Mature Megakaryocytes Research Articles

A modern overview of the process of platelet formation (thrombocytopoiesis) and its dependence on several factors.

Structural and functional alterations in platelets are an actual problem that requires more attention. The treatment of these illnesses proves challenging, inefficient and heavily relies on platelet donations. A difficult task confronting science is producing platelets in vitro, which calls for meticulous examination of factors affecting platelet generation. It is known that megakaryocytes produce platelets in vitro and in vivo differently: in the laboratory we can get a smaller number of platelets compared to the human body. This review primarily examines the stages of megakaryocyte maturation and the processes involved in platelet formation. The article reflects the results of both fundamental research on the problem and the new results obtained over the past decade. Currently, most scientists accept the pro-platelets theory of platelet formation. This review aims to explore in detail each stage of pro-platelet formation and the platelet formation process. It explains on the processes of polyploidization, endomitosis, and apoptosis, as well as the functions of structural cell components (microtubules, mitochondria, T- and α-granules) and pro-platelet migration. The microenvironment influence is acknowledged for the osteoblastic and vascular niches that affect thrombocytopoiesis. The additional aspect is the contribution of specific proteins to thrombocytopoiesis such as RhoA, β1-tubulin, cytokines IL-6, IL-8, Toll-like receptors, etc.

Oxazolidinone antibiotics impair ex vivo megakaryocyte differentiation from hematopoietic progenitor cells and their maturation into platelets.

Oxazolidinones (linezolid and tedizolid) adverse reactions include thrombocytopenia, the mechanism of which is still largely unknown. In cultured cells, oxazolidinones impair mitochondrial protein synthesis and oxidative metabolism. As mitochondrial activity is essential for megakaryocyte differentiation and maturation into platelets, we examined whether oxazolidinones impair these processes ex vivo and alter, in parallel, the activity of mitochondrial cytochrome c-oxidase (CYTOX; enzyme partly encoded by the mitochondrial genome) and cell morphology. Human CD34+ cells were isolated, incubated with cytokines (up to 14 days) and clinically relevant oxazolidinone concentrations or in control conditions, and used for (i) clonogenic assays [counting of megakaryocyte (CFU-Mk), granulocyte-monocyte (CFU-GM), burst-forming unit-erythroid (BFU-E) colonies]; (ii) the measure of the expression of megakaryocyte surface antigens (CD34 to CD41 and CD42); (iii) counting of proplatelets; (iv) the measurement of CYTOX activity; and (v) cell morphology (optic and electron microscopy). Oxazolidinones caused a significant decrease in BFU-E but not CFU-Mk or CFU-GM colonies. Yet, the megakaryocytic lineage was markedly affected, with a decreased differentiation of CD34+ into CD41+/CD42+ cells, an abolition of proplatelet formation and striking decrease in the numbers of large polylobulated nucleus megakaryocytes, with a complete loss of intracellular demarcation membrane system, disappearance of mitochondria, and suppression of CYTOX activity. These alterations were more marked in cells incubated with tedizolid than linezolid. These data suggest that oxazolidinones may induce thrombocytopenia by impairing megakaryocytic differentiation through mitochondrial dysfunction. Pharmacological interventions to prevent this toxicity might therefore be difficult as mitochondrial toxicity is most probably inherently linked to their antibacterial activity.

Characterization of mesothelin gene expression in dogs and overexpression in canine mesotheliomas.

Canine mesotheliomas are uncommon malignant tumors typically detected late. Minimally invasive diagnostic biomarkers would facilitate diagnosis at earlier stages, thereby improving clinical outcomes. We hypothesized that mesothelin could be used as a reliable diagnostic biomarker for canine mesotheliomas since it has been used as a cancer biomarker for human mesothelioma. We aimed to explore and characterize mesothelin gene expression in dogs and assess its use as a diagnostic biomarker for canine mesotheliomas. We quantified expressed canine mesothelin transcripts via reverse transcription polymerase chain reaction (RT-PCR) and sequenced them using ribonucleic acid (RNA) extracted from a canine mesothelioma cell line. After confirming mesothelin expression, we assessed its levels in major organ tissues and compared them with those in the mesothelioma tissues using quantitative PCR (qPCR). Mesothelin overexpression in mesotheliomas was detected, and we further compared its levels using qPCR between mesotheliomas and non-mesotheliomas using tumor tissues and clinical sample effusions, confirming its significance as a diagnostic biomarker for canine mesothelioma. Mesothelin complementary deoxyribonucleic acid (cDNA) was amplified via RT-PCR, yielding a single band of expected upon DNA electrophoresis. Sequence analyses confirmed it as a predicted canine mesothelin transcript from the genome sequence database. Comparative sequence analysis of the deduced amino acid sequence of the expressed canine mesothelin demonstrated molecular signature similarities with the human mesothelin. However, the pre-sequence of canine mesothelin lacks the mature megakaryocyte potentiating factor (MPF) portion, which is typically cleaved post-translationally with furin. Mesothelin expression was quantified via qPCR revealing low levels in the mesothelial and lung tissues, with negligible expression in the other major organs. Canine mesothelin exhibited significantly higher expression in the canine mesotheliomas than in the noncancerous tissues. Moreover, analysis of clinical samples using qPCR demonstrated markedly elevated mesothelin expression in canine mesotheliomas compared to non-mesothelioma cases. Canine mesothelin exhibits molecular and biological characteristics akin to human mesothelin. It could serve as a vital biomarker for diagnosing canine mesotheliomas, applicable to both tissue- and effusion-based samples.

Trogocytosis: A potential pathogenic mechanism in immune thrombocytopenia leading to impaired megakaryocyte maturation and reduced platelet production

Immune thrombocytopenia (ITP) is an autoimmune disease due to increased peripheral platelets destruction and inappropriate bone marrow production of platelets by megakaryocytes (MKs). The pathophysiology of ITP is complex and multifactorial, which remains not fully understood. However, the production of antiplatelet autoantibodies and the destruction of antibody-coated platelets through antibody-dependent cellular phagocytosis by macrophages (Mθ) expressing Fc gamma receptor (FcγR) are regarded as the key mechanisms. Glycoprotein (GP) Ⅱb/Ⅲa is the main target of antiplatelet antibodies leading to platelet phagocytosis by splenic Mθ, through interactions with FcγR. As GPⅡb/Ⅲa is normally expressed both on platelets and the MKs from which they are derived, we inferred that partial phagocytosis (definitely trogocytosis) of MKs by bone marrow Mθ might exist, leading to the increased amount of granular MKs (or non-platelets-producing MKs) in ITP. And we further hypothesized that the “bitten” MKs cannot sustain their normal function of maturation. Such trogocytosis mechanism may shed new light on the understanding of morphology alterations, maturation defects and decreased platelets production of MKs in ITP, and provide appealing implications in therapeutic modifications targeting the interplay of MKs and Mθ in bone marrow.

Genetic engineering of megakaryocytes from blood progenitor cells using messenger RNA lipid nanoparticles

BackgroundPlatelets are an essential component of hemorrhage control and management, and engineering platelets to express therapeutic proteins could expand their use as a cell therapy. Genetically engineered platelets can be achieved by modifying the platelet precursor cells, megakaryocytes (MKs). Current strategies include transfecting MK progenitors ex vivo with viral vectors harboring lineage-driven transgenes and inducing the production of in vitro modified platelets. The use of viruses, however, poses challenges in clinical implementation, and no methods currently exist to genetically modify MKs with nonviral techniques. Lipid nanoparticles (LNPs) are a nonviral delivery system that could enable a facile strategy to modify MKs with a variety of nucleic acid payloads. ObjectivesTo investigate whether LNP can transfect cultured hematopoietic stem/progenitor cell–derived MKs to express exogenous proteins and induce functional changes. MethodsMK and MK progenitors differentiated from cord blood–derived hematopoietic stem/progenitor cells were treated with LNP formulations containing messenger RNA and resembling the clinically approved LNP formulations. Transfection efficiency was assessed through flow cytometry by expression of enhanced green fluorescent protein. Functional changes to the MKs were assessed through rotational thromboelastometry by expression of exogenous coagulation factor (F)VII, a representative physiologically relevant protein. ResultsLNP enabled transfection efficiencies of 99% in MKs and did not impair MK maturation, viability, and morphology. MKs engineered to express exogenous FVII decreased clotting time in FVII-deficient plasma following clot initiation. ConclusionThis approach provides an easy-to-use modular platform to genetically modify MK and MK progenitors, which can be potentially extended to producing genetically modified cultured platelets.

YAP1 regulates thrombopoiesis by binding to MYH9 in immune thrombocytopenia

AbstractImmune thrombocytopenia (ITP) is a complicated bleeding disease characterized by a sharp platelet reduction. As a dominating element involved in ITP, megakaryocytes (MKs) are responsible for thrombopoiesis. However, the mechanism underlying the dysregulation of thrombopoiesis that occurs in ITP remains unidentified. In this study, we examined the role of Yes-associated protein 1 (YAP1) in thrombopoiesis during ITP. We observed reduced YAP1 expression with cytoskeletal actin misalignment in MKs from patients with ITP. Using an experimental ITP mouse model, we showed that reduced YAP1 expression induced aberrant MK distribution, reduced the percentage of late MKs among the total MKs, and caused submaximal platelet recovery. Mechanistically, YAP1 upregulation by binding of GATA-binding protein 1 to its promoter promoted MK maturation. Phosphorylated YAP1 promoted cytoskeletal activation by binding its WW2 domain to myosin heavy chain 9, thereby facilitating thrombopoiesis. Targeting YAP1 with its activator XMU-MP-1 was sufficient to rescue cytoskeletal defects and thrombopoiesis dysregulation in YAP1+/− mice with ITP and patients. Taken together, these results demonstrate the crucial role of YAP1 in thrombopoiesis, providing potential for the development of diagnostic markers and therapeutic options for ITP.

Megakaryocyte maturation involves activation of the adaptive unfolded protein response.

Endoplasmic reticulum stress triggers the unfolded protein response (UPR) to promote cell survival or apoptosis. Transient endoplasmic reticulum stress activation has been reported to trigger megakaryocyte production, and UPR activation has been reported as a feature of megakaryocytic cancers. However, the role of UPR signaling in megakaryocyte biology is not fully understood. We studied the involvement of UPR in human megakaryocytic differentiation using PMA (phorbol 12-myristate 13-acetate)-induced maturation of megakaryoblastic cell lines and thrombopoietin-induced differentiation of human peripheral blood-derived progenitors. Our results demonstrate that an adaptive UPR is a feature of megakaryocytic differentiation and that this response is not associated with ER stress-induced apoptosis. Differentiation did not alter the response to the canonical endoplasmic reticulum stressors DTT or thapsigargin. However, thapsigargin, but not DTT, inhibited differentiation, consistent with the involvement of Ca2+ signaling in megakaryocyte differentiation.

Thrombopoietin receptor agonists regulate myeloid-derived suppressor cell-mediated immunomodulatory effects in ITP.

TPO receptor agonists (TPO-RAs) are a class of clinical second-line regimens for the treatment of primary immune thrombocytopenia (ITP). It can promote megakaryocyte maturation and increase platelet production, but its effect on immunosuppressive cells in patients with ITP has not been explored. Sixty-two ITP patients and 34 healthy controls (HCs) were included in this study. The proportion and functions of myeloid-derived immunosuppressive cells (MDSCs) in ITP patients and HCs were investigated. We found that the proportion and function of MDSCs in ITP patients treated with TPO-RAs were significantly higher than those treated with glucocorticoids (GCs), which was correlated with the clinical efficacy. The proportion and function of cytotoxic Th1 cells and CD8+T cells decreased, while the proportion and immunosuppressive function of Treg cells increased in ITP patients treated with TPO-RAs. We further proved, through MDSC depletion tests, that the inhibitory effect of MDSCs on Th1 cells and the promotion of Treg cells in the original immune micro-environment of GCs-treated ITP patients were impaired; however, these MDSCs' functions were improved in TPO-RAs-treated patients. Finally, we found that the KLF9 gene in MDSCs cells of ITP patients treated with TPO-RAs was down-regulated, which contribute to the higher mRNA expression of GADD34 gene and improved function of MDSCs. These results demonstrate a novel mechanism of TPO-RAs for the treatment of ITP through the assessment of MDSCs and their subsequent impact on T cells, which provides a new basis for TPO-RAs as first-line treatment approach to the treatment of ITP.

Inherited thrombocytopenia associated with a variant in the FLI1 binding site in the 5' UTR of ANKRD26.

Variants in the 5' UTR of ANKRD26 are a common cause of inherited thrombocytopenia (ANKRD26-RT), and are associated with sustained ANKRD26 expression, which inhibits megakaryocyte maturation and proplatelet formation. ANKRD26 expression is controlled by the binding of a RUNX1/FLI1 complex to the 5' UTR. To date, all reported ANKRD26-RD associated variants have been within the RUNX1 binding site and a 22 base pair flanking region. Here, we report a novel variant in the 5' UTR of ANKRD26, c.-107C>T. This variant is in the FLI1 binding site, and is predicted to disrupt FLI1 binding due to loss of a hydrogen bond with FLI1. Differentiated PBMCs from affected family members showed impaired megakaryocyte maturation and proplatelet formation and sustained expression of ANKRD26, and platelets from affected family members had higher ANKRD26 expression than control platelets. The variant increased activity of the ANKRD26 promotor in a reporter assay. We also provide evidence that the previously reported c.-140C>G ANKRD26 5' UTR variant is benign and not associated with thrombocytopenia. Identification of the c.-107C>T variant extends the range of the regulatory region in the 5' UTR of ANKRD26 that is associated with ANKRD26-RT.

Daucosterol regulates JAK2-STAT3 signaling pathway to promote megakaryocyte differentiation

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

Notch1 regulates hepatic thrombopoietin production

AbstractNotch signaling regulates cell-fate decisions in several developmental processes and cell functions. However, the role of Notch in hepatic thrombopoietin (TPO) production remains unclear. We noted thrombocytopenia in mice with hepatic Notch1 deficiency and so investigated TPO production and other features of platelets in these mice. We found that the liver ultrastructure and hepatocyte function were comparable between control and Notch1-deficient mice. However, the Notch1-deficient mice had significantly lower plasma TPO and hepatic TPO messenger RNA levels, concomitant with lower numbers of platelets and impaired megakaryocyte differentiation and maturation, which were rescued by addition of exogenous TPO. Additionally, JAK2/STAT3 phosphorylation was significantly inhibited in Notch1-deficient hepatocytes, consistent with the RNA-sequencing analysis. JAK2/STAT3 phosphorylation and TPO production was also impaired in cultured Notch1-deficient hepatocytes after treatment with desialylated platelets. Consistently, hepatocyte-specific Notch1 deletion inhibited JAK2/STAT3 phosphorylation and hepatic TPO production induced by administration of desialylated platelets in vivo. Interestingly, Notch1 deficiency downregulated the expression of HES5 but not HES1. Moreover, desialylated platelets promoted the binding of HES5 to JAK2/STAT3, leading to JAK2/STAT3 phosphorylation and pathway activation in hepatocytes. Hepatocyte Ashwell-Morell receptor (AMR), a heterodimer of asialoglycoprotein receptor 1 [ASGR1] and ASGR2, physically associates with Notch1, and inhibition of AMR impaired Notch1 signaling activation and hepatic TPO production. Furthermore, blockage of Delta-like 4 on desialylated platelets inhibited hepatocyte Notch1 activation and HES5 expression, JAK2/STAT3 phosphorylation, and subsequent TPO production. In conclusion, our study identifies a novel regulatory role of Notch1 in hepatic TPO production, indicating that it might be a target for modulating TPO level.

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

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

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

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

Abstract 1381: Characterization of the 12-lipoxygenase expression in the megakaryoblastic cell line, Dami

Abstract Megakaryocytes are myeloid cells produced primarily in the bone marrow and are best known for releasing platelets in the blood stream. In the platelet production process, megakaryocytes transfer their bioactive content, including the 12-lipoxygenase (12-LO) enzyme, into newly formed platelets. The 12-LO has been shown to be implicated in platelet activation and is overexpressed in several chronic inflammatory conditions, including several types of cancers. The 12-LO is responsible for the conversion of the arachidonic acid into the 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), a key lipid mediator implicated in several steps of the metastatic cascade. While 12-LO is expressed in mature human megakaryocytes, we recently identified two megakaryoblast cell lines isolated from leukemia patients lacking 12-LO expression. However, only one of the two cell lines expresses functional 12-LO protein upon megakaryocyte maturation. The goal of this study is to characterize the 12-LO expression during the megakaryocyte maturation process. We hypothesize that 12-LO expression is increased during cell maturation and that inhibition of the 12-LO expression will result in dysfunctional platelets. Both the Dami cell and MEG-01 cells were induced with the co-incubation of both phorbol myristate acetate and eltrombopag, a thrombopoietin receptor agonist. The maturation and viability of the cells were confirmed by flow cytometry. Expression of the 12-LO was assessed by RT-qPCR and immunoblot. Quantification of the 12(S)-HETE was performed by reversed-phase high performance liquid chromatography. Platelet release was quantified by flow cytometry using CD41 fluorescent labelled antibodies. We observed a 3-fold increase in 12-LO protein expression following cell maturation in the Dami cell line. Interestingly, mRNA expression between the undifferentiated Dami cells and mature Dami cells remained unchanged. Furthermore, we did not detect any 12-LO protein expression in the MEG-01 after the cell’s maturation. Finally, we identified a specific 12-LO antisens non-coding RNA that is implicated in the regulation of the protein expression in megakaryocytes. Our study reports the first mechanistic regulation of 12-LO expression implicated in the megakaryocyte maturation process. Since both platelets and platelet-specific 12-LO play an important role in cancer biology, it is important to better understand how megakaryocytes package the enzyme inside these small cells. It remains elusive whether 12-LO enrichment in platelet correlates with the disease severity. Citation Format: Luc H. Boudreau, Vanessa L. Gauvin, Jaël Richard, Marie-France N. Soucy, Mathieu P.A. Hébert, Eric P. Allain. Characterization of the 12-lipoxygenase expression in the megakaryoblastic cell line, Dami [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1381.

Essential thrombocythaemia.

Essential thrombocythemia (ET) is a chronic Philadelphia-negative myeloproliferative neoplasm that has its main involvement in the megakaryopoietic lineage, generating sustained thrombocytosis in peripheral blood and an increase in the number of mature megakaryocytes in the bone marrow. In addition to marked thrombocytosis, it is characterized by increased thrombotic or hemorrhagic risk and the presence of constitutional symptoms. Patients with ET have a low but known risk of disease progression to myelofibrosis and/or acute leukemia. The diagnosis is made based on the 2016 WHO criteria. At present, available treatments for patients with ET are mainly aimed at minimizing the risk of thrombosis and/or bleeding.

An Innovative Inducer of Platelet Production, Isochlorogenic Acid A, Is Uncovered through the Application of Deep Neural Networks.

(1) Background: Radiation-induced thrombocytopenia (RIT) often occurs in cancer patients undergoing radiation therapy, which can result in morbidity and even death. However, a notable deficiency exists in the availability of specific drugs designed for the treatment of RIT. (2) Methods: In our pursuit of new drugs for RIT treatment, we employed three deep learning (DL) algorithms: convolutional neural network (CNN), deep neural network (DNN), and a hybrid neural network that combines the computational characteristics of the two. These algorithms construct computational models that can screen compounds for drug activity by utilizing the distinct physicochemical properties of the molecules. The best model underwent testing using a set of 10 drugs endorsed by the US Food and Drug Administration (FDA) specifically for the treatment of thrombocytopenia. (3) Results: The Hybrid CNN+DNN (HCD) model demonstrated the most effective predictive performance on the test dataset, achieving an accuracy of 98.3% and a precision of 97.0%. Both metrics surpassed the performance of the other models, and the model predicted that seven FDA drugs would exhibit activity. Isochlorogenic acid A, identified through screening the Chinese Pharmacopoeia Natural Product Library, was subsequently subjected to experimental verification. The results indicated a substantial enhancement in the differentiation and maturation of megakaryocytes (MKs), along with a notable increase in platelet production. (4) Conclusions: This underscores the potential therapeutic efficacy of isochlorogenic acid A in addressing RIT.

Thrombopoietin receptor agonists use and risk of thrombotic events in patients with immune thrombocytopenic purpura: A systematic review and meta‑analysis of randomized controlled trials.

Thrombopoietin receptor agonists (TPO-RAs) have a role in second-line immune thrombocytopenic purpura (ITP) treatment, binding to and activating thrombopoietin receptors on megakaryocyte membranes in the bone marrow. This promotes megakaryocyte maturation and increases platelet production. Despite a 2-6% incidence of thrombotic events during TPO-RA treatment, it remains uncertain whether TPO-RAs elevate thrombosis rates. A comprehensive search of electronic databases was conducted using the relevant search criteria. To assess the risk of bias, the included studies were assessed using the revised Cochrane Risk of Bias Assessment Tool 2.0, and a meta-analysis was performed using RevMan 5.4.1. A total of 1,698 patients with ITP were included from randomized controlled trials (RCTs). There were 26 thromboembolic events in the TPO-RAs group and 4 in the control group. However, there was no significant difference in the incidence of thrombotic events between the two groups [odds ratio (OR)=1.76, 95% confidence interval (CI): 0.78-4.00, P=0.18], even if the duration of treatment was >12 weeks (OR=2.46, 95% CI: 0.81-7.43, P=0.11). Subgroup analysis showed that none of the four drugs significantly increased the incidence of thrombotic events (romiplostim: OR=0.92, 95% CI: 0.14-6.13, P=0.93; eltrombopag: OR=2.32, 95% CI: 0.64-8.47, P=0.20; avatrombopag: OR=4.15, 95% CI: 0.20-85.23, P=0.36; and hetrombopag: OR=0.76, 95% CI: 0.03-18.76, P=0.87). There was also no significant difference in the results of the double-blinded placebo-controlled RCTs (OR=1.21, 95% CI: 0.41-3.58, P=0.73). Compared to patients with ITP who did not receive TPO-RA treatment, those receiving TPO-RA treatment did not exhibit a significantly increased risk of thrombotic events.

Interleukins in Platelet Biology: Unraveling the Complex Regulatory Network.

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.

AC-73 and Syrosingopine Inhibit SARS-CoV-2 Entry into Megakaryocytes by Targeting CD147 and MCT4.

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.

Newly identified roles for PIEZO1 mechanosensor in controlling normal megakaryocyte development and in primary myelofibrosis.

Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in mouse and human Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Yet, a role for PIEZO1 in megakaryopoiesis and proplatelet formation has never been investigated. Here, we show that activation of PIEZO1 increases the number of immature Mks in mice, while the number of mature Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its activation reduces Mk size, ploidy, maturation, and proplatelet extension. Resulting effects of PIEZO1 activation on Mks resemble the profile in Primary Myelofibrosis (PMF). Intriguingly, Mks derived from Jak2V617F PMF mice show significantly elevated PIEZO1 expression, compared to wild-type controls. Accordingly, Mks isolated from bone marrow aspirates of JAK2V617F PMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2V617F PMF patients are rescued upon PIEZO1 inhibition. Together, our data suggest that PIEZO1 places a brake on Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease.