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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 -
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 -
Journal of Thrombosis and Haemostasis :... Sep 2023Germline mutations in the ETV6 transcription factor gene are responsible for familial thrombocytopenia and leukemia predisposition syndrome. Although previous studies...
BACKGROUND
Germline mutations in the ETV6 transcription factor gene are responsible for familial thrombocytopenia and leukemia predisposition syndrome. Although previous studies have shown that ETV6 plays an important role in megakaryocyte (MK) maturation and platelet formation, the mechanisms by which ETV6 dysfunction promotes thrombocytopenia remain unclear.
OBJECTIVES
To decipher the transcriptional mechanisms and gene regulatory network linking ETV6 germline mutations and thrombocytopenia.
METHODS
Presuming that ETV6 mutations result in selective effects at a particular cell stage, we applied single-cell RNA sequencing to understand gene expression changes during megakaryopoiesis in peripheral CD34 cells from healthy controls and patients with ETV6-related thrombocytopenia.
RESULTS
Analysis of gene expression and regulon activity revealed distinct clusters partitioned into 7 major cell stages: hematopoietic stem/progenitor cells, common-myeloid progenitors (CMPs), MK-primed CMPs, granulocyte-monocyte progenitors, MK-erythroid progenitors (MEPs), progenitor MKs/mature MKs, and platelet-like particles. We observed a differentiation trajectory in which MEPs developed directly from hematopoietic stem/progenitor cells and bypassed the CMP stage. ETV6 deficiency led to the development of aberrant cells as early as the MEP stage, which intensified at the progenitor MK/mature MK stage, with a highly deregulated core "ribosome biogenesis" pathway. Indeed, increased translation levels have been documented in patient CD34-derived MKs with overexpression of ribosomal protein S6 and phosphorylated ribosomal protein S6 in both CD34-derived MKs and platelets. Treatment of patient MKs with the ribosomal biogenesis inhibitor CX-5461 resulted in an increase in platelet-like particles.
CONCLUSION
These findings provide novel insight into both megakaryopoiesis and the link among ETV6, translation, and platelet production.
Topics: Humans; Cell Differentiation; Megakaryocytes; Ribosomal Protein S6; Single-Cell Analysis; Thrombocytopenia; Thrombopoiesis; Antigens, CD34; ETS Translocation Variant 6 Protein
PubMed: 37085035
DOI: 10.1016/j.jtha.2023.04.007 -
Blood Advances Aug 2023Erythrocytes undergo a well-defined switch from fetal to postnatal circulation, which is mainly reflected by the stage-specific expression of hemoglobin chains....
Erythrocytes undergo a well-defined switch from fetal to postnatal circulation, which is mainly reflected by the stage-specific expression of hemoglobin chains. Perinatal alterations in thrombopoiesis are poorly understood. We assessed the ontogenesis of platelet phenotype and function from early prematurity to adulthood. We recruited 64 subjects comprising 7 extremely preterm (27-31 weeks gestational age), 25 moderately preterm (32-36 weeks), 10 term neonates, 8 infants (<2 years), 5 children (2-13 years), and 9 adults (>13 years). Blood was withdrawn at up to 3 different time points in neonates (t1: 0-2, t2: 3-7, and t3: 8-14 days after birth). We found that the expression levels of the major surface receptors for fibrinogen, collagen, vWF, fibronectin, and laminin were reduced but correlated with decreased platelet size, indicating a normal surface density. Although CD62P and CD63 surface exposure upon stimulation with TRAP-6, ADP, or U46619 was unaltered or only slightly reduced in neonates, GPIIb/IIIa inside-out and outside-in activation was blunted but showed a continuous increase until adulthood, correlating with the expression of the GPIIb/IIIa regulating tetraspanin CD151. Platelet subpopulation analysis using automated clustering revealed that neonates presented with a CD63+/PAC-1- pattern, followed by a continuous increase in CD63+/PAC-1+ platelets until adulthood. Our findings revealed that the number of platelet-monocyte and platelet-neutrophil aggregates, but not platelet-lymphocyte aggregates, is increased in neonates and that neonatal aggregate formation depends in part on CD62P activation. Our PLatelets In Neonatal Infants Study (PLINIUS) provides several lines of evidence that the platelet phenotype and function evolve continuously from neonates to adulthood.
Topics: Humans; Pregnancy; Female; Infant, Newborn; Blood Platelets; Platelet Activation; Platelet Glycoprotein GPIIb-IIIa Complex; Infant, Premature; 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid
PubMed: 37042931
DOI: 10.1182/bloodadvances.2023009824 -
Platelets Dec 2023Noncoding RNAs (ncRNAs) are a group of RNA molecules that cannot encode proteins, and a better understanding of the complex interaction networks coordinated by ncRNAs... (Review)
Review
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 per day, and are renewed every 8-9 days. The process of thrombopoiesis is affected by multiple factors, in which ncRNAs also exert a significant regulatory role. This article reviewed the regulatory roles of ncRNAs, mainly microRNAs (miRNAs), circRNAs (circular RNAs), and long non-coding RNAs (lncRNAs), in thrombopoiesis in recent years as well as their roles in primary immune thrombocytopenia (ITP).
Topics: Humans; Thrombopoiesis; Blood Platelets; Megakaryocytes; MicroRNAs; RNA, Untranslated
PubMed: 36550091
DOI: 10.1080/09537104.2022.2157382 -
Blood Advances Jul 2023GATA1 mutations that result in loss of the N-terminal 83 amino acids are a feature of myeloid leukemia in children with Down syndrome, rare familial cases of...
GATA1 mutations that result in loss of the N-terminal 83 amino acids are a feature of myeloid leukemia in children with Down syndrome, rare familial cases of dyserythropoietic anemia, and a subset of cases of Diamond-Blackfan anemia. The Gata1s mouse model, which expresses only the short GATA1 isoform that begins at methionine 84, has been shown to have a defect in hematopoiesis, especially impaired erythropoiesis with expanded megakaryopoiesis, during gestation. However, these mice reportedly did not show any postnatal phenotype. Here, we demonstrate that Gata1s mutant mice display macrocytic anemia and features of aberrant megakaryopoiesis throughout life, culminating in profound splenomegaly and bone marrow fibrosis. These data support the use of this animal model for studies of GATA1 deficiencies.
Topics: Animals; Mice; Cell Lineage; Down Syndrome; Erythropoiesis; Protein Isoforms; Thrombopoiesis
PubMed: 36350717
DOI: 10.1182/bloodadvances.2022008124 -
Thrombosis Research Nov 2023Autophagy, the continuous recycling of intracellular building blocks, molecules, and organelles is necessary to preserve cellular function and homeostasis. In this... (Review)
Review
Autophagy, the continuous recycling of intracellular building blocks, molecules, and organelles is necessary to preserve cellular function and homeostasis. In this context, it was demonstrated that autophagy plays an important role in megakaryopoiesis, the development and differentiation of hematopoietic progenitor cells into megakaryocytes. Furthermore, in recent years, autophagic proteins were detected in platelets, anucleate cells generated by megakaryocytes, responsible for hemostasis, thrombosis, and a key cell in inflammation and host immune responses. In the last decade studies have indicated the occurrence of autophagy in platelets. Moreover, autophagy in platelets was subsequently demonstrated to be involved in platelet aggregation, adhesion, and thrombus formation. Here, we review the current knowledge about autophagy in platelets, its function, and clinical implications. However, at the advent of platelet autophagy research, additional discoveries derived from evolving work will be required to precisely define the contributions of autophagy in platelets, and to expand the ever increasing physiologic and pathologic roles these remarkable and versatile blood cells play.
Topics: Humans; Blood Platelets; Megakaryocytes; Thrombopoiesis; Thrombosis; Autophagy; Biology
PubMed: 36058760
DOI: 10.1016/j.thromres.2022.08.019