key: cord-0702981-lzm2y1bl
authors: Prasannan, Nithya; Heightman, Melissa; Hillman, Toby; Wall, Emma; Bell, Robert; Kessler, Anna; Neave, Lucy; Doyle, Andrew; Devaraj, Akshitha; Singh, Deepak; Dehbi, Hakim-Moulay; Scully, Marie
title: Impaired exercise capacity in post-COVID syndrome: the role of VWF-ADAMTS13 axis
date: 2022-05-13
journal: Blood Adv
DOI: 10.1182/bloodadvances.2021006944
sha: aabddb75e9468906e881eac99d538a888eda4529
doc_id: 702981
cord_uid: lzm2y1bl

Post-COVID syndrome (PCS) or Long-COVID is an increasingly recognised complication of acute SARS-CoV-2 infection, characterised by persistent fatigue, reduced exercise tolerance chest pain, shortness of breath and cognitive slowing. Acute COVID-19 is strongly linked with increased risk of thrombosis; a prothrombotic state, quantified by elevated Von Willebrand Factor (VWF) Antigen (Ag):ADAMTS13 ratio, and is associated with severity of acute COVID-19 infection. We investigated if patients with PCS also had evidence of a pro-thrombotic state associating with symptom severity. In a large cohort of patients referred to a dedicated post-COVID-19 clinic, thrombotic risk including VWF(Ag):ADAMTS13 ratio, was investigated. An elevated VWF(Ag):ADAMTS13 ratio (≥1.5) was raised in nearly one-third of the cohort and four times more likely in patients with impaired exercise capacity as evidenced by desaturation ≥3% and/or rise in lactate level more than 1 from baseline on 1-minute sit to stand test and/or 6-minute walk test (p<0.0001). 20% (56/276) had impaired exercise capacity, of which 55% (31/56) had a raised VWF(Ag):ADAMTS13 ratio ≥1.5 (p<0.0001). FVIII and VWF(Ag) were elevated in 26% and 18% respectively and support a hypercoagulable state in some patients with PCS. These findings suggest possible ongoing microvascular/endothelial dysfunction in the pathogenesis of PCS and highlight a potential role for antithrombotic therapy in the management of these patients.

The emergence of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to significant morbidity and mortality on a global level with 257,662,742 confirmed cases of COVID-19 and 5,166,671 deaths 1 at the time of writing. Significant advances have been made in the clinical management of severe COVID-19 disease in the hospitalised population 2 . However, just under 10% of patients with COVID-19 are admitted to hospital, the majority of people infected with COVID-19 are not hospitalised 3 . A proportion of cases experience ongoing symptoms, dominated by fatigue, chest pain, shortness of breath and cognitive slowing. This has been named Long-COVID by patient groups, and termed post-COVID syndrome (PCS) by the National Institute for Health and Care Excellence (NICE) 4 .

Estimates from the Office for National Statistics suggest that 1.1 million people in the UK are suffering from PCS 5 . The NICE guidance on the long term effects of COVID-19 provide both definitions for illness and a framework for clinical management in PCS clinics, recently endorsed by the WHO 6 . It has been defined as a condition that "occurs in individuals with a history of probable or confirmed SARS CoV-2 infection, usually 3 months from the onset of COVID-19 with symptoms and that last for at least 2 months and cannot be explained by an alternative diagnosis" 4 .

Acute SARS-CoV-2 infection generates a hypercoagulable state, particularly evident in hospitalised patients 7 . Raised D-dimer levels which has been associated with poor outcomes 7 , as well as elevated factor VIII (FVIII) and Von Willebrand factor (VWF) levels further support the hypercoagulability hypothesis 8 . VWF plays a major role in primary haemostasis and multimeric size is regulated by ADAMTS13 (A Disintegrin and Metalloprotease with Thrombospondin 1 repeats, number 13). In acute COVID-19 infected patients, a rise in VWF:Ag (antigen) which may be associated with a relative reduction in ADAMTS13 and therefore an increase in VWF(Ag):ADAMTS13 ratio is recognised and this is likely to contribute to the hypercoagulable state and risk of microvascular thrombosis in these patients. 8, 9 Enhanced thrombin generation, decreased fibrinolytic activity, raised levels of FVIII and plasminogen-activator inhibitor type 1 (PAI-1), 10,11 high endothelin-1 levels, 12 and raised D-dimer 13 has also been presented in PCS patients. These suggest a role for endothelial cell dysfunction and hypercoagulability.

We hypothesised that the pro-thrombotic state is implicated in the pathogenesis of PCS. We investigated coagulation parameters associated with reduced exercise capacity in patients with PCS, identifying the utility of these parameters in PCS to determine ongoing disease activity. We further investigated if an association exists between elevated VWF(Ag):ADAMTS13 ratio and impaired exercise capacity in patients with PCS at a dedicated clinical service for PCS.

Of the 1471 face to face appointments seen in a dedicated outpatient post-COVID clinic between July 2020 to May 2021, baseline demographics were assessed retrospectively in patients who had VWF(Ag):ADAMTS13 ratio tested. Of the 413 patient episodes, 352 were more than 3 months after acute infection with COVID-19. 22 episodes were repeat measurements performed over time and excluded from analysis unless specified, leaving 330 patients for full analysis. Trends in patients with repeat measurements are shown separately. Patients followed up in the clinic had proven or presumed (when PCR testing was not widely available during initial stages of pandemic) COVID-19 infection. A clinical syndrome compatible with COVID-19 infection was agreed by two independent senior clinicians in those with presumed COVID-19 infection. Referral to the post COVID clinic included both patients post hospitalisation and referrals from community non-hospitalised cases. Patients attending the clinic did not have blood tests performed if they were completed by their general practitioner as part of the referral to the post-COVID clinic, or if recent blood test results were available through attendance to the hospital emergency or other departments. Blood tests were not repeated in patients reporting symptom improvement and this decision was not influenced by any other baseline characteristics such as age.

ADAMTS13 activity was measured using FRETS VWF73 assay 14 . Von Willebrand screen (VWF antigen and VWF activity) was measured using standard automated immunoturbimetric assay in a Sysmex CS-2500 analyser with a Siemens kit (VWF:Ag and INNOVANCE VWF activity, Siemens Healthcare Diagnostics, Marburg, Germany), and a clotting based assay using Factor VIII (FVIII) deficient plasma (reference OTXW17) and

Dade Actin FS Activated PTT reagent (reference B4218-100) was used to measure FVIII. All analysis was performed on plasma derived from peripheral blood samples collected in sodium citrate tubes, as part of routine assessment in the post COVID clinic which included D-dimer. VWF(Ag):ADAMTS13 ratio was calculated on 50 voluntary normal controls (Medical Research Ethics Committee Numbers 08/H0810/54 and 08/H0716/72). The VWF(Ag):ADAMTS13 ratio was also calculated in the PCS patient cohort and correlated with symptoms including exercise tolerance as assessed by 1 minute sit-to-stand test and/or 6 minute walk test. The 6 minute walk test (6MWT) and 1 minute sit-to-stand (STS) test were performed to assess abnormalities in components of exercise testing and therefore as an indicator of impaired exercise capacity. The 1 minute STS test involves going from sit to stand as many times as possible in 1 minute. Desaturation and number of repetitions completed as part of the STS test were examined. The 6MWT, as outlined in the American Thoracic Society (ATS) guidelines 15 , is a simple practical test which requires a 100 feet hallway but without the requirement for complex or additional equipment. It measures the distance that a patient can quickly walk on a flat surface in 6 minutes. Partial pressure of oxygen, pH and capillary lactate were measured from an ear-prick blood test pre and post exercise during the 6MWT, in parallel with continuous peripheral oxygen saturation measurement. Peripheral oxygen desaturation ≥3% for 6MWT and STS test, as well as an increase in lactate >1 from baseline during 6MWT were taken as markers of impaired exercise capacity. Reference values for the 6MWT has been assessed in healthy subjects aged 20-50 years and the mean oxygen saturation after walking was 97±1.3% with a mean change in saturation of 1±1.1% 16 . 293/330 patients had STS testing performed and these were analysed in more detail with focus on the number of repetitions completed. 1 minute STS testing in COPD patients has previously shown 19±6 repetitions on average 17 and similarly 21±6 in interstitial lung disease 18 . This test has also been used to evaluate physical capacity and exertional desaturation one month post discharge in patients who survived COVID-19 pneumonia. The average number of repetitions was 20.9±4.8 in these patients 19 . Repetitions ≤20 was therefore considered as an indicator of fatigue. Correlation between these components of the sit-to-stand test and VWF(Ag):ADAMTS13 ratio was assessed. Potential associations with D-dimer results were also investigated.

All statistical analysis was performed using Graphpad Prism 9. Continuous data was summarised as median and interquartile range (IQR) with use of the Mann Whitney test to compare ranks across 2 groups, and Kruskal-Wallis testing was used to compare ranks across 3 groups. Gaussian distribution was not assumed for the comparison between groups in this paper, as variables showed signs on non-normality, in particular a long right tail for VWF(Ag):ADAMTS13 ratio ( Figure 1a ). VWF(Ag):ADAMTS13 ratio was calculated by (VWF(Ag)/ADAMTS13 activity) X 100 in each set of results for patients and normal controls.

Number and percentage was used to summarise categorical data. Chi squared/Fishers exact test was used to assess statistical significance in categorical variables. Odds ratios (OR) and 95% confidence intervals (CI) were estimated using logistic regression models. A p-value<0.05 was considered statistically significant.

The baseline demographics of 330 patients were analysed. 60% were female and 40% were male, with a median age of 46 years (range 18-88 years). Patients followed up in clinic varied from 3 to 15 months post proven or presumed COVID-19 infection. 320/330 (97%) had symptoms greater than 3 months following acute COVID-19 infection, of which respiratory symptoms and fatigue were commonest (Table 1) . 85/330 (26%) of PCS patients had high FVIII ranging from 2.1 to 5.1IU/ml (normal range (NR) 0.5-2IU/ml) and 60/330 (18%) had high VWF(Ag) ranging from 1.7-3.3IUml (NR 0.5-1.6 IU/ml). ADAMTS13 activity ranged from 68.2 to 159.3IU/dL (NR 60-146IU/dL). VWF(Ag):ADAMTS13 ratio was divided into two groups: <1.5 (normal) and ≥1. 5 (abnormal) and assessed separately against patient symptoms and results of exercise tolerance testing via the 1 minute sit-to-stand exercise test and/or 6 minute walk test. Of the 97% of symptomatic patients, 72% (230/320) had VWF(Ag):ADAMTS13 ratio <1.5 and 28%

(90/320) had a ratio ≥1.5. 3% of the total cohort (10/330) was asymptomatic and had a median VWF(Ag):ADAMTS13 ratio of 1.1. Therefore, VWF(Ag):ADAMTS13 ratio did not correlate with presenting clinical symptoms However, VWF(Ag):ADAMTS13 ratio≥1.5 did correlate with abnormal exercise intolerance.

Of the 330 patients assessed, 54 (16%) did not undergo exercise testing or failed to complete the test. 276 (84%) completed exercise testing (Figure 1b) . 220/276 (80%) were able to perform these tasks without impairment. Within this group, 171/220 (78%) had a normal VWF(Ag):ADAMTS13 ratio <1.5 and 49/220 (22%) had a VWF(Ag):ADAMTS13 ratio>1.5. 56/276 (20%) had evidence of abnormal exercise testing as confirmed by desaturation ≥3% and/or an increase in lactate levels >1 above baseline. Of these, 31/56 (55%) had a high ratio ≥1.5 (Table 2 ). This was equivalent to 11% of the whole cohort.

Abnormal exercise testing was associated with a higher VWF(Ag):ADAMTS13 ratio with a median of 1.5 (IQR 1.2-1.7) compared with 1.1 (IQR 0.9-1.4) in those with normal exercise test (p<0.0001, figure 1c) . The risk of a raised VWF(Ag):ADAMTS13 ratio was found to be 4 times more likely in those with an abnormal exercise test (OR 4.3 [95% CI 2.3-7.9], p<0.0001), demonstrating a strong association by univariate analysis. The OR was maintained (4 [95% CI 2.1-7.8]) by multivariate analysis after adjustment for age, gender and other co-morbidities as potential confounders.

Analysis of the 1 minute sit-to-stand test was performed focussing particularly on the number of repetitions completed. Repetitions ≤20 was considered indicative of fatigue. Of the 293 STS tests analysed, 228 were more than 3 months since acute COVID-19 infection and were used for analysis. 12 patients did not have repetitions recorded, leaving full analysis to be completed in 216 patients (no repeat measurements were included). 156/216 patients completed more than 20 repetitions and of these only 35/156 (22%) had a VWF(Ag):ADAMTS13 ratio ≥1.5. Conversely, 60 patients completed ≤20 repetitions and 19/60 (32%) had a VWF(Ag):ADAMTS13 ratio ≥1.5. However, no significant difference was found between these groups (p=0.17).

VWF(Ag):ADAMTS13 ratio and significant desaturation (≥3%) was analysed in relation to number of repetitions completed in the STS test (Table 3 ). 15% (9/60) of those with ≤20

repetitions had a significant drop in oxygen saturation ≥3%, of which 67% ( 6/9) had a VWF(Ag):ADAMTS13 ratio ≥1.5. The median ratio was 1.6 (IQR 1-1.8) compared to a normal ratio of 1.2 (IQR 0.9-1.5) in the 85% (51/60) who did not have a significant drop in saturations (p=0.07)). Comparably, in those who completed more than 20 repetitions, 13% The split of abnormalities in STS and 6MWT were assessed. Desaturation ≥3% was the most frequent abnormality in 29/56 (52%) patients, followed by lactate rise in 16/56 (29%) and desaturation plus lactate rise in 11/56 (20%). Median VWF(Ag):ADAMTS13 ratios were 1.5 (IQR 1-1.7), 1.3 (IQR 0.9-1.5) and 1.6 (IQR 1.6-1.9) respectively. 9/11 (82%) of those with desaturation >3% and a rise in lactate had a VWF(Ag):ADAMTS13 ratio≥ 1.5 compared to 17/29 (59%) in those that desaturated and 6/16 (38%) in those who had a rise in lactate alone (p=0.07). Elevated D-dimer levels (>550ug/l FEU) did not associate with desaturation or lactate rise (p=0.50). VWF(Ag):ADAMTS13 ratio and exercise test results were analysed according to 3 age groups: ≤40 years, 41-60 years and >60 years. The median VWF(Ag):ADAMTS13 ratio was found to be 1 (IQR 0.8-1.3), 1.2 (IQR 1-1.5) and 1.6 (IQR 1.2-2) respectively (p<0.0001), showing a difference in ratio across these age groups. Exercise testing within these age groups showed whilst fewer patients had evidence of abnormal compared to normal exercise testing (abnormal exercise test in 16% (14/86) of patients <40 years, 17% (26/150) of patients 40-60 years and 41% (16/39) in patients >60 years), this was seen in a greater proportion of patients over 60 years suggesting an age effect (p=0.003).

Pre-existing lung co-morbidities were also analysed in relation to VWF(Ag):ADAMTS13 ratio and exercise test results. 45/330 (14%) had a pre-existing diagnosis of lung disease. No difference in median VWF(Ag):ADAMTS13 ratio was found in those with and without preexisting lung disease -1.1.(IQR 0.9-1.7) and 1.2 (IQR 0.9-1.5) respectively (p=0.80). Of the 56 patients with abnormal exercise testing, only 7 (13%) with an abnormal exercise test had pre-existing lung disease compared to 49/56 (88%) who did not. In the 7 patients with known lung disease -4 had asthma (all with good control except 1 patient), 2 had chronic obstructive pulmonary disease (both were on treatment) and 1 had recent respiratory tuberculosis (TB) infection. Of the 220 patients with normal exercise testing, 28/220 (13%) had known lung disease compared to 192/220 (87%) who did not (p>0.99). Therefore, no significance effect of pre-existing lung disease was associated with an abnormal exercise test.

Median FVIII and VWF antigen levels were higher in patients with abnormal exercise tolerance (Table 4 ). No significant difference in D-dimer levels above 550ug/l FEU was found between those with normal and abnormal exercise tolerance (780ug/l FEU and 780ug/l FEU respectively, p=0.5). Finally, changes in VWF(Ag):ADAMTS13 ratio and exercise test results in 18 patients who had repeat assessments performed over time were analysed (Table 5 ). Findings were variable from a stable VWF(Ag):ADAMTS13 ratio to a reduction in ratio. Exercise testing was not repeated in all patients.

The longer term impact of COVID-19 infection is now recognised to be associated with ongoing symptomatology beyond 3 months following acute infection; 22.1% of patients experience at least one symptom at five weeks following COVID-19 infection and 9.8% at twelve weeks 20 and high symptom burden post infection in non-hospitalised as well as posthospitalised patients have been shown 21 . A thrombo-inflammatory basis to the pathogenesis of COVID-19 infection has been implicated. A raised FVIII, VWF and D-dimer are the primary haemostasis factors supportive of a hypercoagulable state. Analysis of these coagulation parameters led to investigation of VWF(Ag):ADAMTS 13 ratio and a raised ratio was particularly seen in the most severe COVID-19 cases 8, 9 . We extended analysis of the VWF(Ag):ADAMTS13 ratio to patients with PCS and identified nearly a third of the cohort had an increased ratio.

The pathophysiology of PCS has not been adequately addressed. This is the first report to our knowledge to identify and report an association between a raised VWF(Ag):ADAMTS13 ratio and impaired exercise capacity, specifically related to desaturation >3% and a rise in lactate levels more than 1 above baseline. We found an association between a raised VWF(Ag):ADAMTS13 ratio and limited exercise capacity, in both hospital and non-hospital cases. A raised ratio was found to be four times more likely in patients with an abnormal exercise test. These findings suggest an ongoing prothrombotic state in PCS, demonstrated by measurable laboratory and clinical parameters, therefore adding confirmation to the hypothesis of microvascular/endothelial dysfunction in the pathogenesis of PCS. We hypothesise the persistence of a hypercoagulable state which may be associated with endothelial dysfunction and microthrombi in the capillary bed of large muscles, causing a reduction in oxygen capacity during exercise, resulting in anaerobic respiration and fatigue. Fogarty et al 26 demonstrated a role for sustained endotheliopathy in PCS, supported by increased endogenous thrombin potential and peak thrombin levels, as well as raised VWF antigen, VWF propeptide and FVIII. They showed a significant inverse correlation between 6MWT distances and both VWF antigen and VWF propeptide, which was however lost after adjustment for age, sex and severity of initial infection. Our results provide further support to the role of endothelial dysfunction in the pathogenesis of PCS. However, in addition to this, we have provided evidence of an association between a raised VWF(Ag):ADAMTS13 ratio≥1.5 and impaired exercise capacity as evidenced by desaturation≥3% and/or rise in lactate levels. Desaturation ≥3% on a 1 minute STS test was specifically associated with a high VWF(Ag):ADAMTS13 ratio These findings support screening patients referred with PCS for VWF(Ag):ADAMTS13 ratio and exercise testing. In a time where the pathogenesis of this newly arisen phenomenon is not yet clear, it is important from a research perspective to do these screening tests. The underlying mechanism that leads to abnormalities of components of exercise testing in this group of patients with a VWF(Ag):ADAMTS13 ratio ≥1.5 remain unanswered. Importantly, it also suggests a potential role for antithrombotic therapy in this cohort of patients and requires large scale trials to evaluate this as a management option in PCS sufferers. 

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Nil.

N.P. analyzed data and wrote manuscript. The remaining authors declare no competing financial interests.

 Figure 1a ,b,c