Endotheliopathy in COVID-19-associated coagulopathy: evidence from a single-centre, cross-sectional study

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Salient features:

This article focuses on:

  • Objective
  • Methods
  • Study design and participants
  • Procedures
  • Statistical analysis
  • Results
  • Discussion
  • Limitations
  • Conclusion

Detailed summary:

Objective:

  • Primary objective: To do exploratory analyses of haemostatic factors and markers of endothelial cell and platelet activation in patients with COVID-19 admitted to the intensive care unit (ICU) and those with COVID-19 not admitted to ICU.
  • Secondary objective: To assess the relationship between these markers and clinical outcomes, including in-hospital mortality and discharges from the hospital.

Methods :

Study design and participants

  • A cross-sectional study was done of adult (≥18 years) patients hospitalised in Yale-New Haven Hospital between April 13 and April 24, 2020, with a confirmed diagnosis of COVID-19, as measured using PCR assays on nasopharyngeal swab samples.
  • Patients were categorised according to whether they had been admitted to a medical ICU or a specialised non-ICU COVID-19 floor in our hospital.
  • To ensure a maximum separation of severity of illness between the ICU and non-ICU cohort and to reduce bias where possible, ICU patients who were intubated and non-ICU patients with minimal oxygen support requirement, were included.
  • Most ICU patients required ventilator support at the time laboratory measurements were taken, whereas all non-ICU patients were on no more than 3 L of supplemental oxygen.
  • Patient characteristics including age, sex, ethnicity, major comorbidities, laboratory studies, and treatments were recorded.
  • Clinical outcomes including hospital discharge and in-hospital death were assessed on May 23, 2020.
  • An additional 13 asymptomatic, non-hospitalised individuals who volunteered to serve as controls for a subset of plasma biomarker measurements were also included.

Procedures:

  • Venous blood was collected from hospitalised patients and non-hospitalised controls and processed according to standard laboratory techniques.
  • Due to diurnal variations in plasminogen activator inhibitor-1 (PAI-1), for hospitalised patients, blood specimens were collected with the first scheduled morning draw, which in all but five patients occurred between 0300 h and 0700 h.
  • Blood collection from non-hospitalised controls was not subject to the same time restrictions and occurred between 1100 h and 1400 h.
  • For coagulation and endothelial cell and platelet marker measurements, blood was collected in 3·2% sodium citrate tubes and centrifuged at 4000 rpm for 20 min.
  • Measurements of D-dimer and fibrinogen were done at our institution’s clinical laboratory using a BCS XP System with manufacturer reagents and controls per laboratory protocol.
  • VWF antigen and VWF activity were measured using a latex enhanced immunoassay; the VWF antigen assay used polystyrene particles coated with rabbit polyclonal antibody directed against VWF, while the VWF activity assay used a lyophilised suspension of polystyrene latex particles coated with anti-VWF mouse monoclonal antibody directed against the platelet-binding epitope of VWF.
  • Factor VIII activity was measured using a one-stage partial thromboplastin time-based test in which patient plasma was diluted and added to reagent or manufacturer lyophilised, factor VIII-deficient plasma, with correction of the partial thromboplastin time being proportional to the factor VIII activity percentage, as derived from a calibration curve.
  • For measurements of PAI-1 and thrombinantithrombin complexes (TAT), plasma supernatant was frozen, then sent to national Clinical Laboratory Improvement Amendments-certified reference laboratories, where testing for these analytes was done by ELISA. Soluble P-selectin, soluble CD40 ligand (sCD40L), and soluble thrombomodulin were measured using ELISA assays.
  • These assays were run on samples that had adequate volume remaining after the in-hospital testing described previously was completed.
  • For soluble P-selectin and soluble thrombomodulin, samples were diluted in a 1:4 ratio before addition to ELISA plates.
  • For sCD40L, samples were undiluted. Assays were done in duplicates according to the manufacturer’s instructions. Biomarkers are presented with their standard reference range.
  • This includes VWF antigen and activity, which are reported as percentages compared with calibration curves using values obtained from the standardised reference population used for clinical laboratory testing throughout our hospital system.

Statistical analysis:

  • The differences between coagulation studies and endothelial cell markers in ICU patients and nonICU patients, and controls where applicable, using the unpaired two-sided t test if samples conformed to the normal distribution, with Welch correction for unequal variances.
  • For samples that did not satisfy the normal distribution, unpaired two-tailed MannWhitney U test was used.
  • D’Agostino-Pearson and Anderson-Darling tests of normality was also used.
  • Endothelial cell parameters in ICU patients, non-ICU patients, and healthy controls were compared using one-way ANOVA with posthoc multiple comparisons of means for samples with the normal distribution and Kruskal-Wallis rank test with post-hoc multiple comparisons of mean ranks for samples that did not conform to the normal distribution.
  • The associations of endothelial cell markers with each other and with mortality was evaluated using non-parametric Spearman correlation to account for non-normal distribution of all interrogated parameters except soluble P-selectin.
  • Given the specificity of soluble thrombomodulin to endothelial cells and findings on correlation analysis, patients were segregated into two groups according to their soluble thrombomodulin test results.
  • The median soluble thrombomodulin value was chosen for the entire cohort as a classification cutoff because there were no predefined upper and lower limits for the soluble thrombomodulin assay.
  • Kaplan–Meier analyses was done and compared in-hospital survival of patients with high or low soluble thrombomodulin values using the log-rank test for both full and ICU cohorts.
  • The rates of hospital discharges in patients with high and low soluble thrombomodulin was compared using the χ² test of proportions.
  • All statistical analysis was done using GraphPad Prism.

Results :

  • 68 adult patients (48 ICU, 20 non-ICU) were included in the final study cohort.
  • 13 non-hospitalised, asymptomatic controls were included as a comparator group for measurements of specific endothelial cell and platelet biomarkers that did not have standard reference ranges.
  • All coagulation parameters were measured in all 68 patients, whereas endothelial cell and platelet biomarkers were measured in 50 patients for whom blood remained after coagulation testing.
  • Obesity, hypertension, hyperlipidaemia, and diabetes were common, affecting up to a half of all patients.
  • Three patients had active malignancy and three had cirrhosis, while one patient had a history of cirrhosis that resolved after liver transplant; all such patients were in the ICU cohort.
  • 41 patients were on prophylactic-dose anticoagulation, 13 on intermediate-dose anticoagulation and 11 on therapeutic anticoagulation; three patients were not anticoagulated.
  • 48 patients, including all but two ICU patients, had received tocilizumab before their coagulation factors were measured.
  • Among all patient characteristics analysed, only sex showed a significant difference in distribution between ICU and non-ICU subgroups.
  • In terms of the measurement of coagulation factors, endogenous anticoagulants, and fibrinolytic enzymes, there were near-universal elevations in PAI-1, with preserved α2-antiplasmin activity among both ICU and non-ICU patients.
  • Non-ICU patients had elevated VWF antigen, VWF activity, and factor VIII activity, with 16 patients, 15 patients, and 18 patients above the normal range, respectively.
  • These levels were further elevated in ICU patients, with 35 ICU patients having VWF activities above the limit of detection.
  • For endogenous anticoagulants, antithrombin activity, protein C activity, and protein S activity in ICU and non-ICU patients were generally preserved.
  • Five patients had an antithrombin activity below 70%, all of whom were in the ICU and had cirrhosis or bacterial or fungal sepsis.
  • D-dimer and TAT concentrations were elevated in the entire study cohort, with significantly higher values in ICU patients compared with non-ICU patients.
  • D-dimer and TAT were also significantly correlated in the entire study cohort.
  • Soluble P-selectin, sCD40L and soluble thrombomodulin in 40 ICU and 10 non-ICU patients and in 13 non-COVID-19, non-hospitalised controls were measured.
  • Both soluble P-selectin and sCD40L were significantly elevated in ICU patients compared with controls.
  • Soluble P-selectin concentrations were also significantly higher in ICU patients than in non-ICU patient.
  • The increase in soluble thrombomodulin in ICU patients compared with controls was not significant.
  • Concentrations of both soluble P-selectin and soluble thrombomodulin were significantly correlated with VWF antigen, consistent with their common source from endothelial cells.
  • Both VWF antigen and soluble thrombomodulin were significantly correlated with mortality among all patients and soluble thrombomodulin remained significantly correlated with mortality when the analysis was restricted to ICU patients.
  • Of the 50 patients in whom soluble thrombomodulin was measured, the median value was 3·26 ng/mL which was used to define two groups of 25 patients each: low soluble thrombomodulin and high soluble thrombomodulin.
  • 22 patients with low soluble thrombomodulin were discharged from the hospital compared with 13 patients with high soluble thrombomodulin.
  • Among the 40 ICU patients in whom soluble thrombomodulin was measured, patients with low soluble thrombomodulin were also more likely than patients with high soluble thrombomodulin to be discharged from the hospital.
  • In a survival analysis, in-hospital mortality was significantly lower among patients with low soluble thrombomodulin compared with patients with high soluble thrombomodulin in the entire cohort and among ICU patients.

Discussion :

  • In this study of laboratory features of COVID-19- associated coagulopathy, several findings were reported and suggest that endotheliopathy and platelet activation are important features of COVID-19 in hospitalised patients.
  • VWF is also elevated in non-critically ill patients with COVID-19.
  • Critical illness is associated with further elevations in VWF, as well as increases in soluble P-selectin and sCD40L when compared with controls.
  • Together, these results provide biochemical evidence that endotheliopathy and platelet activation are ubiquitous in COVID-19- associated coagulopathy and might play key roles in the progression of disease.
  • Soluble thrombomodulin is an endothelial transmembrane glycoprotein that is released upon endothelial disruption or injury.
  • Among healthy individuals, circulating thrombomodulin is produced by physiological cleavage and shedding of membrane-bound thrombomodulin.
  • In hyperinflammatory states, elevated soluble thrombomodulin concentrations are thought to be secondary to direct endothelial cell damage.
  • Soluble thrombomodulin is a predictive marker of mortality in COVID-19, coupled with the observed increases in VWF and soluble P-selectin specifically in critically ill patients, support a model in which endotheliopathy is an important event in the transition to critical illness and death in patients with COVID-19.
  • The finding of preserved antifibrinolytic α2-antiplasmin activity in patients with COVID-19, together with the preserved endogenous anticoagulant activity (antithrombin activity, protein C activity, and protein S activity) observed both here and in other studies, supports the notion that COVID-19-associated coagulopathy is mechanistically distinct from disseminated intravascular coagulation.
  • PAI-1 has been linked to elevated IL-6 in other contexts and has been suggested to play a role in severe acute respiratory distress syndrome.
  • The finding of elevated PAI-1 in critically ill and non-critically ill patients with COVID-19 further supports a picture of endotheliopathy, given the role of endothelial cells as the primary source of PAI-1.
  • Elevation of PAI-1 also suggests the possibility that classical fibrinolysis might be inhibited in COVID-19- associated coagulopathy.
  • Measurements of tissue and urokinase-type plasminogen activator and plasminogen levels might help to further assess this, although the interpretation of such levels might not be relevant if the observed elevation in PAI-1 is enough to overcome a possible increase in plasminogen activator.
  • COVID-19-associated coagulopathy is an endotheliopathy that results in augmented VWF release, platelet activation, and hypercoagulability, leading to the clinical prothrombotic manifestations of COVID-19-associated coagulopathy, which can include venous, arterial, and microvascular thrombosis.
  • The factors responsible for this endotheliopathy and platelet activation are uncertain but could include direct viral infection of endothelial cells, collateral damage to the tissue as a result of immune infiltration and activation, complement activation, or any number of inflammatory cytokines believed to play a role in COVID-19 disease.
  • A central role for endothelial cells and platelets has been characterised in other forms of critical illnesses, including septic shock, acute respiratory distress syndrome, and veno-occlusive disease following haematopoietic stem-cell transplantation, where increased markers of endotheliopathy and platelet activation are observed.
  • Disseminated intravascular coagulation also involves substantial endothelial cell and platelet activation but is mechanistically distinct from COVID-19-associated coagulopathy, in part as a result of unmitigated activation of the coagulation cascade and resultant endogenous anticoagulant consumption in disseminated intravascular coagulation.
  • In models of septic shock, elevated VWF antigen and activity can be accompanied by reductions in the ADAMTS13 metalloproteinase responsible for cleaving ultra-large VWF multimers into smaller VWF forms, leading to thrombotic microangiopathy via a mechanism distinct from disseminated intravascular coagulation.
  • The findings of endotheliopathy and platelet activation point to antiplatelet therapy or endothelial cell modification as potential therapeutic targets in addition to traditional anticoagulation targeting thrombin generation.
  • Dipyridamole, defibrotide, eculizumab, and other agents with endothelial cell-modifying effects might have therapeutic potential.
  • In light of the preliminary finding that soluble thrombomodulin might predict mortality in patients with COVID-19, measurements of soluble thrombomodulin could be useful in identifying patients who might benefit the most from these therapies.

Limitations:

  • A small sample size
  • The heterogeneity of patient groups, with a wide range of ages, comorbidities, length of hospitalisation; and the fact that each patient’s laboratory measures for the purposes of this study were assessed once during their hospital stay, as well as limited generalisability of the findings in a single-centre study.
  • Further studies incorporating serial sampling of relevant biomarkers over time will yield additional mechanistic insights into the biology of endotheliopathy in COVID-19.
  • The widespread use of tocilizumab, particularly among ICU patients, might have led to an underestimation of the degree of endotheliopathy in ICU patients in this study, as previous literature has reported an association of IL-6 signalling with endothelial cell activation and VWF release, and other studies have shown that IL-6 blockade reduces TAT and PAI-1 concentrations.
  • Five patients had blood samples drawn outside of the 0300–0700 h window, which could potentially affect PAI-1 concentrations given the circadian fluctuations of this enzyme; however, in all but one of these instances, PAI-1 was high, indicating that the time of PAI-1 measurement did not have an impact on PAI-1 concentration.
  • Further evidence of platelet activation, beyond increased circulating concentrations of soluble P-selectin and sCD40L, will be needed to characterise the extent of platelet activation in COVID-19.

Conclusion :

  • Measurements of VWF, PAI-1, soluble thrombomodulin, soluble P-selectin, and sCD40L, found that endotheliopathy and platelet activation might be important factors in the pathophysiology of COVID-19-associated coagulopathy.
  • Additional studies are required to elucidate the factors that drive endotheliopathy in critical illness and to explore the possible therapeutic effects of adding antiplatelet or endothelial cell-modifying therapy

Reference Link: https://www.thelancet.com/journals/lanhae/article/PIIS2352-3026(20)30216-7/fulltext

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