A case report of vaccine-induced immune thrombotic thrombocytopenia (VITT) with genetic analysis.

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.

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.

Preclinical development of kinetin as a safe error-prone SARS-CoV-2 antiviral able to attenuate virus-induced inflammation.

Orally available antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are necessary because of the continuous circulation of new variants that challenge immunized individuals. Because severe COVID-19 is a virus-triggered immune and inflammatory dysfunction, molecules endowed with both antiviral and anti-inflammatory activity are highly desirable. We identified here that kinetin (MB-905) inhibits the in vitro replication of SARS-CoV-2 in human hepatic and pulmonary cell lines. On infected monocytes, MB-905 reduced virus replication, IL-6 and TNFα levels. MB-905 is converted into its triphosphate nucleotide to inhibit viral RNA synthesis and induce error-prone virus replication. Coinhibition of SARS-CoV-2 exonuclease, a proofreading enzyme that corrects erroneously incorporated nucleotides during viral RNA replication, potentiated the inhibitory effect of MB-905. MB-905 shows good oral absorption, its metabolites are stable, achieving long-lasting plasma and lung concentrations, and this drug is not mutagenic nor cardiotoxic in acute and chronic treatments. SARS-CoV-2-infected hACE-mice and hamsters treated with MB-905 show decreased viral replication, lung necrosis, hemorrhage and inflammation. Because kinetin is clinically investigated for a rare genetic disease at regimens beyond the predicted concentrations of antiviral/anti-inflammatory inhibition, our investigation suggests the opportunity for the rapid clinical development of a new antiviral substance for the treatment of COVID-19.

Agathisflavone, a natural biflavonoid that inhibits SARS-CoV-2 replication by targeting its proteases.

Despite the fast development of vaccines, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still circulates through variants of concern (VoC) and escape the humoral immune response. SARS-CoV-2 has provoked over 200,000 deaths/months since its emergence and only a few antiviral drugs showed clinical benefit up to this moment. Thus, chemical structures endowed with anti-SARS-CoV-2 activity are important for continuous antiviral development and natural products represent a fruitful source of substances with biological activity. In the present study, agathisflavone (AGT), a biflavonoid from Anacardium occidentale was investigated as a candidate anti-SARS-CoV-2 compound. In silico and enzymatic analysis indicated that AGT may target mainly the viral main protease (Mpro) and not the papain-like protease (PLpro) in a non-competitive way. Cell-based assays in type II pneumocytes cell lineage (Calu-3) showed that SARS-CoV-2 is more susceptible to AGT than to apigenin (APG, monomer of AGT), in a dose-dependent manner, with an EC50 of 4.23 ± 0.21 µM and CC50 of 61.3 ± 0.1 µM and with a capacity to inhibit the level of pro-inflammatory mediator tumor necrosis factor-alpha (TNF-α). These results configure AGT as an interesting chemical scaffold for the development of novel semisynthetic antivirals against SARS-CoV-2.

Increased Lung Immune Metabolic Activity in COVID-19 Survivors.

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.

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.

Generalized Edema and Pseudothrombocytopenia After ChAdOx1 nCoV-19 COVID-19 Vaccination: A Case Report.

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).

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.

Commercially Available Flavonols Are Better SARS-CoV-2 Inhibitors than Isoflavone and Flavones.

Despite the fast development of vaccines, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still circulating and generating variants of concern (VoC) that escape the humoral immune response. In this context, the search for anti-SARS-CoV-2 compounds is still essential. A class of natural polyphenols known as flavonoids, frequently available in fruits and vegetables, is widely explored in the treatment of different diseases and used as a scaffold for the design of novel drugs. Therefore, herein we evaluate seven flavonoids divided into three subclasses, isoflavone (genistein), flavone (apigenin and luteolin) and flavonol (fisetin, kaempferol, myricetin, and quercetin), for COVID-19 treatment using cell-based assays and in silico calculations validated with experimental enzymatic data. The flavonols were better SARS-CoV-2 inhibitors than isoflavone and flavones. The increasing number of hydroxyl groups in ring B of the flavonols kaempferol, quercetin, and myricetin decreased the 50% effective concentration (EC50) value due to their impact on the orientation of the compounds inside the target. Myricetin and fisetin appear to be preferred candidates; they are both anti-inflammatory (decreasing TNF-α levels) and inhibit SARS-CoV-2 mainly by targeting the processability of the main protease (Mpro) in a non-competitive manner, with a potency comparable to the repurposed drug atazanavir. However, fisetin and myricetin might also be considered hits that are amenable to synthetic modification to improve their anti-SARS-CoV-2 profile by inhibiting not only Mpro, but also the 3'-5' exonuclease (ExoN).

Generalized Edema and Pseudothrombocytopenia After ChAdOx1 nCoV-19 COVID-19 Vaccination: A Case Report

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).

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.

Atazanavir Is a Competitive Inhibitor of SARS-CoV-2 Mpro, Impairing Variants Replication In Vitro and In Vivo.

Atazanavir (ATV) has already been considered as a potential repurposing drug to 2019 coronavirus disease (COVID-19); however, there are controversial reports on its mechanism of action and effectiveness as anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Through the pre-clinical chain of experiments: enzymatic, molecular docking, cell-based and in vivo assays, it is demonstrated here that both SARS-CoV-2 B.1 lineage and variant of concern gamma are susceptible to this antiretroviral. Enzymatic assays and molecular docking calculations showed that SARS-CoV-2 main protease (Mpro) was inhibited by ATV, with Morrison's inhibitory constant (Ki) 1.5-fold higher than GC376 (a positive control) dependent of the catalytic water (H2Ocat) content. ATV was a competitive inhibitor, increasing the Mpro's Michaelis-Menten (Km) more than sixfold. Cell-based assays indicated that different lineages of SARS-CoV-2 is susceptible to ATV. Using oral administration of ATV in mice to reach plasmatic exposure similar to humans, transgenic mice expression in human angiotensin converting enzyme 2 (K18-hACE2) were partially protected against lethal challenge with SARS-CoV-2 gamma. Moreover, less cell death and inflammation were observed in the lung from infected and treated mice. Our studies may contribute to a better comprehension of the Mpro/ATV interaction, which could pave the way to the development of specific inhibitors of this viral protease.