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

Development of inactive modified antithrombin as an antidote to heparin anticoagulants

Les héparines regroupant les héparines standards (HNF), les héparines de bas poids moléculaire(HBPM), et le fondaparinux, sont des médicaments anticoagulants. Ils potentialisent l’antithrombine (AT) : un inhibiteur physiologique de la coagulation. Leur utilisation en thérapeutique est associée à un risque hémorragique majeur. Actuellement, le sulfate de protamine est le seul antidote disponible vis-à-vis des HNF. Il est partiellement efficace vis-à-vis des HBPM, et n’a aucun effet contre le fondaparinux, qui n’a pas d’antidote jusqu’à présent. C’est dans ce contexte que nous proposons des AT modifiées inactives, mais capables de se lier aux molécules d’héparines. Ces AT déplaceraient les molécules d’héparines de l’AT plasmatique, et neutraliseraient leur effet anticoagulant. Pour produire de telles AT, nous avons choisi une approche recombinante et une approche chimique. Dans la première approche, nous avons exprimé le variant AT-N135Q-Pro394. Ce variant possède une activité anti-Xa ou anti-IIa inférieure à 0,02% en présence de dérivés hépariniques, et une affinité à l’héparine 3 fois meilleure, comparée à l’AT plasmatique. En revanche, dans l’approche chimique, nous avons modifié l’AT plasmatique par la 2,3-butanedione (AT-BD), un réactif chimique de caractérisation des arginines. Contrairement au variant, cette AT-BD a une perte d’activité anticoagulante modérée, puis une affinité à l’héparine 20 fois meilleure, comparée à l’AT plasmatique. Malgré ces différences de propriétés biochimiques, ces 2 AT modifiées neutralisent d’une façon similaire les héparines in vitro et sur un modèle murin. Par ailleurs, à l’inverse du sulfate de protamine, nos antidotes n’ont pas d’activité anticoagulante propre sur un test de céphaline activée. Ainsi, ce travail de thèse a permis non seulement de proposer les premiers et les seuls antidotes spécifiques au fondaparinux décrits, mais aussi des antidotes alternatifs pour tous les anticoagulants hépariniques.Unfractionnated heparin (UFH), low molecular weight heparins (LMWH), and fondaparinux are used therapeutically as anticoagulants. They potentiate antithrombin (AT): a physiological inhibitor of coagulation. Their therapeutic use is associated with a major risk of bleeding. Currently, protamine sulfate is the only antidote available for UFH. It is partially effective for LMWH, and has no effect against fondaparinux, which has no antidote. So, we propose modified inactive AT, but able to bind heparin molecules as antidote of these heparins. These molecules would compete with plasmatic AT for binding to heparins, and neutralize their anticoagulant effect. To produce that AT, we realized a genetic approach and a chemical approach. In the first approach, we expressed the variant AT-N135Q-Pro394 that had an anti-Xa or anti-IIa activity below 0.02% in the presence of heparins, and heparin affinity three times higher, compared to the plasmatic AT. In the chemical approach, we modified the plasmatic AT by 2,3-butanedione (AT-BD), a chemical reagent for arginin’s characterization. The AT-BD had a moderate loss of anticoagulant activity, and a heparin affinity 20 times higher, compared to the plasmatic AT. Despite these differences in biochemical properties, these two modified AT neutralize similarly heparins in vitro and in a mouse model. Moreover, unlike protamine sulfate, our antidotes had not an intrinsic anticoagulant effect in activated partial thromboplastin test. Thus, this PhD-work offers the first and the only specific antidote described to fondaparinux, and it can be used too alternatively for all anticoagulant heparins

Développement d’une antithrombine modifiée inactive comme antidote des anticoagulants hépariniques

Unfractionnated heparin (UFH), low molecular weight heparins (LMWH), and fondaparinux are used therapeutically as anticoagulants. They potentiate antithrombin (AT): a physiological inhibitor of coagulation. Their therapeutic use is associated with a major risk of bleeding. Currently, protamine sulfate is the only antidote available for UFH. It is partially effective for LMWH, and has no effect against fondaparinux, which has no antidote. So, we propose modified inactive AT, but able to bind heparin molecules as antidote of these heparins. These molecules would compete with plasmatic AT for binding to heparins, and neutralize their anticoagulant effect. To produce that AT, we realized a genetic approach and a chemical approach. In the first approach, we expressed the variant AT-N135Q-Pro394 that had an anti-Xa or anti-IIa activity below 0.02% in the presence of heparins, and heparin affinity three times higher, compared to the plasmatic AT. In the chemical approach, we modified the plasmatic AT by 2,3-butanedione (AT-BD), a chemical reagent for arginin’s characterization. The AT-BD had a moderate loss of anticoagulant activity, and a heparin affinity 20 times higher, compared to the plasmatic AT. Despite these differences in biochemical properties, these two modified AT neutralize similarly heparins in vitro and in a mouse model. Moreover, unlike protamine sulfate, our antidotes had not an intrinsic anticoagulant effect in activated partial thromboplastin test. Thus, this PhD-work offers the first and the only specific antidote described to fondaparinux, and it can be used too alternatively for all anticoagulant heparins.Les héparines regroupant les héparines standards (HNF), les héparines de bas poids moléculaire(HBPM), et le fondaparinux, sont des médicaments anticoagulants. Ils potentialisent l’antithrombine (AT) : un inhibiteur physiologique de la coagulation. Leur utilisation en thérapeutique est associée à un risque hémorragique majeur. Actuellement, le sulfate de protamine est le seul antidote disponible vis-à-vis des HNF. Il est partiellement efficace vis-à-vis des HBPM, et n’a aucun effet contre le fondaparinux, qui n’a pas d’antidote jusqu’à présent. C’est dans ce contexte que nous proposons des AT modifiées inactives, mais capables de se lier aux molécules d’héparines. Ces AT déplaceraient les molécules d’héparines de l’AT plasmatique, et neutraliseraient leur effet anticoagulant. Pour produire de telles AT, nous avons choisi une approche recombinante et une approche chimique. Dans la première approche, nous avons exprimé le variant AT-N135Q-Pro394. Ce variant possède une activité anti-Xa ou anti-IIa inférieure à 0,02% en présence de dérivés hépariniques, et une affinité à l’héparine 3 fois meilleure, comparée à l’AT plasmatique. En revanche, dans l’approche chimique, nous avons modifié l’AT plasmatique par la 2,3-butanedione (AT-BD), un réactif chimique de caractérisation des arginines. Contrairement au variant, cette AT-BD a une perte d’activité anticoagulante modérée, puis une affinité à l’héparine 20 fois meilleure, comparée à l’AT plasmatique. Malgré ces différences de propriétés biochimiques, ces 2 AT modifiées neutralisent d’une façon similaire les héparines in vitro et sur un modèle murin. Par ailleurs, à l’inverse du sulfate de protamine, nos antidotes n’ont pas d’activité anticoagulante propre sur un test de céphaline activée. Ainsi, ce travail de thèse a permis non seulement de proposer les premiers et les seuls antidotes spécifiques au fondaparinux décrits, mais aussi des antidotes alternatifs pour tous les anticoagulants hépariniques

Développement d'une antithrombine modifiée inactive comme antidote des anticoagulants hépariniques

Les héparines regroupant les héparines standards (HNF), les héparines de bas poids moléculaire(HBPM), et le fondaparinux, sont des médicaments anticoagulants. Ils potentialisent l antithrombine (AT) : un inhibiteur physiologique de la coagulation. Leur utilisation en thérapeutique est associée à un risque hémorragique majeur. Actuellement, le sulfate de protamine est le seul antidote disponible vis-à-vis des HNF. Il est partiellement efficace vis-à-vis des HBPM, et n a aucun effet contre le fondaparinux, qui n a pas d antidote jusqu à présent. C est dans ce contexte que nous proposons des AT modifiées inactives, mais capables de se lier aux molécules d héparines. Ces AT déplaceraient les molécules d héparines de l AT plasmatique, et neutraliseraient leur effet anticoagulant. Pour produire de telles AT, nous avons choisi une approche recombinante et une approche chimique. Dans la première approche, nous avons exprimé le variant AT-N135Q-Pro394. Ce variant possède une activité anti-Xa ou anti-IIa inférieure à 0,02% en présence de dérivés hépariniques, et une affinité à l héparine 3 fois meilleure, comparée à l AT plasmatique. En revanche, dans l approche chimique, nous avons modifié l AT plasmatique par la 2,3-butanedione (AT-BD), un réactif chimique de caractérisation des arginines. Contrairement au variant, cette AT-BD a une perte d activité anticoagulante modérée, puis une affinité à l héparine 20 fois meilleure, comparée à l AT plasmatique. Malgré ces différences de propriétés biochimiques, ces 2 AT modifiées neutralisent d une façon similaire les héparines in vitro et sur un modèle murin. Par ailleurs, à l inverse du sulfate de protamine, nos antidotes n ont pas d activité anticoagulante propre sur un test de céphaline activée. Ainsi, ce travail de thèse a permis non seulement de proposer les premiers et les seuls antidotes spécifiques au fondaparinux décrits, mais aussi des antidotes alternatifs pour tous les anticoagulants hépariniques.Unfractionnated heparin (UFH), low molecular weight heparins (LMWH), and fondaparinux are used therapeutically as anticoagulants. They potentiate antithrombin (AT): a physiological inhibitor of coagulation. Their therapeutic use is associated with a major risk of bleeding. Currently, protamine sulfate is the only antidote available for UFH. It is partially effective for LMWH, and has no effect against fondaparinux, which has no antidote. So, we propose modified inactive AT, but able to bind heparin molecules as antidote of these heparins. These molecules would compete with plasmatic AT for binding to heparins, and neutralize their anticoagulant effect. To produce that AT, we realized a genetic approach and a chemical approach. In the first approach, we expressed the variant AT-N135Q-Pro394 that had an anti-Xa or anti-IIa activity below 0.02% in the presence of heparins, and heparin affinity three times higher, compared to the plasmatic AT. In the chemical approach, we modified the plasmatic AT by 2,3-butanedione (AT-BD), a chemical reagent for arginin s characterization. The AT-BD had a moderate loss of anticoagulant activity, and a heparin affinity 20 times higher, compared to the plasmatic AT. Despite these differences in biochemical properties, these two modified AT neutralize similarly heparins in vitro and in a mouse model. Moreover, unlike protamine sulfate, our antidotes had not an intrinsic anticoagulant effect in activated partial thromboplastin test. Thus, this PhD-work offers the first and the only specific antidote described to fondaparinux, and it can be used too alternatively for all anticoagulant heparins.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

The von Willebrand factor - ADAMTS-13 axis in malaria

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%. </p

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

Investigating the clearance of VWF A-domains using site-directed PEGylation and novel N-linked glycosylation

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.</p

Aptamer BT200 blocks interaction of K1405-1408 in the VWF-A1 domain with macrophage LRP1

Rondaptivan pegol (previously BT200) is a PEGylated RNA aptamer that binds to the A1 domain of VWF. Recent clinical trials demonstrated that BT200 significantly increased plasma VWF-FVIII levels by attenuating VWF clearance. The biological mechanism(s) through which BT200 attenuates in vivo clearance of VWF have not been defined. We hypothesized that BT200 interaction with the VWF-A1 domain may increase plasma VWF levels by attenuating macrophage-mediated clearance. We observed that full length- and VWF-A1A2A3 binding to macrophages, and VWF-A1 domain binding to LRP1 cluster II and cluster IV, were concentration-dependently inhibited by BT200. Additionally, full length VWF binding to LRP1 expressed on HEK293T (HEK-LRP1) cells was also inhibited by BT200. Importantly, BT200 interacts with the VWF-A1 domain in proximity to a conserved cluster of four lysine residues (K1405, K1406, K1407 and K1408). Alanine mutagenesis of this K1405-K1408 cluster (VWF-4A) significantly (p&amp;lt;0.001) attenuated binding of VWF to both LRP1 clusters II and IV. Furthermore, in vivo clearance of VWF-4A was significantly (p&amp;lt;0.001) reduced compared to wild type VWF. BT200 did not significantly inhibit binding of VWF-4A to LRP1 cluster IV or HEK-LRP1 cells. Finally, BT200 interaction with the VWF-A1 domain also inhibited binding to macrophage galactose lectin (MGL) and the SR-AI scavenger receptor. Collectively, our findings demonstrate that BT200 prolongs VWF half-life by attenuating macrophage-mediated clearance and specifically the interaction of K1405-1408 in the VWF-A1 domain with macrophage LRP1. These data support the concept that targeted inhibition of VWF clearance pathways represent a novel therapeutic approach for VWD and hemophilia A