Acute to post-acute COVID-19 thromboinflammation persistence: Mechanisms and potential consequences.

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.

Human endogenous retrovirus K in the respiratory tract is associated with COVID-19 physiopathology.

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.

Proteomic Profile of Procoagulant Extracellular Vesicles Reflects Complement System Activation and Platelet Hyperreactivity of Patients with Severe COVID-19.

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.

Platelet proteome reveals features of cell death, antiviral response and viral replication in covid-19.

Coronavirus disease 2019 (COVID-19) has affected over 400 million people worldwide, leading to 6 million deaths. Among the complex symptomatology of COVID-19, hypercoagulation and thrombosis have been described to directly contribute to lethality, pointing out platelets as an important SARS-CoV-2 target. In this work, we explored the platelet proteome of COVID-19 patients through a label-free shotgun proteomics approach to identify platelet responses to infection, as well as validation experiments in a larger patient cohort. Exclusively detected proteins (EPs) and differentially expressed proteins (DEPs) were identified in the proteomic dataset and thus classified into biological processes to map pathways correlated with pathogenesis. Significant changes in the expression of proteins related to platelet activation, cell death, and antiviral response through interferon type-I were found in all patients. Since the outcome of COVID-19 varies highly among individuals, we also performed a cross-comparison of proteins found in survivors and nonsurvivors. Proteins belonging to the translation pathway were strongly highlighted in the nonsurvivor group. Moreover, the SARS-CoV-2 genome was fully sequenced in platelets from five patients, indicating viral internalization and preprocessing, with CD147 as a potential entry route. In summary, platelets play a significant role in COVID-19 pathogenesis via platelet activation, antiviral response, and disease severity.

Platelet-leukocyte interactions in COVID-19: Contributions to hypercoagulability, inflammation, and disease severity.

A State of the Art lecture titled "Platelet-leukocyte interactions in COVID-19: Contributions to hypercoagulability, inflammation and disease severity" was presented at the International Society for Thrombosis and Hemostasis (ISTH) congress in 2021. Severe coronavirus disease 2019 (COVID-19) has been associated with a high incidence of coagulopathy and thromboembolic events that contributes to disease severity and poor outcomes. Therefore, understanding the mechanisms of COVID-19-associated hypercoagulability and thromboinflammation has gained great interest. Here, we review the mechanisms involved in platelet activation and platelet interactions with leukocytes during COVID-19. We highlight recent evidence that platelet activation, platelet-monocyte, and platelet-neutrophil interactions in COVID-19 support pathological thromboinflammation, including in driving tissue factor expression and NETosis, which have been associated with thromboembolic complication and poor outcomes in critically ill patients. The contributions of platelet-leukocyte interactions to COVID-19 immunoregulation, inflammation, and hypercoagulability, as well as their potential implications in disease severity and therapeutic strategies, will be discussed. Finally, we summarize relevant new data on this topic presented during the 2021 ISTH Congress.

Platelet-monocyte interaction amplifies thromboinflammation through tissue factor signaling in COVID-19.

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.

Platelet-leukocyte interactions in the pathogenesis of viral infections.

Evolving evidence demonstrates that platelets have major roles in viral syndromes through previously unrecognized viral sensing and effector functions. Activated platelets and increased platelet-leukocyte aggregates are observed in clinical and experimental viral infections. The mechanisms and outcomes of platelet-leukocyte interactions depend on the interacting leukocyte as well as on the pathogen and pathological conditions. In this review, we discuss the mechanisms involved in platelet interactions with leukocytes and its functions during viral infections. We focus on the contributions of human platelet-leukocyte interactions to pathophysiological and protective responses during viral infections of major global health relevance, including acquired immunodeficiency syndrome (AIDS), dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), influenza pneumonia, and COVID-19.

VIP plasma levels associate with survival in severe COVID-19 patients, correlating with protective effects in SARS-CoV-2-infected cells.

Infection by SARS-CoV-2 may elicit uncontrolled and damaging inflammatory responses. Thus, it is critical to identify compounds able to inhibit virus replication and thwart the inflammatory reaction. Here, we show that the plasma levels of the immunoregulatory neuropeptide VIP are elevated in patients with severe COVID-19, correlating with reduced inflammatory mediators and with survival on those patients. In vitro, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), highly similar neuropeptides, decreased the SARS-CoV-2 RNA content in human monocytes and viral production in lung epithelial cells, also reducing cell death. Both neuropeptides inhibited the production of proinflammatory mediators in lung epithelial cells and in monocytes. VIP and PACAP prevented in monocytes the SARS-CoV-2-induced activation of NF-kB and SREBP1 and SREBP2, transcriptions factors involved in proinflammatory reactions and lipid metabolism, respectively. They also promoted CREB activation, a transcription factor with antiapoptotic activity and negative regulator of NF-kB. Specific inhibition of NF-kB and SREBP1/2 reproduced the anti-inflammatory, antiviral, and cell death protection effects of VIP and PACAP. Our results support further clinical investigations of these neuropeptides against COVID-19.

Simvastatin Downregulates the SARS-CoV-2-Induced Inflammatory Response and Impairs Viral Infection Through Disruption of Lipid Rafts.

Coronavirus disease 2019 (COVID-19) is currently a worldwide emergency caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). In observational clinical studies, statins have been identified as beneficial to hospitalized patients with COVID-19. However, experimental evidence of underlying statins protection against SARS-CoV-2 remains elusive. Here we reported for the first-time experimental evidence of the protective effects of simvastatin treatment both in vitro and in vivo. We found that treatment with simvastatin significantly reduced the viral replication and lung damage in vivo, delaying SARS-CoV-2-associated physiopathology and mortality in the K18-hACE2-transgenic mice model. Moreover, simvastatin also downregulated the inflammation triggered by SARS-CoV-2 infection in pulmonary tissue and in human neutrophils, peripheral blood monocytes, and lung epithelial Calu-3 cells in vitro, showing its potential to modulate the inflammatory response both at the site of infection and systemically. Additionally, we also observed that simvastatin affected the course of SARS-CoV-2 infection through displacing ACE2 on cell membrane lipid rafts. In conclusion, our results show that simvastatin exhibits early protective effects on SARS-CoV-2 infection by inhibiting virus cell entry and inflammatory cytokine production, through mechanisms at least in part dependent on lipid rafts disruption.

Fundamentals in Covid-19-Associated Thrombosis: Molecular and Cellular Aspects.

The novel coronavirus disease (COVID-19) is associated with a high incidence of coagulopathy and venous thromboembolism that may contribute to the worsening of the clinical outcome in affected patients. Marked increased D-dimer levels are the most common laboratory finding and have been repeatedly reported in critically ill COVID-19 patients. The infection caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is followed by a massive release of pro-inflammatory cytokines, which mediate the activation of endothelial cells, platelets, monocytes, and neutrophils in the vasculature. In this context, COVID-19-associated thrombosis is a complex process that seems to engage vascular cells along with soluble plasma factors, including the coagulation cascade, and complement system that contribute to the establishment of the prothrombotic state. In this review, we summarize the main findings concerning the cellular mechanisms proposed for the establishment of COVID-19-associated thrombosis.

The Weight of Obesity in Immunity from Influenza to COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged in December 2019 and rapidly outspread worldwide endangering human health. The coronavirus disease 2019 (COVID-19) manifests itself through a wide spectrum of symptoms that can evolve to severe presentations as pneumonia and several non-respiratory complications. Increased susceptibility to COVID-19 hospitalization and mortality have been linked to associated comorbidities as diabetes, hypertension, cardiovascular diseases and, recently, to obesity. Similarly, individuals living with obesity are at greater risk to develop clinical complications and to have poor prognosis in severe influenza pneumonia. Immune and metabolic dysfunctions associated with the increased susceptibility to influenza infection are linked to obesity-associated low-grade inflammation, compromised immune and endocrine systems, and to high cardiovascular risk. These preexisting conditions may favor virological persistence, amplify immunopathological responses and worsen hemodynamic instability in severe COVID-19 as well. In this review we highlight the main factors and the current state of the art on obesity as risk factor for influenza and COVID-19 hospitalization, severe respiratory manifestations, extrapulmonary complications and even death. Finally, immunoregulatory mechanisms of severe influenza pneumonia in individuals with obesity are addressed as likely factors involved in COVID-19 pathophysiology.

Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators.

Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism, energy homeostasis and intracellular transport, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection were seen to modulate pathways of lipid synthesis and uptake as monitored by testing for CD36, SREBP-1, PPARγ, and DGAT-1 expression in monocytes and triggered LD formation in different human cell lines. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected Vero cells. Electron microscopy (EM) analysis of SARS-CoV-2 infected Vero cells show viral particles colocalizing with LDs, suggestive that LDs might serve as an assembly platform. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of mediators pro-inflammatory response. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.

Platelet activation and platelet-monocyte aggregate formation trigger tissue factor expression in patients with severe COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent pathogen responsible for the coronavirus disease 2019 (COVID-19). Since its emergence, the novel coronavirus has rapidly achieved pandemic proportions causing remarkably increased morbidity and mortality around the world. A hypercoagulability state has been reported as a major pathologic event in COVID-19, and thromboembolic complications listed among life-threatening complications of the disease. Platelets are chief effector cells of hemostasis and pathological thrombosis. However, the participation of platelets in the pathogenesis of COVID-19 remains elusive. This report demonstrates that increased platelet activation and platelet-monocyte aggregate formation are observed in severe COVID-19 patients, but not in patients presenting mild COVID-19 syndrome. In addition, exposure to plasma from severe COVID-19 patients increased the activation of control platelets ex vivo. In our cohort of COVID-19 patients admitted to the intensive care unit, platelet-monocyte interaction was strongly associated with tissue factor (TF) expression by the monocytes. Platelet activation and monocyte TF expression were associated with markers of coagulation exacerbation as fibrinogen and D-dimers, and were increased in patients requiring invasive mechanical ventilation or patients who evolved with in-hospital mortality. Finally, platelets from severe COVID-19 patients were able to induce TF expression ex vivo in monocytes from healthy volunteers, a phenomenon that was inhibited by platelet P-selectin neutralization or integrin αIIb/ß3 blocking with the aggregation inhibitor abciximab. Altogether, these data shed light on new pathological mechanisms involving platelet activation and platelet-dependent monocyte TF expression, which were associated with COVID-19 severity and mortality.