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Platelets, small hemostatic blood cells, are derived from megakaryocytes. Both bone marrow and lung are principal sites of thrombopoiesis although underlying mechanisms remain unclear. Outside the body, however, our ability to generate large number of functional platelets is poor. Here we show that perfusion of megakaryocytes ex vivo through the mouse lung vasculature generates substantial platelet numbers, up to 3000 per megakaryocyte. Despite their large size, megakaryocytes are able repeatedly to passage through the lung vasculature, leading to enucleation and subsequent platelet generation intravascularly. Using ex vivo lung and an in vitro microfluidic chamber we determine how oxygenation, ventilation, healthy pulmonary endothelium and the microvascular structure support thrombopoiesis. We also show a critical role for the actin regulator Tropomyosin 4 in the final steps of platelet formation in lung vasculature. This work reveals the mechanisms of thrombopoiesis in lung vasculature and informs approaches to large-scale generation of platelets.

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.

The production of platelets is tightly regulated by thrombopoietin (THPO). Mutations in the THPO gene cause thrombocytopenia. Although mice lacking Thpo present with thrombocytopenia, predicting phenotypes and pathogenicity of novel THPO mutations in mice is limited. Zebrafish can be a powerful tool for fast validation and study of candidate genes of human hematological diseases and have already been used as a model of human thrombocytopenia.We aim to investigate the role of Thpo in zebrafish thrombopoiesis and to establish a Thpo-deficient zebrafish model. The model could be applied for illustrating the clinically discovered human THPO variants of which the clinical significance is not known and to evaluate the effect of THPO receptor agonists (THPO-Ras), as well as a screening platform for new drugs.We generated a thpo loss-of-function zebrafish model using CRISPR/Cas9. After disruption of zebrafish thpo, thpoZebrafish with the mutation thpo

A common genetic variant within the T cell receptor alpha (TCRA)-T cell receptor delta (TCRD) locus (rs2204985) has been recently found to associate with thymic function. Aim of this study was to investigate the potential impact of donor rs2204985 genotype on patient’s outcome after unrelated hematopoietic stem cell transplantation (uHSCT). 2016 adult patients were retrospectively analyzed. rs2204985 genotyping was performed by next generation sequencing, p < 0.05 was considered significant and donor rs2204985 GG/AG genotypes were set as reference vs. the AA genotype. Multivariate analysis of the combined cohort regarding the impact of donor’s rs2204985 genotype indicated different risk estimates in 10/10 and 9/10 HLA matched transplantations. A subanalysis on account of HLA incompatibility revealed that donor AA genotype in single HLA mismatched cases (n = 624) associated with significantly inferior overall- (HR: 1.48, p = 0.003) and disease-free survival (HR: 1.50, p = 0.001). This effect was driven by a combined higher risk of relapse incidence (HR: 1.40, p = 0.026) and non-relapse mortality (HR: 1.38, p = 0.042). This is the first study to explore the role of rs2204985 in a clinical uHSCT setting. Our data suggest that donor rs2204985 AA genotype in combination with single HLA mismatches may adversely impact post-HSCT outcome and should thus be avoided., publishedVersion

Fetal and neonatal megakaryocyte progenitors are hyperproliferative compared with adult progenitors and generate a large number of small, low-ploidy megakaryocytes. Historically, these developmental differences have been interpreted as “immaturity.” However, more recent studies have demonstrated that the small, low-ploidy fetal and neonatal megakaryocytes have all the characteristics of adult polyploid megakaryocytes, including the presence of granules, a well-developed demarcation membrane system, and proplatelet formation. Thus, rather than immaturity, the features of fetal and neonatal megakaryopoiesis reflect a developmentally unique uncoupling of proliferation, polyploidization, and cytoplasmic maturation, which allows fetuses and neonates to populate their rapidly expanding bone marrow and blood volume. At the molecular level, the features of fetal and neonatal megakaryopoiesis are the result of a complex interplay of developmentally regulated pathways and environmental signals from the different hematopoietic niches. Over the past few years, studies have challenged traditional paradigms about the origin of the megakaryocyte lineage in both fetal and adult life, and the application of single-cell RNA sequencing has led to a better characterization of embryonic, fetal, and adult megakaryocytes. In particular, a growing body of data suggests that at all stages of development, the various functions of megakaryocytes are not fulfilled by the megakaryocyte population as a whole, but rather by distinct megakaryocyte subpopulations with dedicated roles. Finally, recent studies have provided novel insights into the mechanisms underlying developmental disorders of megakaryopoiesis, which either uniquely affect fetuses and neonates or have different clinical presentations in neonatal compared with adult life.

Platelets (small, anucleate cell fragments) derive from large precursor cells, megakaryocytes (MKs), that reside in the bone marrow. MKs emerge from hematopoietic stem cells in a complex differentiation process that involves cytoplasmic maturation, including the formation of the demarcation membrane system, and polyploidization. The main function of MKs is the generation of platelets, which predominantly occurs through the release of long, microtubule-rich proplatelets into vessel sinusoids. However, the idea of a 1-dimensional role of MKs as platelet precursors is currently being questioned because of advances in high-resolution microscopy and single-cell omics. On the one hand, recent findings suggest that proplatelet formation from bone marrow–derived MKs is not the only mechanism of platelet production, but that it may also occur through budding of the plasma membrane and in distant organs such as lung or liver. On the other hand, novel evidence suggests that MKs not only maintain physiological platelet levels but further contribute to bone marrow homeostasis through the release of extracellular vesicles or cytokines, such as transforming growth factor β1 or platelet factor 4. The notion of multitasking MKs was reinforced in recent studies by using single-cell RNA sequencing approaches on MKs derived from adult and fetal bone marrow and lungs, leading to the identification of different MK subsets that appeared to exhibit immunomodulatory or secretory roles. In the following article, novel insights into the mechanisms leading to proplatelet formation in vitro and in vivo will be reviewed and the hypothesis of MKs as immunoregulatory cells will be critically discussed.

The process of proplatelet formation (PPF) requires coordinated interaction between megakaryocytes (MKs) and the extracellular matrix (ECM), followed by a dynamic reorganization of the actin and microtubule cytoskeleton. Localized fluxes of intracellular calcium ions (Ca2+) facilitate MK-ECM interaction and PPF. Glutamate-gated N-methyl-D-aspartate receptor (NMDAR) is highly permeable to Ca2+. NMDAR antagonists inhibit MK maturation ex vivo; however, there are no in vivo data. Using the Cre-loxP system, we generated a platelet lineage–specific knockout mouse model of reduced NMDAR function in MKs and platelets (Pf4-Grin1−/− mice). Effects of NMDAR deletion were examined using well-established assays of platelet function and production in vivo and ex vivo. We found that Pf4-Grin1−/− mice had defects in megakaryopoiesis, thrombopoiesis, and platelet function, which manifested as reduced platelet counts, lower rates of platelet production in the immune model of thrombocytopenia, and prolonged tail bleeding time. Platelet activation was impaired to a range of agonists associated with reduced Ca2+ responses, including metabotropic like, and defective platelet spreading. MKs showed reduced colony and proplatelet formation. Impaired reorganization of intracellular F-actin and α-tubulin was identified as the main cause of reduced platelet function and production. Pf4-Grin1−/− MKs also had lower levels of transcripts encoding crucial ECM elements and enzymes, suggesting NMDAR signaling is involved in ECM remodeling. In summary, we provide the first genetic evidence that NMDAR plays an active role in platelet function and production. NMDAR regulates PPF through a mechanism that involves MK-ECM interaction and cytoskeletal reorganization. Our results suggest that NMDAR helps guide PPF in vivo.

We frequently encounter newborn infants with thrombocytosis in the neonatal intensive care unit. However, neonatal thrombocytosis is not yet fully understood. Thrombocytosis is more frequently identified in newborns and young infants, notably more often in those younger than 2 years than in older children or adults. The production of megakaryocytes (megakaryopoiesis) and platelets (thrombopoiesis) is mainly regulated by thrombopoietin (TPO). Increased TPO levels during infection or inflammation can stimulate megakaryopoiesis, resulting in thrombopoiesis. TPO concentrations are higher in newborn infants than in adults. Levels increase after birth, peak on the second day after birth, and start decreasing at 1 month of age. Initial platelet counts at birth increase with gestational age. Thus, preterm infants have lower initial platelet counts at birth than late-preterm or term infants. Postnatal thrombocytosis is more frequently observed in preterm infants than in term infants. A high TPO concentration and low TPO receptor expression on platelets leading to elevated plasma-free TPO, increased sensitivity of megakaryocyte precursor cells to TPO, a decreased red blood cell count, and immaturity of platelet regulation are speculated to induce thrombocytosis in preterm infants. Thrombocytosis in newborn infants is considered a reactive process (secondary thrombocytosis) following infection, acute/chronic inflammation, or anemia. Thrombocytosis in newborn infants is benign, resolves spontaneously, and, unlike in adults, is rarely associated with hemorrhagic and thromboembolic complications.

The process of platelet production has so far been understood to be a 2-stage process: megakaryocyte maturation from hematopoietic stem cells followed by proplatelet formation, with each phase regulating the peripheral blood platelet count. Proplatelet formation releases into the bloodstream beads-on-a-string preplatelets, which undergo fission into mature platelets. For the first time, we show that preplatelet maturation is a third, tightly regulated, critical process akin to cytokinesis that regulates platelet count. We show that deficiency in cytokine receptor-like factor 3 (CRLF3) in mice leads to an isolated and sustained 25% to 48% reduction in the platelet count without any effect on other blood cell lineages. We show that Crlf3−/− preplatelets have increased microtubule stability, possibly because of increased microtubule glutamylation via the interaction of CRLF3 with key members of the Hippo pathway. Using a mouse model of JAK2 V617F essential thrombocythemia, we show that a lack of CRLF3 leads to long-term lineage-specific normalization of the platelet count. We thereby postulate that targeting CRLF3 has therapeutic potential for treatment of thrombocythemia.

BACKGROUND: Thrombocytopenia leading to life-threatening excessive bleeding can be treated by platelet transfusion. Currently, such treatments are totally dependent on donor-derived platelets. To support future applications in the use of in vitro-derived platelets, we sought to identify genes whose manipulation might improve the efficiency of megakaryocyte production and resulting hemostatic effectiveness. Disruption of Lyn kinase has previously been shown to improve cell survival, megakaryocyte ploidy and TPO-mediated activation in mice, but its role in human megakaryocytes and platelets has not been examined. METHODS: To analyze the role of Lyn at defined differentiation stages during human megakaryocyte differentiation, conditional Lyn-deficient cells were generated using CRISPR/Cas9 technology in iPS cells. The efficiency of Lyn-deficient megakaryocytes to differentiate and become activated in response to a range of platelet agonists was analyzed in iPSC-derived megakaryocytes. RESULTS: Temporally controlled deletion of Lyn improved the in vitro differentiation of hematopoietic progenitor cells into mature megakaryocytes, as measured by the rate and extent of appearance of CD41(+)CD42(+) cells. Lyn-deficient megakaryocytes also demonstrated improved hemostatic effectiveness, as reported by their ability to mediate clot formation in rotational thromboelastometry. Finally, Lyn-deficient megakaryocytes produced increased numbers of platelet-like particles (PLP) in vitro. CONCLUSIONS: Conditional deletion of Lyn kinase increases the hemostatic effectiveness of megakaryocytes and their progeny as well as improving their yield. Adoption of this system during generation of in vitro-derived platelets may contribute to both their efficiency of production and their ability to support hemostasis.

Noncoding RNAs (ncRNAs) are a group of RNA molecules that cannot encode proteins, and a better understanding of the complex interaction networks coordinated by ncRNAs will provide a theoretical basis for the development of therapeutics targeting the regulatory effects of ncRNAs. Platelets are produced upon the differentiation of hematopoietic stem cells into megakaryocytes, 10

Bone marrow puncture (BMP), is usually performed to identify thrombopoiesis activity and is still the gold standard to determine the etiology of thrombocytopenia. This diagnostic test is invasive hence it may cause discomfort to the patient. One of noninvasive test to determine the etiology of thrombocytopenia is by measuring immature platelet fraction (IPF). IPF is highly correlated to the activity of thrombopoiesis and by understanding the value, clinicians are able to use it in determining whether or not invasive examination is needed and more importantly avoiding unnecessary platelet transfusion. This research was a cross-sectional descriptive observational study aimed to evaluate IPF value in thrombocytopenic inpatients in the Department of Internal Medicine, Haji Adam Malik Hospital Medan. 83 people were recruited, 48 were female (57.83%) and 35 were male (42.16%). IPF values ranged 0,5-59,6% using Sysmex XN-1000 (reference range 1-4,8%). There were 5 (6.02%) low IPF values, 29 (34.93%) normal IPF values and 49 (59.03%) high IPF values. Evaluation of IPF in thrombocytopenic patients is a promising tool to discriminate central from peripheral thrombocytopenia.

SummarySystemic lupus erythematosus (SLE) is an autoimmune condition developing thrombocytopenia in about 10-15% of cases, however, mechanisms leading to low platelet count were not deeply investigated in this illness. Here we studied possible causes of thrombocytopenia, including different mechanisms of platelet clearance and impairment in platelet production. Twenty-five SLE patients with and without thrombocytopenia were included. Platelet apoptosis, assessed by measurement of loss of mitochondrial membrane potential, active caspase 3 and phosphatidylserine exposure, was found to increase in thrombocytopenic patients. Plasma from 67% SLE patients (thrombocytopenic and non-thrombocytopenic) induced loss of sialic acid (Ricinus communis agglutinin I and/or Peanut agglutinin binding) from normal platelet glycoproteins. Concerning platelet production, SLE plasma increased megakaryopoiesis (evaluated using normal human cord blood CD34+ hematopoietic progenitors), but inhibited thrombopoiesis (proplatelet count). Anti-platelet autoantibody depletion from SLE plasma reverted this inhibition. Overall, abnormalities were more frequently observed in thrombocytopenic than non-thrombocytopenic SLE patients and in those with active disease (SLEDAI≥5). In conclusion, platelet clearance due to apoptosis and desialylation, and impaired platelet production mainly due to inhibition of thrombopoiesis, could be relevant mechanisms leading to thrombocytopenia in SLE. These findings could provide a rational basis for the choice of proper therapies to correct platelet counts in these patients.[Figure: see text].

Abivertinib, a third-generation tyrosine kinase inhibitor, is originally designed to target epidermal growth factor receptor (EGFR)-activating mutations. Previous studies have shown that abivertinib has promising antitumor activity and a well-tolerated safety profile in patients with non-small-cell lung cancer. However, abivertinib also exhibited high inhibitory activity against Bruton’s tyrosine kinase and Janus kinase 3. Given that these kinases play some roles in the progression of megakaryopoiesis, we speculate that abivertinib can affect megakaryocyte (MK) differentiation and platelet biogenesis. We treated cord blood CD34+ hematopoietic stem cells, Meg-01 cells, and C57BL/6 mice with abivertinib and observed megakaryopoiesis to determine the biological effect of abivertinib on MK differentiation and platelet biogenesis. Our in vitro results showed that abivertinib impaired the CFU-MK formation, proliferation of CD34+ HSC-derived MK progenitor cells, and differentiation and functions of MKs and inhibited Meg-01-derived MK differentiation. These results suggested that megakaryopoiesis was inhibited by abivertinib. We also demonstrated in vivo that abivertinib decreased the number of MKs in bone marrow and platelet counts in mice, which suggested that thrombopoiesis was also inhibited. Thus, these preclinical data collectively suggested that abivertinib could inhibit MK differentiation and platelet biogenesis and might be an agent for thrombocythemia.

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

Current therapies for immune thrombocytopenia (ITP) are successful in providing a haemostatic platelet count in over two-thirds of patients. Still, some patients have an inadequate response and there is a need for other therapies. A number of novel therapies for ITP are currently being developed based upon the current pathophysiology of ITP. Many therapies are targetted at reducing platelet destruction by decreasing anti-platelet antibody production by immunosuppression with monoclonal antibodies targetted against CD40, CD38 and the immunoproteasome or physically reducing the anti-platelet antibody concentration by inhibition of the neonatal Fc receptor. Others target the phagocytic system by inhibiting FcγR function with staphylococcal protein A, hypersialylated IgG, polymeric Fc fragments, or Bruton kinase. With a recognition that platelet destruction is also mediated by complement, inhibitors of C1s are also being tested. Inhibition of platelet desialylation may also play a role. Other novel therapies promote platelet production with new oral thrombopoietin receptor agonists or the use of low-level laser light to improve mitochondrial activity and prevent megakaryocyte apoptosis. This review will focus on these novel mechanisms for treating ITP and assess the status of treatments currently under development. Successful new treatments for ITP might also provide a pathway to treat other autoimmune disorders.

Platelets play a role not only in hemostasis and thrombosis, but also in inflammation and innate immunity. We previously reported that an activated form of tyrosyl-tRNA synthetase (YRS ACT ) has an extratranslational activity that enhances megakaryopoiesis and platelet production in mice. Here, we report that YRS ACT mimics inflammatory stress inducing a unique megakaryocyte (MK) population with stem cell (Sca1) and myeloid (F4/80) markers through a mechanism dependent on Toll-like receptor (TLR) activation and type I interferon (IFN-I) signaling. This mimicry of inflammatory stress by YRS ACT was studied in mice infected by lymphocytic choriomeningitis virus (LCMV). Using Sca1/EGFP transgenic mice, we demonstrated that IFN-I induced by YRS ACT or LCMV infection suppressed normal hematopoiesis while activating an alternative pathway of thrombopoiesis. Platelets of inflammatory origin (Sca1/EGFP + ) were a relevant proportion of those circulating during recovery from thrombocytopenia. Analysis of these “inflammatory” MKs and platelets suggested their origin in myeloid/MK-biased hematopoietic stem cells (HSCs) that bypassed the classical MK-erythroid progenitor (MEP) pathway to replenish platelets and promote recovery from thrombocytopenia. Notably, inflammatory platelets displayed enhanced agonist-induced activation and procoagulant activities. Moreover, myeloid/MK-biased progenitors and MKs were mobilized from the bone marrow, as evidenced by their presence in the lung microvasculature within fibrin-containing microthrombi. Our results define the function of YRS ACT in platelet generation and contribute to elucidate platelet alterations in number and function during viral infection.

Immune thrombocytopenia (ITP) is an autoimmune disorder, in which megakaryocyte dysfunction caused by an autoimmune reaction can lead to thrombocytopenia, although the underlying mechanisms remain unclear. Here, we performed single-cell transcriptome profiling of bone marrow CD34+ hematopoietic stem and progenitor cells (HSPCs) to determine defects in megakaryopoiesis in ITP. Gene expression, cell-cell interactions, and transcriptional regulatory networks varied in HSPCs of ITP, particularly in immune cell progenitors. Differentially expressed gene (DEG) analysis indicated that there was an impaired megakaryopoiesis of ITP. Flow cytometry confirmed that the number of CD9+ and HES1+ cells from Lin−CD34+CD45RA− HSPCs decreased in ITP. Liquid culture assays demonstrated that CD9+Lin−CD34+CD45RA− HSPCs tended to differentiate into megakaryocytes; however, this tendency was not observed in ITP patients and more erythrocytes were produced. The percentage of megakaryocytes differentiated from CD9+Lin−CD34+CD45RA− HSPCs was 3-fold higher than that of the CD9− counterparts from healthy controls (HCs), whereas, in ITP patients, the percentage decreased to only 1/4th of that in the HCs and was comparable to that from the CD9− HSPCs. Additionally, when co-cultured with pre-B cells from ITP patients, the differentiation of CD9+Lin−CD34+CD45RA− HSPCs toward the megakaryopoietic lineage was impaired. Further analysis revealed that megakaryocytic progenitors (MkP) can be divided into seven subclusters with different gene expression patterns and functions. The ITP-associated DEGs were MkP subtype-specific, with most DEGs concentrated in the subcluster possessing dual functions of immunomodulation and platelet generation. This study comprehensively dissects defective hematopoiesis and provides novel insights regarding the pathogenesis of ITP.

Giemsa staining, phalloidin staining, and flow cytometry were performed to measure the effect of AECRs on the megakaryocyte differentiation in K562 and Meg-01 cells. A radiation-induced thrombocytopenia mouse model was constructed to assess the therapeutic actions of AECRs on thrombocytopenia. Network pharmacology and experimental verification were carried out to clarify its mechanism against thrombocytopenia.AECRs promoted megakaryocyte differentiation in K562 and Meg-01 cells and accelerated platelet recovery and megakaryopoiesis with no systemic toxicity in radiation-induced thrombocytopenia mice. The PI3K/AKT, MEK/ERK, and JAK2/STAT3 signaling pathways contributed to AECR-induced megakaryocyte differentiation. The suppression of the above signaling pathways by their inhibitors blocked AERC-induced megakaryocyte differentiation.AECRs can promote megakaryopoiesis and thrombopoiesis through activating PI3K/AKT, MEK/ERK, and JAK2/STAT3 signaling pathways, which has the potential to treat radiation-induced thrombocytopenia in the clinic.

Platelets are affected by many factors, such as infectious or aseptic inflammation, and different inflammatory states may induce either thrombocytopenia or thrombocytosis. Tumor necrosis factor α (TNFα) is an important inflammatory cytokine that has been shown to affect the activity of hematopoietic stem cells. However, its role in megakaryocyte (MK) development and platelet production remains largely unknown. This study aimed to investigate the effects of TNFα on MK and platelet generation.The ex vivo study with human CD34Our study revealed distinct roles for TNFα in megakaryopoiesis and thrombopoiesis and may provide new insights regarding the treatment for platelet disorders.

PURPOSE OF THE REVIEW This review highlights recent advancements in understanding the regulation of platelet numbers, focusing on mechanisms by which carbohydrates (glycans) link platelet removal with platelet production in the bone marrow in health and disease. RECENT FINDINGS This review is focused on the role of carbohydrates, specifically sialic acid moieties, as a central mediator of platelet clearance. We discuss recently identified novel mechanisms of carbohydrate-mediated platelet removal and carbohydrate-binding receptors that mediate platelet removal. SUMMARY The platelet production rate by megakaryocytes and removal kinetics controls the circulating platelet count. Alterations in either process can lead to thrombocytopenia (low platelet count) or thrombocytosis (high platelet count) are associated with the risk of bleeding or overt thrombus formation and serious complications. Thus, regulation of a steady-state platelet count is vital in preventing adverse events. There are few mechanisms delineated that shed light on carbohydrates' role in the complex and massive platelet removal process. This review focuses on carbohydrate-related mechanisms that contribute to the control of platelet numbers.

Thrombopoiesis had long been a challenging area of study due to the rarity of megakaryocyte precursors in the bone marrow and the incomplete understanding of its regulatory cytokines. A breakthrough was achieved in the early 1990s with the discovery of the thrombopoietin receptor (TpoR) and its ligand thrombopoietin (TPO). This accelerated research in thrombopoiesis, including the uncovering of the molecular basis of myeloproliferative neoplasms (MPN) and the advent of drugs to treat thrombocytopenic purpura. TpoR mutations affecting its membrane dynamics or transport were increasingly associated with pathologies such as MPN and thrombocytosis. It also became apparent that TpoR affected hematopoietic stem cell (HSC) quiescence while priming hematopoietic stem cells (HSCs) towards the megakaryocyte lineage. Thorough knowledge of TpoR surface localization, dimerization, dynamics and stability is therefore crucial to understanding thrombopoiesis and related pathologies. In this review, we will discuss the mechanisms of TpoR traffic. We will focus on the recent progress in TpoR membrane dynamics and highlight the areas that remain unexplored.

This paper aims to emphasize the importance of applying international consensus guidelines to detect qualitative and quantitative abnormalities of megakaryocytes on smears of bone marrow aspirates (BMA) for a shared and harmonized diagnostic path between different laboratories. Careful evaluation of megakaryocytes on BMA smears represents a cornerstone in the diagnosis of most clonal and nonclonal hematological diseases. Images associated with the detailed morphologic description of normal, reactive, abnormal, and dysplastic megakaryocytes are also reported together with examples of similar cells that, if not promptly identified, can lead to a morphological misdiagnosis.

Human platelet lysate (HPL) has been proposed as a safe and efficient xeno-free alternative to fetal bovine serum (FBS) for large-scale culturing of cell-based medicinal products. However, the use of blood derivatives poses a potential risk of pathogen transmission. To mitigate this risk, different pathogen reduction treatment (PRT) practices can be applied on starting materials or on final products, but these methods might modify the final composition and the quality of the products.We evaluated the impact of applying a PRT based on riboflavin and ultraviolet irradiation on the raw materials used to manufacture an improved Good Manufacturing Practices (GMP)-grade HPL product in a public blood center. Growth promotion and the levels of growth factors and proteins were compared between an inactivated product (HPL4-i) and a non-inactivated product (HPL4). Stability studies were performed at 4°C, -20°C, and -80°C.The application of a PRT on the starting materials significantly altered the protein composition of HPL4-i as compared with HPL4. Despite this, the growth promoting rates were unaffected when compared with FBS used as a control. While all products were stable at -20°C and -80°C for 24 months, a significant decrease in the activity of HPL4-i was observed when stored at 4°C.Our results show that the application of a PRT based on riboflavin and ultraviolet light on starting materials used in the manufacture of HPL modifies the final composition of the product, yet its cell growth promoting activity is maintained at levels similar to those of non-inactivated products.

No data; International audience; The role of iron in the formation and functioning of erythrocytes, and to a lesser degree of white blood cells, is well established, but the relationship between iron and platelets is less documented. Physiologically, iron plays an important role in hematopoiesis, including thrombopoiesis; iron levels direct, together with genetic factors, the lineage commitment of megakaryocytic/erythroid progenitors toward either megakaryocyte or erythroid progenitors. Megakaryocytic iron contributes to cellular machinery, especially energy production in platelet mitochondria. Thrombocytosis, possibly favoring vascular thrombosis, is a classical feature observed with abnormally low total body iron stores (mainly due to blood losses or decreased duodenal iron intake), but thrombocytopenia can also occur in severe iron deficiency anemia. Iron sequestration, as seen in inflammatory conditions, can be associated with early thrombocytopenia due to platelet consumption and followed by reactive replenishment of the platelet pool with possibility of thrombocytosis. Iron overload of genetic origin (hemochromatosis), despite expected mitochondrial damage related to ferroptosis, has not been reported to cause thrombocytopenia (except in case of high degree of hepatic fibrosis), and iron-related alteration of platelet function is still a matter of debate. In acquired iron overload (of transfusional and/or dyserythropoiesis origin), quantitative or qualitative platelet changes are difficult to attribute to iron alone due to the interference of the underlying hematological conditions; likewise, hematological improvement, including increased blood platelet counts, observed under iron oral chelation is likely to reflect mechanisms other than the sole beneficial impact of iron depletion.

Background Several platelet-derived microRNAs are associated with platelet reactivity (PR) and clinical outcome in cardiovascular patients. We previously showed an association between miR-204-5p and PR in stable cardiovascular patients, but data on functional mechanisms are lacking. Aims To validate miR-204-5p as a regulator of PR in platelet-like structures (PLS) derived from human megakaryocytes and to address mechanistic issues. Methods Human hematopoietic stem cells were differentiated into megakaryocytes, enabling the transfection of miR-204-5p and the recovery of subsequent PLS. The morphology of transfected megakaryocytes and PLS was characterized using flow cytometry and microscopy. The functional impact of miR-204-5p was assessed using a flow assay, the quantification of the activated form of the GPIIbIIIa receptor, and a fibrinogen-binding assay. Quantitative polymerase chain reaction and western blot were used to evaluate the impact of miR-204-5p on a validated target, CDC42. The impact of CDC42 modulation was investigated using a silencing strategy. Results miR-204-5p transfection induced cytoskeletal changes in megakaryocytes associated with the retracted protrusion of proPLS, but it had no impact on the number of PLS released. Functional assays showed that the PLS produced by megakaryocytes transfected with miR-204-5p were more reactive than controls. This phenotype is mediated by the regulation of GPIIbIIIa expression, a key contributor in platelet–fibrinogen interaction. Similar results were obtained after CDC42 silencing, suggesting that miR-204-5p regulates PR, at least in part, via CDC42 downregulation. Conclusion We functionally validated miR-204-5p as a regulator of the PR that occurs through CDC42 downregulation and regulation of fibrinogen receptor expression.

Lysophosphatidic acid (LPA) is one of the lipids identified to be involved in stem cell differentiation. It exerts various functions through activation of G protein-coupled lysophosphatidic acid receptors (LPARs). In previous studies, we have demonstrated that activation of LPA receptor 3 (LPA3) promotes erythropoiesis of human hematopoietic stem cells (HSCs) and zebrafish using molecular and pharmacological approaches. Our results show that treatment with lysophosphatidic acid receptor 2 (LPA2) agonist suppressed erythropoiesis, whereas activation of LPA3 by 1-oleoyl-2-methyl-sn-glycero-3-phosphothionate (2S-OMPT) promoted it, both in vitro and in vivo. Furthermore, we have demonstrated the inhibitory role of LPA3 during megakaryopoiesis. However, the mechanism underlying these observations remains elusive. In the present study, we suggest that the expression pattern of LPARs may be correlated with the transcriptional factors GATA-1 and GATA-2 at different stages of myeloid progenitors. We determined that manipulation of GATA factors affected the expression levels of LPA2 and LPA3 in K562 leukemia cells. Using luciferase assays, we demonstrate that the promoter regions of LPAR2 and LPAR3 genes were regulated by these GATA factors in HEK293T cells. Mutation of GATA-binding sites in these regions abrogated luciferase activity, suggesting that LPA2 and LPA3 are regulated by GATA factors. Moreover, physical interaction between GATA factors and the promoter region of LPAR genes was verified in K562 cells using chromatin immunoprecipitation (ChIP) studies. Taken together, our results suggest that balance between LPA2 and LPA3 expression, which may be determined by GATA factors, is a regulatory switch for lineage commitment in myeloid progenitors. The expression-level balance of LPA receptor subtypes represents a novel mechanism regulating erythropoiesis and megakaryopoiesis.

Thrombocytopenia is a common complication of human cytomegalovirus (HCMV) infection in immunocompromised hosts, which contributes to poor prognosis even in patients receiving antiviral treatment. Here, we investigated the megakaryo/thrombopoiesis process, including the involvement of the c-Mpl/IEX-1 pathway, after HCMV infection, identified receptors mediating the interaction between megakaryocytes (MKs) and HCMV, and explored novel therapeutic targets. Our data shows that HCMV directly infects megakaryocytes in patients with HCMV DNAemia and influences megakaryopoiesis via the c-Mpl/IEX-1 pathway throughout megakaryocyte maturation, apoptosis, and platelet generation in vivo and in vitro. After treatment with inhibitors of PDGFRα and αvβ3, the HCMV infection rate in MKs was significantly reduced, suggesting that IMC-3G3 and anti-αvβ3 are potential therapeutic alternatives for viral infection. In summary, our study proposes a possible mechanism and potential treatments for thrombocytopenia caused by HCMV infection and other viral diseases associated with abnormal hemostasis.

RUNX1-related disorder (RUNX1-RD) is caused by germline variants affecting the RUNX1 gene. This rare, heterogeneous disorder has no specific clinical or laboratory phenotype, making genetic diagnosis necessary. Although international recommendations have been established to classify the pathogenicity of variants, identifying the causative alteration remains a challenge in RUNX1-RD. Murine models may be useful not only for definitively settling the controversy about the pathogenicity of certain RUNX1 variants, but also for elucidating the mechanisms of molecular pathogenesis. Therefore, we developed a knock-in murine model, using the CRISPR/Cas9 system, carrying the RUNX1 p.Leu43Ser variant (mimicking human p.Leu56Ser) to study its pathogenic potential and mechanisms of platelet dysfunction. A total number of 75 mice were generated; 25 per genotype (RUNX1WT/WT, RUNX1WT/L43S, and RUNX1L43S/L43S). Platelet phenotype was assessed by flow cytometry and confocal microscopy. On average, RUNX1L43S/L43S and RUNX1WT/L43S mice had a significantly longer tail-bleeding time than RUNX1WT/WT mice, indicating the variant's involvement in hemostasis. However, only homozygous mice displayed mild thrombocytopenia. RUNX1L43S/L43S and RUNX1WT/L43S displayed impaired agonist-induced spreading and α-granule release, with no differences in δ-granule secretion. Levels of integrin αIIbβ3 activation, fibrinogen binding, and aggregation were significantly lower in platelets from RUNX1L43S/L43S and RUNX1WT/L43S using phorbol 12-myristate 13-acetate (PMA), adenosine diphosphate (ADP), and high thrombin doses. Lower levels of PKC phosphorylation in RUNX1L43S/L43S and RUNX1WT/L43S suggested that the PKC-signaling pathway was impaired. Overall, we demonstrated the deleterious effect of the RUNX1 p.Leu56Ser variant in mice via the impairment of integrin αIIbβ3 activation, aggregation, α-granule secretion, and platelet spreading, mimicking the phenotype associated with RUNX1 variants in the clinical setting., This work was partially supported by grants from Instituto de Salud Carlos III (ISCIII) and Feder (PI17/01311, PI17/01966, and CB15/00055), Fundación Séneca (19873/GERM/15), Gerencia Regional de Salud (GRS 2061A/19 and 1647/A/17), Fundación Mutua Madrileña (FMM, AP172142019), and Sociedad Española de Trombosis y Hemostasia (SETH-FETH; Premio López Borrasca 2019 and Ayuda a Grupos de Trabajo en Patología Hemorrágica 2019). The authors' research on IPDs is conducted in accordance with the aims of the Functional and Molecular Characterization of Patients with Inherited Platelet Disorders Project, which is supported by the Hemorrhagic Diathesis Working Group of the Spanish Society of Thrombosis and Haemostasis. A.M.-Q., C.F.-I., and L.H.-C. were supported by predoctoral grants from the Junta de Castilla y León, Spain. E.V. was supported by the predoctoral grant from the University of Salamanca, Spain. IG-T and RB were supported by "Contratos postdoctorales Programa II) from the University of Salamanca, Spain.

Background information Megakaryocytes (MKs) follow a unique cell cycle duplication process, called endomitosis, resulting in polyploidisation of cells. It is hypothesised that polyploidy, as well as an increment in cytoplasm volume, allow more efficient platelets generation from MKs. Although polyploidy leads to an increase in the DNA amount, which impacts gene expression, little is known about ribosomal biogenesis in these polylobulated polyploid cells. Results The nucleolus acts as a hub for ribosomal biogenesis, which in turn governs the protein synthesis rate of the cells. We therefore estimated the size and activity of the nucleolus in polyploid cells during megakaryopoiesis in vitro. Polyploid megakaryocytic cell lines and in vitro cultured MKs, which were obtained from human cord blood-derived CD 34+ cells, revealed that miRNA 146b regulated the activity of nucleolar and coiled-body phosphoprotein 1, which plays an integral role in determining nucleolar size and activity. Additionally, miRNA-146b was up-regulated during endomitosis and was found to promote megakaryopoiesis. Conclusion We propose that miRNA 146b regulates not only nucleolar size and activity, but also megakaryopoiesis. Significance This study highlights the importance of nucleolar activity and miRNA in the progression of megakaryopoiesis and thrombopoiesis.

Immune thrombocytopenia (ITP) is an autoimmune disease that is characterized by a significant reduction in the number of circulating platelets and frequently associated with bleeding. Although the pathogenesis of ITP is still not completely elucidated, it is largely recognized that the low platelet count observed in ITP patients is due to multiple alterations of the immune system leading to increased platelet destruction as well as impaired thrombopoiesis. The clinical manifestations and patients' response to different treatments are very heterogeneous suggesting that ITP is a group of disorders sharing common characteristics, namely, loss of immune tolerance toward platelet (and megakaryocyte) antigens and dysfunctional primary hemostasis. Management of ITP is challenging and requires intensive communication between patients and caregivers. The decision to initiate treatment should be based on the platelet count level, age of the patient, bleeding manifestation, and other factors that influence the bleeding risk in individual patients. In this review, we present recent data on the mechanisms that lead to platelet destruction in ITP with a particular focus on current findings concerning alterations of thrombopoiesis. In addition, we give an insight into the efficacy and safety of current therapies and management of ITP bleeding emergencies.

In specialized cells, the expression of specific tubulin isoforms and their subsequent post-translational modifications drive and coordinate unique morphologies and behaviors. The mechanisms by which β1-tubulin, the platelet and megakaryocyte (MK) lineage restricted tubulin isoform, drives platelet production and function remains poorly understood. We investigated the roles of two key post-translational tubulin polymodifications (polyglutamylation and polyglycylation) on these processes using a cohort of thrombocytopenic patients, human induced pluripotent stem cell derived MK, and healthy human donor platelets. We find distinct patterns of polymodification in MK and platelets, mediated by the antagonistic activities of the cell specific expression of tubulin tyrosine ligase like enzymes and cytosolic carboxypeptidase enzymes. The resulting microtubule patterning spatially regulates motor proteins to drive proplatelet formation in megakaryocytes, and the cytoskeletal reorganization required for thrombus formation. This work is the first to show a reversible system of polymodification by which different cell specific functions are achieved.

Ex vivo production of human platelets have been pursued as an alternative measure to resolve limitations in the supply and safety of current platelet transfusion products. To this end, induced pluripotent stem cells (iPSCs) are considered an ideal global source, since they are not only pluripotent and self-renewing, but also are available from basically any person, have relatively few ethical issues, and are easy to manipulate. From human iPSCs, megakaryocyte (MK) lines with robust proliferation capacity have been established by the introduction of specified sets of genes. These expandable MKs are also cryopreservable and thus would be suitable as master cells for good manufacturing practice (GMP) grade production of platelets, assuring availability on demand and safety against blood-borne infections. Meanwhile, developments in bioreactors that physically mimic the in vivo environment and discovery of substances that promote thrombopoiesis have yielded competent platelets with improved efficiency. The derivation of platelets from iPSCs could further resolve transfusion-related alloimmune complications through the manufacturing of autologous products and human leukocyte antigen (HLA)-compatible platelets by manipulation of HLAs and human platelet antigens (HPAs). Considering these key advances in the field, HLA-deleted platelets could become a universal product that is manufactured at industrial level to safely fulfill almost all demands. In this review, we overview the ex vivo production of iPSC-derived platelets towards clinical applications, a production that would revolutionize the blood transfusion system.

[Background]: Rare inherited thrombocytopenias are caused by alterations in genes involved in megakaryopoiesis, thrombopoiesis and/or platelet release. Diagnosis is challenging due to poor specificity of platelet laboratory assays, large numbers of culprit genes, and difficult assessment of the pathogenicity of novel variants. [Objectives]: To characterize the clinical and laboratory phenotype, and identifying the underlying molecular alteration, in a pedigree with thrombocytopenia of uncertain etiology. [Patients/Methods]: Index case was enrolled in our Spanish multicentric project of inherited platelet disorders due to lifelong thrombocytopenia and bleeding. Bleeding score was recorded by ISTH‐BAT. Laboratory phenotyping consisted of blood cells count, blood film, platelet aggregation and flow cytometric analysis. Genotyping was made by whole‐exome sequencing (WES). Cytoskeleton proteins were analyzed in resting/spreading platelets by immunofluorescence and immunoblotting. [Results]: Five family members displayed lifelong mild thrombocytopenia with a high number of enlarged platelets in blood film, and mild bleeding tendency. Patient's platelets showed normal aggregation and granule secretion response to several agonists. WES revealed a novel nonsense variant (c.322C>T; p.Gln108*) in TPM4 (NM_003290.3), the gene encoding for tropomyosin‐4 (TPM4). This variant led to impairment of platelet spreading capacity after stimulation with TRAP‐6 and CRP, delocalization of TPM4 in activated platelets, and significantly reduced TPM4 levels in platelet lysates. Moreover, the index case displayed up‐regulation of TPM2 and TPM3 mRNA levels. [Conclusions]: This study identifies a novel TPM4 nonsense variant segregating with macrothrombocytopenia and impaired platelet cytoskeletal remodeling and spreading. These findings support the relevant role of TPM4 in thrombopoiesis and further expand our knowledge of TPM4‐related thrombocytopenia., This work was partially supported by grants from Instituto de Salud Carlos III (ISCIII) and Feder (PI17/01966, PI20/00926), Gerencia Regional de Salud (GRS2061A/19, GRS2135/A/2020, GRS2314/A/2021), Fundación Mutua Madrileña (FMM, AP172142019) and Sociedad Española de Trombosis y Hemostasia (SETHFETH; Premio López Borrasca 2019 and Ayuda a Grupos de Trabajo en Patología Hemorrágica 2020 and 2021).

Platelet homeostasis is dependent on a tight regulation of both platelet production and clearance. The small GTPase Rap1 mediates platelet adhesion and hemostatic plug formation. However, Rap1 signaling is also critical for platelet homeostasis as both Rap1 deficiency and uninhibited Rap1 signaling lead to marked thrombocytopenia in mice. Here, we investigated the mechanism by which deficiency in Rasa3, a critical negative regulator of Rap1, causes macrothrombocytopenia in mice. Despite marked morphological and ultrastructural abnormalities, megakaryocytes in hypomorphic Rasa3hlb/hlb (R3hlb/hlb) or Rasa3-/- mice demonstrated robust proplatelet formation in vivo, suggesting that defective thrombopoiesis is not the main cause of thrombocytopenia. Rather, we observed that R3hlb/hlb platelets became trapped in the spleen marginal zone/red pulp interface, with evidence of platelet phagocytosis by macrophages. Clearance of mutant platelets was also observed in the liver, especially in splenectomized mice. Platelet count and platelet life span in Rasa3-mutant mice were restored by genetic or pharmacological approaches to inhibit the Rap1/talin1/αIIbβ3 integrin axis. A similar pattern of splenic clearance was observed in mice injected with anti-αIIbβ3 but not anti-glycoprotein Ibα platelet-depleting antibodies. In summary, we describe a potentially novel, integrin-based mechanism of platelet clearance that could be critical for our understanding of select inherited and acquired thrombocytopenias.

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. CXCR4

Intravascular neutrophils and platelets collaborate in maintaining host integrity, but their interaction can also trigger thrombotic complications. We report here that cooperation between neutrophil and platelet lineages extends to the earliest stages of platelet formation by megakaryocytes in the bone marrow. Using intravital microscopy, we show that neutrophils "plucked" intravascular megakaryocyte extensions, termed proplatelets, to control platelet production. Following CXCR4-CXCL12-dependent migration towards perisinusoidal megakaryocytes, plucking neutrophils actively pulled on proplatelets and triggered myosin light chain and extracellular-signal-regulated kinase activation through reactive oxygen species. By these mechanisms, neutrophils accelerate proplatelet growth and facilitate continuous release of platelets in steady state. Following myocardial infarction, plucking neutrophils drove excessive release of young, reticulated platelets and boosted the risk of recurrent ischemia. Ablation of neutrophil plucking normalized thrombopoiesis and reduced recurrent thrombosis after myocardial infarction and thrombus burden in venous thrombosis. We establish neutrophil plucking as a target to reduce thromboischemic events.

The number of platelets is used to assess thrombopoiesis; it varies greatly between individuals, although it is relatively steady in healthy people, with a modest drop with age. The severity of thrombocytopenia is a considerable factor in determining bleeding risk. However, while some patients with platelet numbers less than 10,000 L-1 have a little bleeding, others with platelet numbers above 50,000 L-1 have large hemorrhages, indicating that platelet count alone may not necessarily predict the occurrence of bleeding in patients with a marked decrease in platelet number. Platelet function testing in thrombocytopenia may be useful as a diagnostic tool and as guidance to treatment for thrombocytopenic patients. Severe thrombocytopenia is a major risk factor for hemorrhage, but platelet function and bleeding risk at very low platelet counts are poorly understood due to the limitations of platelet function testing at very low platelet counts. Using flow cytometry is generally independent of platelet number and hence possible in blood from individuals with thrombocytopenia.

Background Blood platelets are anucleate cell fragments that prevent bleeding and minimize blood vessel injury. They are formed from the cytoplasm of megakaryocytes located in the bone marrow. For successful platelet production, megakaryocyte fragments must pass through the sinusoid endothelial barrier by a cell biology process unique to these giant cells as compared with erythrocytes and leukocytes. Currently, the mechanisms by which megakaryocytes interact and progress through the endothelial cells are not understood, resulting in a significant gap in our knowledge of platelet production. Objective The aim of this study was to investigate how megakaryocytes interact and progress through the endothelial cells of mouse bone marrow sinusoids. Methods We used a combination of fluorescence, electron, and three-dimensional microscopy to characterize the cellular events between megakaryocytes and endothelial cells. Results We identified protrusive, F-actin-based podosome-like structures, called in vivo-MK podosomes, which initiate the formation of pores through endothelial cells. These structures present a collective and spatial organization through their interconnection via a contractile network of actomyosin, essential to regulate the endothelial openings. This ensures proper passage of megakaryocyte-derived processes into the blood circulation to promote thrombopoiesis. Conclusion This study provides novel insight into the in vivo function of podosomes of megakaryocytes with critical importance to platelet production.

Ex vivo (EV)-derived megakaryocytes (MKs) have shown great promise as a substitute for platelets in transfusion medicine to alleviate a severe shortage of donor-platelets. Challenges remain that include poor efficiency, a limited scale of production, and undefined short-term storage conditions of EV-derived MKs. This study aims to develop a high-efficiency system for large-scale production of Good Manufacturing Practice (GMP)-grade MKs and determine the short-term storage condition for the MKs. A roller-bottle culture system was introduced to produce GMP-grade MKs from small-molecule/cytokine cocktail expanded hematopoietic stem cells. Various buffer systems and temperatures for the short-term storage of MKs were assessed by cell viability, biomarker expression, and DNA ploidy levels. MKs stored for 24 hours were transplanted into sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice to confirm their platelet-releasing and tissue-homing ability in vivo. A yield of ~ 2.5 × 104 CD41a+ /CD42b+ MKs with purity of ~ 80% was achieved from one original cord blood CD34+ cell. Compared with the static culture, the roller-bottle culture system significantly enhanced megakaryopoiesis, as shown by the cell size, DNA ploidy, and megakaryopoiesis-related gene expression. The optimal storage condition for the MKs was defined as normal saline with 10% human serum albumin at 22℃. Stored MKs were capable of rapidly producing functional platelets and largely distributing in the lungs of NOD/SCID mice. The novel development of efficient production and storage system for GMP-grade MKs represents a significant step toward application of these MKs in the clinic.

The mechanisms that regulate platelet biogenesis remain unclear; factors that trigger megakaryocytes (MKs) to initiate platelet production are poorly understood. Platelet formation begins with proplatelets which are cellular extensions that originate from the MK cell body. Proplatelet formation is a highly dynamic and asynchronous process which poses unique challenges for researchers to accurately capture and analyze. We have designed an open-source, high-content, high-throughput, label-free analysis platform. Phase-contrast images of live, primary MKs are captured over a 24-hour period. Pixel-based machine-learning classification done by ilastik generates probability maps of key cellular features (circular MKs and branching proplatelets), which are then processed by a customized CellProfiler pipeline to identify and filter structures of interest based on morphologic parameters. CellProfiler Analyst, provides a final, supervised, machine learning classification to bolster accurate identification of cellular structures. This entire workflow yields the percent of proplatelet production, area, and count of proplatelets and MKs, as well as other statistics including skeletonization information for measuring proplatelet branching and length. We propose using a combination of these analyzed metrics, in particular the area measurements of MKs and proplatelets, when assessing in-vitro proplatelet production. Accuracy was validated against manually counted images and an existing algorithm. We then used the new platform to test compounds known to cause thrombocytopenia, including bromodomain inhibitors, and uncovered previously unrecognized effects of drugs on proplatelet formation, thus demonstrating the utility of our analysis platform. This advance in creating unbiased data analysis will increase the scale and scope of proplatelet production studies and potentially serve as a valuable resource for investigating molecular mechanisms of thrombocytopenia.

Ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase, has a favorable safety profile in patients with B cell-related malignancies. A primary adverse effect of ibrutinib is thrombocytopenia in the early stages of treatment, but platelet counts increase or recover as treatment continues. Currently, the effects of ibrutinib on megakaryopoiesis remain unclear. In this study, we investigated the mechanism by which ibrutinib induces thrombocytopenia using cord blood CD34+ hematopoietic stem cells (HSCs), a human megakaryoblastic cell line (SET-2), and C57BL/6 mice. We show that treatment with ibrutinib can suppress CD34+ HSC differentiation into megakaryocytes (MKs) and decrease the number of colony-forming unit-MKs (CFU-MKs). The ibrutinib-dependent inhibition of early megakaryopoiesis seems to mainly involve impaired proliferation of progenitor cells without induction of apoptosis. The effects of ibrutinib on late-stage megakaryopoiesis, in contrast to early-stage megakaryopoiesis, include enhanced MK differentiation, ploidy, and proplatelet formation in CD34+ HSC-derived MKs and SET-2 cells. We also demonstrated that MK adhesion and spreading, but not migration, were inhibited by ibrutinib. Furthermore, we revealed that integrin αIIbβ3 outside-in signaling in MKs was inhibited by ibrutinib. Consistent with previous clinical observations, in C57BL/6 mice treated with ibrutinib, platelet counts decreased by days 2 to 7 and recovered to normal levels by day 15. Together, these results reveal the pathogenesis of ibrutinib-induced transient thrombocytopenia. In conclusion, ibrutinib suppresses early megakaryopoiesis, as evidenced by inhibition of MK progenitor cell proliferation and CFU-MK formation. Ibrutinib enhances MK differentiation, ploidy, and proplatelet formation, while it impairs integrin αIIbβ3 outside-in signaling.

Aging is associated with a higher risk of developing malignant diseases, including myelodysplastic syndromes, clonal disorders characterised by chronic cytopenias (anaemia, neutropenia and thrombocytopenia) and abnormal cellular maturation. Myelodysplastic syndromes arising in older subjects are influenced by combinations of acquired somatic genetic lesions driving evolution from clonal haematopoiesis to myelodysplastic syndromes and from myelodysplastic syndromes to acute leukaemia. A different pattern of mutations has been identified in a small subset of myelodysplastic syndromes arising in young patients with familial syndromes. In particular, dysregulation of ANKRD26, RUNX1 and ETV6 genes plays a role in familial thrombocytopenia with predisposition to myelodysplastic syndromes and acute leukaemia. Whether these genes affect thrombopoiesis in sporadic myelodysplastic syndrome with thrombocytopenia is still undefined. Thirty-one myelodysplastic syndromes subjects and 27 controls subjects were investigated. Genomic DNA was used for mutation screening (ETV6, RUNX1, 5'UTR ANKRD26 genes). Functional studies were performed in the MEG-01-akaryoblastic cell line. We found four novel variants of RUNX1 gene, all in elderly myelodysplastic syndromes subjects with thrombocytopenia. Functional studies of the variant p.Pro103Arg showed no changes in RUNX1 expression, but the variant was associated with deregulated high transcriptional activity of ANKRD26 in MEG-01 cells. RUNX1 variant p.Pro103Arg was also associated with increased viability and reduced apoptosis of MEG-01, as well as impaired platelet production. Our findings are consistent with dysregulation of ANKRD26 in RUNX1 haploinsufficiency. Lack of repression of ANKRD26 expression may contribute to thrombocytopenia of subjects with sporadic myelodysplastic syndromes.

Ras-related protein 1 (Rap1) is a major convergence point of the platelet-signaling pathways that result in talin-1 binding to the integrin β cytoplasmic domain and consequent integrin activation, platelet aggregation, and effective hemostasis. The nature of the connection between Rap1 and talin-1 in integrin activation is an important remaining gap in our understanding of this process. Previous work identified a low-affinity Rap1-binding site in the talin-1 F0 domain that makes a small contribution to integrin activation in platelets. We recently identified an additional Rap1-binding site in the talin-1 F1 domain that makes a greater contribution than F0 in model systems. Here we generated mice bearing point mutations, which block Rap1 binding without affecting talin-1 expression, in either the talin-1 F1 domain (R118E) alone, which were viable, or in both the F0 and F1 domains (R35E,R118E), which were embryonic lethal. Loss of the Rap1–talin-1 F1 interaction in platelets markedly decreases talin-1–mediated activation of platelet β1- and β3-integrins. Integrin activation and platelet aggregation in mice whose platelets express only talin-1(R35E, R118E) are even more impaired, resembling the defect seen in platelets lacking both Rap1a and Rap1b. Although Rap1 is important in thrombopoiesis, platelet secretion, and surface exposure of phosphatidylserine, loss of the Rap1–talin-1 interaction in talin-1(R35E, R118E) platelets had little effect on these processes. These findings show that talin-1 is the principal direct effector of Rap1 GTPases that regulates platelet integrin activation in hemostasis.

Clinical studies have shown that melatonin lowers the frequency of thrombocytopenia in patients with cancer undergoing radiotherapy or chemotherapy. Here, we investigated the mechanisms by which melatonin promotes platelet formation and survival. Our results show that melatonin exerted protective effects on serum-free induced apoptosis of CHRF megakaryocytes (MKs). Melatonin promoted the formation of MK colony forming units (CFUs) in a dose-dependent manner. Using doxorubicin-treated CHRF cells, we found that melatonin rescued G2/M cell cycle arrest and cell apoptosis induced by doxorubicin. The expression of p-AKT was increased by melatonin treatment, an effect that was abolished by melatonin receptor blocker. In addition, we demonstrated that melatonin enhanced the recovery of platelets in an irradiated mouse model. Megakaryopoiesis was largely preserved in melatonin-treated mice. We obtained the same results in vivo from bone marrow histology and CFU-MK formation assays. Melatonin may exert these protective effects by directly stimulating megakaryopoiesis and inhibiting megakaryocyte apoptosis through activation of its receptors and AKT signaling.

Immune thrombocytopenia (ITP) is an acquired bleeding disorder characterized by antibody-mediated platelet destruction. Different mechanisms have been suggested to explain accelerated platelet clearance and impaired thrombopoiesis, but the pathophysiology of ITP has yet to be fully delineated. In this study, we tested 2 mouse models of immune-mediated thrombocytopenia using the rat anti-mouse GPIbα monoclonal antibody 5A7, generated in our laboratory. After a single IV administration of high-dose (2 mg/kg) 5A7, opsonized platelets were rapidly cleared from the circulation into the spleen and liver; this was associated with rapid upregulation of thrombopoietin (TPO) messenger RNA. In contrast, subcutaneous administration of low-dose 5A7 (0.08-0.16 mg/kg) every 3 days gradually lowered the platelet count; in this case, opsonized platelets were observed only in the spleen, and TPO levels remained unaltered. Interestingly, in both models, the 5A7 antibody was found on the surface of, as well as internalized to, bone marrow megakaryocytes. Consequently, platelets generated in the chronic phase of repeated subcutaneous 5A7 administration model showed reduced GPIbα membrane expression on their surface. Our findings indicate that evaluation of platelet surface GPIbα relative to platelet size may be a useful marker to support the diagnosis of anti-GPIbα antibody–induced ITP.

Over the last 100 years the role of platelets in hemostatic events and their production by megakaryocytes have gradually been defined. Progressively, thrombocytopenia was recognized as a cause of bleeding, first through an acquired immune disorder; then, since 1948, when Bernard-Soulier syndrome was first described, inherited thrombocytopenia became a fascinating example of Mendelian disease. The platelet count is often severely decreased and platelet size variable; associated platelet function defects frequently aggravate bleeding. Macrothrombocytopenia with variable proportions of enlarged platelets is common. The number of circulating platelets will depend on platelet production, consumption and lifespan. The bulk of macrothrombocytopenias arise from defects in megakaryopoiesis with causal variants in transcription factor genes giving rise to altered stem cell differentiation and changes in early megakaryocyte development and maturation. Genes encoding surface receptors, cytoskeletal and signaling proteins also feature prominently and Sanger sequencing associated with careful phenotyping has allowed their early classification. It quickly became apparent that many inherited thrombocytopenias are syndromic while others are linked to an increased risk of hematologic malignancies. In the last decade, the application of next-generation sequencing, including whole exome sequencing, and the use of gene platforms for rapid testing have greatly accelerated the discovery of causal genes and extended the list of variants in more common disorders. Genes linked to an increased platelet turnover and apoptosis have also been identified. The current challenges are now to use next-generation sequencing in first-step screening and to define bleeding risk and treatment better.

Background Regulated protein synthesis is essential for megakaryocyte (MK) and platelet functions, including platelet production and activation. PDK1 (phosphoinositide-dependent kinase 1) regulates platelet functional responses and has been associated with circulating platelet counts. Whether PDK1 also directly regulates protein synthetic responses in MKs and platelets, and platelet production by MKs, remains unknown. Objective To determine if PDK1 regulates protein synthesis in MKs and platelets. Methods Pharmacologic PDK1 inhibitors (BX-795) and mice where PDK1 was selectively ablated in MKs and platelets (PDK1-/- ) were used. PDK1 signaling in MKs and platelets (human and murine) were assessed by immunoblots. Activation-dependent translation initiation and protein synthesis in MKs and platelets was assessed by probing for dissociation of eIF4E from 4EBP1, and using m7-GTP pulldowns and S35 methionine incorporation assays. Proplatelet formation by MKs, synthesis of Bcl-3 and MARCKs protein, and clot retraction were employed for functional assays. Results Inhibiting or ablating PDK1 in MKs and platelets abolished the phosphorylation of 4EBP1 and eIF4E by preventing activation of the PI3K and MAPK pathways. Inhibiting PDK1 also prevented dissociation of eIF4E from 4EBP1, decreased binding of eIF4E to m7GTP (required for translation initiation), and significantly reduced de novo protein synthesis. Inhibiting PDK1 reduced proplatelet formation by human MKs and blocked MARCKs protein synthesis. In both human and murine platelets, PDK1 controlled Bcl-3 synthesis. Inhibition of PDK1 led to complete failure of clot retraction in vitro. Conclusions PDK1 is a previously unidentified translational regulator in MKs and platelets, controlling protein synthetic responses, proplatelet formation, and clot retraction.