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.
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.
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).
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.
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.
The plasma multimeric glycoprotein von Willebrand factor (VWF) plays a critical role in primary hemostasis by tethering platelets to exposed collagen at sites of vascular injury. Recent studies have ...identified additional biological roles for VWF, and in particular suggest that VWF may play an important role in regulating inflammatory responses. However, the molecular mechanisms through which VWF exerts its immuno-modulatory effects remain poorly understood. In this study, we report that VWF binding to macrophages triggers downstream MAP kinase signaling, NF-κB activation and production of pro-inflammatory cytokines and chemokines. In addition, VWF binding also drives macrophage M1 polarization and shifts macrophage metabolism towards glycolysis in a p38-dependent manner. Cumulatively, our findings define an important biological role for VWF in modulating macrophage function, and thereby establish a novel link between primary hemostasis and innate immunity.
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.
The von Willebrand factor – ADAMTS‐13 axis in malaria O’Donnell, Andrew S.; Fazavana, Judicael; O’Donnell, James S.
Research and practice in thrombosis and haemostasis,
January 2022, Letnik:
6, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Cerebral malaria (CM) continues to be associated with major morbidity and mortality, particularly in children aged <5 years in sub‐Saharan Africa. Although the biological mechanisms underpinning ...severe malaria pathophysiology remain incompletely understood, studies have shown that cytoadhesion of malaria‐infected erythrocytes to endothelial cells (ECs) within the cerebral microvasculature represents a key step in this process. Furthermore, these studies have also highlighted that marked EC activation, with secretion of Weibel‐Palade bodies (WPBs), occurs at a remarkably early stage following malaria infection. As a result, plasma levels of proteins normally stored within WPBs (including high‐molecular‐weight von Willebrand factor VWF multimers, VWF propeptide, and angiopoietin‐2) are significantly elevated. In this review, we provide an overview of recent studies that have identified novel roles through which these secreted WPB glycoproteins may directly facilitate malaria pathogenesis through a number of different platelet‐dependent and platelet‐independent pathways. Collectively, these emerging insights suggest that hemostatic dysfunction, and in particular disruption of the normal VWF–ADAMTS‐13 axis, may be of specific importance in triggering cerebral microangiopathy. Defining the molecular mechanisms involved may offer the opportunity to develop novel targeted therapeutic approaches, which are urgently needed as the mortality rate associated with CM remains in the order of 20%.
Although most plasma FVIII (Factor VIII) circulates in complex with VWF (von Willebrand factor), a minority (3%-5%) circulates as free-FVIII, which is rapidly cleared. Consequently, 20% of total ...FVIII may be cleared as free-FVIII. Critically, the mechanisms of free-FVIII clearance remain poorly understood. However, recent studies have implicated the MGL (macrophage galactose lectin) in modulating VWF clearance.
Since VWF and FVIII share similar glycosylation, we investigated the role of MGL in FVIII clearance. FVIII binding to MGL was assessed in immunosorbent and cell-based assays. In vivo, FVIII clearance was assessed in
and
mice.
In vitro-binding studies identified MGL as a novel macrophage receptor that binds free-FVIII in a glycan-dependent manner.
and
mice who received an anti-MGL1/2 blocking antibody both showed significantly increased endogenous FVIII activity compared with wild-type mice (
=0.036 and
<0.0001, respectively). MGL inhibition also prolonged the half-life of infused FVIII in
mice. To assess whether MGL plays a role in the clearance of free FVIII in a VWF-independent manner, in vivo clearance experiments were repeated in dual
mice. Importantly, the rapid clearance of free FVIII in
mice was significantly (
=0.012) prolonged in the presence of anti-MGL1/2 antibodies. Finally, endogenous plasma FVIII levels in
mice were significantly increased following MGL inhibition (
=0.016).
Cumulatively, these findings demonstrate that MGL plays an important role in regulating macrophage-mediated clearance of both VWF-bound FVIII and free-FVIII in vivo. We propose that this novel FVIII clearance pathway may be of particular clinical importance in patients with type 2N or type 3 Von Willebrand disease.
Previous studies have reported marked interindividual variation in factor VIII (FVIII) clearance in patients with hemophilia (PWH) and proposed a number of factors that influence this heterogeneity.
...To investigate the importance of the clearance rates of endogenous von Willebrand factor (VWF) compared with those of other FVIII half-life modifiers in adult PWH.
The half-life of recombinant FVIII was determined in a cohort of 61 adult PWH. A range of reported modifiers of FVIII clearance was assessed (including plasma VWF:antigen and VWF propeptide levels; VWF-FVIII binding capacity; ABO blood group; and nonneutralizing anti-FVIII antibodies). The FVIII-binding region of the VWF gene was sequenced. Finally, the effects of variation in FVIII half-life on clinical phenotype were investigated.
We demonstrated that heterogeneity in the clearance of endogenous plasma VWF is a key determinant of variable FVIII half-life in PWH. Both ABO blood group and age significantly impact FVIII clearance. The effect of ABO blood group on FVIII half-life in PWH is modulated entirely through its effect on the clearance rates of endogenous VWF. In contrast, the age-related effect on FVIII clearance is, at least in part, VWF independent. In contrast to previous studies, no major effects of variation in VWF-FVIII binding affinity on FVIII clearance were observed. Although high-titer immunoglobulin G antibodies (≥1:80) were observed in 26% of PWH, these did not impact FVIII half-life. Importantly, the annual FVIII usage (IU/kg/y) was significantly (p = .0035) increased in patients with an FVIII half-life of <12 hours.
Our data demonstrate that heterogeneity in the half-life of FVIII concentrates in patients with hemophilia A is primarily attributable to variability in the clearance of endogenous VWF.
•The half-life of factor VIII (FVIII) in patients with hemophilia (PWH) is influenced by plasma levels of von Willebrand factor (VWF):antigen, ABO blood group, and age.•Heterogeneity in the clearance of endogenous plasma VWF is a key determinant of variable FVIII half-life in PWH.•The effect of ABO blood group on FVIII half-life in PWH is modulated through its effect on the clearance rates of endogenous VWF.•The effects of age on FVIII clearance in PWH are, at least in part, VWF independent.
Background
Previous studies have demonstrated that the A1A2A3 domains of von Willebrand factor (VWF) play a key role in regulating macrophage‐mediated clearance in vivo. In particular, the A1‐domain ...has been shown to modulate interaction with macrophage low‐density lipoprotein receptor‐related protein‐1 (LRP1) clearance receptor. Furthermore, N‐linked glycans within the A2‐domain have been shown to protect VWF against premature LRP1‐mediated clearance. Importantly, however, the specific regions within A1A2A3 that enable macrophage binding have not been defined.
Objective and Methods
To address this, we utilized site‐directed PEGylation and introduced novel targeted N‐linked glycosylation within A1A2A3‐VWF and subsequently examined VWF clearance.
Results
Conjugation with a 40‐kDa polyethylene glycol (PEG) moiety significantly extended the half‐life of A1A2A3‐VWF in VWF−/− mice in a site‐specific manner. For example, PEGylation at specific sites within the A1‐domain (S1286) and A3‐domain (V1803, S1807) attenuated VWF clearance in vivo, compared to wild‐type A1A2A3‐VWF. Furthermore, PEGylation at these specific sites ablated binding to differentiated THP‐1 macrophages and LRP1 cluster II and cluster IV in‐vitro. Conversely, PEGylation at other positions (Q1353‐A1‐domain and M1545‐A2‐domain) had limited effects on VWF clearance or binding to LRP1.Novel N‐linked glycan chains were introduced at N1803 and N1807 in the A3‐domain. In contrast to PEGylation at these sites, no significant extension in half‐life was observed with these N‐glycan variants.
Conclusions
These novel data demonstrate that site specific PEGylation but not site specific N‐glycosylation modifies LRP1‐dependent uptake of the A1A2A3‐VWF by macrophages. This suggests that PEGylation, within the A1‐ and A3‐domains in particular, may be used to attenuate LRP1‐mediated clearance of VWF.
Introduction
Deficiencies of both von Willebrand Factor (VWF) and FVIII are associated with significant bleeding phenotypes. Consequently, patients with VWD or hemophilia A commonly require ...replacement therapy with coagulation factor concentrates. However, as infused VWF and FVIII have relatively short plasma half-lives, patient therapy generally necessitates frequent re-dosing. Development of a long-acting rVWF therapy thus represents an important unmet clinical need. We and others have previously demonstrated that the A1A2A3 domains of VWF play a critical role in regulating macrophage-mediated clearance of VWF in vivo. Importantly, crystal structures of the A-domains have also well characterized. In this study, we sought to utilize this data to investigate the hypothesis that site-specific PEGylation within the A1A2A3 domains could be used as a novel strategy to inhibit macrophage-mediated clearance, and thereby inform development of a rVWF molecule with extended plasma half-life.
Methodology
Site-directed mutagenesis was used to engineer novel surface cysteine residues at selected sites within A1A2A3-VWF. Following purification and characterization, individual A1A2A3 cysteine variants were PEGylated using 40kDa PEG maleimide. Clearance of unPEGylated and PEGylated A1A2A3 variants were assessed in VWF-/- mice. VWF-macrophage interactions were quantified in vitro using differentiated THP-1 macrophages. VWF binding to LRP1 clearance receptor was assessed using both immunosorbant assays and Surface Plasmon Resonance.
Results
Novel single cysteine residues were introduced at stringently selected sites within A1A2A3-VWF. These sites spanned all 3 A-domains and included; S1286C, Q1353C, M1545C, L1591C, V1636C, Q1652C, V1803C and S1807C. Interestingly, the introduction of these novel cysteine residues in both the A1 and A3 domains of VWF did not alter the rate of VWF clearance compared to WT A1A2A3-VWF. Conversely however, the A2 domain was less tolerant for the insertion of cysteines, with L1591C and V1636C variants demonstrating a significantly reduced VWF plasma half-life of approx. 1.5 fold versus WT-A1A2A3 (p<0.05).
Subsequently, the engineered cysteine residues were modified by covalent attachment of a 40kDa branched PEG molecule. All variants achieved greater than 80% PEG conjugation efficiency, except V1636C which was eliminated from further study. Remarkably, PEG conjugation displayed site-specific effects on the in vivo half-life of A1A2A3-VWF. For example, PEGylation at S1286C within the A1 domain resulted in a marked increased in VWF half-life compared to WT-A1A2A3 VWF (92.4±6 vs 18.3±0.9 mins, respectively, p<0.001). Conversely, PEGylation at the adjacent site in the A1 domain, Q1353C, or downstream at M1545C within A2 had no significant effect on VWF half-life (23.3±1 and 20.8±3 mins, respectively). Interestingly, despite the fact that no previous roles have been described for the A3 domain of VWF in regulating its clearance, we observed a significant extension in VWF half-life for PEGylated variants within the A3 domain, V1803C and S1807C, (93.3±9 mins and 58.0±5 mins, respectively, p<0.05).
Macrophage LDL receptor related protein 1 (LRP1) has been implicated as key cellular mediator of VWF clearance in vivo. Interestingly, in keeping with the reduced clearance observed for PEGylated VWF variants S1286C, V1803C and S1807C, binding of these variants to clearance receptor LRP1 cluster II and IV was ablated. Conversely, PEGylated variants which failed to extend VWF half-life (Q1353C and M1545C) displayed LRP1 binding that was comparable to WT-A1A2A3 VWF. Interestingly, PEGylation at specific sites in A2 (L1591C and Q1652C) which served to increased VWF half-life displayed normal binding to LRP1 cluster IV. However, binding of these variants to LRP1 cluster II was reduced by 90% compared to WT-A1A2A3.
Conclusion
Collectively, our novel data demonstrate that cysteine-directed PEGylation at specific sites within the A1 (S1286C), A2 (L1591C, Q1652C) and A3 (V1803C and S1807C) domains of A1A2A3-VWF inhibits binding to macrophage clearance receptor LRP1 in vitro. Consequently, these PEGylated A1A2A3-VWF variants demonstrate an extended circulatory half-life in vivo compared to wild type A1A2A3-VWF. Taken together, these results support the use of site-specific PEGylation as a potential approach to develop long-acting full length rVWF molecules.
Cooke:Pfizer: Employment. Terraube:Pfizer: Employment. Cohen:Pfizer: Employment. Pittman:Pfizer: Employment. Cunningham:Pfizer: Employment. Lambert:Pfizer: Employment. O'Donnell:Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; CSL Behring: Consultancy; Octapharma: Speakers Bureau; Leo Pharma: Speakers Bureau; Novo Nordisk: Research Funding, Speakers Bureau; Bayer: Research Funding, Speakers Bureau; Baxter: Research Funding, Speakers Bureau; Shire: Research Funding, Speakers Bureau.
Heparin derivative-based therapy has evolved from unfractionated heparin (UFH) to low-molecular-weight heparins (LMWHs) and now fondaparinux, a synthetic pentasaccharide. Contrary to UFH or LMWHs, ...fondaparinux is not neutralized by protamine sulfate, and no antidote is available to counteract bleeding disorders associated with overdosing. To make the use of fondaparinux safer, we developed an antithrombin (AT) variant as a potent antidote to heparin derivatives. This variant (AT-N135Q-Pro394) combines 2 mutations: substitution of Asn135 by a Gln to remove a glycosylation site and increase affinity for heparins, and the insertion of a Pro between Arg393 and Ser394 to abolish its anticoagulant activity. As expected, AT-N135Q-Pro394 anticoagulant activity was almost abolished, and it exhibited a 3-fold increase in fondaparinux affinity. AT-N135Q-Pro394 was shown to reverse fondaparinux overdosing in vitro in a dose-dependent manner through a competitive process with plasma AT for fondaparinux binding. This antidote effect was also observed in vivo: administration of AT-N135Q-Pro394 in 2.5-fold molar excess versus plasma AT neutralized 86% of the anti-Xa activity within 5 minutes in mice treated with fondaparinux. These results clearly demonstrate that AT-N135Q-Pro394 can reverse the anticoagulant activity of fondaparinux and thus could be used as an antidote for this drug.
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