Platelet activation and coronavirus disease 2019 mortality: Insights from coagulopathy, antiplatelet therapy and inflammation.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is associated with an inflammatory cytokine burst and a prothrombotic coagulopathy. Platelets may contribute to microthrombosis, and constitute a therapeutic target in COVID-19 therapy. AIM: To assess if platelet activation influences mortality in COVID-19. METHODS: We explored two cohorts of patients with COVID-19. Cohort A included 208 ambulatory and hospitalized patients with varying clinical severities and non-COVID patients as controls, in whom plasma concentrations of the soluble platelet activation biomarkers CD40 ligand (sCD40L) and P-selectin (sP-sel) were quantified within the first 48hours following hospitalization. Cohort B was a multicentre cohort of 2878 patients initially admitted to a medical ward. In both cohorts, the primary outcome was in-hospital mortality. RESULTS: In cohort A, median circulating concentrations of sCD40L and sP-sel were only increased in the 89 critical patients compared with non-COVID controls: sP-sel 40,059 (interquartile range 26,876-54,678)pg/mL; sCD40L 1914 (interquartile range 1410-2367)pg/mL (P<0.001 for both). A strong association existed between sP-sel concentration and in-hospital mortality (Kaplan-Meier log-rank P=0.004). However, in a Cox model considering biomarkers of immunothrombosis, sP-sel was no longer associated with mortality, in contrast to coagulopathy evaluated with D-dimer concentration (hazard ratio 4.86, 95% confidence interval 1.64-12.50). Moreover, in cohort B, a Cox model adjusted for co-morbidities suggested that prehospitalization antiplatelet agents had no significant impact on in-hospital mortality (hazard ratio 1.05, 95% CI 0.80-1.37; P=0.73). CONCLUSIONS: Although we observed an association between excessive biomarkers of platelet activation and in-hospital mortality, our findings rather suggest that coagulopathy is more central in driving disease progression, which may explain why prehospitalization antiplatelet drugs were not a protective factor against mortality in our multicentre cohort.

Defective activation and regulation of type I interferon immunity is associated with increasing COVID-19 severity.

Host immunity to infection with SARS-CoV-2 is highly variable, dictating diverse clinical outcomes ranging from asymptomatic to severe disease and death. We previously reported reduced type I interferon in severe COVID-19 patients preceded clinical worsening. Further studies identified genetic mutations in loci of the TLR3- or TLR7-dependent interferon-I pathways, or neutralizing interferon-I autoantibodies as risk factors for development of COVID-19 pneumonia. Here we show in patient cohorts with different severities of COVID-19, that baseline plasma interferon α measures differ according to the immunoassay used, timing of sampling, the interferon α subtype measured, and the presence of autoantibodies. We also show a consistently reduced induction of interferon-I proteins in hospitalized COVID-19 patients upon immune stimulation, that is not associated with detectable neutralizing autoantibodies against interferon α or interferon ω. Intracellular proteomic analysis shows increased monocyte numbers in hospitalized COVID-19 patients but impaired interferon-I response after stimulation. We confirm this by ex vivo whole blood stimulation with interferon-I which induces transcriptomic responses associated with inflammation in hospitalized COVID-19 patients, that is not seen in controls or non-hospitalized moderate cases. These results may explain the dichotomy of the poor clinical response to interferon-I based treatments in late stage COVID-19, despite the importance of interferon-I in early acute infection and may guide alternative therapeutic strategies.

Severe COVID-19 is associated with hyperactivation of the alternative complement pathway

Background Severe coronavirus disease 2019 (COVID-19) is characterized by impaired type I interferon activity and a state of hyperinflammation leading to acute respiratory distress syndrome. The complement system has recently emerged as a key player in triggering and maintaining the inflammatory state, but the role of this molecular cascade in severe COVID-19 is still poorly characterized. Objective We aimed at assessing the contribution of complement pathways at both protein and transcriptomic levels. Methods To this end, we systematically assessed RNA levels of 28 complement genes in circulating whole blood of COVID-19 patients and healthy controls, including genes of the alternative pathway, for which data remain scarce. Results We found differential expression of genes involved in the complement system, yet with various expression patterns: while patients displaying moderate disease had elevated expression of classical pathway genes, severe disease was associated with increased lectin and alternative pathway activation, which correlated with inflammation and coagulopathy markers. Additionally, properdin, a pivotal positive regulator of the alternative pathway, showed high RNA expression but was found at low protein concentrations in severe and critical patients, suggesting its deposition at the sites of complement activation. Notably, low properdin levels were significantly associated with the use of mechanical ventilation (AUC = 0.82, p = 0.002). Conclusion This study sheds light on the role of the alternative pathway in severe COVID-19 and provides additional rationale for the testing of drugs inhibiting the alternative pathway of the complement system. We show that activation of the alternative complement pathway characterizes COVID-19 severity. Specifically, low properdin levels were associated with use of mechanical ventilation. This work provides a rationale for the specific inhibition of the alternative complement pathway.

Lupus Anticoagulant Single Positivity During the Acute Phase of COVID-19 Is Not Associated With Venous Thromboembolism or In-Hospital Mortality.

OBJECTIVE: The clinical relevance of antiphospholipid antibodies (aPLs) in COVID-19 is controversial. This study was undertaken to investigate the prevalence and prognostic value of conventional and nonconventional aPLs in patients with COVID-19. METHODS: This was a multicenter, prospective observational study in a French cohort of patients hospitalized with suspected COVID-19. RESULTS: Two hundred forty-nine patients were hospitalized with suspected COVID-19, in whom COVID-19 was confirmed in 154 and not confirmed in 95. We found a significant increase in lupus anticoagulant (LAC) positivity among patients with COVID-19 compared to patients without COVID-19 (60.9% versus 23.7%; P < 0.001), while prevalence of conventional aPLs (IgG and IgM anti-ß2 -glycoprotein I and IgG and IgM anticardiolipin isotypes) and nonconventional aPLs (IgA isotype of anticardiolipin, IgA isotype of anti-ß2 -glycoprotein I, IgG and IgM isotypes of anti-phosphatidylserine/prothrombin, and IgG and IgM isotypes of antiprothrombin) was low in both groups. Patients with COVID-19 who were positive for LAC, as compared to patients with COVID-19 who were negative for LAC, had higher levels of fibrinogen (median 6.0 gm/liter [interquartile range 5.0-7.0] versus 5.3 gm/liter [interquartile range 4.3-6.4]; P = 0.028) and C-reactive protein (CRP) (median 115.5 mg/liter [interquartile range 66.0-204.8] versus 91.8 mg/liter [interquartile range 27.0-155.1]; P = 0.019). Univariate analysis did not show any association between LAC positivity and higher risks of venous thromboembolism (VTE) (odds ratio 1.02 [95% confidence interval 0.44-2.43], P = 0.95) or in-hospital mortality (odds ratio 1.80 [95% confidence interval 0.70-5.05], P = 0.24). With and without adjustment for CRP level, age, and sex, Kaplan-Meier survival curves according to LAC positivity confirmed the absence of an association with VTE or in-hospital mortality (unadjusted P = 0.64 and P = 0.26, respectively; adjusted hazard ratio 1.13 [95% confidence interval 0.48-2.60] and 1.80 [95% confidence interval 0.67-5.01], respectively). CONCLUSION: Patients with COVID-19 have an increased prevalence of LAC positivity associated with biologic markers of inflammation. However, LAC positivity at the time of hospital admission is not associated with VTE risk and/or in-hospital mortality.

Distinct systemic and mucosal immune responses during acute SARS-CoV-2 infection.

Coordinated local mucosal and systemic immune responses following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection either protect against coronavirus disease 2019 (COVID-19) pathologies or fail, leading to severe clinical outcomes. To understand this process, we performed an integrated analysis of SARS-CoV-2 spike-specific antibodies, cytokines, viral load and bacterial communities in paired nasopharyngeal swabs and plasma samples from a cohort of clinically distinct patients with COVID-19 during acute infection. Plasma viral load was associated with systemic inflammatory cytokines that were elevated in severe COVID-19, and also with spike-specific neutralizing antibodies. By contrast, nasopharyngeal viral load correlated with SARS-CoV-2 humoral responses but inversely with interferon responses, the latter associating with protective microbial communities. Potential pathogenic microorganisms, often implicated in secondary respiratory infections, were associated with mucosal inflammation and elevated in severe COVID-19. Our results demonstrate distinct tissue compartmentalization of SARS-CoV-2 immune responses and highlight a role for the nasopharyngeal microbiome in regulating local and systemic immunity that determines COVID-19 clinical outcomes.

Immune checkpoint inhibitors increase T cell immunity during SARS-CoV-2 infection.

The COVID-19 pandemic has spread worldwide, yet the role of antiviral T cell immunity during infection and the contribution of immune checkpoints remain unclear. By prospectively following a cohort of 292 patients with melanoma, half of which treated with immune checkpoint inhibitors (ICIs), we identified 15 patients with acute or convalescent COVID-19 and investigated their transcriptomic, proteomic, and cellular profiles. We found that ICI treatment was not associated with severe COVID-19 and did not alter the induction of inflammatory and type I interferon responses. In-depth phenotyping demonstrated expansion of CD8 effector memory T cells, enhanced T cell activation, and impaired plasmablast induction in ICI-treated COVID-19 patients. The evaluation of specific adaptive immunity in convalescent patients showed higher spike (S), nucleoprotein (N), and membrane (M) antigen-specific T cell responses and similar induction of spike-specific antibody responses. Our findings provide evidence that ICI during COVID-19 enhanced T cell immunity without exacerbating inflammation.

Regulation of the acetylcholine/α7nAChR anti-inflammatory pathway in COVID-19 patients.

The cholinergic system has been proposed as a potential regulator of COVID-19-induced hypercytokinemia. We investigated whole-blood expression of cholinergic system members and correlated it with COVID-19 severity. Patients with confirmed SARS-CoV-2 infection and healthy aged-matched controls were included in this non-interventional study. A whole blood sample was drawn between 9-11 days after symptoms onset, and peripheral leukocyte phenotyping, cytokines measurement, RNA expression and plasma viral load were determined. Additionally, whole-blood expression of native alpha-7 nicotinic subunit and its negative dominant duplicate (CHRFAM7A), choline acetyltransferase and acetylcholine esterase (AchE) were determined. Thirty-seven patients with COVID-19 (10 moderate, 11 severe and 16 with critical disease) and 14 controls were included. Expression of CHRFAM7A was significantly lower in critical COVID-19 patients compared to controls. COVID-19 patients not expressing CHRFAM7A had higher levels of CRP, more extended pulmonary lesions and displayed more pronounced lymphopenia. COVID-19 patients without CHRFAM7A expression also showed increased TNF pathway expression in whole blood. AchE was also expressed in 30 COVID-19 patients and in all controls. COVID-19-induced hypercytokinemia is associated with decreased expression of the pro-inflammatory dominant negative duplicate CHRFAM7A. Expression of this duplicate might be considered before targeting the cholinergic system in COVID-19 with nicotine.

Platelet activation in critically ill COVID-19 patients.

BACKGROUND: Microvascular, arterial and venous thrombotic events have been largely described during severe coronavirus disease 19 (COVID-19). However, mechanisms underlying hemostasis dysregulation remain unclear. METHODS: We explored two independent cross-sectional cohorts to identify soluble markers and gene-expression signatures that discriminated COVID-19 severity and outcomes. RESULTS: We found that elevated soluble (s)P-selectin at admission was associated with disease severity. Elevated sP-selectin was predictive of intubation and death (ROC AUC = 0.67, p = 0.028 and AUC = 0.74, p = 0.0047, respectively). An optimal cutoff value was predictive of intubation with 66% negative predictive value (NPV) and 61% positive predictive value (PPV), and of death with 90% NPV and 55% PPV. An unbiased gene set enrichment analysis revealed that critically ill patients had increased expression of genes related to platelet activation. Hierarchical clustering identified ITG2AB, GP1BB, PPBP and SELPLG to be upregulated in a grade-dependent manner. ROC curve analysis for the prediction of intubation was significant for SELPLG and PPBP (AUC = 0.8, p = 0.046 for both). An optimal cutoff value for PBPP was predictive of intubation with 100% NPV and 45% PPV, and for SELPLG with 100% NPV and 50% PPV. CONCLUSION: We provide evidence that platelets contribute to COVID-19 severity. Plasma sP-selectin level was associated with severity and in-hospital mortality. Transcriptional analysis identified PPBP/CXCL7 and SELPLG as biomarkers for intubation. These findings provide additional evidence for platelet activation in driving critical COVID-19. Specific studies evaluating the performance of these biomarkers are required.

Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients.

Coronavirus disease 2019 (COVID-19) is characterized by distinct patterns of disease progression that suggest diverse host immune responses. We performed an integrated immune analysis on a cohort of 50 COVID-19 patients with various disease severity. A distinct phenotype was observed in severe and critical patients, consisting of a highly impaired interferon (IFN) type I response (characterized by no IFN-ß and low IFN-α production and activity), which was associated with a persistent blood viral load and an exacerbated inflammatory response. Inflammation was partially driven by the transcriptional factor nuclear factor-κB and characterized by increased tumor necrosis factor-α and interleukin-6 production and signaling. These data suggest that type I IFN deficiency in the blood could be a hallmark of severe COVID-19 and provide a rationale for combined therapeutic approaches.

Angiopoietin-2 as a marker of endothelial activation is a good predictor factor for intensive care unit admission of COVID-19 patients.

BACKGROUND: Coronavirus disease-2019 (COVID-19), a respiratory disease has been associated with ischemic complications, coagulation disorders, and an endotheliitis. OBJECTIVES: To explore endothelial damage and activation-related biomarkers in COVID-19 patients with criteria of hospitalization for referral to intensive care unit (ICU) and/or respiratory worsening. METHODS: Analysis of endothelial and angiogenic soluble markers in plasma from patients at admission. RESULTS: Study enrolled 40 consecutive COVID-19 patients admitted to emergency department that fulfilled criteria for hospitalization. Half of them were admitted in conventional wards without any ICU transfer during hospitalization; whereas the 20 others were directly transferred to ICU. Patients transferred in ICU were more likely to have lymphopenia, decreased SpO2 and increased D-dimer, CRP and creatinine levels. In those patients, soluble E-selectin and angiopoietin-2 were significantly increased (p value at 0.009 and 0.003, respectively). Increase in SELE gene expression (gene coding for E-selectin protein) was confirmed in an independent cohort of 32 patients using a whole blood gene expression profile analysis. In plasma, we found a strong association between angiopoetin-2 and CRP, creatinine and D-dimers (with p value at 0.001, 0.001 and 0.003, respectively). ROC curve analysis identified an Angiopoietin-2 cut-off of 5000 pg/mL as the best predictor for ICU outcome (Se = 80.1%, Sp = 70%, PPV = 72.7%, NPV = 77%), further confirmed in multivariate analysis after adjustment for creatinine, CRP or D-dimers. CONCLUSION: Angiopoietin-2 is a relevant predictive factor for ICU direct admission in COVID-19 patients. This result showing an endothelial activation reinforces the hypothesis of a COVID-19-associated microvascular dysfunction.