RESUMEN
BACKGROUND: It has been described in mice models that myeloproliferative neoplasm (MPN) with JAK2-V617F mutation has an increased expression of programmed death-ligand 1 (PD-L1) in megakaryocytes leading to cancer immune evasion by inhibiting the T-lymphocytes. AIMS: To quantify and compare the PD-L1 expression on bone marrow (BM) of patients with MPN JAK2 positive, negative, and normal controls. METHODS: We collected BM of patients with MPN JAK2 positive, negative and normal controls from 1990 to 2019. We also created a scoring system to quantify PD-L1 expression in megakaryocytes. RESULTS: We obtained 14 BM with JAK2 positive PMF, 5 JAK2 negative PMF, and 10 patients with normal BM biopsies. PD-L1 expression was higher in the JAK2 positive group compared with the control group with a score of 212.6 versus 121.1 (t-value 2.05, p-value 0.025). In addition, the score was higher in the PMF group regardless of JAK2 mutational status when compared with the control group with score of 205.9 versus 121.1 (t-value 2.12, p-value 0.021). There was no difference in the PD-L1 score between the JAK2 negative versus the control group 187.2 versus 121.1 (t-value 1.02, p-value 0.162). CONCLUSION: These findings suggest that PMF patients with a JAK2 mutation have a higher PD-L1 expression in megakaryocytes compared with the control group. We postulate that the combination of checkpoint and JAK2 inhibitors may be an active treatment option in JAK2 mutated PMF given the higher PD-L1 expression.
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Antígeno B7-H1 , Janus Quinasa 2 , Mielofibrosis Primaria , Humanos , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Mielofibrosis Primaria/genética , Mielofibrosis Primaria/metabolismo , Mielofibrosis Primaria/patología , Masculino , Femenino , Persona de Mediana Edad , Anciano , Adulto , Mutación , Médula Ósea/patología , Megacariocitos/patología , Megacariocitos/metabolismo , Anciano de 80 o más Años , Estudios de Casos y ControlesRESUMEN
OBJECTIVE: To investigate the effects of platelet-specific Rictor knockout on platelet activation and thrombus formation in mice. METHODS: PF4-Cre and Rictorfl/fl transgenic mice were crossed to obtain platelet-specific Rictor knockout (Rictor-KO) mice and wild-type mice (n=65), whose expression levels of Rictor, protein kinase B (AKT) and p-AKT were detected using Western blotting. Platelet counts of the mice were determined using routine blood tests, and hemostatic function was assessed by tail vein hemorrhage test. Venous thrombosis models were established in the mice to evaluate the effect of Rictor knockout on thrombosis. Platelet aggregation induced by ADP and thrombin was observed in Rictor-KO and wild-type mice, and flow cytometry was used to analyze the expression levels of integrin αIIbß3 and CD62P in resting and activated platelets. Plasma PF4 levels were determined with ELISA. Megakaryocytes from Rictor-KO and wild-type mice were incubated by vWF immunohistochemical antibody and APC-CD41 antibody to detect the number and ploidy of megakaryocytes, respectively. Platelet elongation on collagen surface was observed with scanning electron microscopy. RESULTS: Compared with the wild-type mice, Rictor-KO mice showed significantly decreased AKT phosphorylation, decreased platelet production, reduced thrombosis, and decreased platelet activation in response to ADP and thrombin stimulation. The Rictor-KO mice also showed lowered expression level of P-selectin protein and activation of integrin αIIbß3 with suppression of platelet extension, reduced plasma PF4 level and decreased number of megakaryocytes in the bone marrow. The ploidy of megakaryocytes and the mean area of proplatelets were both significantly decreased in Rictor-KO mice. CONCLUSION: Platelet-specific Rictor knockout inhibits platelet generation and activation to result in decreased thrombus formation in mice, suggesting the potential of mTORC2 activity inhibition as an efficient antithrombotic strategy.
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Plaquetas , Megacariocitos , Ratones Noqueados , Activación Plaquetaria , Proteínas Proto-Oncogénicas c-akt , Proteína Asociada al mTOR Insensible a la Rapamicina , Trombosis , Animales , Ratones , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Plaquetas/metabolismo , Trombosis/metabolismo , Trombosis/prevención & control , Megacariocitos/metabolismo , Megacariocitos/citología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Agregación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Selectina-P/metabolismo , Recuento de PlaquetasRESUMEN
Blood platelets are produced by megakaryocytes (MKs), their parent cells, which are in the bone marrow. Once mature, MK pierces through the sinusoid vessel, and the initial protrusion further elongates as proplatelet or buds to release platelets. The mechanisms controlling the decision to initiate proplatelet and platelet formation are unknown. Here, we show that the mechanical properties of the microenvironment prevent proplatelet and platelet release in the marrow stroma while allowing this process in the bloodstream. Loss of marrow confinement following myelosuppression led to inappropriate proplatelet and platelet release into the extravascular space. We further used an inert viscoelastic hydrogel to evaluate the impact of compressive stress. Transcriptional analysis showed that culture in three-dimensional gel induced upregulation of genes related to the Rho-GTPase pathway. We found higher Rho-GTPase activation, myosin light chain phosphorylation and F-actin under mechanical constraints while proplatelet formation was inhibited. The use of latrunculin-A to decrease F-actin promoted microtubule-dependent budding and proplatelet extension inside the gel. Additionally, ex vivo exposure of intact bone marrow to latrunculin-A triggered proplatelet extensions in the interstitial space. In vivo, this confinement-mediated high intracellular tension is responsible for the formation of the peripheral zone, a unique actin-rich structure. Cytoskeleton reorganization induces the disappearance of the peripheral zone upon reaching a liquid milieu to facilitate proplatelet and platelet formation. Hence, our data provide insight into the mechanisms preventing ectopic platelet release in the marrow stroma. Identifying such pathways is especially important for understanding pathologies altering marrow mechanics such as chemotherapy or myelofibrosis.
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Plaquetas , Megacariocitos , Plaquetas/metabolismo , Plaquetas/efectos de los fármacos , Megacariocitos/metabolismo , Megacariocitos/efectos de los fármacos , Megacariocitos/citología , Animales , Ratones , Actinas/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Ratones Endogámicos C57BL , Compuestos Bicíclicos Heterocíclicos con Puentes , TiazolidinasRESUMEN
Thrombocytopenia, which is associated with thrombopoietin (TPO) deficiency, presents very limited treatment options and can lead to life-threatening complications. Discovering new therapeutic agents against thrombocytopenia has proven to be a challenging task using traditional screening approaches. Fortunately, machine learning (ML) techniques offer a rapid avenue for exploring chemical space, thereby increasing the likelihood of uncovering new drug candidates. In this study, we focused on computational modeling for drug-induced megakaryocyte differentiation and platelet production using ML methods, aiming to gain insights into the structural characteristics of hematopoietic activity. We developed 112 different classifiers by combining eight ML algorithms with 14 molecule features. The top-performing model achieved good results on both 5-fold cross-validation (with an accuracy of 81.6% and MCC value of 0.589) and external validation (with an accuracy of 83.1% and MCC value of 0.642). Additionally, by leveraging the Shapley additive explanations method, the best model provided quantitative assessments of molecular properties and structures that significantly contributed to the predictions. Furthermore, we employed an ensemble strategy to integrate predictions from multiple models and performed in silico predictions for new molecules with potential activity against thrombocytopenia, sourced from traditional Chinese medicine and the Drug Repurposing Hub. The findings of this study could offer valuable insights into the structural characteristics and computational prediction of thrombopoiesis inducers.
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Aprendizaje Automático , Trombocitopenia , Trombocitopenia/tratamiento farmacológico , Humanos , Descubrimiento de Drogas/métodos , Megacariocitos/metabolismo , Megacariocitos/efectos de los fármacos , Megacariocitos/citología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Simulación por Computador , AlgoritmosRESUMEN
Platelets are among the most abundant cells within the circulation. Given that the platelet lifespan is 7 to 10 days in humans, a constant production of around 100 billion platelets per day is required. Platelet production from precursor cells called megakaryocytes is one of the most enigmatic processes in human biology. Although it has been studied for over a century, there is still controversy about the exact mechanisms leading to platelet release into circulation. The formation of proplatelet extensions from megakaryocytes into bone marrow sinusoids is the best-described mechanism explaining the origin of blood platelets. However, using powerful imaging techniques, several emerging studies have recently raised challenging questions in the field, suggesting that small platelet-sized structures called buds might also contribute to the circulating platelet pool. How and whether these structures differ from microvesicles or membrane blebs, which have previously been described to be released from megakaryocytes, is still a matter of discussion. In this review, we will summarize what the past and present have revealed about platelet production and whether mature blood platelets might emerge via different mechanisms.
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Plaquetas , Megacariocitos , Trombopoyesis , Humanos , Plaquetas/metabolismo , Megacariocitos/citología , Megacariocitos/metabolismo , Animales , Trombopoyesis/fisiologíaRESUMEN
GATA1 is a highly conserved hematopoietic transcription factor (TF), essential for normal erythropoiesis and megakaryopoiesis, that encodes a full-length, predominant isoform and an amino (N) terminus-truncated isoform GATA1s. It is consistently expressed throughout megakaryocyte development and interacts with its target genes either independently or in association with binding partners such as FOG1 (friend of GATA1). While the N-terminus and zinc finger have classically been demonstrated to be necessary for the normal regulation of platelet-specific genes, murine models, cell-line studies, and human case reports indicate that the carboxy-terminal activation domain and zinc finger also play key roles in precisely controlling megakaryocyte growth, proliferation, and maturation. Murine models have shown that disruptions to GATA1 increase the proliferation of immature megakaryocytes with abnormal architecture and impaired terminal differentiation into platelets. In humans, germline GATA1 mutations result in variable cytopenias, including macrothrombocytopenia with abnormal platelet aggregation and excessive bleeding tendencies, while acquired GATA1s mutations in individuals with trisomy 21 (T21) result in transient abnormal myelopoiesis (TAM) and myeloid leukemia of Down syndrome (ML-DS) arising from a megakaryocyte-erythroid progenitor (MEP). Taken together, GATA1 plays a key role in regulating megakaryocyte differentiation, maturation, and proliferative capacity. As sequencing and proteomic technologies expand, additional GATA1 mutations and regulatory mechanisms contributing to human diseases of megakaryocytes and platelets are likely to be revealed.
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Plaquetas , Factor de Transcripción GATA1 , Megacariocitos , Trombopoyesis , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Humanos , Animales , Plaquetas/metabolismo , Trombopoyesis/genética , Megacariocitos/metabolismo , Megacariocitos/citología , Mutación , Trombocitopenia/genética , Trombocitopenia/patología , Trombocitopenia/metabolismo , Diferenciación Celular/genética , RatonesRESUMEN
DIAPH1, a member of the formins family and a Rho effector, was found to be involved in thrombocytopoiesis, and the process of MDS in mice with unknown pathogenesis. In this study, we reported a preliminary study about the heterogeneity in the clinical features and outcomes of DIAPH1 mutations in MDS. DIAPH1 frameshift mutations were identified in 20 out of 88 MDS patients, including 11 frameshift mutations locating at 140892588-141000567 (5q31.3), which causes structure changes at FH1 domain. DIAPH1 mutated cases were correlated with lower megakaryocyte dysplasia in lower-risk patients (IPSS-M score <0) at first diagnosis, and higher megakaryocyte counts pre-transplant. The megakaryopoiesis-related genes: GP1BA and SETBP1 mutation were positively and negatively associated with DIAPH1 mutation, respectively. DIAPH1 mutated cases showed superior overall survival of all patients and low-risk cohorts. In conclusion, we found DIAPH1 frameshift mutations are implicated in megakaryopoiesis of MDS and correlated with superior prognosis.
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Proteínas Adaptadoras Transductoras de Señales , Forminas , Mutación del Sistema de Lectura , Síndromes Mielodisplásicos , Humanos , Forminas/genética , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/patología , Femenino , Masculino , Persona de Mediana Edad , Anciano , Pronóstico , Proteínas Adaptadoras Transductoras de Señales/genética , Adulto , Megacariocitos/patología , Megacariocitos/metabolismo , Proteínas Nucleares/genética , Proteínas Portadoras/genética , Anciano de 80 o más Años , MutaciónRESUMEN
Despite increased understanding of the genomic landscape of Myeloproliferative Neoplasms (MPNs), the pathological mechanisms underlying abnormal megakaryocyte (Mk)-stromal crosstalk and fibrotic progression in MPNs remain unclear. We conducted mass spectrometry-based proteomics on mice with Romiplostim-dependent myelofibrosis to reveal alterations in signaling pathways and protein changes in Mks, platelets, and bone marrow (BM) cells. The chemokine Platelet Factor 4 (PF4)/Cxcl4 was up-regulated in all proteomes and increased in plasma and BM fluids of fibrotic mice. High TPO concentrations sustained in vitro PF4 synthesis and secretion in cultured Mks, while Ruxolitinib restrains the abnormal PF4 expression in vivo. We discovered that PF4 is rapidly internalized by stromal cells through surface glycosaminoglycans (GAGs) to promote myofibroblast differentiation. Cxcl4 gene silencing in Mks mitigated the profibrotic phenotype of stromal cells in TPO-saturated co-culture conditions. Consistently, extensive stromal PF4 uptake and altered GAGs deposition were detected in Romiplostim-treated, JAK2V617F mice and BM biopsies of MPN patients. BM PF4 levels and Mk/platelet CXCL4 expression were elevated in patients, exclusively in overt fibrosis. Finally, pharmacological inhibition of GAGs ameliorated in vivo fibrosis in Romiplostim-treated mice. Thus, our findings highlight the critical role of PF4 in the fibrosis progression of MPNs and substantiate the potential therapeutic strategy of neutralizing PF4-GAGs interaction.
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Factor Plaquetario 4 , Mielofibrosis Primaria , Proteómica , Factor Plaquetario 4/metabolismo , Factor Plaquetario 4/genética , Animales , Ratones , Mielofibrosis Primaria/metabolismo , Mielofibrosis Primaria/patología , Mielofibrosis Primaria/tratamiento farmacológico , Mielofibrosis Primaria/genética , Humanos , Proteómica/métodos , Megacariocitos/metabolismo , Megacariocitos/patología , Ratones Endogámicos C57BL , Diferenciación CelularRESUMEN
A key step in platelet production is the migration of megakaryocytes to the vascular sinusoids within the bone marrow. This homing is mediated by the chemokine CXCL12 and its receptor CXCR4. CXCR4 is also a positive regulator of platelet activation and thrombosis. Pim-1 kinase has been shown to regulate CXCR4 signalling in other cell types, and we have previously described how Pim kinase inhibitors attenuate platelet aggregation to CXCL12. However, the mechanism by which Pim-1 regulates CXCR4 signalling in platelets and megakaryocytes has yet to be elucidated. Using human platelets, murine bone marrow-derived megakaryocytes, and the megakaryocyte cell line MEG-01, we demonstrate that pharmacological Pim kinase inhibition leads to reduced megakaryocyte and platelet function responses to CXCL12, including reduced megakaryocyte migration and platelet granule secretion. Attenuation of CXCL12 signalling was found to be attributed to the reduced surface expression of CXCR4. The decrease in CXCR4 surface levels was found to be mediated by rapid receptor internalisation, in the absence of agonist stimulation. We demonstrate that pharmacological Pim kinase inhibition disrupts megakaryocyte and platelet function by reducing constitutive CXCR4 surface expression, decreasing the number of receptors available for agonist stimulation and signalling. These findings have implications for the development and use of Pim kinase inhibitors for the treatment of conditions associated with elevated circulating levels of CXCL12/SDF1α and increased thrombotic risk.
Asunto(s)
Plaquetas , Quimiocina CXCL12 , Megacariocitos , Proteínas Proto-Oncogénicas c-pim-1 , Receptores CXCR4 , Transducción de Señal , Receptores CXCR4/metabolismo , Plaquetas/metabolismo , Plaquetas/efectos de los fármacos , Megacariocitos/metabolismo , Megacariocitos/efectos de los fármacos , Megacariocitos/citología , Humanos , Transducción de Señal/efectos de los fármacos , Animales , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Quimiocina CXCL12/metabolismo , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Movimiento Celular/efectos de los fármacos , Línea CelularRESUMEN
In contrast to most hematopoietic lineages, megakaryocytes (MKs) can derive rapidly and directly from hematopoietic stem cells (HSCs). The underlying mechanism is unclear, however. Here, we show that DNA damage induces MK markers in HSCs and that G2 arrest, an integral part of the DNA damage response, suffices for MK priming followed by irreversible MK differentiation in HSCs, but not in progenitors. We also show that replication stress causes DNA damage in HSCs and is at least in part due to uracil misincorporation in vitro and in vivo. Consistent with this notion, thymidine attenuated DNA damage, improved HSC maintenance, and reduced the generation of CD41+ MK-committed HSCs. Replication stress and concomitant MK differentiation is therefore one of the barriers to HSC maintenance. DNA damage-induced MK priming may allow rapid generation of a lineage essential to immediate organismal survival, while also removing damaged cells from the HSC pool.
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Diferenciación Celular , Daño del ADN , Células Madre Hematopoyéticas , Megacariocitos , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Animales , Ratones , Megacariocitos/metabolismo , Megacariocitos/citología , Trombopoyesis , Puntos de Control de la Fase G2 del Ciclo Celular , Ratones Endogámicos C57BL , HumanosRESUMEN
Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by heterozygous germline mutations in RUNX1. It is characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematological malignancies. Although FPDMM is a precursor for diseases involving abnormal DNA methylation, the DNA methylation status in FPDMM remains unknown, largely due to a lack of animal models and challenges in obtaining patient-derived samples. Here, using genome editing techniques, we established two lines of human induced pluripotent stem cells (iPSCs) with different FPDMM-mimicking heterozygous RUNX1 mutations. These iPSCs showed defective differentiation of hematopoietic progenitor cells (HPCs) and megakaryocytes (Mks), consistent with FPDMM. The FPDMM-mimicking HPCs showed DNA methylation patterns distinct from those of wild-type HPCs, with hypermethylated regions showing the enrichment of ETS transcription factor (TF) motifs. We found that the expression of FLI1, an ETS family member, was significantly downregulated in FPDMM-mimicking HPCs with a RUNX1 transactivation domain (TAD) mutation. We demonstrated that FLI1 promoted binding-site-directed DNA demethylation, and that overexpression of FLI1 restored their megakaryocytic differentiation efficiency and hypermethylation status. These findings suggest that FLI1 plays a crucial role in regulating DNA methylation and correcting defective megakaryocytic differentiation in FPDMM-mimicking HPCs with a RUNX1 TAD mutation.
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Diferenciación Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Metilación de ADN , Células Madre Pluripotentes Inducidas , Megacariocitos , Mutación , Proteína Proto-Oncogénica c-fli-1 , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Humanos , Megacariocitos/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Diferenciación Celular/genética , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Trastornos de las Plaquetas Sanguíneas/genética , Trastornos de las Plaquetas Sanguíneas/metabolismo , Trastornos de las Plaquetas Sanguíneas/patología , Activación Transcripcional , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Leucemia Mieloide Aguda , Trastornos de la Coagulación Sanguínea HeredadosRESUMEN
Platelets are blood cells derived from megakaryocytes that play a central role in regulating haemostasis and vascular integrity. The microtubule cytoskeleton of megakaryocytes undergoes a critical dynamic reorganization during cycles of endomitosis and platelet biogenesis. Quiescent platelets have a discoid shape maintained by a marginal band composed of microtubule bundles, which undergoes remarkable remodelling during platelet activation, driving shape change and platelet function. Disrupting or enhancing this process can cause platelet dysfunction such as bleeding disorders or thrombosis. However, little is known about the molecular mechanisms underlying the reorganization of the cytoskeleton in the platelet lineage. Recent studies indicate that the emergence of a unique platelet tubulin code and specific pathogenic tubulin mutations cause platelet defects and bleeding disorders. Frequently, these mutations exhibit dominant negative effects, offering valuable insights into both platelet disease mechanisms and the functioning of tubulins. This review will highlight our current understanding of the role of the microtubule cytoskeleton in the life and death of platelets, along with its relevance to platelet disorders.
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Plaquetas , Citoesqueleto , Megacariocitos , Microtúbulos , Humanos , Plaquetas/metabolismo , Megacariocitos/metabolismo , Megacariocitos/citología , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/genética , Animales , Trastornos de las Plaquetas Sanguíneas/metabolismo , Trastornos de las Plaquetas Sanguíneas/genética , Trastornos de las Plaquetas Sanguíneas/patología , MutaciónRESUMEN
Platelet-producing megakaryocytes (MKs) primarily reside in the bone marrow, where they duplicate their DNA content with each cell cycle resulting in polyploid cells with an intricate demarcation membrane system. While key elements of the cytoskeletal reorganizations during proplatelet formation have been identified, what initiates the release of platelets into vessel sinusoids remains largely elusive. Using a cell cycle indicator, we observed a unique phenomenon, during which amplified centrosomes in MKs underwent clustering following mitosis, closely followed by proplatelet formation, which exclusively occurred in G1 of interphase. Forced cell cycle arrest in G1 increased proplatelet formation not only in vitro but also in vivo following short-term starvation of mice. We identified that inhibition of the centrosomal protein kinesin family member C1 (KIFC1) impaired clustering and subsequent proplatelet formation, while KIFC1-deficient mice exhibited reduced platelet counts. In summary, we identified KIFC1- and cell cycle-mediated centrosome clustering as an important initiator of proplatelet formation from MKs.
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Plaquetas , Ciclo Celular , Centrosoma , Cinesinas , Megacariocitos , Centrosoma/metabolismo , Animales , Megacariocitos/metabolismo , Megacariocitos/citología , Ratones , Plaquetas/metabolismo , Cinesinas/metabolismo , Cinesinas/genética , Ratones Noqueados , Humanos , MitosisRESUMEN
Patients with COVID-19 have coagulation and platelet disorders, with platelet alterations and thrombocytopenia representing negative prognostic parameters associated with severe forms of the disease and increased lethality. METHODS: The aim of this study was to study the expression of platelet glycoprotein IIIa (CD61), playing a critical role in platelet aggregation, together with TRL-2 as a marker of innate immune activation. RESULTS: A total of 25 patients were investigated, with the majority (24/25, 96%) having co-morbidities and dying from a fatal form of SARS-CoV-2(+) infection (COVID-19+), with 13 men and 12 females ranging in age from 45 to 80 years. When compared to a control group of SARS-CoV-2 (-) negative lungs (COVID-19-), TLR-2 expression was up-regulated in a subset of patients with deadly COVID-19 fatal lung illness. The proportion of Spike-1 (+) patients found by PCR and ISH correlates to the proportion of Spike-S1-positive cases as detected by digital pathology examination. Furthermore, CD61 expression was considerably higher in the lungs of deceased patients. In conclusion, we demonstrate that innate immune prolonged hyperactivation is related to platelet/megakaryocyte over-expression in the lung. CONCLUSIONS: Microthrombosis in deadly COVID-19+ lung disease is associated with an increase in the number of CD61+ platelets and megakaryocytes in the pulmonary interstitium, as well as their functional activation; this phenomenon is associated with increased expression of innate immunity TLR2+ cells, which binds the SARS-CoV-2 E protein, and significantly with the persistence of the Spike-S1 viral sequence.
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COVID-19 , Pulmón , Megacariocitos , SARS-CoV-2 , Trombosis , Receptor Toll-Like 2 , Regulación hacia Arriba , Humanos , COVID-19/patología , COVID-19/inmunología , COVID-19/metabolismo , Masculino , Femenino , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 2/genética , Megacariocitos/metabolismo , Megacariocitos/patología , Megacariocitos/virología , Anciano , Persona de Mediana Edad , Anciano de 80 o más Años , Pulmón/patología , Pulmón/virología , Pulmón/metabolismo , Regulación hacia Arriba/genética , Trombosis/patología , Integrina beta3/metabolismo , Integrina beta3/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Neumonía Viral/patología , Neumonía Viral/inmunología , Neumonía Viral/mortalidad , Neumonía Viral/virología , Neumonía Viral/metabolismo , Inmunidad Innata , PandemiasRESUMEN
ABSTRACT: Fibrinolytics delivered into the general circulation lack selectivity for nascent thrombi, reducing efficacy and increasing the risk of bleeding. Urokinase-type plasminogen activator (uPA) transgenically expressed within murine platelets provided targeted thromboprophylaxis without causing bleeding but is not clinically feasible. Recent advances in generating megakaryocytes prompted us to develop a potentially clinically relevant means to produce "antithrombotic" platelets from CD34+ hematopoietic stem cell-derived in vitro-grown megakaryocytes. CD34+ megakaryocytes internalize and store in alpha granules (α-granules) single-chain uPA (scuPA) and a plasmin-resistant thrombin-activatable variant (uPAT). Both uPAs colocalized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3, but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+ megakaryocytes was mediated, in part, via LRP1 and αIIbß3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV but not endogenous VWF in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid artery injury model in nonobese diabetic-severe combined immunodeficiency IL2rγnull (NSG) mice homozygous for VWFR1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein Ibα) to test whether platelets derived from scuPA- or uPAT-megakaryocytes would prevent thrombus formation. NSG/VWFR1326H mice exhibited a lower thrombotic burden after carotid artery injury compared with NSG mice unless infused with human platelets or megakaryocytes, whereas intravenous injection of uPA-megakaryocytes generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies describe the use of in vitro-generated megakaryocytes as a potential platform for delivering uPA or other ectopic proteins within platelet α-granules to sites of vascular injury.
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Megacariocitos , Activador de Plasminógeno de Tipo Uroquinasa , Megacariocitos/metabolismo , Megacariocitos/citología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Humanos , Animales , Ratones , Fibrinólisis/efectos de los fármacos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Plaquetas/metabolismo , Trombosis/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Gránulos Citoplasmáticos/metabolismo , Antígenos CD34/metabolismoRESUMEN
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.
Asunto(s)
Regiones no Traducidas 5' , Proteína Proto-Oncogénica c-fli-1 , Trombocitopenia , Humanos , Trombocitopenia/genética , Trombocitopenia/patología , Regiones no Traducidas 5'/genética , Sitios de Unión , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Masculino , Femenino , Linaje , Megacariocitos/metabolismo , Megacariocitos/patología , Plaquetas/metabolismo , Plaquetas/patología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Unión Proteica , Predisposición Genética a la Enfermedad , Péptidos y Proteínas de Señalización IntercelularRESUMEN
BACKGROUND: Hematopoietic stem cell (HSC) regeneration underlies hematopoietic recovery from myelosuppression, which is a life-threatening side effect of cytotoxicity. HSC niche is profoundly disrupted after myelosuppressive injury, while if and how the niche is reshaped and regulates HSC regeneration are poorly understood. METHODS: A mouse model of radiation injury-induced myelosuppression was built by exposing mice to a sublethal dose of ionizing radiation. The dynamic changes in the number, distribution and functionality of HSCs and megakaryocytes were determined by flow cytometry, immunofluorescence, colony assay and bone marrow transplantation, in combination with transcriptomic analysis. The communication between HSCs and megakaryocytes was determined using a coculture system and adoptive transfer. The signaling mechanism was investigated both in vivo and in vitro, and was consolidated using megakaryocyte-specific knockout mice and transgenic mice. RESULTS: Megakaryocytes become a predominant component of HSC niche and localize closer to HSCs after radiation injury. Meanwhile, transient insulin-like growth factor 1 (IGF1) hypersecretion is predominantly provoked in megakaryocytes after radiation injury, whereas HSCs regenerate paralleling megakaryocytic IGF1 hypersecretion. Mechanistically, HSCs are particularly susceptible to megakaryocytic IGF1 hypersecretion, and mTOR downstream of IGF1 signaling not only promotes activation including proliferation and mitochondrial oxidative metabolism of HSCs, but also inhibits ferritinophagy to restrict HSC ferroptosis. Consequently, the delicate coordination between proliferation, mitochondrial oxidative metabolism and ferroptosis ensures functional HSC expansion after radiation injury. Importantly, punctual IGF1 administration simultaneously promotes HSC regeneration and hematopoietic recovery after radiation injury, representing a superior therapeutic approach for myelosuppression. CONCLUSIONS: Our study identifies megakaryocytes as a last line of defense against myelosuppressive injury and megakaryocytic IGF1 as a novel niche signal safeguarding HSC regeneration.
Asunto(s)
Ferroptosis , Células Madre Hematopoyéticas , Factor I del Crecimiento Similar a la Insulina , Megacariocitos , Regeneración , Animales , Células Madre Hematopoyéticas/metabolismo , Megacariocitos/metabolismo , Megacariocitos/efectos de la radiación , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Ferroptosis/genética , Ratones , Ratones Endogámicos C57BL , Traumatismos por Radiación/metabolismo , Traumatismos por Radiación/patología , Traumatismos por Radiación/genética , Transducción de Señal/efectos de la radiaciónRESUMEN
Lung cancer (LC) is the leading cause of cancer-associated deaths worldwide, among which non-small-cell lung cancer (NSCLC) accounts for 80%. Stromal cell-derived factor-1 (SDF-1) inhibition results in a significant depletion of NSCLC metastasis. Additionally, SDF-1 is the only natural chemokine known to bind and activate the receptor CXCR4. Thus, we attempted to clarify the molecular mechanism of SDF-1 underlying NSCLC progression. Transwell migration, adhesion, and G-LISA assays were used to assess megakaryocytic chemotaxis in vitro and in vivo in terms of megakaryocytic migration, adherence, and RhoA activation, respectively. Western blotting was used to assess PI3K/Akt-associated protein abundances in MEG-01 cells and primary megakaryocytes under the indicated treatment. A hematology analyzer and flow cytometry were used to assess platelet counts in peripheral blood and newly formed platelet counts in Lewis LC mice under different treatments. Immunochemistry and flow cytometry were used to measure CD41+ megakaryocyte numbers in Lewis LC mouse tissue under different treatments. ELISA was used to measure serum TPO levels, and H&E staining was used to detect NSCLC metastasis.SDF-1 receptor knockdown suppressed megakaryocytic chemotaxis in Lewis LC mice. SDF-1 receptor inhibition suppressed megakaryocytic chemotaxis via the PI3K/Akt pathway. SDF-1 receptor knockdown suppressed CD41+ megakaryocyte numbers in vivo through PI3K/Akt signaling. SDF-1 receptor inhibition suppressed CD41+ megakaryocytes to hinder NSCLC metastasis. SDF-1 facilitates NSCLC metastasis by enhancing the chemoattraction of megakaryocytes via the PI3K/Akt signaling pathway, which may provide a potential new direction for seeking therapeutic plans for NSCLC.