key: cord-0960985-rxx0w56j
authors: Fogarty, Helen; Townsend, Liam; Morrin, Hannah; Ahmad, Azaz; Comerford, Claire; Karampini, Ellie; Englert, Hanna; Byrne, Mary; Bergin, Colm; O’Sullivan, Jamie M.; Martin‐Loeches, Ignacio; Nadarajan, Parthiban; Bannan, Ciaran; Mallon, Patrick W.; Curley, Gerard F.; Preston, Roger J.S.; Rehill, Aisling M.; McGonagle, Dennis; Ni Cheallaigh, Cliona; Baker, Ross I.; Renné, Thomas; Ward, Soracha E.; O’ Donnell, James S.
title: Persistent Endotheliopathy in the Pathogenesis of Long COVID Syndrome
date: 2021-08-10
journal: J Thromb Haemost
DOI: 10.1111/jth.15490
sha: 07db5124388d1273ce4d8c292dbc195a3437313c
doc_id: 960985
cord_uid: rxx0w56j

BACKGROUND: Persistent symptoms including breathlessness, fatigue and decreased exercise tolerance have been reported in patients after acute SARS‐CoV‐2 infection. The biological mechanisms underlying this ‘Long COVID’ syndrome remain unknown. However, autopsy studies have highlighted the key roles played by pulmonary endotheliopathy and microvascular immunothrombosis in acute COVID‐19. We hypothesized that endothelial cell activation may be sustained in convalescent COVID‐19 patients and contribute to Long COVID pathogenesis. PATIENTS AND METHODS: Fifty patients were reviewed at a median of 68 days following SARS‐CoV‐2 infection. In addition to clinical workup, acute phase markers, EC activation and NETosis parameters and thrombin generation were assessed. RESULTS: Thrombin generation assays revealed significantly shorter lag times (p<0.0001, 95% CI ‐2.57– ‐1.02min), increased endogenous thrombin potential (ETP) (p=0.04, 95% CI 15–416nM/min) and peak thrombin (p<0.0001, 95% CI 39–93nM) in convalescent COVID‐19 patients. These pro‐thrombotic changes were independent of ongoing acute phase response or active NETosis. Importantly, EC biomarkers including VWF:Ag, VWF propeptide (VWFpp) and Factor VIII (FVIII:C) were significantly elevated in convalescent COVID‐19 compared to controls (p=0.004, 95% CI 0.09–0.57IU/ml; p=0.009, 95% CI 0.06–0.5IU/ml; p=0.04, 95% CI 0.03–0.44IU/ml, respectively). In addition, plasma soluble thrombomodulin (sTM) levels were significantly elevated in convalescent COVID‐19 (p=0.02, 95% CI 0.01–2.7ng/ml). Sustained endotheliopathy was more frequent in older, comorbid patients and those requiring hospitalization. Finally, both plasma VWF:Ag and VWFpp levels correlated inversely with 6‐minute walk tests. CONCLUSIONS: Collectively, our findings demonstrate that sustained endotheliopathy is common in convalescent COVID‐19 and raise the intriguing possibility that this may contribute to Long COVID pathogenesis.

Recent studies have reported sustained symptoms in a significant proportion of patients following acute SARS-CoV-2 infection. 1,2 Patients with this 'Long COVID' syndrome complain of persistent breathlessness, fatigue and decreased exercise tolerance. 2 Although the biological mechanisms underlying these ongoing symptoms remain unknown, we recently reported that persistent increased D-dimer levels were present in approximately 25% of convalescent COVID-19 patients up to 4 months following the apparent resolution of their acute infection. 3 Importantly, these increased D-dimers were seen in a significant number of both hospitalized and nonhospitalized COVID-19 patients respectively. Similarly, von Meijenfeldt et al also observed persistently elevated D-dimers in convalescent COVID-19 patients at 4 months after hospital discharge. 4 Moreover, sustained prothrombotic changes in thrombin-generating capacity were also reported. Critically however, the biological mechanisms underlying these persistent procoagulant effects following acute COVID-19 remain poorly understood.

Post-mortem studies in acute COVID-19 have demonstrated disseminated thrombosis throughout the pulmonary vasculature. 5-7 These thrombi are platelet-and fibrin-rich, and also contain neutrophils, neutrophil extracellular traps (NETs) and activated FXII that triggers the contact pathway. 7, 8 Current evidence suggests pulmonary thrombi in patients with severe COVID-19 likely arise in situ within the lungs, rather than being embolic in origin. 7, 9 Autopsy studies have consistently highlighted that marked pulmonary endotheliopathy is a characteristic feature of severe COVID-19. 5,7-9 Consistent with these pathological findings, plasma markers of endothelial cell (EC) activation including von Willebrand factor antigen (VWF:Ag), 10-15 VWF propeptide (VWFpp) 16 and soluble thrombomodulin (sTM) 10 are all markedly elevated in patients with severe COVID-19. Importantly, these EC biomarkers also correlate with disease severity. 10, 12, 16, 17 Given the key roles of endotheliopathy and immuno-thrombosis in modulating the pathogenesis of acute SARS-CoV-2, 18 we hypothesized that persistent EC activation might be important in modulating ongoing pro-coagulant effects in convalescent COVID-19 patients and thereby contribute to the pathogenesis underpinning Long COVID.

This article is protected by copyright. All rights reserved METHODS Consecutive adult patients were enrolled from the post-COVID-19 review clinic in St James's Hospital (SJH), Dublin between May and September 2020. Patient were assessed at a minimum of 6 weeks following either symptom resolution in non-hospitalized patients or hospital discharge for those requiring admission. 3 Informed written consent was obtained from all participants and ethical approval was obtained from the SJH Research Ethics Committee. A control group of nonhospitalized asymptomatic controls (n=17, mean age 47 ± 12 years) were also recruited. Plasma VWF:Ag, VWF propeptide (VWFpp), Factor VIII activity (FVIII:C) and soluble thrombomodulin (sTM) were analysed as previously described. 16 Thrombin generation was performed in a Fluouroskan Ascent Fluorometer with Thrombinoscope software (Stago) using PPP Low reagent (1pM tissue factor, 4mM phospholipids) as before. 19 Additionally, the release of extracellular DNA (exDNA) was measured using the fluorescent DNA-intercalating dye Sytox Green (Invitrogen) and DNase activity was assessed by an in vitro NET degradation assay. 20, 21 Activation of the contact factor pathway was evaluated by photometric measurement using conversion of the chromogenic substrate S-2302 (Chromogenix). 22 Clinical assessment at time of outpatient review included: chest x-ray, 6-minute walk test (6MWT), measuring distance covered, lowest arterial oxygen saturation and maximal exertion (using a Modified Borg Scale). Fatigue scores were assessed using the Chalder fatigue scale. 23 Statistical analyses were performed using the Mann-Whitney U tests and the Spearman rank correlation in GraphPad Prism 9.0 (GraphPad Software, USA) with a p-value of <0.05 considered statistically significant.

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Fifty convalescent COVID-19 patients (mean age 50 ± 17 years) were assessed at a median of 68 (IQR 61.3 -72) days following COVID-19 symptom resolution or hospital discharge (Supp. and comorbidities were apparent in 31 patients (31/50, 62%) (Supp. Table 1 ). All hospitalized patients received weight-and renally-adjusted LMWH prophylaxis during their inpatient stay.

Conversely, non-hospitalized and discharged patients did not receive thromboprophylaxis and none of the cohort was on anticoagulation at time of convalescent clinic review. Convalescent COVID-19 patients also demonstrated a shorter time to peak compared with controls (median 9.8 min versus 13.8 min, p<0.0001, 95% CI -4.8 --2.7) (Fig. 1E) . To investigate whether ongoing contact pathway activation was responsible for the effects on thrombin generation, FXII activation was assessed. No significant differences in systemic FXIIa levels were seen between a subset of convalescent patients (n=20) and controls (n=17) (p=0.16, 95% CI -0.15-0.03) (Supp. Fig. 1C ). Elevated plasma FVIII:C levels have previously been reported in patients with acute COVID-19 and are known to influence thrombin generation. In the convalescent COVID-19 patient cohort, plasma FVIII:C levels remained significantly increased compared to controls (median 1.53 IU/ml versus 1.13 IU/ml; p=0.04, 95% CI 0.03-0.44) with 14 (28%) patients having FVIII:C levels > 1.5 IU/ml (Fig. 1F) . Notably, one patient with FVIII:C of 2.7 IU/ml at follow-up 84 days post-acute illness was later re-admitted with pulmonary embolism. In addition, FVIII:C levels correlated with peak thrombin generation (p=0.001) (Fig. 1G) . Together, these findings suggest that persistent increases in plasma FVIII levels contribute to ongoing increased thrombin generation potential in a significant proportion of convalescent COVID-19 patients.

We next investigated the mechanism(s) responsible for the sustained elevation in plasma FVIII:C levels in convalescent COVID-19 patients. Increased FVIII levels are associated with acute phase responses. 24 However, in contrast to the significant elevation seen in FVIII:C levels

This article is protected by copyright. All rights reserved in convalescent COVID-19 patients, acute phase markers (including C-reactive protein, neutrophil and white cell counts, IL-6 and sCD25 levels) had normalized in most patients (Supp . Table 1 Figs. 1 F-G) .

In normal plasma, the majority of FVIII circulates in high-affinity complex with VWF. Moreover, both FVIII and VWF are predominantly synthesized within EC. 25 We observed that plasma VWF:Ag levels were also significantly increased in convalescent COVID-19 patients compared to controls (median 1.1 IU/ml versus 0.84 IU/ml; p=0.004, 95% CI 0.09-0.57) (Fig. 1H) . Marked inter-individual variation was observed, with VWF:Ag levels ranging from 0.48 to 3.4 IU/mL in convalescence. Notably, VWF:Ag levels above the upper limit of normal (ULN) were observed in 15 patients (30%) with median VWF:Ag 2.0 IU/mL in this subgroup (Fig. 1H) . In addition, FVIII:C levels also correlated strongly with VWF:Ag levels (r=0.87; p<0.0001) (Fig. 1I) . During posttranslational modification within ECs, an N-terminal 741 residue VWF propeptide (VWFpp) is cleaved from each VWF monomer. VWF:Ag and VWFpp are subsequently stored together within WPB and co-secreted in equimolar amounts following EC activation. We recently reported markedly elevated VWFpp levels in acute COVID-19, and found that these levels correlated inversely with clinical outcome. 16 Interestingly, VWFpp levels were also significantly elevated in convalescent COVID-19 patients compared to controls (p=0.009, 95% CI 0.06-0.5) (Fig. 2A) .

Inter-patient variation was again observed, with VWFpp levels being above the upper limit of normal (ULN) in 10 patients (20%). Consistent with the concept of ongoing endotheliopathy, VWFpp levels also correlated strongly with VWF:Ag levels (r=0.87; p<0.0001) (Fig. 2B) .

Collectively, these data demonstrate that persistent endotheliopathy is a common finding in convalescent COVID-19 patients.

Thrombomodulin (TM) is an EC surface receptor that facilitates thrombin-induced activation of protein C on EC surfaces. 26 Recently, Giri et al reported that TM plays a key role in maintaining EC quiescence. 27 Of particular relevance, they showed that VWF expression and secretion was markedly increased in TM-deficient ECs. Interestingly, Goshua et al also demonstrated increased shedding of TM from EC in patients with acute COVID-19. Furthermore, soluble thrombomodulin (sTM) levels were found to represent an independent prognostic biomarker. 10 Given these data,

This article is protected by copyright. All rights reserved we proceeded to investigate sTM levels in our cohort of convalescent COVID-19 patients. We observed that sTM levels remained significantly elevated in convalescent COVID-19 compared to controls (median 5.3 versus 4.1 ng/ml; p=0.02, 95% CI 0.01-2.7) (Fig. 2C) . Interestingly, the highest sTM level (14.4ng/ml) was observed in a patient who did not require hospitalization, suggesting that sustained endotheliopathy during convalescence is not restricted to those who experienced severe COVID-19. Consistent with the concept that loss of TM may be associated with reduced EC quiescence, we observed a significant correlation between sTM levels and plasma VWF:Ag levels (Fig. 2D) . Highest levels of all the EC activation parameters studied were consistently observed in the convalescent COVID cohort (Fig 2E) .

To examine factors that influence sustained endotheliopathy following COVID-19, clinical parameters including age, comorbidities and severity of acute illness were assessed. On univariate analysis, plasma VWF:Ag, VWFpp, FVIII:C and sTM were all significantly higher in patients who had required hospital admission during their acute COVID-19; in patients aged ≥50 years and in patients with ≥2 comorbidities (Fig. 2F & Supp. Fig. 2 A-C) . These data are consistent with our previous observations that these same parameters also correlate with persistent elevated D-dimer levels in convalescent COVID-19 patients. 3 Finally, we investigated the relationship between markers of sustained endotheliopathy and clinical symptoms associated with Long COVID syndrome in our cohort. Interestingly, significant inverse correlations were observed on univariate analysis between 6MWT distances versus both VWF:Ag and VWFpp, respectively (Figs. 2G-H) . Given this association we sought to further evaluate using multivariable linear regression analysis including the common confounders of age, sex and severity of initial infection. Following adjustment however, a significant relationship between 6MWT distance and VWF:Ag (beta coefficient -4.4, 95% CI -65.5 -56.7, p=0.89) and VWFpp (beta coefficient -64.2, 95% CI -157.8 -29.3, p=0.17) was no longer seen.

Our study has some limitations. These include the small number of cases, the limited period after the acute infection, as well as the observational and retrospective design. In addition, the relationship between clinical outcome measures such as the 6MWT may be confounded by old age and co-morbidities. Finally, EC activation is not unique to COVID-19. It is important to emphasize that EC activation and dysfunction have also been described to play important roles in the pathogenesis of other severe viral illnesses including influenza. 28, 29 However, specific differences in vascular perturbance between acute COVID-19 and influenza have also been described. Ackermann et al showed that at autopsy, alveolar capillary microthrombi were 9 times more prevalent in patients with COVID-19 compared to patients with influenza. 5 Moreover, new pulmonary vessel formation was also significantly higher in COVID-19 patients, with prominent intussusceptive angiogenesis. In addition, Stals et al recently reported that thrombotic

This article is protected by copyright. All rights reserved complications were significantly higher in hospitalized patients with acute COVID-19 compared to influenza. 30 Collectively, these data suggest that are some similarities but also important differences vis a vis pathogenesis, endotheliopathy and immunothrombosis between acute COVID-19 and other acute viral infections. To better understand the potential translational significance of our findings, additional studies including "omics" and imaging that directly compare EC activation and dysfunction in convalescent COVID-19 patients as opposed to patients recovering from other types of severe viral illness will be essential.

In conclusion, our data demonstrate for the first time that sustained endothelial cell (EC) activation is common up to 10 weeks following acute SARS-CoV-2 infection. Importantly, this persistent endotheliopathy appears to occur independently of ongoing acute phase response or NETosis and is associated with enhanced thrombin generation potential. We postulate that shedding of TM from EC may play a role in modulating the loss of normal EC quiescence. These findings are interesting given the critical role played by endotheliopathy in the pathogenesis of acute COVID-19. However, further adequately powered clinical trials will be required to determine whether this sustained EC activation and coagulation activation has a role in (i) stratifying patients at increased risk of thrombotic events after resolution of acute SARS-CoV-2 infection who may benefit from extended duration post-discharge thromboprophylaxis and/or (ii) the pathogenesis of Long COVID syndrome.

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This article is protected by copyright. All rights reserved

This article is protected by copyright. All rights reserved

Accepted Article