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1.
J Thromb Haemost ; 2024 Jun 06.
Article in English | MEDLINE | ID: mdl-38849085

ABSTRACT

BACKGROUND: Aging is an independent risk factor for the development of cardiovascular, thrombotic, and other chronic diseases. However, mechanisms of platelet hyperactivation in aging remain poorly understood. OBJECTIVES: Here, we examine whether and how aging alters intracellular signaling in platelets to support platelet hyperactivity and thrombosis. METHODS: Quantitative mass spectrometry with tandem mass tag labeling systematically measured protein phosphorylation in platelets from healthy aged (>65 years) and young human (<45 years) subjects. The role of platelet mechanistic target of rapamycin (mTOR) in aging-induced platelet hyperreactivity was assessed using pharmacologic mTOR inhibition and a platelet-specific mTOR-deficient mouse model (mTORplt-/-). RESULTS: Quantitative phosphoproteomics uncovered differential site-specific protein phosphorylation within mTOR, Rho GTPase, and MAPK pathways in platelets from aged donors. Western blot confirmed constitutive activation of the mTOR pathway in platelets from both aged humans and mice, which was associated with increased aggregation compared with that in young controls. Inhibition of mTOR with either Torin 1 in aged humans or genetic deletion in aged mice reversed platelet hyperreactivity. In a collagen-epinephrine pulmonary thrombosis model, aged wild-type (mTORplt+/+) mice succumbed significantly faster than young controls, while time to death of aged mTORplt-/- mice was similar to that of young mTORplt+/+ mice. Mechanistically, we noted increased Rac1 activation and levels of mitochondrial reactive oxygen species in resting platelets from aged mice, as well as increased p38 phosphorylation upstream of thromboxane generation following agonist stimulation. CONCLUSION: Aging-related changes in mTOR phosphorylation enhance Rac1 and p38 activation to enhance thromboxane generation, platelet hyperactivity, and thrombosis.

2.
Circ Res ; 134(2): 143-161, 2024 01 19.
Article in English | MEDLINE | ID: mdl-38156445

ABSTRACT

BACKGROUND: Single-nucleotide polymorphisms linked with the rs1474868 T allele (MFN2 [mitofusin-2] T/T) in the human mitochondrial fusion protein MFN2 gene are associated with reduced platelet MFN2 RNA expression and platelet counts. This study investigates the impact of MFN2 on megakaryocyte and platelet biology. METHODS: Mice with megakaryocyte/platelet deletion of Mfn2 (Mfn2-/- [Mfn2 conditional knockout]) were generated using Pf4-Cre crossed with floxed Mfn2 mice. Human megakaryocytes were generated from cord blood and platelets isolated from healthy subjects genotyped for rs1474868. Ex vivo approaches assessed mitochondrial morphology, function, and platelet activation responses. In vivo measurements included endogenous/transfused platelet life span, tail bleed time, transient middle cerebral artery occlusion, and pulmonary vascular permeability/hemorrhage following lipopolysaccharide-induced acute lung injury. RESULTS: Mitochondria was more fragmented in megakaryocytes derived from Mfn2-/- mice and from human cord blood with MFN2 T/T genotype compared with control megakaryocytes. Human resting platelets of MFN2 T/T genotype had reduced MFN2 protein, diminished mitochondrial membrane potential, and an increased rate of phosphatidylserine exposure during ex vivo culture. Platelet counts and platelet life span were reduced in Mfn2-/- mice accompanied by an increased rate of phosphatidylserine exposure in resting platelets, especially aged platelets, during ex vivo culture. Mfn2-/- also decreased platelet mitochondrial membrane potential (basal) and activated mitochondrial oxygen consumption rate, reactive oxygen species generation, calcium flux, platelet-neutrophil aggregate formation, and phosphatidylserine exposure following dual agonist activation. Ultimately, Mfn2-/- mice showed prolonged tail bleed times, decreased ischemic stroke infarct size after cerebral ischemia-reperfusion, and exacerbated pulmonary inflammatory hemorrhage following lipopolysaccharide-induced acute lung injury. Analysis of MFN2 SNPs in the iSPAAR study (Identification of SNPs Predisposing to Altered ALI Risk) identified a significant association between MFN2 and 28-day mortality in patients with acute respiratory distress syndrome. CONCLUSIONS: Mfn2 preserves mitochondrial phenotypes in megakaryocytes and platelets and influences platelet life span, function, and outcomes of stroke and lung injury.


Subject(s)
Acute Lung Injury , Lipopolysaccharides , Aged , Animals , Humans , Mice , Acute Lung Injury/metabolism , Blood Platelets/metabolism , Hemorrhage/metabolism , Mitochondria/metabolism , Phosphatidylserines/metabolism
3.
Blood Adv ; 7(24): 7621-7623, 2023 12 26.
Article in English | MEDLINE | ID: mdl-38147360
5.
Blood ; 140(23): 2477-2489, 2022 12 08.
Article in English | MEDLINE | ID: mdl-35930749

ABSTRACT

The MAPK-interacting kinase (Mnk) family includes Mnk1 and Mnk2, which are phosphorylated and activated in response to extracellular stimuli. Mnk1 contributes to cellular responses by regulating messenger RNA (mRNA) translation, and mRNA translation influences platelet production and function. However, the role of Mnk1 in megakaryocytes and platelets has not previously been studied. The present study investigated Mnk1 in megakaryocytes and platelets using both pharmacological and genetic approaches. We demonstrate that Mnk1, but not Mnk2, is expressed and active in human and murine megakaryocytes and platelets. Stimulating human and murine megakaryocytes and platelets induced Mnk1 activation and phosphorylation of eIF4E, a downstream target of activated Mnk1 that triggers mRNA translation. Mnk1 inhibition or deletion significantly diminished protein synthesis in megakaryocytes as measured by polysome profiling and [35S]-methionine incorporation assays. Depletion of Mnk1 also reduced megakaryocyte ploidy and proplatelet forming megakaryocytes in vitro and resulted in thrombocytopenia. However, Mnk1 deletion did not affect the half-life of circulating platelets. Platelets from Mnk1 knockout mice exhibited reduced platelet aggregation, α granule secretion, and integrin αIIbß3 activation. Ribosomal footprint sequencing indicated that Mnk1 regulates the translation of Pla2g4a mRNA (which encodes cPLA2) in megakaryocytes. Consistent with this, Mnk1 ablation reduced cPLA2 activity and thromboxane generation in platelets and megakaryocytes. In vivo, Mnk1 ablation protected against platelet-dependent thromboembolism. These results provide previously unrecognized evidence that Mnk1 regulates mRNA translation and cellular activation in platelets and megakaryocytes, endomitosis and thrombopoiesis, and thrombosis.


Subject(s)
RNA, Messenger , Humans , Animals , Mice
6.
PLoS One ; 16(12): e0261633, 2021.
Article in English | MEDLINE | ID: mdl-34936674

ABSTRACT

We investigated the contributions of platelet microRNAs (miRNAs) to the rate of growth and regulation of gene expression in primary ectopic tumors using mouse models. We previously identified an inhibitory role for platelets in solid tumor growth, mediated by tumor infiltration of platelet microvesicles (microparticles) which are enriched in platelet-derived miRNAs. To investigate the specific roles of platelet miRNAs in tumor growth models, we implanted pancreatic ductal adenocarcinoma cells as a bolus into mice with megakaryocyte-/platelet-specific depletion of mature miRNAs. We observed an ~50% increase in the rate of growth of ectopic primary tumors in these mice compared to controls including at early stages, associated with reduced apoptosis in the tumors, in particular in tumor cells associated with platelet microvesicles-which were depleted of platelet-enriched miRNAs-demonstrating a specific role for platelet miRNAs in modulation of primary tumor growth. Differential expression RNA sequencing of tumor cells isolated from advanced primary tumors revealed a broad cohort of mRNAs modulated in the tumor cells as a function of host platelet miRNAs. Altered genes comprised 548 up-regulated transcripts and 43 down-regulated transcripts, mostly mRNAs altogether spanning a variety of growth signaling pathways-notably pathways related to epithelial-mesenchymal transition-in tumor cells from platelet miRNA-deleted mice compared with those from control mice. Tumors in platelet miRNA-depleted mice showed more sarcomatoid growth and more advanced tumor grade, indicating roles for host platelet miRNAs in tumor plasticity. We further validated increased protein expression of selected genes associated with increased cognate mRNAs in the tumors due to platelet miRNA depletion in the host animals, providing proof of principle of widespread effects of platelet miRNAs on tumor cell functional gene expression in primary tumors in vivo. Together, these data demonstrate that platelet-derived miRNAs modulate solid tumor growth in vivo by broad-spectrum restructuring of the tumor cell transcriptome.


Subject(s)
Blood Platelets/metabolism , MicroRNAs/genetics , Pancreatic Neoplasms/genetics , RNA, Messenger/genetics , Animals , Blood Platelets/pathology , Carcinogenesis/genetics , Carcinogenesis/pathology , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Mice , Mice, Inbred C57BL , Pancreatic Neoplasms/pathology , Transcriptome
7.
Blood ; 138(5): 401-416, 2021 08 05.
Article in English | MEDLINE | ID: mdl-33895821

ABSTRACT

Circulating platelets interact with leukocytes to modulate host immune and thrombotic responses. In sepsis, platelet-leukocyte interactions are increased and have been associated with adverse clinical events, including increased platelet-T-cell interactions. Sepsis is associated with reduced CD8+ T-cell numbers and functional responses, but whether platelets regulate CD8+ T-cell responses during sepsis remains unknown. In our current study, we systemically evaluated platelet antigen internalization and presentation through major histocompatibility complex class I (MHC-I) and their effects on antigen-specific CD8+ T cells in sepsis in vivo and ex vivo. We discovered that both human and murine platelets internalize and proteolyze exogenous antigens, generating peptides that are loaded onto MHC-I. The expression of platelet MHC-I, but not platelet MHC-II, is significantly increased in human and murine platelets during sepsis and in human megakaryocytes stimulated with agonists generated systemically during sepsis (eg, interferon-γ and lipopolysaccharide). Upregulation of platelet MHC-I during sepsis increases antigen cross-presentation and interactions with CD8+ T cells in an antigen-specific manner. Using a platelet lineage-specific MHC-I-deficient mouse strain (B2Mf/f-Pf4Cre), we demonstrate that platelet MHC-I regulates antigen-specific CD8+ T-cell proliferation in vitro, as well as the number and functional responses of CD8+ T cells in vivo, during sepsis. Loss of platelet MHC-I reduces sepsis-associated mortality in mice in an antigen-specific setting. These data identify a new mechanism by which platelets, through MHC-I, process and cross-present antigens, engage antigen-specific CD8+ T cells, and regulate CD8+ T-cell numbers, functional responses, and outcomes during sepsis.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Histocompatibility Antigens Class I/immunology , Immune Tolerance , Sepsis/immunology , Adult , Animals , Cell Proliferation , Female , Histocompatibility Antigens Class I/genetics , Humans , Male , Mice , Mice, Knockout , Prospective Studies , Sepsis/genetics
8.
J Neuroinflammation ; 18(1): 60, 2021 Feb 25.
Article in English | MEDLINE | ID: mdl-33632243

ABSTRACT

BACKGROUND: The term sepsis is used to designate a systemic condition of infection and inflammation associated with hemodynamic changes that result in organic dysfunction. Gestational sepsis can impair the development of the central nervous system and may promote permanent behavior alterations in the offspring. The aim of our work was to evaluate the effects of maternal sepsis on inflammatory cytokine levels and synaptic proteins in the hippocampus, neocortex, frontal cortex, and cerebellum of neonatal, young, and adult mice. Additionally, we analyzed the motor development, behavioral features, and cognitive impairments in neonatal, young and adult offspring. METHODS: Pregnant mice at the 14th embryonic day (E14) were intratracheally instilled with saline 0.9% solution (control group) or Klebsiella spp. (3 × 108 CFU) (sepsis group) and started on meropenem after 5 h. The offspring was sacrificed at postnatal day (P) 2, P8, P30, and P60 and samples of liver, lung, and brain were collected for TNF-α, IL-1ß, and IL-6 measurements by ELISA. Synaptophysin, PSD95, and ß-tubulin levels were analyzed by Western blot. Motor tests were performed at all analyzed ages and behavioral assessments were performed in offspring at P30 and P60. RESULTS: Gestational sepsis induces a systemic pro-inflammatory response in neonates at P2 and P8 characterized by an increase in cytokine levels. Maternal sepsis induced systemic downregulation of pro-inflammatory cytokines, while in the hippocampus, neocortex, frontal cortex, and cerebellum an inflammatory response was detected. These changes in the brain immunity were accompanied by a reduction of synaptophysin and PSD95 levels in the hippocampus, neocortex, frontal cortex, and cerebellum, in all ages. Behavioral tests demonstrated motor impairment in neonates, and depressive-like behavior, fear-conditioned memory, and learning impairments in animals at P30 and P60, while spatial memory abilities were affected only at P60, indicating that gestational sepsis not only induces an inflammatory response in neonatal mouse brains, but also affects neurodevelopment, and leads to a plethora of behavioral alterations and cognitive impairments in the offspring. CONCLUSION: These data suggest that maternal sepsis may be causatively related to the development of depression, learning, and memory impairments in the litter.


Subject(s)
Brain/immunology , Prenatal Exposure Delayed Effects/immunology , Sepsis/immunology , Animals , Behavior, Animal , Brain/metabolism , Cognitive Dysfunction/etiology , Female , Inflammation , Mice , Motor Activity/physiology , Pregnancy , Prenatal Exposure Delayed Effects/metabolism , Sepsis/complications , Synapses/metabolism
9.
J Thromb Haemost ; 19(5): 1319-1330, 2021 05.
Article in English | MEDLINE | ID: mdl-33587773

ABSTRACT

BACKGROUND: Heparanase (HPSE) is the only known mammalian enzyme that can degrade heparan sulfate. Heparan sulfate proteoglycans are essential components of the glycocalyx, and maintain physiological barriers between the blood and endothelial cells. HPSE increases during sepsis, which contributes to injurious glyocalyx degradation, loss of endothelial barrier function, and mortality. OBJECTIVES: As platelets are one of the most abundant cellular sources of HPSE, we sought to determine whether HPSE expression and activity increases in human platelets during clinical sepsis. We also examined associations between platelet HPSE expression and clinical outcomes. PATIENTS/METHODS: Expression and activity of HPSE was determined in platelets isolated from septic patients (n = 59) and, for comparison, sex-matched healthy donors (n = 46) using complementary transcriptomic, proteomic, and functional enzymatic assays. Septic patients were followed for the primary outcome of mortality, and clinical data were captured prospectively for septic patients. RESULTS: The mRNA expression of HPSE was significantly increased in platelets isolated from septic patients. Ribosomal footprint profiling, followed by [S35] methionine labeling assays, demonstrated that HPSE mRNA translation and HPSE protein synthesis were significantly upregulated in platelets during sepsis. While both the pro- and active forms of HPSE protein increased in platelets during sepsis, only the active form of HPSE protein significantly correlated with sepsis-associated mortality. Consistent with transcriptomic and proteomic upregulation, HPSE enzymatic activity was also increased in platelets during sepsis. CONCLUSIONS: During clinical sepsis HPSE, translation, and enzymatic activity are increased in platelets. Increased expression of the active form of HPSE protein is associated with sepsis-associated mortality.


Subject(s)
Blood Platelets/enzymology , Glucuronidase/metabolism , Sepsis , Endothelial Cells , Glucuronidase/genetics , Humans , Proteomics
10.
Thromb Res ; 198: 204-209, 2021 02.
Article in English | MEDLINE | ID: mdl-33360636

ABSTRACT

BACKGROUND: The ability to measure changes in platelet reactivity is important to identify novel aspects of platelet biology and develop targeted therapeutics to prevent bleeding or thrombosis. Current platelet function testing allows for single agonist analysis at a time. The ability to phenotype platelets in a single assay with multiple agonists and adhesion substrates could yield more insights into altered pathways than are feasible with current approaches. We hypothesized platelet electrical resistance (PER) could be used for more comprehensive phenotyping of platelets. METHODS: Platelets were isolated from male and female healthy donors (age 39.6 ± 6.9) and septic patients (age 44.0 ± 13.5). PER 96-well plates were coated with various substrates, including fibrinogen and collagen. Platelets were added to the coated plates in the presence or absence of thrombin or convulxin. Platelet activation and spreading was monitored by measuring changes in electrical impedance. RESULTS: Platelets adhesion to fibrinogen and collagen increased impedance. In addition, impedance increased in response to thrombin or convulxin. No changes in impedance were observed in the absence of platelets or when wells were uncoated, indicating changes in impedance were directly due to platelet adhesion and activation. Inhibiting integrin αIIbß3 decreased impedance when fibrinogen was used as a substrate, consistent with platelet-dependent effects. Platelets from septic patients caused increased impedance compared to healthy donors, demonstrating this assay can be used to assess platelet hyperreactivity. CONCLUSION: PER can be applied as a high throughput tool to measure platelet reactivity in health and disease, where platelet activation is increased.


Subject(s)
Blood Platelets , Platelet Activation , Adult , Electric Impedance , Female , Fibrinogen , Humans , Male , Middle Aged , Platelet Adhesiveness , Platelet Aggregation , Platelet Glycoprotein GPIIb-IIIa Complex
11.
J Leukoc Biol ; 109(5): 915-930, 2021 05.
Article in English | MEDLINE | ID: mdl-33070381

ABSTRACT

Alpha-1-acid glycoprotein (AGP-1) is a positive acute phase glycoprotein with uncertain functions. Serum AGP-1 (sAGP-1) is primarily derived from hepatocytes and circulates as 12-20 different glycoforms. We isolated a glycoform secreted from platelet-activating factor (PAF)-stimulated human neutrophils (nAGP-1). Its peptide sequence was identical to hepatocyte-derived sAGP-1, but nAGP-1 differed from sAGP-1 in its chromatographic behavior, electrophoretic mobility, and pattern of glycosylation. The function of these 2 glycoforms also differed. sAGP-1 activated neutrophil adhesion, migration, and neutrophil extracellular traps (NETosis) involving myeloperoxidase, peptidylarginine deiminase 4, and phosphorylation of ERK in a dose-dependent fashion, whereas nAGP-1 was ineffective as an agonist for these events. Furthermore, sAGP-1, but not nAGP-1, inhibited LPS-stimulated NETosis. Interestingly, nAGP-1 inhibited sAGP-1-stimulated neutrophil NETosis. The discordant effect of the differentially glycosylated AGP-1 glycoforms was also observed in platelets where neither of the AGP-1 glycoforms alone stimulated aggregation of washed human platelets, but sAGP-1, and not nAGP-1, inhibited aggregation induced by PAF or ADP, but not by thrombin. These functional effects of sAGP-1 correlated with intracellular cAMP accumulation and phosphorylation of the protein kinase A substrate vasodilator-stimulated phosphoprotein and reduction of Akt, ERK, and p38 phosphorylation. Thus, the sAGP-1 glycoform limits platelet reactivity, whereas nAGP-1 glycoform also limits proinflammatory actions of sAGP-1. These studies identify new functions for this acute phase glycoprotein and demonstrate that the glycosylation of AGP-1 controls its effects on 2 critical cells of acute inflammation.


Subject(s)
Blood Platelets/metabolism , Neutrophils/metabolism , Orosomucoid/metabolism , Adenosine Diphosphate/pharmacology , Biomarkers/metabolism , Blood Platelets/drug effects , Cyclic AMP/metabolism , Extracellular Traps/metabolism , Glycosylation/drug effects , Humans , Models, Biological , Neutrophil Activation/drug effects , Neutrophils/drug effects , Orosomucoid/agonists , Peptides/metabolism , Platelet Activating Factor/pharmacology , Platelet Aggregation/drug effects , Polysaccharides/metabolism , Protein Isoforms/metabolism
12.
Blood ; 136(15): 1760-1772, 2020 10 08.
Article in English | MEDLINE | ID: mdl-32844999

ABSTRACT

There is heritability to interindividual variation in platelet count, and better understanding of the regulating genetic factors may provide insights for thrombopoiesis. MicroRNAs (miRs) regulate gene expression in health and disease, and megakaryocytes (MKs) deficient in miRs have lower platelet counts, but information about the role of miRs in normal human MK and platelet production is limited. Using genome-wide miR profiling, we observed strong correlations among human bone marrow MKs, platelets, and differentiating cord blood-derived MK cultures, and identified MK miR-125a-5p as associated with human platelet number but not leukocyte or hemoglobin levels. Overexpression and knockdown studies showed that miR-125a-5p positively regulated human MK proplatelet (PP) formation in vitro. Inhibition of miR-125a-5p in vivo lowered murine platelet counts. Analyses of MK and platelet transcriptomes identified LCP1 as a miR-125a-5p target. LCP1 encodes the actin-bundling protein, L-plastin, not previously studied in MKs. We show that miR-125a-5p directly targets and reduces expression of MK L-plastin. Overexpression and knockdown studies show that L-plastin promotes MK progenitor migration, but negatively correlates with human platelet count and inhibits MK PP formation (PPF). This work provides the first evidence for the actin-bundling protein, L-plastin, as a regulator of human MK PPF via inhibition of the late-stage MK invagination system, podosome and PPF, and PP branching. We also provide resources of primary and differentiating MK transcriptomes and miRs associated with platelet counts. miR-125a-5p and L-plastin may be relevant targets for increasing in vitro platelet manufacturing and for managing quantitative platelet disorders.


Subject(s)
Blood Platelets/metabolism , Gene Expression Regulation, Developmental , Megakaryocytes/cytology , Megakaryocytes/metabolism , Membrane Glycoproteins/genetics , MicroRNAs/genetics , Microfilament Proteins/genetics , Thrombopoiesis/genetics , Actins/metabolism , Biomarkers , Gene Knockdown Techniques , Humans , Membrane Glycoproteins/metabolism , Microfilament Proteins/metabolism , RNA Interference
13.
Malar J ; 19(1): 234, 2020 Jul 01.
Article in English | MEDLINE | ID: mdl-32611348

ABSTRACT

BACKGROUND: Malaria-triggered lung injury can occur in both severe and non-severe cases. Platelets may interact with parasitized erythrocytes, leukocytes and endothelium. These interactions can lead to microvessel obstructions and induce release of inflammatory mediators. Induction of the haem oxygenase enzyme is important in the host's response to free haem and to several other molecules generated by infectious or non-infectious diseases. In addition, an important role for the haem oxygenase-1 isotype has been demonstrated in experimental cerebral malaria and in clinical cases. Therefore, the present work aims to determine the influence of haem oxygenase in thrombocytopaenia and acute pulmonary injury during infection with Plasmodium berghei strain NK65. METHODS: C57BL/6 mice were infected with P. berghei and analysed 7-10 days post-infection. For each experiment, Cobalt Protoporphyrin IX/CoPPIX or saline were administered. Bronchoalveolar lavage fluid was used for total and differential leukocyte count and for protein measurement. Lungs were used for histological analyses or for analysis of cytokines and western blotting. The lung permeability was analysed by Evans blue dye concentration. Platelet-leukocyte aggregate formation was assayed using the flow cytometer. RESULTS: Plasmodium berghei NK65 infection generated an intense lung injury, with increased levels of inflammatory mediators, oedema, and cell migration into the lung. Plasmodium berghei infection was also accompanied by marked thrombocytopaenia and formation of platelet-leukocyte aggregates in peripheral blood. Treatment with the HO-1 inducer cobalt protoporphyrin IX (CoPPIX) modified the inflammatory response but did not affect the evolution of parasitaemia. Animals treated with CoPPIX showed an improvement in lung injury, with decreased inflammatory infiltrate in the lung parenchyma, oedema and reduced thrombocytopaenia. CONCLUSION: Data here presented suggest that treatment with CoPPIX inducer leads to less severe pulmonary lung injury and thrombocytopaenia during malaria infection, thus increasing animal survival.


Subject(s)
Acute Lung Injury/drug therapy , Heme Oxygenase-1/pharmacology , Malaria/complications , Membrane Proteins/pharmacology , Protective Agents/pharmacology , Thrombocytopenia/drug therapy , Acute Lung Injury/etiology , Animals , Bronchoalveolar Lavage Fluid/chemistry , Female , Leukocyte Count , Lung/pathology , Male , Mice , Mice, Inbred C57BL , Plasmodium berghei/physiology , Thrombocytopenia/etiology
14.
Blood ; 136(10): 1169-1179, 2020 09 03.
Article in English | MEDLINE | ID: mdl-32597954

ABSTRACT

COVID-19 affects millions of patients worldwide, with clinical presentation ranging from isolated thrombosis to acute respiratory distress syndrome (ARDS) requiring ventilator support. Neutrophil extracellular traps (NETs) originate from decondensed chromatin released to immobilize pathogens, and they can trigger immunothrombosis. We studied the connection between NETs and COVID-19 severity and progression. We conducted a prospective cohort study of COVID-19 patients (n = 33) and age- and sex-matched controls (n = 17). We measured plasma myeloperoxidase (MPO)-DNA complexes (NETs), platelet factor 4, RANTES, and selected cytokines. Three COVID-19 lung autopsies were examined for NETs and platelet involvement. We assessed NET formation ex vivo in COVID-19 neutrophils and in healthy neutrophils incubated with COVID-19 plasma. We also tested the ability of neonatal NET-inhibitory factor (nNIF) to block NET formation induced by COVID-19 plasma. Plasma MPO-DNA complexes increased in COVID-19, with intubation (P < .0001) and death (P < .0005) as outcome. Illness severity correlated directly with plasma MPO-DNA complexes (P = .0360), whereas Pao2/fraction of inspired oxygen correlated inversely (P = .0340). Soluble and cellular factors triggering NETs were significantly increased in COVID-19, and pulmonary autopsies confirmed NET-containing microthrombi with neutrophil-platelet infiltration. Finally, COVID-19 neutrophils ex vivo displayed excessive NETs at baseline, and COVID-19 plasma triggered NET formation, which was blocked by nNIF. Thus, NETs triggering immunothrombosis may, in part, explain the prothrombotic clinical presentations in COVID-19, and NETs may represent targets for therapeutic intervention.


Subject(s)
Coronavirus Infections/complications , Extracellular Traps/immunology , Neutrophils/immunology , Pneumonia, Viral/complications , Thrombosis/complications , Adult , Aged , Betacoronavirus/immunology , Blood Platelets/immunology , Blood Platelets/pathology , Blood Proteins/immunology , COVID-19 , Coronavirus Infections/immunology , Coronavirus Infections/pathology , Female , Humans , Male , Middle Aged , Neutrophil Infiltration , Neutrophils/pathology , Pandemics , Peroxidase/immunology , Pneumonia, Viral/immunology , Pneumonia, Viral/pathology , Prospective Studies , SARS-CoV-2 , Thrombosis/immunology , Thrombosis/pathology
15.
Blood ; 136(11): 1317-1329, 2020 09 10.
Article in English | MEDLINE | ID: mdl-32573711

ABSTRACT

There is an urgent need to understand the pathogenesis of coronavirus disease 2019 (COVID-19). In particular, thrombotic complications in patients with COVID-19 are common and contribute to organ failure and mortality. Patients with severe COVID-19 present with hemostatic abnormalities that mimic disseminated intravascular coagulopathy associated with sepsis, with the major difference being increased risk of thrombosis rather than bleeding. However, whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection alters platelet function to contribute to the pathophysiology of COVID-19 remains unknown. In this study, we report altered platelet gene expression and functional responses in patients infected with SARS-CoV-2. RNA sequencing demonstrated distinct changes in the gene-expression profile of circulating platelets of COVID-19 patients. Pathway analysis revealed differential gene-expression changes in pathways associated with protein ubiquitination, antigen presentation, and mitochondrial dysfunction. The receptor for SARS-CoV-2 binding, angiotensin-converting enzyme 2 (ACE2), was not detected by messenger RNA (mRNA) or protein in platelets. Surprisingly, mRNA from the SARS-CoV-2 N1 gene was detected in platelets from 2 of 25 COVID-19 patients, suggesting that platelets may take-up SARS-COV-2 mRNA independent of ACE2. Resting platelets from COVID-19 patients had increased P-selectin expression basally and upon activation. Circulating platelet-neutrophil, -monocyte, and -T-cell aggregates were all significantly elevated in COVID-19 patients compared with healthy donors. Furthermore, platelets from COVID-19 patients aggregated faster and showed increased spreading on both fibrinogen and collagen. The increase in platelet activation and aggregation could partially be attributed to increased MAPK pathway activation and thromboxane generation. These findings demonstrate that SARS-CoV-2 infection is associated with platelet hyperreactivity, which may contribute to COVID-19 pathophysiology.


Subject(s)
Betacoronavirus/isolation & purification , Blood Coagulation Disorders/pathology , Blood Platelets/pathology , Coronavirus Infections/complications , Pneumonia, Viral/complications , Transcriptome , Biomarkers , Blood Coagulation Disorders/genetics , Blood Coagulation Disorders/metabolism , Blood Coagulation Disorders/virology , Blood Platelets/metabolism , Blood Platelets/virology , COVID-19 , Case-Control Studies , Coronavirus Infections/genetics , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Female , Follow-Up Studies , Gene Expression Profiling , Humans , Male , Middle Aged , Pandemics , Pneumonia, Viral/genetics , Pneumonia, Viral/metabolism , Pneumonia, Viral/virology , Prognosis , Prospective Studies , SARS-CoV-2
16.
J Thromb Haemost ; 18(5): 1183-1196, 2020 05.
Article in English | MEDLINE | ID: mdl-31997536

ABSTRACT

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


Subject(s)
Blood Platelets , Megakaryocytes , Animals , Clot Retraction , Mice , Signal Transduction , Thrombopoiesis
17.
Circ Res ; 126(4): 501-516, 2020 02 14.
Article in English | MEDLINE | ID: mdl-31852401

ABSTRACT

RATIONALE: Longitudinal studies are required to distinguish within versus between-individual variation and repeatability of gene expression. They are uniquely positioned to decipher genetic signal from environmental noise, with potential application to gene variant and expression studies. However, longitudinal analyses of gene expression in healthy individuals-especially with regards to alternative splicing-are lacking for most primary cell types, including platelets. OBJECTIVE: To assess repeatability of gene expression and splicing in platelets and use repeatability to identify novel platelet expression quantitative trait loci (QTLs) and splice QTLs. METHODS AND RESULTS: We sequenced the transcriptome of platelets isolated repeatedly up to 4 years from healthy individuals. We examined within and between individual variation and repeatability of platelet RNA expression and exon skipping, a readily measured alternative splicing event. We find that platelet gene expression is generally stable between and within-individuals over time-with the exception of a subset of genes enriched for the inflammation gene ontology. We show an enrichment among repeatable genes for associations with heritable traits, including known and novel platelet expression QTLs. Several exon skipping events were also highly repeatable, suggesting heritable patterns of splicing in platelets. One of the most repeatable was exon 14 skipping of SELP. Accordingly, we identify rs6128 as a platelet splice QTL and define an rs6128-dependent association between SELP exon 14 skipping and race. In vitro experiments demonstrate that this single nucleotide variant directly affects exon 14 skipping and changes the ratio of transmembrane versus soluble P-selectin protein production. CONCLUSIONS: We conclude that the platelet transcriptome is generally stable over 4 years. We demonstrate the use of repeatability of gene expression and splicing to identify novel platelet expression QTLs and splice QTLs. rs6128 is a platelet splice QTL that alters SELP exon 14 skipping and soluble versus transmembrane P-selectin protein production.


Subject(s)
Alternative Splicing , Blood Platelets/metabolism , P-Selectin/genetics , Quantitative Trait Loci/genetics , RNA-Seq/methods , Transcriptome/genetics , Exons/genetics , Gene Ontology , Humans , Polymorphism, Single Nucleotide
18.
PLoS One ; 14(12): e0224610, 2019.
Article in English | MEDLINE | ID: mdl-31869339

ABSTRACT

Malaria is an infectious disease of major worldwide clinical importance that causes a variety of severe, or complicated, syndromes including cerebral malaria, which is often fatal. Leukocyte integrins are essential for host defense but also mediate physiologic responses of the innate and adaptive immune systems. We previously showed that targeted deletion of the αD subunit (αD-/-) of the αDß2 integrin, which is expressed on key leukocyte subsets in mice and humans, leads to absent expression of the integrin heterodimer on murine macrophages and reduces mortality in mice infected with Plasmodium berghei ANKA (P. berghei ANKA). To further identify mechanisms involved in the protective effect of αD deletion in this model of severe malaria we examined wild type C57BL/6 (WT) and αD-/- mice after P. berghei ANKA infection and found that vessel plugging and leukocyte infiltration were significantly decreased in the brains of αD-/- animals. Intravital microscopy demonstrated decreased rolling and adhesion of leukocytes in cerebral vessels of αD-/- mice. Flow cytometry analysis showed decreased T-lymphocyte accumulation in the brains of infected αD-/- animals. Evans blue dye exclusion assays demonstrated significantly less dye extravasation in the brains of αD-/- mice, indicating preserved blood-brain barrier integrity. WT mice that were salvaged from P. berghei ANKA infection by treatment with chloroquine had impaired aversive memory, which was not observed in αD-/- mice. We conclude that deletion of integrin αDß2 alters the natural course of experimental severe malaria, demonstrating previously unrecognized activities of a key leukocyte integrin in immune-inflammatory responses that mediate cerebral involvement.


Subject(s)
CD11 Antigens/metabolism , Integrin alpha Chains/metabolism , Malaria/physiopathology , Animals , Blood-Brain Barrier/metabolism , Brain/metabolism , Brain Edema/metabolism , Brain Edema/physiopathology , CD11 Antigens/physiology , Chloroquine/metabolism , Disease Models, Animal , Inflammation/metabolism , Integrin alpha Chains/physiology , Integrins/immunology , Integrins/metabolism , Leukocyte Count , Leukocytes/metabolism , Leukocytes/physiology , Macrophages/metabolism , Malaria/genetics , Malaria, Cerebral/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Plasmodium berghei/metabolism
19.
Blood ; 134(12): 911-923, 2019 09 19.
Article in English | MEDLINE | ID: mdl-31366617

ABSTRACT

There is increasing recognition that platelets have a functional role in the pathophysiology of sepsis, though this role has not been precisely defined. Whether sepsis alters the human platelet transcriptome and translational landscape has never been established. We used parallel techniques of RNA sequencing and ribosome footprint profiling to interrogate the platelet transcriptome and translatome in septic patients and healthy donors. We identified 1806 significantly differentially expressed (false discovery rate <0.05) transcripts in platelets from septic patients. Platelet translational events during sepsis were also upregulated. To explore the relevance of a murine model of sepsis, cecal ligation and puncture (CLP), we compared sepsis-induced changes in platelet gene expression between septic patients and mice subjected to CLP. Platelet transcriptional (ρ = 0.42, P = 3.2 × 10-285) and translational (ρ = 0.65, P = 1.09 × 10-56) changes were significantly correlated between septic patients and mice. We focused on ITGA2B, tracking and validating the expression, regulation, and functional impact of changes in ITGA2B during sepsis. Increased ITGA2B was identified in bone marrow megakaryocytes within 24 hours of sepsis onset. Subsequent increases in ITGA2B were seen in circulating platelets, suggesting dynamic trafficking of the messenger RNA. Transcriptional changes in ITGA2B were accompanied by de novo protein synthesis of αIIb and integrin αIIbß3 activation. Increased αIIb was associated with mortality in humans and mice. These findings provide previously unrecognized evidence that human and murine sepsis similarly alters the platelet transcriptional and translational landscape. Moreover, ITGA2B is upregulated and functional in sepsis due to trafficking from megakaryocytes and de novo synthesis in platelets and is associated with increased mortality.


Subject(s)
Blood Platelets/metabolism , Sepsis/genetics , Sepsis/metabolism , Animals , Blood Platelets/pathology , Blood Proteins/analysis , Blood Proteins/genetics , Blood Proteins/metabolism , Case-Control Studies , Female , Gene Expression Profiling , Humans , Male , Mice , Protein Biosynthesis , Proteome/analysis , Proteomics , Sepsis/blood , Sepsis/pathology , Severity of Illness Index , Transcription, Genetic , Transcriptome
20.
Elife ; 82019 05 01.
Article in English | MEDLINE | ID: mdl-31042146

ABSTRACT

Bone marrow megakaryocytes engulf neutrophils in a phenomenon termed emperipolesis. We show here that emperipolesis is a dynamic process mediated actively by both lineages, in part through the ß2-integrin/ICAM-1/ezrin pathway. Tethered neutrophils enter in membrane-bound vesicles before penetrating into the megakaryocyte cytoplasm. Intracytoplasmic neutrophils develop membrane contiguity with the demarcation membrane system, thereby transferring membrane to the megakaryocyte and to daughter platelets. This phenomenon occurs in otherwise unmanipulated murine marrow in vivo, resulting in circulating platelets that bear membrane from non-megakaryocytic hematopoietic donors. Transit through megakaryocytes can be completed as rapidly as minutes, after which neutrophils egress intact. Emperipolesis is amplified in models of murine inflammation associated with platelet overproduction, contributing to platelet production in vitro and in vivo. These findings identify emperipolesis as a new cell-in-cell interaction that enables neutrophils and potentially other cells passing through the megakaryocyte cytoplasm to modulate the production and membrane content of platelets.


Subject(s)
Blood Platelets/metabolism , Emperipolesis/genetics , Inflammation/genetics , Megakaryocytes/metabolism , Animals , Bone Marrow Cells/metabolism , CD18 Antigens/genetics , Cell Communication , Cytoplasm/genetics , Cytoplasm/metabolism , Cytoskeletal Proteins/genetics , Humans , Inflammation/blood , Inflammation/pathology , Intercellular Adhesion Molecule-1/genetics , Membrane Transport Proteins/genetics , Mice , Neutrophils/metabolism
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