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Journal of Thrombosis and Haemostasis :... Nov 2023Megakaryocyte differentiation and platelet production disorders are the main causes of thrombocythemia and thrombocytopenia and lead to thrombosis or hemorrhage....
BACKGROUND
Megakaryocyte differentiation and platelet production disorders are the main causes of thrombocythemia and thrombocytopenia and lead to thrombosis or hemorrhage. Branched-chain amino acids (BCAAs) are essential nutrients that regulate important metabolic signals. BCAA administration could also increase platelet activation and promote the risk of thrombosis.
OBJECTIVES
To unveil the role of BCAAs in thrombocytopoiesis.
METHODS
BCAA-fed mice and megakaryocyte/platelet-specific branched-chain α-keto acid dehydrogenase E1α subunit-deficient mice were used to study the role of BCAAs in thrombocytopoiesis.
RESULTS
In this study, we found that BCAA diet could facilitate megakaryocyte differentiation and platelet production. Meanwhile, megakaryocyte/platelet-specific branched-chain α-keto acid dehydrogenase E1α subunit-deficient mice developed thrombocythemia, which was mainly caused by the excessive differentiation of megakaryocytes and proplatelet biogenesis. Moreover, the use of BT2, the agonist of BCAA catabolism, could affect proplatelet formation (PPF) and megakaryocyte polyploidization, as well as ameliorating the thrombocythemia of BCAA-fed mice.
CONCLUSION
We found that deficiency in BCAA catabolism led to the activation of p70S6K/mammalian target of rapamycin (mTOR) signaling, megakaryocyte over differentiation, and the acceleration of PPF. Activating BCAA metabolism with BT2 could inhibit mTOR signaling, reduce PPF, and ameliorate thrombocythemia in BCAA-fed mice. Therefore, this study reveals a novel role of BCAAs in megakaryocyte differentiation and platelet production, suggesting that targeting BCAA-mediated p70S6K/mTOR signaling may be a potential strategy for the treatment of thrombocytopenia or thrombocythemia.
Topics: Mice; Animals; Amino Acids, Branched-Chain; Ribosomal Protein S6 Kinases, 70-kDa; Thrombopoiesis; 3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); TOR Serine-Threonine Kinases; Thrombocytosis; Thrombocytopenia; Thrombosis; Mammals
PubMed: 37473846
DOI: 10.1016/j.jtha.2023.06.039 -
Veterinary Clinical Pathology Sep 2023Immature platelets (IPs) are newly formed platelets released into circulation that have been demonstrated as good markers of thrombopoiesis. Although many flow...
BACKGROUND
Immature platelets (IPs) are newly formed platelets released into circulation that have been demonstrated as good markers of thrombopoiesis. Although many flow cytometric and fully automated-based methods are available, the latest Sysmex XN-V hematology analyzer for veterinary use is equipped with a specific fluorescent platelet channel (PLT-F) that detects platelets using a platelet-specific dye.
OBJECTIVES
The aims of this study were to evaluate the performance of the Sysmex XSN-1000 V in determining the IPF (immature platelet fraction) and other selected PLT-F channel parameters and to propose IPF reference intervals (RIs) for canine blood samples.
METHODS
Canine EDTA blood samples were analyzed on the Sysmex XN-1000 V to assess linearity, imprecision, carryover, stability, and the effect of platelet clumping on selected platelet parameters from the PLT-F channel. We also reported the de novo generated RIs for the IPF in dogs.
RESULTS
Imprecision was acceptable (CV <10%) for all parameters except for the absolute IPF values (IPF#), in which the reproducibility was 12.15% for the normal-low concentration samples. Linearity and carryover were excellent for all variables. Relative IPF values (IPF %) and IPF# remained stable for both storage conditions for up to 48 hours; however, a nonsignificant progressive increase in these parameters was observed from 12 hours at 4°C. We observed a statistical increase in IPF% and IPF# and a statistically significant decrease in PLT-F counts after intentional in vitro platelet aggregation. RIs were generated for all reference samples (n = 69) and for samples with (n = 25) or without (n = 44) platelet clumps.
CONCLUSIONS
The performance of the new PLT-F channel-derived variables for dogs was excellent. Specific RIs for IPF should be used when platelet aggregates are present.
Topics: Dogs; Animals; Blood Platelets; Platelet Count; Reference Values; Reproducibility of Results; Hematology
PubMed: 37468992
DOI: 10.1111/vcp.13241 -
New Biotechnology Nov 2023In vitro production of blood platelets for transfusion purposes is gaining interest. While platelet production is now possible on a laboratory scale, the challenge is to...
Development of an efficient, ready to use, blood platelet-release device based on two new flow regime parameters: The periodic hydrodynamic loading and the shear stress accumulation.
In vitro production of blood platelets for transfusion purposes is gaining interest. While platelet production is now possible on a laboratory scale, the challenge is to move towards industrial production. Attaining this goal calls for the development of platelet release devices capable of producing large quantities of platelets. To this end, we have developed a continuous-flow platelet release device composed of five spherical chambers each containing two calibrated cones placed in a staggered configuration. Following perfusion of proplatelet-bearing cultured megakaryocytes, the device achieves a high yield of about 100 bona-fide platelets/megakaryocyte, at a flow rate of ∼80 mL/min. Performances and operating conditions comply with the requirements of large-scale platelet production. Moreover, this device enabled an in-depth analysis of the flow regimes through Computational Fluid Dynamics (CFD). This revealed two new universal parameters to be taken into account for an optimal platelet release: i.e. a periodic hydrodynamic load and a sufficient accumulation of shear stress. An efficient 16 Pa.s shear stress accumulation is obtained in our system at a flow rate of 80 mL/min.
Topics: Blood Platelets; Hydrodynamics; Megakaryocytes; Thrombopoiesis
PubMed: 37442418
DOI: 10.1016/j.nbt.2023.07.002 -
Nature Communications Jul 2023Platelets, small hemostatic blood cells, are derived from megakaryocytes. Both bone marrow and lung are principal sites of thrombopoiesis although underlying mechanisms...
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.
Topics: Mice; Animals; Blood Platelets; Microfluidics; Megakaryocytes; Thrombopoiesis; Lung
PubMed: 37419900
DOI: 10.1038/s41467-023-39598-9 -
Journal of Thrombosis and Haemostasis :... Nov 2023Glucocorticoids are widely known for their immunomodulatory action. Their synthetic analogs are used to treat several autoimmune diseases, including immune...
BACKGROUND
Glucocorticoids are widely known for their immunomodulatory action. Their synthetic analogs are used to treat several autoimmune diseases, including immune thrombocytopenia. However, their efficacy and mechanisms of action in immune thrombocytopenia are not fully understood.
OBJECTIVES
To investigate the mechanism of glucocorticoid actions on platelet production.
METHODS
The actions of glucocorticoids on platelet production were studied combining in vivo, ex vivo and in vitro approaches.
RESULTS
Dexamethasone reduced bleeding in mice and rapidly increased circulating young platelet counts. In vitro glucocorticoid treatment stimulated proplatelet formation by megakaryocytes and platelet-like particle release. This effect was blocked by glucocorticoid receptor antagonist RU486, indicating a glucocorticoid receptor-dependent mechanism. Genome-wide analysis revealed that dexamethasone regulates the expression of >1000 genes related to numerous cellular functions, including predominant cytoplasm and cytoskeleton reorganization. Dexamethasone and other glucocorticoids induced the expression of Gda (the gene encoding guanine deaminase), which has been reported to have a role in dendrite development. Inhibition of guanine deaminase enzymatic activity blocked dexamethasone stimulation of proplatelet formation, implicating a critical role for this enzyme in glucocorticoid-mediated platelet production.
CONCLUSION
Our findings identify glucocorticoids as new regulators of thrombopoiesis.
Topics: Mice; Animals; Megakaryocytes; Thrombopoiesis; Glucocorticoids; Purpura, Thrombocytopenic, Idiopathic; Receptors, Glucocorticoid; Guanine Deaminase; Transcriptome; Blood Platelets; Thrombocytopenia; Dexamethasone
PubMed: 37336437
DOI: 10.1016/j.jtha.2023.06.012 -
BMC Cancer May 2023Thrombocytopenia is a common complication in cancer patients undergoing chemotherapy. Chemotherapy-induced thrombocytopenia (CIT) leads to dose reduction and treatment...
BACKGROUND
Thrombocytopenia is a common complication in cancer patients undergoing chemotherapy. Chemotherapy-induced thrombocytopenia (CIT) leads to dose reduction and treatment delays, lowering chemotherapy efficacy and survival rate. Thus, rapid recovery and continuous maintenance of platelet count during chemotherapy cycles are crucial in patients with CIT. Thrombopoietin (TPO) and its receptor, myeloid proliferative leukemia (MPL) protein, play a major role in platelet production. Although several MPL agonists have been developed to regulate thrombopoiesis, none have been approved for the management of CIT due to concerns regarding efficacy or safety. Therefore, the development of effective MPL agonists for treating CIT needs to be further expanded.
METHODS
Anti-MPL antibodies were selected from the human combinatorial antibody phage libraries using phage display. We identified 2R13 as the most active clone among the binding antibodies via cell proliferation assay using BaF3/MPL cells. The effect of 2R13 on megakaryocyte differentiation was evaluated in peripheral blood CD34 cells by analyzing megakaryocyte-specific differentiation markers (CD41a and CD42b) and DNA ploidy using flow cytometry. The 2R13-induced platelet production was examined in 8- to 10-week-old wild-type BALB/c female mice and a thrombocytopenia mouse model established by intraperitoneal injection of 5-fluorouracil (150 mg/kg). The platelet counts were monitored twice a week over 14 days post-initiation of treatment with a single injection of 2R13, or recombinant human TPO (rhTPO) for seven consecutive days.
RESULTS
We found that 2R13 specifically interacted with MPL and activated its signaling pathways. 2R13 stimulated megakaryocyte differentiation, evidenced by increasing the proportion of high-ploidy (≥ 8N) megakaryocytes in peripheral blood-CD34 cells. The platelet count was increased by a single injection of 2R13 for up to 14 days. Injection of 5-fluorouracil considerably reduced the platelet count by day 4, which was recovered by 2R13. The platelets produced by 2R13 sustained a higher count than that achieved using seven consecutive injections of rhTPO.
CONCLUSIONS
Our findings suggest that 2R13 is a promising therapeutic agent for CIT treatment.
Topics: Mice; Animals; Humans; Female; Receptors, Thrombopoietin; Blood Platelets; Thrombopoiesis; Antibodies; Recombinant Proteins; Antigens, CD34; Fluorouracil; Thrombocytopenia; Antineoplastic Agents
PubMed: 37259024
DOI: 10.1186/s12885-023-10975-3 -
Thrombosis Research Nov 2023Platelets and their parent cell, the megakaryocyte (MK), are increasingly recognized for their roles during infection and inflammation. The MK residing in the bone...
Platelets and their parent cell, the megakaryocyte (MK), are increasingly recognized for their roles during infection and inflammation. The MK residing in the bone marrow or arising from precursors trafficked to other organs for development go on to form platelets through thrombopoiesis. Infection, by direct and indirect mechanisms, can alter the transcriptional profile of MKs. The altered environment, whether mediated by inflammatory cytokines or other signaling mechanisms results in an altered platelet transcriptome. Platelets released into the circulation, in turn, interact with each other, circulating leukocytes and endothelial cells and contribute to the clearance of pathogens or the potentiation of pathophysiology through such mechanisms as immunothrombosis. In this article we hope to identify key contributions that explore the impact of an altered transcriptomic landscape during severe, systemic response to infection broadly defined as sepsis, and viral infections, including SARS-CoV2. We include current publications that outline the role of MKs from bone-marrow and extra-medullary sites as well as the circulating platelet. The underlying diseases result in thrombotic complications that exacerbate organ dysfunction and mortality. Understanding the impact of platelets on the pathophysiology of disease may drive therapeutic advances to improve the morbidity and mortality of these deadly afflictions.
Topics: Humans; Megakaryocytes; Transcriptome; Endothelial Cells; RNA, Viral; COVID-19; SARS-CoV-2; Blood Platelets; Thrombopoiesis; Sepsis
PubMed: 37258336
DOI: 10.1016/j.thromres.2023.05.015 -
International Journal of Molecular... May 2023Megakaryocytes are the main members of the hematopoietic system responsible for regulating vascular homeostasis through their progeny platelets, which are generally... (Review)
Review
Megakaryocytes are the main members of the hematopoietic system responsible for regulating vascular homeostasis through their progeny platelets, which are generally known for maintaining hemostasis. Megakaryocytes are characterized as large polyploid cells that reside in the bone marrow but may also circulate in the vasculature. They are generated directly or through a multi-lineage commitment step from the most primitive progenitor or Hematopoietic Stem Cells (HSCs) in a process called "megakaryopoiesis". Immature megakaryocytes enter a complicated development process defined as "thrombopoiesis" that ultimately results in the release of extended protrusions called proplatelets into bone marrow sinusoidal or lung microvessels. One of the main mediators that play an important modulatory role in hematopoiesis and hemostasis is nitric oxide (NO), a free radical gas produced by three isoforms of nitric oxide synthase within the mammalian cells. In this review, we summarize the effect of NO and its signaling on megakaryopoiesis and thrombopoiesis under both physiological and pathophysiological conditions.
Topics: Animals; Megakaryocytes; Nitric Oxide; Blood Platelets; Thrombopoiesis; Hematopoietic Stem Cells; Mammals
PubMed: 37175857
DOI: 10.3390/ijms24098145 -
Research Square Apr 2023Previous studies have shown that human platelets and megakaryocytes carry microRNAs suggesting their role in platelet function and megakaryocyte development,...
Previous studies have shown that human platelets and megakaryocytes carry microRNAs suggesting their role in platelet function and megakaryocyte development, respectively. However, a comprehensive study on the microRNAs and their targets has not been undertaken. Zebrafish thrombocytes could be used as a model to study their role in megakaryocyte maturation and platelet function because thrombocytes have both megakaryocyte features and platelet properties. In our laboratory, we identified 15 microRNAs in thrombocytes using single-cell RNA sequencing. We knocked down each of these 15 microRNAs by the piggyback method and found knockdown of three microRNAs, , and in adult zebrafish led to an increase in the percentage of thrombocytes. Functional thrombocyte analysis using plate tilt assay showed no modulatory effect of the three microRNAs on thrombocyte aggregation/agglutination. We also found enhanced thrombosis using arterial laser thrombosis assay in a group of zebrafish larvae after , and knockdowns. These results suggested , and are repressors for thrombocyte production. We then explored miRWalk database for downstream targets and then selected those that are expressed in thrombocytes, and from this list based on their role in differentiation selected 14 genes, , and that encode transcriptional regulators. The qRT-PCR analysis of expression levels of the above genes following knockdown showed changes in the expression of 13 targets. We then studied the effect of the 13 targets on thrombocyte production and identified 5 genes, , and that showed thrombocytosis and one gene, that showed thrombocytopenia. Furthermore, we tested whether regulates any of the above 13 transcription factors after knockdown using qRT-PCR. Six of the 13 genes showed similar gene expression as observed with knockdown and 7 genes showed opposing results. Thus, our results suggested a possible regulatory network in common with both and . We also identified that , , and play a role in thrombopoiesis. Since the gene showed a differential expression profile in and knockdowns but resulted in thrombocytopenia in knockdown in both adults and larvae we also studied an mutant and showed the mutant had thrombocytopenia. Taken together, these studies showed that thrombopoiesis is controlled by a network of transcription regulators that are regulated by multiple microRNAs in both positive and negative manner resulting in overall inhibition of thrombopoiesis.
PubMed: 37162944
DOI: 10.21203/rs.3.rs-2807790/v1 -
Biomedicine & Pharmacotherapy =... Jul 2023Thrombocytopenia is a common hematological disease caused by many factors. It usually complicates critical diseases and increases morbidity and mortality. The treatment...
BACKGROUND
Thrombocytopenia is a common hematological disease caused by many factors. It usually complicates critical diseases and increases morbidity and mortality. The treatment of thrombocytopenia remains a great challenge in clinical practice, however, its treatment options are limited. In this study, the active monomer xanthotoxin (XAT) was screened out to explore its medicinal value and provide novel therapeutic strategies for the clinical treatment of thrombocytopenia.
METHODS
The effects of XAT on megakaryocyte differentiation and maturation were detected by flow cytometry, Giemsa and phalloidin staining. RNA-seq identified differentially expressed genes and enriched pathways. The signaling pathway and transcription factors were verified through WB and immunofluorescence staining. Tg (cd41: eGFP) transgenic zebrafish and mice with thrombocytopenia were used to evaluate the biological activity of XAT on platelet formation and the related hematopoietic organ index in vivo.
RESULTS
XAT promoted the differentiation and maturation of Meg-01 cells in vitro. Meanwhile, XAT could stimulate platelet formation in transgenic zebrafish and recover platelet production and function in irradiation-induced thrombocytopenia mice. Further RNA-seq prediction and WB verification revealed that XAT activates the IL-1R1 target and MEK/ERK signaling pathway, and upregulates the expression of transcription factors related to the hematopoietic lineage to promote megakaryocyte differentiation and platelet formation.
CONCLUSION
XAT accelerates megakaryocyte differentiation and maturation to promote platelet production and recovery through triggering IL-1R1 and activating the MEK/ERK signaling pathway, providing a new pharmacotherapy strategy for thrombocytopenia.
Topics: Mice; Animals; Thrombopoiesis; Blood Platelets; Megakaryocytes; Methoxsalen; Zebrafish; Thrombocytopenia; Transcription Factors; Signal Transduction; Mitogen-Activated Protein Kinase Kinases
PubMed: 37156117
DOI: 10.1016/j.biopha.2023.114811