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.

Dynamics of circulating calprotectin accurately predict the outcome of moderate COVID-19 patients.

BACKGROUND: Severe COVID-19 is associated with a high circulating level of calprotectin, the S100A8/S100A9 alarmin heterodimer. Baseline calprotectin amount measured in peripheral blood at diagnosis correlates with disease severity. The optimal use of this biomarker along COVID-19 course remains to be delineated. METHODS: We focused on patients with a WHO-defined moderate COVID-19 requiring hospitalization in a medical ward. We collected plasma and serum from three independent cohorts (N = 626 patients) and measured calprotectin amount at admission. We performed longitudinal measures of calprotectin in 457 of these patients (1461 samples) and used a joint latent class mixture model in which classes were defined by age, body mass index and comorbidities to identify calprotectin trajectories predicting the risk of transfer into an intensive care unit or death. FINDINGS: After adjustment for age, sex, body mass index and comorbidities, the predictive value of baseline calprotectin in patients with moderate COVID19 could be refined by serial monitoring of the biomarker. We discriminated three calprotectin trajectories associated with low, moderate, and high risk of poor outcome, and we designed an algorithm available as online software (https://calpla.gustaveroussy.fr:8443/) to monitor the probability of a poor outcome in individual patients with moderate COVID-19. INTERPRETATION: These results emphasize the clinical interest of serial monitoring of calprotectin amount in the peripheral blood to anticipate the risk of poor outcomes in patients with moderate COVID-19 hospitalized in a standard care unit. FUNDING: The study received support (research grants) from ThermoFisher immunodiagnostics (France) and Gustave Roussy Foundation.

What level of D-dimers can safely exclude pulmonary embolism in COVID-19 patients presenting to the emergency department?

OBJECTIVES: To identify which level of D-dimer would allow the safe exclusion of pulmonary embolism (PE) in COVID-19 patients presenting to the emergency department (ED). METHODS: This retrospective study was conducted on the COVID database of Assistance Publique - Hôpitaux de Paris (AP-HP). COVID-19 patients who presented at the ED of AP-HP hospitals between March 1 and May 15, 2020, and had CTPA following D-dimer dosage within 48h of presentation were included. The D-dimer sensitivity, specificity, and positive and negative predictive values were calculated for different D-dimer thresholds, as well as the false-negative and failure rates, and the number of CTPAs potentially avoided. RESULTS: A total of 781 patients (mean age 62.0 years, 53.8% men) with positive RT-PCR for SARS-Cov-2 were included and 60 of them (7.7%) had CTPA-confirmed PE. Their median D-dimer level was significantly higher than that of patients without PE (4,013 vs 1,198 ng·mL-1, p < 0.001). Using 500 ng·mL-1, or an age-adjusted cut-off for patients > 50 years, the sensitivity and the NPV were above 90%. With these thresholds, 17.1% and 31.5% of CTPAs could have been avoided, respectively. Four of the 178 patients who had a D-dimer below the age-adjusted cutoff had PE, leading to an acceptable failure rate of 2.2%. Using higher D-dimer cut-offs could have avoided more CTPAs, but would have lowered the sensitivity and increased the failure rate. CONCLUSION: The same D-Dimer thresholds as those validated in non-COVID outpatients should be used to safely rule out PE. KEY POINTS: • The median D-dimer level was significantly higher in COVID-19 patients with PE as compared to those without PE (4,013 ng·mL-1 vs 1,198 ng·mL-1 respectively, p < 0.001). • Using 500 ng·mL-1, or an age-adjusted D-dimer cut-off to exclude pulmonary embolism, the sensitivity and negative predictive value were above 90%. • Higher cut-offs would lead to a reduction in the sensitivity below 85% and an increase in the failure rate, especially for patients under 50 years.

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.

Prolonged SARS-CoV-2 RNA virus shedding and lymphopenia are hallmarks of COVID-19 in cancer patients with poor prognosis.

Patients with cancer are at higher risk of severe coronavirus infectious disease 2019 (COVID-19), but the mechanisms underlying virus-host interactions during cancer therapies remain elusive. When comparing nasopharyngeal swabs from cancer and noncancer patients for RT-qPCR cycle thresholds measuring acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in 1063 patients (58% with cancer), we found that malignant disease favors the magnitude and duration of viral RNA shedding concomitant with prolonged serum elevations of type 1 IFN that anticorrelated with anti-RBD IgG antibodies. Cancer patients with a prolonged SARS-CoV-2 RNA detection exhibited the typical immunopathology of severe COVID-19 at the early phase of infection including circulation of immature neutrophils, depletion of nonconventional monocytes, and a general lymphopenia that, however, was accompanied by a rise in plasmablasts, activated follicular T-helper cells, and non-naive Granzyme B+FasL+, EomeshighTCF-1high, PD-1+CD8+ Tc1 cells. Virus-induced lymphopenia worsened cancer-associated lymphocyte loss, and low lymphocyte counts correlated with chronic SARS-CoV-2 RNA shedding, COVID-19 severity, and a higher risk of cancer-related death in the first and second surge of the pandemic. Lymphocyte loss correlated with significant changes in metabolites from the polyamine and biliary salt pathways as well as increased blood DNA from Enterobacteriaceae and Micrococcaceae gut family members in long-term viral carriers. We surmise that cancer therapies may exacerbate the paradoxical association between lymphopenia and COVID-19-related immunopathology, and that the prevention of COVID-19-induced lymphocyte loss may reduce cancer-associated death.

COVID-19 is a systemic vascular hemopathy: insight for mechanistic and clinical aspects.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is presenting as a systemic disease associated with vascular inflammation and endothelial injury. Severe forms of SARS-CoV-2 infection induce acute respiratory distress syndrome (ARDS) and there is still an ongoing debate on whether COVID-19 ARDS and its perfusion defect differs from ARDS induced by other causes. Beside pro-inflammatory cytokines (such as interleukin-1 ß [IL-1ß] or IL-6), several main pathological phenomena have been seen because of endothelial cell (EC) dysfunction: hypercoagulation reflected by fibrin degradation products called D-dimers, micro- and macrothrombosis and pathological angiogenesis. Direct endothelial infection by SARS-CoV-2 is not likely to occur and ACE-2 expression by EC is a matter of debate. Indeed, endothelial damage reported in severely ill patients with COVID-19 could be more likely secondary to infection of neighboring cells and/or a consequence of inflammation. Endotheliopathy could give rise to hypercoagulation by alteration in the levels of different factors such as von Willebrand factor. Other than thrombotic events, pathological angiogenesis is among the recent findings. Overexpression of different proangiogenic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF-2) or placental growth factors (PlGF) have been found in plasma or lung biopsies of COVID-19 patients. Finally, SARS-CoV-2 infection induces an emergency myelopoiesis associated to deregulated immunity and mobilization of endothelial progenitor cells, leading to features of acquired hematological malignancies or cardiovascular disease, which are discussed in this review. Altogether, this review will try to elucidate the pathophysiology of thrombotic complications, pathological angiogenesis and EC dysfunction, allowing better insight in new targets and antithrombotic protocols to better address vascular system dysfunction. Since treating SARS-CoV-2 infection and its potential long-term effects involves targeting the vascular compartment and/or mobilization of immature immune cells, we propose to define COVID-19 and its complications as a systemic vascular acquired hemopathy.

Lack of antibodies against seasonal coronavirus OC43 nucleocapsid protein identifies patients at risk of critical COVID-19.

BACKGROUND: The vast majority of COVID-19 patients experience a mild disease. However, a minority suffers from critical disease with substantial morbidity and mortality. OBJECTIVES: To identify individuals at risk of critical COVID-19, the relevance of a seroreactivity against seasonal human coronaviruses was analyzed. METHODS: We conducted a multi-center non-interventional study comprising 296 patients with confirmed SARS-CoV-2 infections from four tertiary care referral centers in Germany and France. The ICU group comprised more males, whereas the outpatient group contained a higher percentage of females. For each patient, the serum or plasma sample obtained closest after symptom onset was examined by immunoblot regarding IgG antibodies against the nucleocapsid protein (NP) of HCoV 229E, NL63, OC43 and HKU1. RESULTS: Median age was 60 years (range 18-96). Patients with critical disease (n=106) had significantly lower levels of anti-HCoV OC43 nucleocapsid protein (NP)-specific antibodies compared to other COVID-19 inpatients (p=0.007). In multivariate analysis (adjusted for age, sex and BMI), OC43 negative inpatients had an increased risk of critical disease (adjusted odds ratio (AOR) 2.68 [95% CI 1.09 - 7.05]), higher than the risk by increased age or BMI, and lower than the risk by male sex. A risk stratification based on sex and OC43 serostatus was derived from this analysis. CONCLUSIONS: Our results suggest that prior infections with seasonal human coronaviruses can protect against a severe course of COVID-19. Therefore, anti-OC43 antibodies should be measured for COVID-19 inpatients and considered as part of the risk assessment for each patient. Hence, we expect individuals tested negative for anti-OC43 antibodies to particularly benefit from vaccination against SARS-CoV-2, especially with other risk factors prevailing.

Placental growth factor level in plasma predicts COVID-19 severity and in-hospital mortality.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a respiratory disease associated with vascular inflammation and endothelial injury. OBJECTIVES: To correlate circulating angiogenic markers vascular endothelial growth factor A (VEGF-A), placental growth factor (PlGF), and fibroblast growth factor 2 (FGF-2) to in-hospital mortality in COVID-19 adult patients. METHODS: Consecutive ambulatory and hospitalized patients with COVID-19 infection were enrolled. VEGF-A, PlGF, and FGF-2 were measured in each patient ≤48 h following admission. RESULTS: The study enrolled 237 patients with suspected COVID-19: 208 patients had a positive diagnostic for COVID-19, of whom 23 were mild outpatients and 185 patients hospitalized after admission. Levels of VEGF-A, PlGF, and FGF-2 significantly increase with the severity of the disease (P < .001). Using a logistic regression model, we found a significant association between the increase of FGF-2 or PlGF and mortality (odds ratio [OR] 1.11, 95% confidence interval [CI; 1.07-1.16], P < .001 for FGF-2 and OR 1.07 95% CI [1.04-1.10], P < .001 for PlGF) while no association were found for VEGF-A levels. Receiver operating characteristic curve analysis was performed and we identified PlGF above 30 pg/ml as the best predictor of in-hospital mortality in COVID-19 patients. Survival analysis for PlGF confirmed its interest for in-hospital mortality prediction, by using a Kaplan-Meier survival curve (P = .001) and a Cox proportional hazard model adjusted to age, body mass index, D-dimer, and C-reactive protein (3.23 95% CI [1.29-8.11], P = .001). CONCLUSION: Angiogenic factor PlGF is a relevant predictive factor for in-hospital mortality in COVID-19 patients. More than a biomarker, we hypothesize that PlGF blocking strategies could be a new interesting therapeutic approach in COVID-19.

Metabolomic analyses of COVID-19 patients unravel stage-dependent and prognostic biomarkers.

The circulating metabolome provides a snapshot of the physiological state of the organism responding to pathogenic challenges. Here we report alterations in the plasma metabolome reflecting the clinical presentation of COVID-19 patients with mild (ambulatory) diseases, moderate disease (radiologically confirmed pneumonitis, hospitalization and oxygen therapy), and critical disease (in intensive care). This analysis revealed major disease- and stage-associated shifts in the metabolome, meaning that at least 77 metabolites including amino acids, lipids, polyamines and sugars, as well as their derivatives, were altered in critical COVID-19 patient's plasma as compared to mild COVID-19 patients. Among a uniformly moderate cohort of patients who received tocilizumab, only 10 metabolites were different among individuals with a favorable evolution as compared to those who required transfer into the intensive care unit. The elevation of one single metabolite, anthranilic acid, had a poor prognostic value, correlating with the maintenance of high interleukin-10 and -18 levels. Given that products of the kynurenine pathway including anthranilic acid have immunosuppressive properties, we speculate on the therapeutic utility to inhibit the rate-limiting enzymes of this pathway including indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase.

Circulating Von Willebrand factor and high molecular weight multimers as markers of endothelial injury predict COVID-19 in-hospital mortality.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a respiratory disease associated with endotheliitis and microthrombosis. OBJECTIVES: To correlate endothelial dysfunction to in-hospital mortality in a bi-centric cohort of COVID-19 adult patients. METHODS: Consecutive ambulatory and hospitalized patients with laboratory-confirmed COVID-19 were enrolled. A panel of endothelial biomarkers and von Willebrand factor (VWF) multimers were measured in each patient ≤ 48 h following admission. RESULTS: Study enrolled 208 COVID-19 patients of whom 23 were mild outpatients and 189 patients hospitalized after admission. Most of endothelial biomarkers tested were found increased in the 89 critical patients transferred to intensive care unit. However, only von Willebrand factor antigen (VWF:Ag) scaled according to clinical severity, with levels significantly higher in critical patients (median 507%, IQR 428-596) compared to non-critical patients (288%, 230-350, p < 0.0001) or COVID-19 outpatients (144%, 133-198, p = 0.007). Moreover, VWF high molecular weight multimers (HMWM) were significantly higher in critical patients (median ratio 1.18, IQR 0.86-1.09) compared to non-critical patients (0.96, 1.04-1.39, p < 0.001). Among all endothelial biomarkers measured, ROC curve analysis identified a VWF:Ag cut-off of 423% as the best predictor for in-hospital mortality. The accuracy of VWF:Ag was further confirmed in a Kaplan-Meier estimator analysis and a Cox proportional Hazard model adjusted on age, BMI, C-reactive protein and D-dimer levels. CONCLUSION: VWF:Ag is a relevant predictive factor for in-hospital mortality in COVID-19 patients. More than a biomarker, we hypothesize that VWF, including excess of HMWM forms, drives microthrombosis in COVID-19.

No impact of cancer and plague-relevant FPR1 polymorphisms on COVID-19.

Formyl peptide receptor 1 (FPR1) is a pattern-recognition receptor that detects bacterial as well as endogenous danger-associated molecular patterns to trigger innate immune responses by myeloid cells. A single nucleotide polymorphism, rs867228 (allelic frequency 19-20%), in the gene coding for FPR1 accelerates the manifestation of multiple carcinomas, likely due to reduced anticancer immunosurveillance secondary to a defect in antigen presentation by dendritic cells. Another polymorphism in FPR1, rs5030880 (allelic frequency 12-13%), has been involved in the resistance to plague, correlating with the fact that FPR1 is the receptor for Yersinia pestis. Driven by the reported preclinical effects of FPR1 on lung inflammation and fibrosis, we investigated whether rs867228 or rs5030880 would affect the severity of coronavirus disease-19 (COVID-19). Data obtained on patients from two different hospitals in Paris refute the hypothesis that rs867228 or rs5030880 would affect the severity of COVID-19.

Immune responses during COVID-19 infection.

Over the past 16 years, three coronaviruses (CoVs), severe acute respiratory syndrome CoV (SARS-CoV) in 2002, Middle East respiratory syndrome CoV (MERS-CoV) in 2012 and 2015, and SARS-CoV-2 in 2020, have been causing severe and fatal human epidemics. The unpredictability of coronavirus disease-19 (COVID-19) poses a major burden on health care and economic systems across the world. This is caused by the paucity of in-depth knowledge of the risk factors for severe COVID-19, insufficient diagnostic tools for the detection of SARS-CoV-2, as well as the absence of specific and effective drug treatments. While protective humoral and cellular immune responses are usually mounted against these betacoronaviruses, immune responses to SARS-CoV2 sometimes derail towards inflammatory tissue damage, leading to rapid admissions to intensive care units. The lack of knowledge on mechanisms that tilt the balance between these two opposite outcomes poses major threats to many ongoing clinical trials dealing with immunostimulatory or immunoregulatory therapeutics. This review will discuss innate and cognate immune responses underlying protective or deleterious immune reactions against these pathogenic coronaviruses.

Elevated Calprotectin and Abnormal Myeloid Cell Subsets Discriminate Severe from Mild COVID-19.

Blood myeloid cells are known to be dysregulated in coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2. It is unknown whether the innate myeloid response differs with disease severity and whether markers of innate immunity discriminate high-risk patients. Thus, we performed high-dimensional flow cytometry and single-cell RNA sequencing of COVID-19 patient peripheral blood cells and detected disappearance of non-classical CD14LowCD16High monocytes, accumulation of HLA-DRLow classical monocytes (Human Leukocyte Antigen - DR isotype), and release of massive amounts of calprotectin (S100A8/S100A9) in severe cases. Immature CD10LowCD101-CXCR4+/- neutrophils with an immunosuppressive profile accumulated in the blood and lungs, suggesting emergency myelopoiesis. Finally, we show that calprotectin plasma level and a routine flow cytometry assay detecting decreased frequencies of non-classical monocytes could discriminate patients who develop a severe form of COVID-19, suggesting a predictive value that deserves prospective evaluation.