent of platelet autophagy research, additional discoveries derived from evolving work will be required to precisely define the contributions of autophagy in platelets, and to expand the ever increasing physiologic and pathologic roles these remarkable and versatile blood cells play. • Autophagy plays an important role in megakaryopoiesis. • Autophagosome formation and autophagy are essential in platelet biology. • Autophagy in platelets is involved in platelet aggregation, adhesion, and thrombus formation. • The importance of platelet autophagic functions is demonstrated in clinically relevant conditions and disease states, such as diabetes, hypoxia/ischemia, ITP, and sepsis. • Elaborate methods are available to study autophagy in platelets reliably and in great detail. [ABSTRACT FROM AUTHOR]

Bleeding complications in patients with myeloproliferative neoplasms are known to be related to acquired von Willebrand disease or platelet dysfunction. Here, we describe two very similar cases of chronic phase chronic myeloid leukemia with extreme leukocytosis (>500,000/mcL) and deep tissue bleeding after a minor trauma, who had increased PT and PTT, normal VWF antigen and activity. The patients were found to have acquired factor V deficiency. Factor V deficiency as a cause of bleeding in patients with uncontrolled chronic myeloid leukemia has not yet been reported. Bleeding resolved after correction of hyperleukocytosis, and FV level improved. We conclude that acquired FV deficiency is possible in patients with uncontrolled chronic myeloid leukemia and extreme leukocytosis. [ABSTRACT FROM AUTHOR]

Acute megakaryocytic leukemia (AMKL) is a rare neoplasm caused by abnormal megakaryoblasts. Megakaryoblasts keep dividing and avoid undergoing polyploidization to escape maturation. Small-molecule probes inducing polyploidization of megakaryocytic leukemia cells accelerate the differentiation of megakaryocytes. This study aims to determine that Rho kinase (ROCK) inhibition on megakaryoblasts enhances polyploidization and the inhibition of ROCK1 by fasudil benefits AMKL mice. The study investigated fasudil on the megakaryoblast cells in vitro and in vivo. With the differentiation and apoptosis induction, fasudil was used to treat 6133/MPLW515L mice, and the differentiation level was evaluated. Fasudil could reduce proliferation and promote the polyploidization of megakaryoblasts. Meanwhile, fasudil reduced the disease burden of 6133/MPLW515L AMKL mice at a dose that is safe for healthy mice. Combination therapy of ROCK1 inhibitor fasudil and reported clinical AURKA inhibitor MLN8237 achieved a better antileukemia effect in vivo, which alleviated hepatosplenomegaly and promoted the differentiation of megakaryoblast cells. ROCK1 inhibitor fasudil is a good proliferation inhibitor and polyploidization inducer of megakaryoblast cells and might be a novel rationale for clinical AMKL treatment. [ABSTRACT FROM AUTHOR]

Bone marrow fibrosis represents an important structural change in the marrow that interferes with some of its normal functions. The aetiopathogenesis of fibrosis is not well established except in its primary form. The present review consolidates current understanding of marrow fibrosis. We searched PubMed without time restriction using key words: bone marrow and fibrosis as the main stem against the terms: growth factors, cytokines and chemokines, morphology, megakaryocytes and platelets, myeloproliferative disorders, myelodysplastic syndrome, collagen biosynthesis, mesenchymal stem cells, vitamins and minerals and hormones, and mechanism of tissue fibrosis. Tissue marrow fibrosis-related papers were short listed and analysed for the review. It emerged that bone marrow fibrosis is the outcome of complex interactions between growth factors, cytokines, chemokines and hormones together with their facilitators and inhibitors. Fibrogenesis is initiated by mobilisation of special immunophenotypic subsets of mesenchymal stem cells in the marrow that transform into fibroblasts. Fibrogenic stimuli may arise from neoplastic haemopoietic or non-hematopoietic cells, as well as immune cells involved in infections and inflammatory conditions. Autoimmunity is involved in a small subset of patients with marrow fibrosis. Megakaryocytes and platelets are either directly involved or are important intermediaries in stimulating mesenchymal stem cells. MMPs, TIMPs, TGF-β, PDGRF, and basic FGF and CRCXL4 chemokines are involved in these processes. Genetic and epigenetic changes underlie many of these conditions. Highlights: Fibrosis of the marrow is always secondary to a primary event which may be clonal or non clonal. Megakaryocytes and platelets form the central arm in myelofibrosis. Haemopoietic stem cells, clonal or activated in various inflammatory processes, immune cells and megakaryocytes (clonal or activated) interact with mesenchymal stem cells of GLI+ and Lep+ subsets that differentiate into myofibroblasts, -fibroblasts and fibrocytes. At each of these steps different cytokines, vitamins, hormones and minerals interact to produce the fibrocollagenous matrix. TGF-β and CXCL4 are important growth factors and chemokines assist this process. PDGFα, VEGF, angiopoietin, bFGF-, BMP pathway and inflammatory/oxidative cytokine pathways and tissue oxygen sensing mechanism via HID1α are also involved in the process in specific cases. Vitamins, hormones, minerals, and protease/anti-protease balance finally determine the extent of fibrosis. Many signals converge to produce TGF-β that stimulates marrow fibrosis. There may be a healthy noradrenergic input to the marrow vasculature that prevents fibrosis. Epigenetic regulation of fibrosis is getting increasing attention for understanding the process and directing targeted therapy. Addressing these signals with various targeted therapies alone or in combination are being developed (e.g ruxolotinib in PMF) [ABSTRACT FROM AUTHOR]

Platelets are generated by specialized cells called megakaryocytes (MKs). However, MK's origin and platelet release mode have remained incompletely understood. Here, we established direct visualization of embryonic thrombopoiesis in vivo by combining multiphoton intravital microscopy (MP-IVM) with a fluorescence switch reporter mouse model under control of the platelet factor 4 promoter (Pf4CreRosa26mTmG). Using this microscopy tool, we discovered that fetal liver MKs provide higher thrombopoietic activity than yolk sac MKs. Mechanistically, fetal platelets were released from MKs either by membrane buds or the formation of proplatelets, with the former constituting the key process. In E14.5 c-Myb-deficient embryos that lack definitive hematopoiesis, MK and platelet numbers were similar to wild-type embryos, indicating the independence of embryonic thrombopoiesis from definitive hematopoiesis at this stage of development. In summary, our novel MP-IVM protocol allows the characterization of thrombopoiesis with high spatio-temporal resolution in the mouse embryo and has identified membrane budding as the main mechanism of fetal platelet production. [ABSTRACT FROM AUTHOR]

Recent research has focused on analysing megakaryocyte images to extract the information needed to track the progression of nervous system diseases. Segmentation is a fundamental step in describing and analysing the core contents of megakaryocytes, including the cytoplasm and nucleus. In this study, 45 megakaryocyte images were obtained. A new segmentation image technique was proposed, called the updating fuzzy c-means technique, through the intelligent selection of the centres of each cluster to separate cell components. The first step of this technique (fuzzification) was based on a knowledge analysis of the local parameters (entropy, contrast and standard deviation) that had a substantial influence on the grey-level distribution between the cytoplasm and nucleus. The second important step was the construction of fuzzy rules in terms of the variation in these local parameters to control the intelligent pick-out or update the centroid of each cluster and obtain a successful separation of the cytoplasm and nucleus. The final step was defuzzification to obtain the output images. The results revealed the superiority of the proposed method over recent technique. The accuracy of the segmented nucleus was greater than 7.46%; in the case of the cytoplasm, the accuracy was higher at 18%. These results indicated that this technique may be applied on other biomedical images. [ABSTRACT FROM AUTHOR]