Sustained VWF‐ADAMTS‐13 axis imbalance and endotheliopathy in long COVID syndrome is related to immune dysfunction

Abstract Background Prolonged recovery is common after acute SARS‐CoV‐2 infection; however, the pathophysiological mechanisms underpinning Long COVID syndrome remain unknown. VWF/ADAMTS‐13 imbalance, dysregulated angiogenesis, and immunothrombosis are hallmarks of acute COVID‐19. We hypothesized that VWF/ADAMTS‐13 imbalance persists in convalescence together with endothelial cell (EC) activation and angiogenic disturbance. Additionally, we postulate that ongoing immune cell dysfunction may be linked to sustained EC and coagulation activation. Patients and methods Fifty patients were reviewed at a minimum of 6 weeks following acute COVID‐19. ADAMTS‐13, Weibel Palade Body (WPB) proteins, and angiogenesis‐related proteins were assessed and clinical evaluation and immunophenotyping performed. Comparisons were made with healthy controls (n = 20) and acute COVID‐19 patients (n = 36). Results ADAMTS‐13 levels were reduced (p = 0.009) and the VWF‐ADAMTS‐13 ratio was increased in convalescence (p = 0.0004). Levels of platelet factor 4 (PF4), a putative protector of VWF, were also elevated (p = 0.0001). A non‐significant increase in WPB proteins Angiopoietin‐2 (Ang‐2) and Osteoprotegerin (OPG) was observed in convalescent patients and WPB markers correlated with EC parameters. Enhanced expression of 21 angiogenesis‐related proteins was observed in convalescent COVID‐19. Finally, immunophenotyping revealed significantly elevated intermediate monocytes and activated CD4+ and CD8+ T cells in convalescence, which correlated with thrombin generation and endotheliopathy markers, respectively. Conclusion Our data provide insights into sustained EC activation, dysregulated angiogenesis, and VWF/ADAMTS‐13 axis imbalance in convalescent COVID‐19. In keeping with the pivotal role of immunothrombosis in acute COVID‐19, our findings support the hypothesis that abnormal T cell and monocyte populations may be important in the context of persistent EC activation and hemostatic dysfunction during convalescence.


| INTRODUC TI ON
Prolonged functional impairment following COVID-19 disease, referred to as Long COVID or post-acute COVID-19 syndrome (PACS), is estimated to occur in 30%-40% of infected individuals. 1,2 Typical features include fatigue, breathlessness, and reduced exercise tolerance. However, the clinical phenotype is heterogeneous and proposed definitions vary. 3,4 Despite the global morbidity associated with Long COVID, the underlying pathogenic mechanisms are poorly understood. However, recent studies have demonstrated that, similar to acute illness, persistent coagulation activation is common in convalescent COVID-19 patients. [5][6][7][8][9] In particular, several groups have reported sustained increases in endogenous thrombin potential and D-dimer levels in patients between 4 and 12 months following acute illness. 6,7,9 Ongoing hemostatic dysfunction was more common in patients who required intensive care support during their acute COVID-19 illness and in patients aged >50 years. 6,7  immunothrombosis are hallmarks of acute COVID-19. We hypothesized that VWF/ ADAMTS-13 imbalance persists in convalescence together with endothelial cell (EC) activation and angiogenic disturbance. Additionally, we postulate that ongoing immune cell dysfunction may be linked to sustained EC and coagulation activation. Results: ADAMTS-13 levels were reduced (p = 0.009) and the VWF-ADAMTS-13 ratio was increased in convalescence (p = 0.0004). Levels of platelet factor 4 (PF4), a putative protector of VWF, were also elevated (p = 0.0001). A non-significant increase in WPB proteins Angiopoietin-2 (Ang-2) and Osteoprotegerin (OPG) was observed in convalescent patients and WPB markers correlated with EC parameters.
Enhanced expression of 21 angiogenesis-related proteins was observed in convalescent COVID-19. Finally, immunophenotyping revealed significantly elevated intermediate monocytes and activated CD4+ and CD8+ T cells in convalescence, which correlated with thrombin generation and endotheliopathy markers, respectively.

Conclusion:
Our data provide insights into sustained EC activation, dysregulated angiogenesis, and VWF/ADAMTS-13 axis imbalance in convalescent COVID- 19 Interestingly, however, prolonged increased D-dimer levels and coagulation activation were also seen in some patients who had mild COVID-19 and were managed exclusively as out-patients. 6,7 Post mortem studies have highlighted that acute SARS-CoV-2 infection is associated with specific deleterious effects on endothelial cells (EC). 10,11 Features include loss of normal tight EC junctions leading to increased EC permeability, enhanced EC apoptosis, and abnormal angiogenesis. [11][12][13][14] In keeping with the concept that SARS-CoV-2 infection is associated with marked EC activation, plasma levels of von Willebrand factor antigen (VWF:Ag), VWF propeptide (VWFpp), and Angiopoietin-2 (Ang-2) are all significantly increased and correlate with clinical severity. [15][16][17] In addition, reduced plasma ADAMTS-13 levels and abnormal ultra-large VWF multimers have been observed in acute COVID-19. 18,19 Collectively, these data suggest that acute EC activation and dysregulation of the normal VWF/ ADAMTS-13 axis play important roles in the pathobiology underlying pulmonary immunothrombosis and microvascular occlusion in acute COVID-19. Interestingly, recent studies have further reported that plasma VWF:Ag, VWFpp, and factor VIII (FVIII) levels all remain significantly elevated after 3 months in convalescent COVID-19 patients compared to healthy controls. 6 Based on these data, roles for sustained EC activation in the pathogenesis of persistent hemostatic dysfunction and Long COVID symptomatology have been proposed.
In this study, we sought to further investigate the nature of ongoing endotheliopathy and VWF-ADAMTS-13 axis dysfunction in convalescent COVID-19. Correlations were evaluated using the Spearman rank correlation test. (F) Heatmap visualization indicating angiogenesis-related protein expression detected in each subject (columns) for each protein (rows), comparing a subset of convalescent COVID-19 patients (n = 9, 6/9 males) and healthy controls (n = 3, 2/3 males). This subset of convalescent patients and controls were selected to match the age and sex profile of the overall convalescent cohort. Protein levels were measured via membrane-based antibody array and data are represented by mean pixel intensity with red indicating higher and green indicating lower levels of the protein of interest. Comparisons between groups were assessed using the Mann-Whitney U test.(ns = not significant, ***p <0 .001, ****p <0 .0001).

| ME THODS
Fluorometer with Thrombinoscope software (Stago) using PPP Low reagent (1 pM tissue factor, 4 mM phospholipids) as before. 6,21 Characterization of peripheral blood monocyte and T lymphocyte populations by flow cytometry were performed as previously described. 22 (Table 1). Acute hospitalized COVID-19 patients received weight-and renally adjusted low molecular weight heparin prophylaxis whereas non-hospitalized and convalescent patients did not.

| RE SULTS AND D ISCUSS I ON
In keeping with previous studies, we confirmed significant increases in plasma VWF:Ag levels ( Figure S1) and concurrent reductions in plasma ADAMTS-13 levels ( Figure 1A) in our acute COVID-19 subgroup. As a result, the VWF-ADAMTS-13 ratio was markedly elevated >10 fold in acute COVID-19 patients compared to controls ( Figure 1B). In patients with convalescent COVID-19, we observed that plasma ADAMTS-13 levels were significantly reduced compared to controls (median 598 ng/ml vs. 630 ng/ml, p = 0.009; Figure 1A). Marked inter-individual variation was apparent, with ADAMTS-13 levels ranging from 220 ng/ml to 900 ng/ ml in the convalescent cohort. Pertinently, plasma ADAMTS-13 levels below the lower limit of our local normal reference range (399 ng/ml) were seen in 15/50 (30%) convalescent COVID-19 patients, with a median ADAMTS-13 level of 384 ng/ml in this subgroup. ADAMTS-13 levels were significantly lower in convalescent COVID-19 patients who had required hospitalization compared to those managed entirely as outpatients (p = 0.04; data not shown).
The absolute reduction in ADAMTS-13 levels in convalescent COVID-19 patients was significantly less marked than that seen in patients with acute COVID-19 ( Figure 1A). Importantly, however, the reduction in ADAMTS-13 levels coupled with elevated plasma VWF:Ag levels, meant that the VWF-ADAMTS-13 ratio remained significantly elevated in convalescent COVID-19 patients compared to controls (median 2.3 vs. 1.0, p = .0004; Figure 1B). In fact,
The increases in plasma VWF:Ag, VWFpp, and FVIII:C levels seen in convalescent COVID-19 suggest ongoing EC activation and Weibel Palade Body (WPB) exocytosis. Importantly WPB also store other important pro-inflammatory and pro-angiogenic molecules, including Ang-2 and OPG. 26,27 To further investigate the hypothesis that sustained EC activation may be associated with ongoing exocytosis of WPB constituents in convalescent COVID-19, we next investigated Ang-2 and OPG levels. Overall, plasma Ang-2 levels were non-significantly increased in the convalescent cohort compared to healthy controls (median 1009 pg/ml vs. 859 pg/ ml, p = .23; Figure 2A). However, significant inter-patient variation was observed, with plasma Ang-2 levels above the local normal range observed in 22% (11/50) of convalescent COVID-19 patients (Figure 2A). Similarly, marked inter-individual variability in plasma OPG levels was also seen in the convalescent COVID-19 cohort ( Figure 2B). Overall, there was a non-significant increase in convalescent OPG levels compared to controls (median 835 pg/ml vs. 818 pg/ml, p = 0.9; Figure 2B). However, 14/50 (28%) of convalescent COVID-19 patients had plasma OPG levels above the upper limit of the normal healthy controls. Consistent with the concept of ongoing endotheliopathy and WPB exocytosis in patients with convalescent COVID-19, significant correlations were observed among OPG, VWFpp, FVIII, and Ang-2 ( Figures 2C-E).
angiogenesis in multiple organ sites, with 139 angiogenesis-related proteins being significantly dysregulated, compared to 74 control tissues from non-COVID patients. 28 Subsequent studies have found circulating pro-angiogenic factors (including VEGF-A, PDGF-AA and PDGF-AB/ BB) were significantly elevated in acute COVID-19 and correlated with disease severity. 29 Because VWF, Ang-2, and OPG can influence EC angiogenesis, we next performed a pilot study to assess a panel of 24 angiogenesis-related proteins in a subset (n = 9) of our convalescent COVID-19 patients and healthy controls (n = 3). Significant interpatient heterogeneity was again seen ( Figure 2F). Overall, significantly (p <0 .05) enhanced expression of 21 angiogenesis-related proteins was observed in the convalescent COVID-19 subgroup compared to controls ( Figure 2F). In keeping with previous studies in acute COVID-19, elevated pro-angiogenic markers included Ang-2, as well as members of the VEGF, PDGF, placental growth factor (PGF), and fibroblast growth factor (FGF) families. 17,29 Conversely, plasma levels of endostatin and serpin F1 (both inhibitors of angiogenesis) were significantly reduced in convalescent COVID-19 ( Figure 2F). These preliminary data can serve as a platform for future larger studies exploring the concept that dysregulated angiogenesis is not only a feature of acute COVID-19, but may also be sustained during convalescence in a subset of patients.
The molecular mechanisms responsible for persistent hemostatic abnormalities in convalescent COVID-19 have not been defined. However, recent studies have described persistent immune profile abnormalities in convalescent COVID-19 patients, including abnormal monocyte profiles and T cell activation. 22 We hypothesized that sustained inflammatory cell abnormalities may be important in triggering ongoing thrombin generation and/or endotheliopathy. Consistent with previous reports, immunophenotyping revealed increased HLA-DR + CD14 + CD16+ intermediate monocytes in convalescent (n = 37) and acute COVID-19 patients (n = 32) compared with controls (n = 20; Figure 3A), whereas classical and non-classical monocyte profiles were unchanged in convalescence ( Figure S1). We and others have previously reported sustained increases in endogenous thrombin potential (ETP) and peak thrombin generation in convalescent COVID-19 patients compared to controls. 6 Interestingly, intermediate monocyte percentage correlated significantly (p = 0.007) with ETP and peak thrombin (p = 0.02) in our patient cohort ( Figure 3B,C; Table S1). This is noteworthy as tissue factor induction on intermediate monocytes has been shown to contribute to platelet activation in acute COVID-19. 30 Finally, similar to previous studies, we observed significant reductions in naïve CD4+ and CD8+ T cell subsets in both convalescent (n = 37) and acute COVID-19 patients (n = 32) compared to controls (n = 20; Figure 3D,E) with concomitant elevations in activated CD4+ and CD8+ T cell subsets ( Figure 3F,G).
Importantly, no significant associations were noted between (i) intermediate monocytes and EC activation markers or (ii) T cell activation subsets and thrombin generation parameters (Tables S1 and S2).
In conclusion, our data provide novel insights into the nature of sustained EC activation, WPB exocytosis, and VWF/ADAMTS-13 axis imbalance in convalescent COVID-19. In keeping with the pivotal role of immunothrombosis in acute COVID-19, our findings support the hypothesis that abnormal T cell and monocyte profiles may be important in the context of persistent EC activation and ongoing hemostatic dysfunction during convalescence. Further studies will be required to determine how this prolonged EC and immune activation, together with dysregulated angiogenesis in convalescent patients, may contribute to functional impairment post-COVID-19.