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COVID-19 , Humanos , Activación Plaquetaria , SARS-CoV-2 , Sobrevivientes , Plaquetas/fisiologíaRESUMEN
INTRODUCTION: The Ad26.COV2·S (Janssen/Johnson & Johnson) COVID-19 vaccine, has been rarely associated with vaccine-induced immune thrombocytopenia and thrombosis (VITT). We investigated the prevalence of anti-PF4 antibody positivity, thrombocytopenia, D-dimer elevation, plasmatic thromboinflammatory markers, and platelet functional assays following Ad26.COV2·S vaccination in Rio de Janeiro, Brazil. METHODS: From July to September 2021, participants were assessed prior, 1, and 3 weeks post-vaccination. Platelet count and D-dimer were measured at each visit and anti-PF4 at week 3. A positive anti-PF4 prompted retrospective testing of the sample from week 0. Individuals with new thrombocytopenia or elevated D-dimer, positive anti-PF4, and 38 matched controls without laboratory abnormalities were evaluated for plasmatic p-selectin, tissue factor, and functional platelet activation assays. RESULTS: 630 individuals were included; 306 (48.57%) females, median age 28 years. Forty-two (6.67%) presented ≥1 laboratory abnormality in week 1 or 3. Five (0.79%) had thrombocytopenia, 31 (4.91%) elevated D-dimer, and 9 (1.57%) had positive anti-PF4 at week 3. Individuals with laboratory abnormalities and controls showed a slight increase in plasmatic p-selectin and tissue factor. Ten individuals with laboratory abnormalities yielded increased surface expression of p-selectin, and their ability to activate platelets in a FcγRIIa dependent manner was further evaluated. Two were partially inhibited by high concentrations of heparin and blockage of FcγRII with IV.3 antibody. Plasma obtained before vaccination produced similar results, suggesting a lack of association with vaccination. CONCLUSIONS: Vaccination with Ad26.COV2·S vaccine led to a very low frequency of low-titer positive anti-PF4 antibodies, elevation of D-dimer, and mild thrombocytopenia, with no associated clinically relevant increase in thromboinflammatory markers and platelet activation.
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BACKGROUND: Blood plasma is the main source of extracellular vesicles (EVs) in clinical studies aiming to identify biomarkers and to investigate pathophysiological processes, especially regarding EV roles in inflammation and thrombosis. However, EV isolation from plasma has faced the fundamental issue of lipoprotein contamination, representing an important bias since lipoproteins are highly abundant and modulate cell signaling, metabolism, and thromboinflammation. OBJECTIVES: Here, we aimed to isolate plasma EVs after depleting lipoproteins, thereby improving sample purity and EV thromboinflammatory analysis. METHODS: Density-based gradient ultracentrifugation (G-UC) was used for lipoprotein depletion before EV isolation from plasma through size-exclusion chromatography (SEC) or serial centrifugation (SC). Recovered EVs were analyzed by size, concentration, cellular source, ultrastructure, and bottom-up proteomics. RESULTS: G-UC efficiently separated lipoproteins from the plasma, allowing subsequent EV isolation through SEC or SC. Combined analysis from EV proteomics, cholesterol quantification, and apoB-100 detection confirmed the significant reduction in lipoproteins from isolated EVs. Proteomic analysis identified similar gene ontology and cellular components in EVs, regardless of lipoprotein depletion, which was consistent with similar EV cellular sources, size, and ultrastructure by flow cytometry and transmission electron microscopy. Importantly, lipoprotein depletion increased the detection of less abundant proteins in EV proteome and enhanced thromboinflammatory responses of platelets and monocytes stimulated in vitro with EV isolates. CONCLUSION: Combination of G-UC+SEC significantly reduced EV lipoprotein contamination without interfering in EV cellular source, gene ontology, and ultrastructure, allowing the recovery of highly pure EVs with potential implications for functional assays and proteomic and lipidomic analyses.
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Cromatografía en Gel , Vesículas Extracelulares , Lipoproteínas , Proteómica , Humanos , Vesículas Extracelulares/metabolismo , Proteómica/métodos , Lipoproteínas/sangre , Plaquetas/metabolismo , Centrifugación por Gradiente de Densidad , Inflamación/sangre , Proteoma , Monocitos/metabolismoRESUMEN
Concerns for the long-term effects of COVID-19 infection have grown due to frequently reported persisting symptoms that can affect multiple systems for longer than 4 weeks after initial infection, a condition known as long-COVID-19 or post-acute COVID-19 syndrome (PACS). Even nonhospitalized survivors have an elevated risk for the development of thromboinflammatory-associated events, such as ischemic stroke and heart failure, pulmonary embolism and deep vein thrombosis. Recent findings point to the persistence of many mechanisms of hypercoagulability identified to be associated with disease severity and mortality in the acute phase of the disease, such as sustained inflammation and endotheliopathy, accompanied by abnormal fibrin generation and impaired fibrinolysis. Platelets seem to be central to the sustained hypercoagulable state, displaying hyperreactivity to stimuli and increased adhesive capacity. Platelets also contribute to elevated levels of thromboinflammatory mediators and pro-coagulant extracellular vesicles in individuals with ongoing PACS. Despite new advances in the understanding of mechanisms sustaining thromboinflammation in PACS, little is known about what triggers this persistence. In this graphical review, we provide a schematic representation of the known mechanisms and consequences of persisting thromboinflammation in COVID-19 survivors and summarize the hypothesized triggers maintaining this prothrombotic state.
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Neutrophil extracellular traps (NETs) have been implicated in several hallmarks of cancer. Among the protumor effects, NETs promote epithelial-mesenchymal transition (EMT) in different cancer models. EMT has been linked to an enhanced expression of the clotting-initiating protein, tissue factor (TF), thus favoring the metastatic potential. TF may also exert protumor effects by facilitating the activation of protease-activated receptor 2 (PAR2). Herein, we evaluated whether NETs could induce TF expression in breast cancer cells and further promote procoagulant and intracellular signaling effects via the TF/PAR2 axis. T-47D and MCF7 cell lines were treated with isolated NETs, and samples were obtained for real-time PCR, flow cytometry, Western blotting, and plasma coagulation assays. In silico analyses were performed employing RNA-seq data from breast cancer patients deposited in The Cancer Genome Atlas (TCGA) database. A positive correlation was observed between neutrophil/NETs gene signatures and TF gene expression. Neutrophils/NETs gene signatures and PAR2 gene expression also showed a significant positive correlation in the bioinformatics model. In vitro analysis showed that treatment with NETs upregulated TF gene and protein expression in breast cancer cell lines. The inhibition of ERK/JNK reduced the TF gene expression induced by NETs. Remarkably, the pharmacological or genetic inhibition of the TF/PAR2 signaling axis attenuated the NETs-induced expression of several protumor genes. Also, treatment of NETs with a neutrophil elastase inhibitor reduced the expression of metastasis-related genes. Our results suggest that the TF/PAR2 signaling axis contributes to the pro-cancer effects of NETs in human breast cancer cells.
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The emergence of the rare syndrome called vaccine-induced immune thrombocytopenia and thrombosis (VITT) after adenoviral vector vaccines, including ChAdOx1 nCov-19, raises concern about one's predisposing risk factors. Here we report the case of a 56-year-old white man who developed VITT leading to death within 9 days of symptom onset. He presented with superior sagittal sinus thrombosis, right frontal intraparenchymal hematoma, frontoparietal subarachnoid and massive ventricular hemorrhage, and right lower extremity arterial and venous thrombosis. His laboratory results showed elevated D-dimer, C-reactive protein, tissue factor, P-selectin (CD62p), and positive anti-platelet factor 4. The patient's plasma promoted higher CD62p expression in healthy donors' platelets than the controls. Genetic investigation on coagulation, thrombophilia, inflammation, and type I interferon-related genes was performed. From rare variants in European or African genomic databases, 68 single-nucleotide polymorphisms (SNPs) in one allele and 11 in two alleles from common SNPs were found in the patient genome. This report highlights the possible relationship between VITT and genetic variants. Additional investigations regarding the genetic predisposition of VITT are needed.
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Reports of side effects of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are increasing worldwide. Capillary leak syndrome and vaccine-induced immune thrombotic thrombocytopenia are very rare but life-threatening adverse events that should be identified early and treated. However, isolated thrombocytopenia can indicate pseudothrombocytopenia. In certain people, ethylenediaminetetraacetic acid (EDTA) induces an in vitro platelet aggregation, resulting in misleading underestimation of platelet counts. It is essential to recognize pseudothrombocytopenia to prevent diagnostic errors, overtreatment, anxiety, and unnecessary invasive procedures. We present a case who developed generalized edema and persistent pseudothrombocytopenia after the first dose of the ChAdOx1 nCoV-19 vaccine (AstraZeneca).
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COVID-19 , Trombocitopenia , COVID-19/prevención & control , Vacunas contra la COVID-19/efectos adversos , ChAdOx1 nCoV-19 , Edema , Humanos , SARS-CoV-2 , Trombocitopenia/inducido químicamente , Trombocitopenia/diagnóstico , Vacunación/efectos adversosRESUMEN
PURPOSE: We quantified lung glycolytic metabolic activity, clinical symptoms and inflammation, coagulation, and endothelial activation biomarkers in 2019 coronavirus disease (COVID-19) pneumonia survivors. METHODS: Adults previously hospitalized with moderate to severe COVID-19 pneumonia were prospectively included. Subjects filled out a questionnaire on clinical consequences, underwent chest CT and 18 F-FDG PET/CT, and provided blood samples on the same day. Forty-five volunteers served as control subjects. Analysis of CT images and quantitative voxel-based analysis of PET/CT images were performed for both groups. 18 F-FDG uptake in the whole-lung volume and in high- and low-attenuation areas was calculated and normalized to liver values. Quantification of plasma markers of inflammation (interleukin 6), d -dimer, and endothelial cell activation (angiopoietins 1 and 2, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1) was also performed. RESULTS: We enrolled 53 COVID-19 survivors (62.3% were male; median age, 50 years). All survivors reported at least 1 persistent symptom, and 41.5% reported more than 6 symptoms. The mean lung density was greater in survivors than in control subjects, and more metabolic activity was observed in normal and dense lung areas, even months after symptom onset. Plasma proinflammatory, coagulation, and endothelial activation biomarker concentrations were also significantly higher in survivors. CONCLUSION: We observed more metabolic activity in areas of high and normal lung attenuation several months after moderate to severe COVID-19 pneumonia. In addition, plasma markers of thromboinflammation and endothelial activation persisted. These findings may have implications for our understanding of the in vivo pathogenesis and long-lasting effects of COVID-19 pneumonia.
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COVID-19 , Neumonía , Trombosis , Adulto , Masculino , Humanos , Persona de Mediana Edad , Femenino , Fluorodesoxiglucosa F18 , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , COVID-19/diagnóstico por imagen , Inflamación/diagnóstico por imagen , Pulmón/diagnóstico por imagen , Biomarcadores , SobrevivientesRESUMEN
Background: Extracellular vesicles (EVs) are a valuable source of biomarkers and display the pathophysiological status of various diseases. In COVID-19, EVs have been explored in several studies for their ability to reflect molecular changes caused by SARS-CoV-2. Here we provide insights into the roles of EVs in pathological processes associated with the progression and severity of COVID-19. Methods: In this study, we used a label-free shotgun proteomic approach to identify and quantify alterations in EV protein abundance in severe COVID-19 patients. We isolated plasma extracellular vesicles from healthy donors and patients with severe COVID-19 by size exclusion chromatography (SEC). Then, flow cytometry was performed to assess the origin of EVs and to investigate the presence of circulating procoagulant EVs in COVID-19 patients. A total protein extraction was performed, and samples were analyzed by nLC-MS/MS in a Q-Exactive HF-X. Finally, computational analysis was applied to signify biological processes related to disease pathogenesis. Results: We report significant changes in the proteome of EVs from patients with severe COVID-19. Flow cytometry experiments indicated an increase in total circulating EVs and with tissue factor (TF) dependent procoagulant activity. Differentially expressed proteins in the disease groups were associated with complement and coagulation cascades, platelet degranulation, and acute inflammatory response. Conclusions: The proteomic data reinforce the changes in the proteome of extracellular vesicles from patients infected with SARS-CoV-2 and suggest a role for EVs in severe COVID-19.
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COVID-19 , Vesículas Extracelulares , Vesículas Extracelulares/metabolismo , Humanos , Proteoma/metabolismo , Proteómica/métodos , SARS-CoV-2 , Espectrometría de Masas en TándemRESUMEN
Accumulating evidence into the pathogenesis of COVID-19 highlights a hypercoagulability state with high risk of life-threatening thromboembolic complications. However, the mechanisms of hypercoagulability and their link to hyperinflammation remain poorly understood. Here, we investigate functions and mechanisms of platelet activation and platelet-monocyte interactions in inflammatory amplification during SARS-CoV-2 infection. We used a combination of immunophenotyping, single-cell analysis, functional assays, and pharmacological approaches to gain insights on mechanisms. Critically ill patients with COVID-19 exhibited increased platelet-monocyte aggregates formation. We identified a subset of inflammatory monocytes presenting high CD16 and low HLA-DR expression as the subset mainly interacting with platelets during severe COVID-19. Single-cell RNA-sequencing analysis indicated enhanced fibrinogen receptor Mac-1 in monocytes from patients with severe COVID-19. Monocytes from patients with severe COVID-19 displayed increased platelet binding and hyperresponsiveness to P-selectin and fibrinogen with respect to tumor necrosis factor-α and interleukin-1ß secretion. Platelets were able to orchestrate monocyte responses driving tissue factor (TF) expression, inflammatory activation, and inflammatory cytokines secretion in SARS-CoV-2 infection. Platelet-monocyte interactions ex vivo and in SARS-CoV-2 infection model in vitro reciprocally activated monocytes and platelets, inducing the heightened secretion of a wide panel of inflammatory mediators. We identified platelet adhesion as a primary signaling mechanism inducing mediator secretion and TF expression, whereas TF signaling played major roles in amplifying inflammation by inducing proinflammatory cytokines, especially tumor necrosis factor-α and interleukin-1ß. Our data identify platelet-induced TF expression and activity at the crossroad of coagulation and inflammation in severe COVID-19.
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COVID-19 , Trombofilia , Trombosis , Plaquetas/metabolismo , Citocinas/metabolismo , Humanos , Inflamación/patología , Interleucina-1beta/metabolismo , Monocitos/metabolismo , SARS-CoV-2 , Tromboinflamación , Tromboplastina/metabolismo , Trombosis/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Critically ill 2019 coronavirus disease (COVID-19) patients under invasive mechanical ventilation (IMV) are 10 to 40 times more likely to die than the general population. Although progression from mild to severe COVID-19 has been associated with hypoxia, uncontrolled inflammation, and coagulopathy, the mechanisms involved in the progression to severity are poorly understood. METHODS: The virome of tracheal aspirates (TA) from 25 COVID-19 patients under IMV was assessed through unbiased RNA sequencing (RNA-seq), and correlation analyses were conducted using available clinical data. Unbiased sequences from nasopharyngeal swabs (NS) from mild cases and TA from non-COVID patients were included in our study for further comparisons. RESULTS: We found higher levels and differential expression of human endogenous retrovirus K (HERV-K) genes in TA from critically ill and deceased patients when comparing nasopharyngeal swabs from mild cases to TA from non-COVID patients. In critically ill patients, higher HERV-K levels were associated with early mortality (within 14 days of diagnosis) in the intensive care unit. Increased HERV-K expression in deceased patients was associated with IL-17-related inflammation, monocyte activation, and an increased consumption of clotting/fibrinolysis factors. Moreover, increased HERV-K expression was detected in human primary monocytes from healthy donors after experimental SARS-CoV-2 infection in vitro. CONCLUSION: Our data implicate the levels of HERV-K transcripts in the physiopathology of COVID-19 in the respiratory tract of patients under invasive mechanical ventilation. Video abstract.