Persistent endotheliopathy in the pathogenesis of long COVID syndrome

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. Objectives To assess whether 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, endothelial cell (EC) activation and NETosis parameters and thrombin generation were assessed. Results Thrombin generation assays revealed significantly shorter lag times (p < .0001, 95% CI −2.57 to −1.02 min), increased endogenous thrombin potential (p = .04, 95% CI 15–416 nM/min), and peak thrombin (p < .0001, 95% CI 39–93 nM) in convalescent COVID‐19 patients. These prothrombotic changes were independent of ongoing acute phase response or active NETosis. Importantly, EC biomarkers including von Willebrand factor antigen (VWF:Ag), VWF propeptide (VWFpp), and factor VIII were significantly elevated in convalescent COVID‐19 compared with controls (p = .004, 95% CI 0.09–0.57 IU/ml; p = .009, 95% CI 0.06–0.5 IU/ml; p = .04, 95% CI 0.03–0.44 IU/ml, respectively). In addition, plasma soluble thrombomodulin levels were significantly elevated in convalescent COVID‐19 (p = .02, 95% CI 0.01–2.7 ng/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‐min 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.


| INTRODUC TI ON
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 thrombingenerating capacity were also reported. Critically, however, the biological mechanisms underlying these persistent procoagulant effects following acute COVID-19 remain poorly understood.
Postmortem studies in acute COVID-19 have demonstrated disseminated thrombosis throughout the pulmonary vasculature. [5][6][7] These thrombi are platelet-and fibrin-rich, and also contain neutrophils, neutrophil extracellular traps (NETs) and activated factor XII (FXII) that triggers the contact pathway. 7 However, autopsy studies have highlighted the key roles played by pulmonary endotheliopathy and microvascular immunothrombosis in acute COVID-19.

Objectives:
To assess whether 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, endothelial cell (EC) activation and NETosis parameters and thrombin generation were assessed.

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.  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 immunothrombosis in modulating the pathogenesis of acute SARS-CoV-2, 18 we hypothesized that persistent EC activation might be important in modulating ongoing procoagulant effects in convalescent COVID-19 patients and thereby contribute to the pathogenesis underpinning long COVID. Committee. A control group of nonhospitalized asymptomatic controls (n = 17, mean age 47 ± 12 years) were also recruited. Plasma VWF:Ag, VWFpp, FVIII:C, and sTM were analyzed as previously described. 16 Thrombin generation was performed in a Fluouroskan Ascent Fluorometer with Thrombinoscope software (Stago) using PPP Low reagent (1 pM tissue factor, 4 mM phospholipids) as before. 19 Additionally, the release of extracellular DNA 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-min 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 < .05 considered statistically significant.

| RE SULTS AND D ISCUSS I ON
Fifty convalescent COVID-19 patients (mean age 50 ± 17 years) were assessed at a median of 68 (interquartile range 61.3-72) days following COVID-19 symptom resolution or hospital discharge (Table S1). The majority of patients (37/50, 74%) required hospitalization during their acute COVID-19 illness and eight patients (16%) required intensive care unit admission. Median body mass index was 28 kg/m 2 (interquartile range 25-32) and comorbidities were apparent in 31 patients (31/50, 62%) (Table S1). All hospitalized 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 in convalescent COVID-19 patients, acute phase markers (including C-reactive protein, neutrophil and white cell counts, interleukin-6, and sCD25 levels) had normalized in most patients (Table S1 and Figure S1D,E). Although NETosis has been implicated in acute COVID-19 immunothrombosis and endotheliopathy, 8 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 with controls (median 1.1 IU/ml vs. 0.84 IU/ml; p = .004, 95% CI 0.09-0.57) ( Figure 1H). Marked interindividual 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 were observed in 15 patients (30%) with median VWF:Ag 2.0 IU/ml in this subgroup ( Figure 1H). In addition, FVIII:C levels also correlated strongly with VWF:Ag levels (r = 0.87; p < .0001) ( Figure 1I). During posttranslational modification within ECs, an N-terminal 741 residue 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 with controls (p = .009, 95% CI 0.06-0.5) (Figure 2A). Interpatient variation was again observed, with VWFpp levels being above the upper limit of normal in 10 patients (20%). Consistent with the concept of ongoing endotheliopathy, VWFpp levels also correlated strongly with VWF:Ag levels (r = 0.87; p < .0001) ( Figure 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, sTM levels were found to represent an independent prognostic biomarker. 10 Given these data, 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 with controls (median 5.3 vs. 4.1 ng/ml; p = .02, 95% CI 0.01-2.7) ( Figure 2C). Interestingly, the highest sTM level (14.4 ng/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 ( Figure 2D). Highest levels of all the EC activation parameters studied were consistently observed in the convalescent COVID cohort ( Figure 2E).
To examine factors that influence sustained endotheliopathy Our study has some limitations. These include the small number of cases, the limited period after the acute infection, and the observational and retrospective design. In addition, the relationship between clinical outcome measures such as the 6MWT may be confounded by old age and comorbidities. Finally, EC activation is not unique to COVID-19. 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 with 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 complications were significantly higher in hospitalized patients with acute COVID-19 compared with 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 EC activation is common up to 10 weeks following acute SARS-CoV-2 infection. Importantly, this persistent endotheliopathy (E) Heatmap visualization indicating EC marker levels detected in each subject (columns) for each protein (rows). (F) Convalescent VWF:Ag results are grouped according to whether acute infection was managed as an outpatient or inpatient; patients were aged ≥50 or <50 years and whether comorbidity counts were ≥2 or <2, respectively. Correlations are shown between 6-min walk test distance and plasma levels of (G) VWF:Ag and (H) VWFpp, respectively. Correlations were evaluated using the Spearman rank correlation test. (ns, not significant, *p < .05, **p < .01, ****p < .0001). EC, endothelial cell; VWF:Ag, von Willebrand factor antigen; VWFpp, von Willebrand factor propeptide [Color figure can be viewed at wileyonlinelibrary.com] 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 (1) stratifying patients at increased risk of thrombotic events after resolution of acute SARS-CoV-2 infection who may benefit from extended duration postdischarge thromboprophylaxis and/or (2) the pathogenesis of long COVID syndrome.