Sialylation on O-linked glycans protects von Willebrand factor from macrophage galactose lectin mediated clearance

Terminal sialylation determines the plasma half-life of von Willebrand factor (VWF). A role for macrophage galactose lectin (MGL) in regulating hyposialylated VWF clearance has recently been proposed. In this study, we showed that MGL influences physiological plasma VWF clearance. MGL inhibition was associated with a significantly extended mean residence time and 3-fold increase in endogenous plasma VWF antigen levels (P<0.05). Using a series of VWF truncations, we further demonstrated that the A1 domain of VWF is predominantly responsible for enabling the MGL interaction. Binding of both full-length and VWF-A1-A2-A3 to MGL was significantly enhanced in the presence of ristocetin (P<0.05), suggesting that the MGL-binding site in A1 is not fully accessible in globular VWF. Additional studies using different VWF glycoforms demonstrated that VWF O-linked glycans, clustered at either end of the A1 domain, play a key role in protecting VWF against MGLmediated clearance. Reduced sialylation has been associated with pathological, increased clearance of VWF in patients with von Willebrand disease. Herein, we demonstrate that specific loss of α2-3 linked sialylation from O-glycans results in markedly increased MGL-binding in vitro, and markedly enhanced MGL-mediated clearance of VWF in vivo. Our data further show that the asialoglycoprotein receptor (ASGPR) does not have a significant role in mediating the increased clearance of VWF following loss of O-sialylation. Conversely however, we observed that loss of N-linked sialylation from VWF drives enhanced circulatory clearance predominantly via the ASGPR. Collectively, our data support the hypothesis that in addition to regulating physiological VWF clearance, the MGL receptor works in tandem with ASGPR to modulate enhanced clearance of aberrantly sialylated VWF in the pathogenesis of von Willebrand disease

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

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

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

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

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.</p

Nonclinical evaluation of novel cationically modified polysaccharide antidotes for unfractionated heparin.

Protamine, the only registered antidote of unfractionated heparin (UFH), may produce a number of adverse effects, such as anaphylactic shock or serious hypotension. We aimed to develop an alternative UFH antidote as efficient as protamine, but safer and easier to produce. As a starting material, we have chosen generally non-toxic, biocompatible, widely available, inexpensive, and easy to functionalize polysaccharides. Our approach was to synthesize, purify and characterize cationic derivatives of dextran, hydroxypropylcellulose, pullulan and γ-cyclodextrin, then to screen them for potential heparin-reversal activity using an in vitro assay and finally examine efficacy and safety of the most active polymers in Wistar rat and BALB/c mouse models of experimentally induced arterial and venous thrombosis. Efficacy studies included the measurement of thrombus formation, activated partial thromboplastin time, bleeding time, and anti-factor Xa activity; safety studies included the measurement of hemodynamic, hematologic and immunologic parameters. Linear, high molecular weight dextran substituted with glycidyltrimethylammonium chloride groups at a ratio of 0.65 per glucose unit (Dex40-GTMAC3) is the most potent and the safest UFH inhibitor showing activity comparable to that of protamine while possessing lower immunogenicity. Cationic polysaccharides of various structures neutralize UFH. Dex40-GTMAC3 is a promising and potentially better UFH antidote than protamine