Coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has rapidly spread worldwide since December 2019. Disease severity is strongly associated with abnormal routine coagulation tests (particularly increased D-dimer levels) and thrombocytopenia.[1] These alterations are accompanied by a high prevalence of deep venous thrombosis, pulmonary embolism, and arterial thrombotic events at uncommon sites.[2] Platelets are at the crossroad of thrombosis and immunity and contribute to thrombus formation and disease severity during bacterial sepsis[3] [4] and viral infections.[5] [6] We here investigated platelet activation and responsiveness to relevant stimuli in hospitalized COVID-19 patients. Patients were included in the ELDER-BIOME protocol (clinicaltrials.gov identifier NCT02928367). We prospectively enrolled suspected COVID-19 patients admitted to the ward, in two hospitals in The Netherlands (Academic Medical Center in Amsterdam and Flevo Hospital in Almere). COVID-19 infection was confirmed by polymerase chain reaction (PCR) of material obtained by nasopharyngeal swab. Controls were hospital employees (n = 8) or patients visiting the outpatient clinics without infectious disease symptoms (n = 18); the results obtained in these control subgroups did not differ and were combined. Informed consent was obtained from all participants. Citrated peripheral blood was collected within 48 hours after admission and processed within 4 hours. Platelet-rich plasma (PRP) was obtained by centrifugation (180 × g, 15 minutes, 21°C), mixed with a 1:5 ratio of acid citrate dextrose and subsequently centrifuged (200 × g, 2 minutes) to remove remaining erythrocytes and leukocytes. Platelets in buffer-diluted PRP were stimulated for 30 minutes with cross-linked collagen-related peptide (CRP-XL, CambCol-Laboratory, Cambridge, United Kingdom; 0.1 or 1 µg/mL) or thrombin receptor activator peptide-6 (TRAP-6, Bachem, Bubendorf, Switzerland; 15 µM). Platelet activation was assessed by flow cytometry using anti-CD61-AlexaFluor700 (clone VI-PL2), anti-CD62P-PerCP-Cy5.5 (clone AK4), anti-CD63-PE-Cy7 (clone H5C6), and anti-glycoprotein (GP) IIbIIIa-AlexaFluor647 (clone PAC-1) (Biolegend, San Diego, California, United States). Analysis was performed on FACSCanto II until 10,000 CD61+ events were recorded. Data were analyzed using FlowJo v10 (BD Biosciences, San Jose, California, United States). Platelet-neutrophil complexes were measured in whole blood (without ex vivo stimulation) as previously described,[7] using anti-CD66b-FITC, anti-CD11b-PE, anti-CD61-AlexaFluor700 (Biolegend), and Viability Dye-LFluor780 (eBioscience, San Diego, California, United States). Platelet numbers in PRP were established by flow cytometry, using Precision Count Beads (BD Bioscience). Soluble GPVI was measured in citrated plasma (1,500 × g, 15 minutes) by enzyme-linked immunosorbent assay (ELISA) (Invitrogen, Waltham, Massachusetts, United States). Soluble P-selectin GP ligand-1 (PSGL-1), CD40L, and D-dimer were measured using LEGENDplex Human Thrombosis Panel (Biolegend). Soluble P-selectin and platelet factor (PF) 4 were measured by ELISA (R&D Duoset, Minneapolis, Minnesota, United States). All analyses were done using GraphPad Prism v8.0 (San Diego, California, United States). Results were compared using paired Wilcoxon test or unpaired Mann–Whitney U tests. We applied linear regression to assess the correlation between neutrophil counts and soluble PSGL-1 levels. p-Values < 0.05 defined statistical significance. We enrolled 35 patients and 26 controls between April 10 and May 15, 2020. Five patients had a negative SARS-CoV-2 PCR and were excluded from analyses. Baseline characteristics of patients and controls were comparable, except for body mass index and ethnicity ([Table 1]). Neutrophil counts were higher in COVID-19 patients compared with controls, whereas platelet counts were unaltered. Three patients (10.0%) were subsequently admitted to the intensive care unit and four (13.3%) died during hospital stay. Two patients (6.6%) were diagnosed with pulmonary embolism. To assess circulating platelet activation, we measured the expression of P-selectin, CD63 (reflecting α- and dense granules secretion, respectively), and the fibrinogen-binding conformation of GPIIbIIIa.[8] Platelets from COVID-19 patients showed increased expression of P-selectin and active GPIIbIIIa, while CD63 expression was comparable to that of control platelets ([Fig. 1A]). The plasma levels of α-granule proteins PF4 and soluble CD40L were higher in COVID-19 patients, further indicating activated platelets in vivo ([Fig. 1B]). While earlier studies also reported enhanced α granule release,[9] [10] our study adds to this that dense granules likely are not affected in COVID-19 patients not requiring intensive care. Platelet-neutrophil complexes are linked to platelet hyperactivation in vivo.[3] COVID-19 patients had more platelet-neutrophil complexes compared with controls ([Fig. 1C]). Moreover, neutrophils from COVID-19 patients had higher CD11b expression, suggesting a higher activation status ([Fig. 1D]). Interestingly, neutrophils that were in complex with platelets had the highest CD11b expression, implying that platelet activation and interaction with neutrophils is associated with increased neutrophil activation. Likely, the enhanced platelet P-selectin expression in COVID-19 patients contributed to the interaction with neutrophils.[8] Plasma levels of soluble P-selectin and PSGL-1 (the neutrophil receptor mediating platelet-neutrophil complex formation) were not different between patients and controls ([Fig. 1B]). Soluble PSGL-1 was positively correlated with neutrophil counts in COVID-19 patients (r = 0.53, p = 0.0029) but not in controls (r = 0.35, p = 0.24) ([Fig. 1E]), suggesting that PSGL-1 shedding is associated with neutrophil activation...

Background Activated platelets have been implicated in the proinflammatory and prothrombotic phenotype of coronavirus disease 2019 (COVID-19). While it is increasingly recognized that lipids have important structural and signaling roles in platelets, the lipidomic landscape of platelets during infection has remained unexplored. Objective To investigate the platelet lipidome of patients hospitalized for COVID-19. Methods We performed untargeted lipidomics in platelets of 25 patients hospitalized for COVID-19 and 23 noninfectious controls with similar age and sex characteristics, and with comparable comorbidities. Results Twenty-five percent of the 1,650 annotated lipids were significantly different between the groups. The significantly altered part of the platelet lipidome mostly comprised lipids that were less abundant in patients with COVID-19 (20.4% down, 4.6% up, 75% unchanged). Platelets from COVID-19 patients showed decreased levels of membrane plasmalogens, and a distinct decrease of long-chain, unsaturated triacylglycerols. Conversely, platelets from patients with COVID-19 displayed class-wide higher abundances of bis(monoacylglycero)phosphate and its biosynthetic precursor lysophosphatidylglycerol. Levels of these classes positively correlated with ex vivo platelet reactivity—as measured by P-selectin expression after PAR1 activation—irrespective of disease state. Conclusion Taken together, this investigation provides the first exploration of the profound impact of infection on the human platelet lipidome, and reveals associations between the lipid composition of platelets and their reactivity. These results warrant further lipidomic research in other infections and disease states involving platelet pathophysiology. The data that support the findings of this study are available from the corresponding author. ^* A.R.S. and V.L. are co-first authors. Received: 09 December 2021 Accepted: 30 March 2022 Article published online: 18 July 2022 © 2022. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/) Georg Thieme Verlag KG Rüdigerstraße 14, 70469 Stuttgart, Germany

BACKGROUND: This analysis provides details on baseline and changes in quality of life (QoL) and its components as measured by EQ-5D-5L questionnaire, as well as association with objective outcomes, applying high-intensity heart failure (HF) care in patients with acute HF. METHODS: In STRONG-HF trial (Safety, Tolerability, and Efficacy of Rapid Optimization, Helped by NT-proBNP Testing, of Heart Failure Therapies) patients with acute HF were randomized just before discharge to either usual care or a high-intensity care strategy of guideline-directed medical therapy up-titration. Patients ranked their state of health on the EQ-5D visual analog scale score ranging from 0 (the worst imaginable health) to 100 (the best imaginable health) at baseline and at 90 days follow-up. RESULTS: In 1072 patients with acute HF with available assessment of QoL (539/533 patients assigned high-intensity care/usual care) the mean baseline EQ-visual analog scale score was 59.2 (SD, 15.1) with no difference between the treatment groups. Patients with lower baseline EQ-visual analog scale (meaning worse QoL) were more likely to be women, self-reported Black and non-European ( P <0.001). The strongest independent predictors of a greater improvement in QoL were younger age ( P <0.001), no HF hospitalization in the previous year ( P <0.001), lower NYHA class before hospital admission ( P <0.001) and high-intensity care treatment (mean difference, 4.2 [95% CI, 2.5–5.8]; P <0.001). No statistically significant heterogeneity in the benefits of high-intensity care was seen across patient subgroups of different ages, with left ventricular ejection fraction above or below 40%, NT-proBNP (N-terminal pro-B-type natriuretic peptide) and systolic blood pressure above or below the median value. The treatment effect on the primary end point did not vary significantly across baseline EQ-visual analog scale ( P _interaction =0.87). CONCLUSIONS: Early up-titration of guideline-directed medical therapy significantly improves all dimensions of QoL in patients with HF and improves prognosis regardless of baseline self-assessed health status. The likelihood of achieving optimal doses of HF medications does not depend on baseline QoL. REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT03412201.

Lymphopenia at hospital admission occurs in over one-third of patients with community-acquired pneumonia (CAP), yet its clinical relevance and pathophysiological implications remain underexplored. We evaluated outcomes and immune features of patients with lymphopenic CAP (L-CAP), a previously described immunophenotype characterized by admission lymphocyte count <0.724 × 10⁹ cells/L. Observational study in 149 patients admitted to a general ward for CAP. We measured 34 plasma biomarkers reflective of inflammation, endothelial cell responses, coagulation, and immune checkpoints. We characterized lymphocyte phenotypes in 29 patients using spectral flow cytometry. L-CAP occurred in 45 patients (30.2%) and was associated with prolonged time-to-clinical-stability (median 5 versus 3 days), also when we accounted for competing events for reaching clinical stability and adjusted for baseline covariates (subdistribution hazard ratio 0.63; 95% confidence interval 0.45-0.88). L-CAP patients demonstrated a proportional depletion of CD4 T follicular helper cells, CD4 T effector memory cells, naïve CD8 T cells and IgG+ B cells. Plasma biomarker analyses indicated increased activation of the cytokine network and the vascular endothelium in L-CAP. L-CAP patients have a protracted clinical recovery course and a more broadly dysregulated host response. These findings highlight the prognostic and pathophysiological relevance of admission lymphopenia in patients with CAP.

Alterations in platelet function have been implicated in the pathophysiology of COVID-19 since the beginning of the pandemic. While early reports linked hyperactivated platelets to thromboembolic events in COVID-19, subsequent investigations demonstrated hyporeactive platelets with a procoagulant phenotype. Mitochondria are important for energy metabolism and the function of platelets. Here, we sought to map the energy metabolism of platelets in a cohort of noncritically ill COVID-19 patients and assess platelet mitochondrial function, activation status, and responsiveness to external stimuli. We enrolled hospitalized COVID-19 patients and controls between October 2020 and December 2021. Platelets function and metabolism was analyzed by flow cytometry, metabolomics, glucose fluxomics, electron and fluorescence microscopy and western blot. Platelets from COVID-19 patients showed increased phosphatidylserine externalization indicating a procoagulant phenotype and hyporeactivity to ex vivo stimuli, associated with profound mitochondrial dysfunction characterized by mitochondrial depolarization, lower mitochondrial DNA–encoded transcript levels, an altered mitochondrial morphology consistent with increased mitochondrial fission, and increased pyruvate/lactate ratios in platelet supernatants. Metabolic profiling by untargeted metabolomics revealed NADH, NAD⁺, and ATP among the top decreased metabolites in patients’ platelets, suggestive of energy metabolism failure. Consistently, platelet fluxomics analyses showed a strongly reduced utilization of ¹³C-glucose in all major energy pathways together with a rerouting of glucose to de novo generation of purine metabolites. Patients’ platelets further showed evidence of oxidative stress, together with increased glutathione oxidation and synthesis. Addition of plasma from COVID-19 patients to normal platelets partially reproduced the phenotype of patients’ platelets and disclosed a temporal relationship between mitochondrial decay and (subsequent) phosphatidylserine exposure and hyporeactivity. These data link energy metabolism failure in platelets from COVID-19 patients with a prothrombotic platelet phenotype with features matching cell death.

Community-acquired pneumonia (CAP) is a leading cause of death, with mortality linked to an unbalanced host response. Sirtuin (Sirt)1, a histone deacetylase, regulating metabolism and epigenetics, may be fundamental in activating the innate immune response. Sirt1 mRNA expression was significantly reduced in monocytes from CAP patients (n = 76) upon admission compared to healthy controls (n = 42), with levels returning to normal after 30 days. Pharmacological activation of Sirt1 with SRT1720 decreased LPS- and K. pneumoniae-induced IL-6 release in primary human monocytes and decreased NF-κB activation in THP1 cells. In a mouse K. pneumoniae pneumosepsis model, SRT1720 strongly reduced neutrophil influx and degranulation markers in bronchoalveolar lavage fluid, lowered pulmonary concentrations of IL-6 and TNF-α, and reduced lung pathology scores. Simultaneously, it reduced neutrophil content in liver tissue and plasma transaminase levels, alongside a trend toward reduced liver necrosis. Plasma IL-6 and TNF-α were significantly lower in SRT1720-treated mice at 42 h. Finally, while SRT1720 did not impact bacterial loads in the lungs, it reduced bacterial burden in blood, with a similar trend observed in liver homogenates. In conclusion, the Sirt1 activator SRT1720 exerts anti-inflammatory effects on human monocytes, reduces local and systemic inflammation and organ injury, and diminishes bacterial dissemination in murine pneumosepsis.