Circulating EV and platelet releasate proteomes mirror the hyperactive, pro-inflammatory state of COVID-19 patients and may be adjunctive markers of COVID-19 severity

Background: COVID-19 infection can present with a heightened inflammatory state and platelet hyperactivation, however the roles of circulating extracellular vesicles (cEVs;pro-coagulant and pro-inflammatory mediators of intercellular communication) and the platelet releasate (PR;cargo released from activated platelets) remain uncharacterised. We hypothesised that the cEVs and PR proteomic signatures are altered in COVID-19 patients. Aim(s): To characterise and contrast paired cEVs and PR proteomes from COVID-19 patients and controls. Method(s): Ethical approval for this study was granted by the Mater Misericordiae University Hospital, Ireland. PR and platelet poor plasma (PPP) samples from COVID-19 patients requiring intensive care (severe, n = 6) or hospital care (non-severe, n = 6), and appropriate controls (n = 6 healthy controls and n = 5 COVID-negative hospitalised patients) were obtained with informed consent. cEVs were enriched from PPP by ultrafiltration. Samples were analysed by mass spectrometry and immunoassays. Result(s): Comparative statistical analysis revealed substantial overlap in the proteomic signatures of the cEVs and PR across COVID-19 patients and controls. Profound increases in circulating levels of pro-inflammatory proteins correlated with severity of infection, alongside significant dysregulation of proteins involved in blood coagulation including von Willebrand factor and fibrinogen. We also found evidence of 'spent' platelets in COVID-19 patients, with reduced platelet factor 4 (PF4) levels in the PR and concomitant increase of PF4 in plasma. Strikingly, several platelet-released proteins exhibited strong correlation with routinely obtained haematological parameters and together clearly differentiated between a severe and non-severe hospitalised COVID-19 patients. Conclusion(s): These preliminary data suggest that severe COVID-19 patients have a distinct cEV and PR profile and that proteomic signatures may be a useful tool in assessing the severity of COVID-19 when characterized in larger sample sizes. Our findings may lay the foundations to provide healthcare professionals with an adjunctive and minimally invasive tool towards assessing COVID-19 severity.

Anticoagulation as a therapeutic strategy for hospitalised patients with COVID-19

The COVID-19 pandemic has devastated the global community and continues to cause significant morbidity and mortality worldwide. The development of effective vaccines has represented a major step towards reducing transmission and illness severity but significant challenges remain, particularly in regions where vaccine access has been limited. COVID-19 is associated with hypercoagulability and increased risk of thrombosis, with greatest risk among the critically ill. Interestingly, early observational data suggested that anticoagulant therapy might improve clinical outcomes, aside from thrombotic events, in patients with COVID-19. In this review we summarise data generated from three published randomised clinical trials which have sought to determine the effect of therapeutic heparin anticoagulation on efficacy and safety outcomes in hospitalised patients with COVID-19: the multiplatform REMAP-CAP, ACTIV-4a and ATTACC randomised controlled trials and the RAPID trial. In the multiplatform REMAP-CAP, ACTIV-4a and ATTACC randomised controlled trials, therapeutic heparin was not associated with benefit in critically ill patients with COVID-19 compared with usual care (adjusted proportional odds ratio (OR) for increased organ-support free days up to day 21: 0.83;95% credible interval, 0.67–1.03, posterior probability of futility 99.9%). Conversely, among hospitalised patients without critical illness, therapeutic heparin was associated with an increased probability of organ support-free days alive (adjusted OR, 1.27;95% credible interval, 1.03–1.58). The RAPID trial also evaluated the effect of therapeutic heparin compared with prophylactic heparin in non-critically ill patients. In this study, therapeutic heparin did not significantly reduce the odds of the primary composite outcome (death, mechanical ventilation or intensive care unit admission) (OR 0.69;95% confidence interval [CI], 0.43 to 1.10;p = 0.12) but was associated with a significant reduction in all-cause mortality [OR, 0.22 (95%-CI, 0.07 to 0.65)]. Collectively these studies suggest that therapeutic anticoagulation with heparin may reduce the severity of illness and potentially even confer a survival benefit in hospitalised, non-critically ill patients with COVID-19. No benefit for therapeutic anticoagulation with heparin was evident in critically ill patients with COVID-19. Therefore, while the results of additional studies in this evolving field are pending, it is important to approach decisions regarding therapeutic heparin in moderately ill hospitalised patients with COVID-19 in a measured and individualised manner.

COVID-19 Is Associated with Endothelial Dysfunction and Enhanced Plasma Thrombin Generation Despite Pharmacological Thromboprophylaxis

Background Hospitalised patients with severe COVID-19 (requiring critical care level support) appear to be at increased risk of thrombosis despite standard pharmacological thromboprophylaxis. The magnitude of thrombotic risk in patients with COVID-19 of moderate severity (not requiring critical care) is less clear. The optimal approach to thromboprophylaxis (and the role of intensified thromboprophylaxis) remains to be determined. Evidence of endothelial dysfunction has been widely reported in COVID-19 (particularly in severe COVID) and this may contribute to hypercoagulability. Aim To assess differences in patterns of hypercoagulability and endothelial dysfunction between a group of patients with moderate COVID-19 and a group of age-matched hospitalized patients (SARS-CoV-2 PCR negative) receiving low molecular weight heparin (LMWH) thromboprophylaxis. Methods Blood was collected from individuals admitted to hospital with COVID-19 of moderate severity (not requiring critical care level support) and a group of age-matched patients admitted with infective/inflammatory illness (SARS-CoV-2 PCR negative). All subjects received standard-dose LMWH thromboprophylaxis, with blood drawn at 12 hours post-dose (and with measurement of anti-FXa activity levels). Circulating levels of endothelial & fibrinolytic markers including ICAM, PAI-1, VCAM, soluble thrombomodulin (sTM), and tissue plasminogen activator (tPA) were determined by ELISA. Thrombin generation (TG) in platelet-poor plasma was assessed by calibrated automated thrombography in the presence of tissue factor (Final concentration, 1pM & 5pM), thrombomodulin (TM) (Final concentration, 6.25nM), and an inhibitory anti-tissue factor pathway inhibitor antibody (anti-TFPI;Final concentration 100μg/mL). Results 14 COVID-19 positive subjects and 11 hospitalized controls were recruited. There were no differences in mean age (69.7±4.5 vs 61.6±4.7 years;p= 0.2) or mean Body mass index (25.7±1.1 vs 22.7±1.2 Kg/m2;p=0.1) between groups. No COVID-19 patient or control required critical care support. In the COVID group, radiological evidence of pneumonitis [diffuse (n=3) or peripheral infiltrates (n=7)] was present in the majority of cases. None of the COVID-19 cases were requiring supplemental oxygen at the time of recruitment. All controls were admitted with either respiratory or urinary infection [radiological evidence of pneumonia in 4/11;supplemental oxygen requirement in 2/11, (28-36% FiO2 via nasal cannula)]. Plasma levels of sTM, ICAM, PAI-1 & VCAM were similar in both groups. Levels of t-PA were significantly higher in the COVID group (8.31±4.35 vs 4.91±2.37 ng/mL;p= 0.005). Despite similar plasma anti-Xa activity in both groups (0.06 vs 0.04 IU/mL;p=0.2), mean endogenous thrombin potential (ETP) was significantly higher in the COVID group (1929±119.7 vs 1528±138.9 nM*min;p=0.02), although peak thrombin was similar (173.6±26 vs 161.5±31nM). ETP-TM ratio was similar between groups (0.3±0.1 vs 0.2±0.1;p=0.3). Despite increased ETP, the lag time to thrombin generation was significantly prolonged in the COVID group (8.3±0.6 vs 5.8±0.5 mins, p= 0.006). This pattern has previously been observed in vascular diseases associated with altered plasma tissue factor pathway inhibitor (TFPI) activity. In the presence of an anti-TFPI antibody, the difference in lagtime between groups was attenuated (4.7±0.2 vs 3.5±0.1 mins;p= 0.002) and the difference in overall thrombin generation (delta TG) between both groups became significantly increased (Fig.1). Conclusion Plasma thrombin generation is enhanced in patients with non-severe COVID-19 despite pharmacological thromboprophylaxis. Endothelial dysfunction is also observed in this group and appears to modulate parameters of plasma thrombin generation. The clinical implications of these observations are not known although clinical studies of intensified thromboprophylaxis in attenuating thrombotic risk and other complications are ongoing. Fig 1. Inhibition of TFPI activity enhances thrombin generation in COVID-19. n the presence of an inhibitory anti-TFPI antibody, peak plasma thrombin generation was enhanced in COVID-19 in contrast to that observed among SARS-CoV-2 PCR negative hospitalised patients (339.6+25.2 vs 247.4+10.1, p=0.01). [Formula presented] Disclosures: Maguire: Actelion: Research Funding;Bayer Pharma: Research Funding. Ni Ainle: Daiichi-Sankyo: Research Funding;Actelion: Research Funding;Leo Pharma: Research Funding;Bayer Pharma: Research Funding. Kevane: Leo Pharma: Research Funding.

Tissue factor-stimulated plasma thrombin generation is enhanced in hospitalised patients with COVID-19 despite pharmacological thromboprophylaxis

Background : COVID-19 confers an increased risk of thrombosis however the mechanisms underlying this coagulopathy and the optimal approach to thromboprophylaxis are unknown. Thrombotic risk is likely greatest among patients with severe COVID-19 requiring critical organ support however patients with moderate disease may be at risk and might also benefit from intensified thromboprophylaxis. Aims : To characterise plasma thrombin generation (TG) in patients with COVID-19 of moderate severity, treated with pharmacological thromboprophylaxis. Methods : Blood was collected from individuals admitted to hospital with COVID-19 of moderate severity (not requiring critical care support) and a group of age-matched patients admitted with infective/ inflammatory illness (negative for COVID-19). All subjects received standard dose low molecular weight heparin (LMWH) thromboprophylaxis with samples taken at time of predicted trough levels (confirmed by measuring anti-FXa activity). TG in platelet-poor plasma was determined by calibrated automated thrombography in the presence/absence of tissue factor (TF) (ppp-LOW reagent, 1 pM TF & 4 μM phospholipid;MP-reagent, 4 μM phospholipid;Thrombinoscope BV™). Results : Fourteen COVID-19 positive subjects and 11 hospitalised COVID-19 negative controls were recruited. Mean trough plasma anti-Xa activity was similar in both groups (0.06 vs 0.04 IU/mL;P = 0.2). In the presence of TF, mean endogenous thrombin potential was significantly higher in the COVID group in comparison to controls (1929 ± 119.7 vs 1528 ± 138.9 nM∗min;P = 0.02). Peak thrombin was also higher in COVID-19 (267.3 ± 22.2 vs 208.6 ± 17.8 nM;P = 0.06). Despite increased TG overall, lagtime to TG was significantly prolonged in COVID-19 (8.1 ± 0.5 vs 6.2 ± 0.5 mins;P = 0.02). No difference in any parameter of TG was observed between groups in the absence of TF. Conclusions : Despite pharmacological thromboprophylaxis plasma TG is enhanced in COVID-19. The underlying mechanisms remain to be elucidated. Specific clinical implications of increased TG despite pharmacological thromboprophylaxis have yet to be determined although clinical trials evaluating intensified anticoagulant regimens in a similar population are ongoing.

Impact of COVID-19 on platelet activity and the platelet releasate

Background : Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected over 100 million globally to date. Although high rates of venous thromboembolism and evidence of COVID-19-induced endothelial dysfunction have been reported, the precise aetiology of the increased thrombotic risk associated with COVID-19 infection remains to be fully elucidated. Several studies to date suggest a role for platelets in COVID-19-associated thrombosis. Aims : To assess the impact of COVID-19 on platelet activity and to characterise the proteome of the platelet releasate from COVID-19 patients, compared with healthy controls. Methods : Ethical approval was granted by the Institutional Review Board of the Mater Misericordiae University Hospital. Haematologic parameters of patients with severe COVID-19 disease (requiring intensive care;n = 34), with non-severe disease (not requiring intensive care;n = 20) and in general medical in-patients without COVID-19 ( n = 20) were assessed. Platelet function and activity were evaluated by secretion and platelet marker analysis ( n = 6 each cohort). The proteome of the platelet releasate was assessed using label-free mass spectrometry. Results : We demonstrated agonist-induced ADP release was 30-to-90 fold higher in COVID-19 patients compared with hospitalized controls (Fig. 1) and circulating levels of platelet-factor 4 (PF4), soluble P-selectin (sP-selectin) and thrombopoietin (TPO) were also significantly elevated in COVID-19. This study shows that COVID-19 patients possess hyperactive circulating platelets combined with a decreased activation threshold. Mass spectrometry analysis identified over 400 proteins from the releasate of COVID-19 patients and controls, including a multitude of inflammatory, vasoactive and vesicular proteins. The release of a subset of highly-relevant platelet proteins was modified based on the severity of COVID-19 infection. controls (Fig. 1) and circulating levels of platelet-factor 4 (PF4), soluble P-selectin (sP-selectin) and thrombopoietin (TPO) were also significantly elevated in COVID-19. This study shows that COVID-19 patients possess hyperactive circulating platelets combined with a decreased activation threshold. Mass spectrometry analysis identified over 400 proteins from the releasate of COVID-19 patients and controls, including a multitude of inflammatory, vasoactive and vesicular proteins. The release of a subset of highly-relevant platelet proteins was modified based on the severity of COVID-19 infection.