Von Willebrand factor: how unique structural adaptations support and coordinate its complex function.

Von Willebrand factor (VWF) is a multimeric protein consisting of covalently linked monomers, which share an identical domain architecture. Although involved in processes like inflammation, angiogenesis and cancer metastasis, VWF is mostly known for its role in hemostasis, by acting as a chaperone-protein for coagulation factor VIII (FVIII) and by contributing to the recruitment of platelets during thrombus formation. To serve its role in hemostasis, VWF needs to bind a variety of ligands, including FVIII, platelet-receptor glycoprotein Ib-alpha, VWF-cleaving protease ADAMTS13, sub-endothelial collagen and integrin alpha-IIb/beta-3. Importantly, interactions are differently regulated for each of these ligands. How are these binding events accomplished and coordinated? The basic structures of the domains that constitute the VWF protein are found in hundreds of other proteins of pro- and eukaryotic organisms. However, the determination of the three-dimensional structures of these domains within the VWF context and especially in complex with its ligands reveals that exclusive, VWF-specific structural adaptations have been incorporated in its domains. They provide an explanation of how VWF binds its ligands in a synchronized and timely fashion. In the current review, we have focused on the domains that interact with the main ligands of VWF and discuss how elucidating the three-dimensional structures of these domains has contributed to our understanding of how VWF function is controlled. We further detail how mutations in these domains that are associated with von Willebrand disease modulate the interaction between VWF and its ligands.

Illustrated State-of-the-Art Capsules of the ISTH 2024 Congress.

Here, we present a series of illustrated capsules from the State of the Art (SOA) speakers at the 2024 International Society on Thrombosis and Haemostasis Congress in Bangkok, Thailand. This year's Congress marks the first time that the International Society on Thrombosis and Haemostasis has held its flagship scientific meeting in Southeast Asia and is the first to be organized by an international Planning Committee. The Bangkok program will feature innovative science and clinical updates from around the world, reflecting the diversity and multidisciplinary growth of our field. In these illustrated SOA capsules, you will find an exploration of novel models of thrombosis and bleeding and biomaterial discoveries that can trigger or block coagulation. Thromboinflammation is now understood to drive many disease states, and the SOA speakers cover cellular and coagulation responses to COVID-19 and other infections. The theme of crosstalk between coagulation and inflammation expands with capsules on protein S signaling, complement, and fibrinolytic inhibitors. Novel agents for hemophilia and thrombosis prevention are introduced. Challenging clinical conditions are also covered, such as inherited platelet disorders and antiphospholipid antibody syndrome. The scientific program in Bangkok will also showcase the work of clinicians and scientists from all parts of the world and chronicle real-world challenges. For example, 2 SOA capsules address the diagnosis and management of von Willebrand disease in low-income settings. Take some time to browse through these short illustrated reviews; we're sure that you'll be entertained, educated, and inspired to further explore the world of thrombosis and hemostasis.

Fitusiran reduces bleeding in factor X-deficient mice.

ABSTRACT: Factor X (FX) deficiency is a rare bleeding disorder manifesting a bleeding tendency caused by low FX activity levels. We aim to explore the use of fitusiran (an investigational small interfering RNA that silences antithrombin expression) to increase thrombin generation and the in vivo hemostatic potential under conditions of FX deficiency. We therefore developed a novel model of inducible FX deficiency, generating mice expressing <1% FX activity and antigen (f10low mice). Compared with control f10WT mice, f10low mice had sixfold and fourfold prolonged clotting times in prothrombin time and activated partial prothrombin time assays, respectively (P < .001). Thrombin generation was severely reduced, irrespective of whether tissue factor or factor XIa was used as an initiator. In vivo analysis revealed near-absent thrombus formation in a laser-induced vessel injury model. Furthermore, in 2 distinct bleeding models, f10low mice displayed an increased bleeding tendency compared with f10WT mice. In the tail-clip assay, blood loss was increased from 12 ± 16 µL to 590 ± 335 µL (P < .0001). In the saphenous vein puncture (SVP) model, the number of clots generated was reduced from 19 ± 5 clots every 30 minutes for f10WT mice to 2 ± 2 clots every 30 minutes (P < .0001) for f10low mice. In both models, bleeding was corrected upon infusion of purified FX. Treatment of f10low mice with fitusiran (2 × 10 mg/kg at 1 week interval) resulted in 17 ± 6% residual antithrombin activity and increased thrombin generation (fourfold and twofold to threefold increase in endogenous thrombin potential and thrombin peak, respectively). In the SVP model, the number of clots was increased to 8 ± 6 clots every 30 minutes (P = .0029). Altogether, we demonstrate that reduction in antithrombin levels is associated with improved hemostatic activity under conditions of FX deficiency.

Thrombin generation on vascular cells in the presence of factor VIII and/or emicizumab.

BACKGROUND: The effect of factor VIII (FVIII) or emicizumab on thrombin generation is usually assessed in assays using synthetic phospholipids. Here, we assessed thrombin generation at the surface of human arterial cells (aortic endothelial cells [hAECs] and aortic vascular smooth muscle cells [hVSMCs]). OBJECTIVES: To explore the capacity of hAECs (resting or stimulated) and hVSMCs to support thrombin generation by FVIII or emicizumab. METHODS: Primary hVSMCs and hAECs were analyzed for tissue factor (TF)-activity and antigen, phosphatidylserine (PS)-exposure, tissue factor pathway inhibitor (TFPI)-content and thrombomodulin expression. Cells were incubated with FVIII-deficient plasma spiked with FVIII, emicizumab, activated prothrombin complex concentrate (APCC) or combinations thereof. RESULTS: TF activity and PS-exposure were present on both hVSMCs and hAECs. In contrast, thrombomodulin and TFPI were expressed on hAECs, while virtually lacking on hVSMCs, confirming the procoagulant nature of hVSMCs. Tumor necrosis factor α-mediated stimulation of hAECs increased not only TF antigen, TF activity, and PS-exposure but also TFPI and thrombomodulin expression. As expected, FVIII and emicizumab promoted thrombin generation on nonstimulated hAECs and hVSMCs, with more thrombin being generated on hVSMCs. Unexpectedly, FVIII and emicizumab increased thrombin generation to a lesser extent on stimulated hAECs compared with nonstimulated hAECs. Finally, adding emicizumab to FVIII did not further increase thrombin generation, whereas the addition of emicizumab to APCC resulted in exaggerated thrombin generation. CONCLUSION: Tumor necrosis factor stimulation of hAECs increases both pro- and anticoagulant activity. Unexpectedly, the increased anticoagulant activity is sufficient to limit both FVIII- and emicizumab-induced thrombin generation. This protective effect disappears when emicizumab is combined with APCC.

Differences in venous clot structures between hemophilic mice treated with emicizumab versus factor VIII or factor VIIIFc.

Recombinant factor VIII (rFVIII), rFVIIIFc and emicizumab are established treatment options in the management of hemophilia A. Each has its unique mode of action, which can influence thrombin generation kinetics and therefore also the kinetics of thrombin substrates. Such differences may potentially result in clots with different structural and physical properties. A starting observation of incomplete wound closure in a patient on emicizumab-prophylaxis led us employ a relevant mouse model in which we noticed that emicizumab-induced clots appeared less stable compared to FVIII-induced clots. We thus analyzed fibrin formation in vitro and in vivo. In vitro fibrin formation was faster and more abundant in the presence of emicizumab compared to rFVIII/rFVIIIFc. Furthermore, the time-interval between the initiation of fibrin formation and factor XIII activation was twice as long for emicizumab compared to rFVIII/rFVIIIFc. Scanning-electron microscopy and immunofluorescent spinning-disk confocal-microscopy of in vivo generated clots confirmed increased fibrin formation in the presence of emicizumab. Unexpectedly, we also detected a different morphology between rFVIII/rFVIIIFc- and emicizumab-induced clots. Contrary to the regular fibrin-mesh obtained with rFVIII/rFVIIIFc, fibrin-fibers appeared to be fused into large patches upon emicizumabtreatment. Moreover, fewer red blood cells were detected in regions where these fibrin patches were present. The presence of highly-dense fibrin-structures associated with a diffuse fiber-structure in emicizumab-induced clots was also observed when using superresolution imaging. We hypothesize that the modified kinetics of thrombin, fibrin and factor XIIIa generation contribute to differences in structural and physical properties between clots formed in the presence of FVIII or emicizumab.

Angiopoietin-2 binds to multiple interactive sites within von Willebrand factor.

Background: Biosynthesis of von Willebrand factor (VWF) in endothelial cells drives the formation of storage-organelles known as Weibel-Palade bodies (WPBs). WPBs also contain several other proteins, including angiopoietin-2 (Ang-2). Objectives: At present, the molecular basis of the VWF-Ang-2 interaction is poorly understood. Here, we used immunosorbent-binding assays and specific recombinant VWF fragments to analyze VWF-Ang-2 interactions. Results: We found that VWF bound to immobilized Ang-2 most efficiently (half-maximal binding at 0.5 ± 0.1 µg/mL) under conditions of high CaCl2 (10 mM) and slightly acidic pH (6.4-7.0). Interestingly, several isolated recombinant VWF domains (A1/Fc, A2/Fc, D4/Fc, and D'D3-HPC4) displayed dose-dependent binding to immobilized Ang-2. Binding appeared specific, as antibodies against D'D3, A1, and A2 significantly reduced the binding of these domains to Ang-2. Complexes between VWF and Ang-2 in plasma could be detected by immunoprecipitation- and immunosorbent assays. Unexpectedly, control experiments also revealed complexes between VWF and angiopoietin-1 (Ang-1), a protein structurally homologous to Ang-2. Furthermore, direct binding studies showed dose-dependent binding of VWF to immobilized Ang-1 (half-maximal binding at 1.8 ± 1.0 µg/mL). Interestingly, rather than competing for Ang-1 binding, Ang-2 enhanced the binding of VWF to Ang-1 about 3-fold. Competition experiments further revealed that binding to VWF does not prevent Ang-1 and Ang-2 from binding to Tie-2. Conclusion: Our data show that both Ang-1 and Ang-2 bind to VWF, seemingly using different interactive sites. Ang-2 modulates the binding of VWF to Ang-1, the (patho)-physiological consequences of which remain to be investigated.

Toll-like receptor 4 and macrophage scavenger receptor 1 crosstalk regulates phagocytosis of a fungal pathogen.

The opportunistic fungal pathogen Cryptococcus neoformans causes lethal infections in immunocompromised patients. Macrophages are central to the host response to cryptococci; however, it is unclear how C. neoformans is recognised and phagocytosed by macrophages. Here we investigate the role of TLR4 in the non-opsonic phagocytosis of C. neoformans. We find that loss of TLR4 function unexpectedly increases phagocytosis of non-opsonised cryptococci by murine and human macrophages. The increased phagocytosis observed in Tlr4-/- cells was dampened by pre-treatment of macrophages with oxidised-LDL, a known ligand of scavenger receptors. The scavenger receptor, macrophage scavenger receptor 1 (MSR1) (also known as SR-A1 or CD204) was upregulated in Tlr4-/- macrophages. Genetic ablation of MSR1 resulted in a 75% decrease in phagocytosis of non-opsonised cryptococci, strongly suggesting that it is a key non-opsonic receptor for this pathogen. We go on to show that MSR1-mediated uptake likely involves the formation of a multimolecular signalling complex involving FcγR leading to SYK, PI3K, p38 and ERK1/2 activation to drive actin remodelling and phagocytosis. Altogether, our data indicate a hitherto unidentified role for TLR4/MSR1 crosstalk in the non-opsonic phagocytosis of C. neoformans.

Imlifidase, a new option to optimize the management of patients with hemophilia A on emicizumab.

BACKGROUND: Emicizumab is a bispecific, chimeric, humanized immunoglobulin G (IgG)4 that mimics the procoagulant activity of factor (F) VIII (FVIII). Its long half-life and subcutaneous route of administration have been life-changing in treating patients with hemophilia A (HA) with or without FVIII inhibitors. However, emicizumab only partially mimics FVIII activity; it prevents but does not treat acute bleeds. Emergency management is particularly complicated in patients with FVIII inhibitors receiving emicizumab prophylaxis in whom exogenous FVIII is inefficient. We have shown recently that Imlifidase (IdeS), a bacterial IgG-degrading enzyme, efficiently eliminates human anti-FVIII IgG in a mouse model of severe HA with inhibitors and opens a therapeutic window for the administration of exogenous FVIII. OBJECTIVES: To investigate the impact of IdeS treatment in inhibitor-positive HA mice injected with emicizumab. METHODS: IdeS was injected to HA mice reconstituted with human neutralizing anti-FVIII IgG and treated with emicizumab. RESULTS: IdeS hydrolyzed emicizumab in vitro and in vivo, albeit, at slower rates than another recombinant human monoclonal IgG4. While F(ab')2 fragments were rapidly cleared from the circulation, thus leading to a rapid loss of emicizumab procoagulant activity, low amounts of single-cleaved intermediate IgG persisted for several days. Moreover, the IdeS-mediated elimination of the neutralizing anti-FVIII IgG and restoration of the hemostatic efficacy of exogenous FVIII were not impaired by the presence of emicizumab and polyclonal human IgG in inhibitor-positive HA mice. CONCLUSION: Our results suggest that IdeS could be administered to inhibitor-positive patients with HA receiving emicizumab prophylaxis to improve and ease the management of breakthrough bleeds or programmed major surgeries.

Over-expression of Dyrk1A affects bleeding by modulating plasma fibronectin and fibrinogen level in mice.

Down syndrome is the most common chromosomal abnormality in humans. Patients with Down syndrome have hematologic disorders, including mild to moderate thrombocytopenia. In case of Down syndrome, thrombocytopenia is not associated with bleeding, and it remains poorly characterized regarding molecular mechanisms. We investigated the effects of overexpression of Dyrk1A, an important factor contributing to some major Down syndrome phenotypes, on platelet number and bleeding in mice. Mice overexpressing Dyrk1A have a decrease in platelet number by 20%. However, bleeding time was found to be reduced by 50%. The thrombocytopenia and the decreased bleeding time observed were not associated to an abnormal platelet receptors expression, to a defect of platelet activation by ADP, thrombin or convulxin, to the presence of activated platelets in the circulation or to an abnormal half-life of the platelets. To propose molecular mechanisms explaining this discrepancy, we performed a network analysis of Dyrk1A interactome and demonstrated that Dyrk1A, fibronectin and fibrinogen interact indirectly through two distinct clusters of proteins. Moreover, in mice overexpressing Dyrk1A, increased plasma fibronectin and fibrinogen levels were found, linked to an increase of the hepatic fibrinogen production. Our results indicate that overexpression of Dyrk1A in mice induces decreased bleeding consistent with increased plasma fibronectin and fibrinogen levels, revealing a new role of Dyrk1A depending on its indirect interaction with these two proteins.

Transplacental delivery of therapeutic proteins by engineered immunoglobulin G: a step toward perinatal replacement therapy.

BACKGROUND: Transplacental delivery of maternal immunoglobulin G (IgG) provides humoral protection during the first months of life until the newborn's immune system reaches maturity. The maternofetal interface has been exploited therapeutically to replace missing enzymes in the fetus, as shown in experimental mucopolysaccharidoses, or to shape adaptive immune repertoires during fetal development and induce tolerance to self-antigens or immunogenic therapeutic molecules. OBJECTIVES: To investigate whether proteins that are administered to pregnant mice or endogenously present in their circulation may be delivered through the placenta. METHODS: We engineered monovalent immunoglobulin G (FabFc) specific for different domains of human factor VIII (FVIII), a therapeutically relevant model antigen. FabFc was injected with exogenous FVIII into pregnant severe hemophilia A mice or pregnant mice expressing human FVIII following AAV8-mediated gene therapy. FabFc and FVIII were detected in the pregnant mice and/or fetuses by enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS: Administration of FabFc to pregnant mice allowed the maternofetal delivery of FVIII in a FcRn-dependent manner. FVIII antigen levels achieved in the fetuses represented 10% of normal plasma levels in the human. We identified antigen/FabFc complex stability, antigen size, and shielding of promiscuous protein patches as key parameters to foster optimal antigen delivery. CONCLUSION: Our results pave the way toward the development of novel strategies for the in utero delivery of endogenous maternal proteins to replace genetically deficient fetal proteins or to educate the immune system and favor active immune tolerance upon protein encounter later in life.

Efficacy of platelet-inspired hemostatic nanoparticles on bleeding in von Willebrand disease murine models.

The lack of innovation in von Willebrand disease (VWD) originates from many factors including the complexity and heterogeneity of the disease but also from a lack of recognition of the impact of the bleeding symptoms experienced by patients with VWD. Recently, a few research initiatives aiming to move past replacement therapies using plasma-derived or recombinant von Willebrand factor (VWF) concentrates have started to emerge. Here, we report an original approach using synthetic platelet (SP) nanoparticles for the treatment of VWD type 2B (VWD-2B) and severe VWD (type 3 VWD). SP are liposomal nanoparticles decorated with peptides enabling them to concomitantly bind to collagen, VWF, and activated platelets. In vitro, using various microfluidic assays, we show the efficacy of SPs to improve thrombus formation in VWF-deficient condition (with human platelets) or using blood from mice with VWD-2B and deficient VWF (VWF-KO, ie, type 3 VWD). In vivo, using a tail-clip assay, SP treatment reduced blood loss by 35% in mice with VWD-2B and 68% in mice with VWF-KO. Additional studies using nanoparticles decorated with various combinations of peptides demonstrated that the collagen-binding peptide, although not sufficient by itself, was crucial for SP efficacy in VWD-2B; whereas all 3 peptides appeared necessary for mice with VWF-KO. Clot imaging by immunofluorescence and scanning electron microscopy revealed that SP treatment of mice with VWF-KO led to a strong clot, similar to those obtained in wild-type mice. Altogether, our results show that SP could represent an attractive therapeutic alternative for VWD, especially considering their long half-life and stability.

Development of a dual hybrid AAV vector for endothelial-targeted expression of von Willebrand factor.

Von Willebrand disease (VWD), the most common inherited bleeding disorder in humans, is caused by quantitative or qualitative defects in von Willebrand factor (VWF). VWD represents a potential target for gene therapy applications, as a single treatment could potentially result in a long-term correction of the disease. In recent years, several liver-directed gene therapy approaches have been exploited for VWD, but their efficacy was generally limited by the large size of the VWF transgene and the reduced hemostatic activity of the protein produced from hepatocytes. In this context, we aimed at developing a gene therapy strategy for gene delivery into endothelial cells, the natural site of biosynthesis of VWF. We optimized an endothelial-specific dual hybrid AAV vector, in which the large VWF cDNA was put under the control of an endothelial promoter and correctly reconstituted upon cell transduction by a combination of trans-splicing and homologous recombination mechanisms. In addition, we modified the AAV vector capsid by introducing an endothelial-targeting peptide to improve the efficiency for endothelial-directed gene transfer. This vector platform allowed the reconstitution of full-length VWF transgene both in vitro in human umbilical vein endothelial cells and in vivo in VWD mice, resulting in long-term expression of VWF.

A nanobody against the VWF A3 domain detects ADAMTS13-induced proteolysis in congenital and acquired VWD.

von Willebrand factor (VWF) is a multimeric protein, the size of which is regulated via ADAMTS13-mediated proteolysis within the A2 domain. We aimed to isolate nanobodies distinguishing between proteolyzed and non-proteolyzed VWF, leading to the identification of a nanobody (designated KB-VWF-D3.1) targeting the A3 domain, the epitope of which overlaps the collagen-binding site. Although KB-VWF-D3.1 binds with similar efficiency to dimeric and multimeric derivatives of VWF, binding to VWF was lost upon proteolysis by ADAMTS13, suggesting that proteolysis in the A2 domain modulates exposure of its epitope in the A3 domain. We therefore used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments showed that a loss of 10% intact VWF could be detected using this nanobody. By comparing plasma from volunteers to that from congenital von Willebrand disease (VWD) patients, intact-VWF levels were significantly reduced for all VWD types, and most severely in VWD type 2A-group 2, in which mutations promote ADAMTS13-mediated proteolysis. Unexpectedly, we also observed increased proteolysis in some patients with VWD type 1 and VWD type 2M. A significant correlation (r = 0.51, P < .0001) between the relative amount of high-molecular weight multimers and levels of intact VWF was observed. Reduced levels of intact VWF were further found in plasmas from patients with severe aortic stenosis and patients receiving mechanical circulatory support. KB-VWF-D3.1 is thus a nanobody that detects changes in the exposure of its epitope within the collagen-binding site of the A3 domain. In view of its unique characteristics, it has the potential to be used as a diagnostic tool to investigate whether a loss of larger multimers is due to ADAMTS13-mediated proteolysis.

Polyphenol-rich açaí seed extract exhibits reno-protective and anti-fibrotic activities in renal tubular cells and mice with kidney failure.

The main goal of this study was to evaluate the reno-protective effects of a phenolic-rich Açaí seed extract (ASE) in mice with kidney failure. Kidney failure was induced chemically with an adenine-rich diet (0.25% w/w for 4 weeks) in male CD1 Swiss mice. Mice were then provided daily with ASE (at a dose of ~ 350 mg/kg/day) in drinking water for 4 weeks. Adenine mice exhibited renal dysfunction evidenced by increased proteinuria, increased uremia, extensive tubular atrophy and kidney fibrosis associated with overexpression of pro-fibrotic genes (collagen 1a1, transforming growth factor ß1, TGF-ß1) and markers of tubular injury (such as Kidney injury molecule-1, KIM-1). ASE was able to beneficially counteract all these effects. ASE improved oxidative damage and fibrosis by decreasing carbonylated protein and MDA concentrations, as well as collagen deposition in renal tissue. ASE decreased the expression of TGF-ß1 gene and the abundance of protein TGF-ß1 in kidneys. It further decreased both expression and urinary excretion of tubular injury biomarkers, e.g., KIM-1 and Neutrophil gelatinase-associated lipocalin. CKD ASE-treated mice exhibited higher polyphenol content and total antioxidant capacity compared to control mice. ASE further prevented the expression of profibrotic genes in HK2 human tubular cells exposed to uremic toxins. Taken together, these findings suggest that ASE exerted potent reno-protective and anti-fibrotic effects through its antioxidant activity and the modulation of the TGF-ß1 pathway.

Identification of von Willebrand factor D4 domain mutations in patients of Afro-Caribbean descent: In vitro characterization.

Background: Von Willebrand disease was diagnosed in two Afro-Caribbean patients and sequencing of the VWF gene (VWF) revealed the presence of multiple variants located throughout the gene, including variants located in the D4 domain of VWF: p.(Pro2145Thrfs*5) in one patient and p.(Cys2216Phefs*9) in the other patient. Interestingly, D4 variants have not been studied often. Objectives: Our goal was to characterize how the D4 variants p.(Pro2145Thrfs*5) and p.(Cys2216Phefs*9) influenced VWF biosynthesis/secretion and functions using in vitro assays. Methods: Recombinant VWF (rVWF), mutant or wild-type, was produced via transient transfection of the human embryonic kidney cell line 293T. The use of different tags for the wild-type and the mutant allele allowed us to distinguish between the two forms when measuring VWF antigen in medium and cell lysates. Binding of rVWF to its ligands, collagen, factor VIII, ADAMTS13, and platelet receptors was also investigated. Results: Homozygous expression of the p.(Cys2216Phefs*9)-rVWF mutation resulted in an almost complete intracellular retention of the protein. Heterozygous expression led to secretion of almost exclusively wild-type-rVWF, logically capable of normal interaction with the different ligands. In contrast, the p.(Pro2145Thrfs*5)-rVWF exhibited reduced binding to type III collagen and αIIbß3 integrin compared to wild-type-rVWF. Conclusions: We report two mutations of the D4 domains that induced combined qualitative and quantitative defects.

Antithrombotic potential of a single-domain antibody enhancing the activated protein C-cofactor activity of protein S.

BACKGROUND: Protein S (PS) is a natural anticoagulant acting as a cofactor for activated protein C (APC) in the proteolytic inactivation of activated factors V (FVa) and VIII (FVIIIa), but also for tissue factor pathway inhibitor α (TFPIα) in the inhibition of activated factor X (FXa). OBJECTIVE: For therapeutic purposes, we aimed at generating single-domain antibodies (sdAbs) that could specifically modulate the APC-cofactor activity of PS in vivo. METHODS: A llama-derived immune library of sdAbs was generated and screened on recombinant human PS by phage display. PS binders were tested in a global activated partial thromboplastin time (APTT)-based APC-cofactor activity assay. RESULTS: A PS-specific sdAb (PS003) was found to enhance the APC-cofactor activity of PS in our APTT-based assay, and this enhancing effect was greater for a bivalent form of PS003 (PS003biv). Further characterization of PS003biv demonstrated that PS003biv also enhanced the APC-cofactor activity of PS in a tissue factor (TF)-induced thrombin generation assay and stimulated APC in the inactivation of FVa, but not FVIIIa, in plasma-based assays. Furthermore, PS003biv was directed against the sex hormone-binding globulin (SHBG)-like domain but did not inhibit the binding of PS to C4b-binding protein (C4BP) and did not interfere with the TFPIα-cofactor activity of PS. In mice, PS003biv exerted an antithrombotic effect in a FeCl3 -induced thrombosis model, while not affecting physiological hemostasis in a tail-clip bleeding model. DISCUSSION: Altogether, these results showed that pharmacological enhancement of the APC-cofactor activity of PS through an original anti-PS sdAb might constitute a promising and safe antithrombotic strategy.

The VWF/LRP4/αVß3-axis represents a novel pathway regulating proliferation of human vascular smooth muscle cells.

AIMS: Von Willebrand factor (VWF) is a plasma glycoprotein involved in primary haemostasis, while also having additional roles beyond haemostasis namely in cancer, inflammation, angiogenesis, and potentially in vascular smooth muscle cell (VSMC) proliferation. Here, we addressed how VWF modulates VSMC proliferation and investigated the underlying molecular pathways and the in vivo pathophysiological relevance. METHODS AND RESULTS: VWF induced proliferation of human aortic VSMCs and also promoted VSMC migration. Treatment of cells with a siRNA against αv integrin or the RGT-peptide blocking αvß3 signalling abolished proliferation. However, VWF did not bind to αvß3 on VSMCs through its RGD-motif. Rather, we identified the VWF A2 domain as the region mediating binding to the cells. We hypothesized the involvement of a member of the LDL-related receptor protein (LRP) family due to their known ability to act as co-receptors. Using the universal LRP-inhibitor receptor-associated protein, we confirmed LRP-mediated VSMC proliferation. siRNA experiments and confocal fluorescence microscopy identified LRP4 as the VWF-counterreceptor on VSMCs. Also co-localization between αvß3 and LRP4 was observed via proximity ligation analysis and immuno-precipitation experiments. The pathophysiological relevance of our data was supported by VWF-deficient mice having significantly reduced hyperplasia in carotid artery ligation and artery femoral denudation models. In wild-type mice, infiltration of VWF in intimal regions enriched in proliferating VSMCs was found. Interestingly, also analysis of human atherosclerotic lesions showed abundant VWF accumulation in VSMC-proliferating rich intimal areas. CONCLUSION: VWF mediates VSMC proliferation through a mechanism involving A2 domain binding to the LRP4 receptor and integrin αvß3 signalling. Our findings provide new insights into the mechanisms that drive physiological repair and pathological hyperplasia of the arterial vessel wall. In addition, the VWF/LRP4-axis may represent a novel therapeutic target to modulate VSMC proliferation.

In vitro recovery of FIX clotting activity as a marker of highly functional hepatocytes in a hemophilia B iPSC model.

BACKGROUND AND AIMS: Pluripotent stem cell-derived hepatocytes differentiated in monolayer culture are known to have more fetal than adult hepatocyte characteristics. If numerous studies tend to show that this immature phenotype might not necessarily be an obstacle to their use in transplantation, other applications such as drug screening, toxicological studies, or bioartificial livers are reliant on hepatocyte functionality and require full differentiation of hepatocytes. New technologies have been used to improve the differentiation process in recent years, usually evaluated by measuring the albumin production and CYP450 activity. Here we used the complex production and most importantly the activity of the coagulation factor IX (FIX) produced by mature hepatocytes to assess the differentiation of hemophilia B (HB) patient's induced pluripotent stem cells (iPSCs) in both monolayer culture and organoids. APPROACH AND RESULTS: Indeed, HB is an X-linked monogenic disease due to an impaired activity of FIX synthesized by hepatocytes in the liver. We have developed an in vitro model of HB hepatocytes using iPSCs generated from fibroblasts of a severe HB patient. We used CRISPR/Cas9 technology to target the genomic insertion of a coagulation factor 9 minigene bearing the Padua mutation to enhance FIX activity. Noncorrected and corrected iPSCs were differentiated into hepatocytes under both two-dimensional and three-dimensional differentiation protocols and deciphered the production of active FIX in vitro. Finally, we assessed the therapeutic efficacy of this approach in vivo using a mouse model of HB. CONCLUSIONS: Functional FIX, whose post-translational modifications only occur in fully mature hepatocytes, was only produced in corrected iPSCs differentiated in organoids. Immunohistochemistry analyses of mouse livers indicated a good cell engraftment, and the FIX activity detected in the plasma of transplanted animals confirmed rescue of the bleeding phenotype.

A multiplex serological assay for the characterization of IgG immune response to SARS-CoV-2

BackgroundIn the fight against SARS-COV-2, the development of serological assays based on different antigenic domains represent a versatile tool to get a comprehensive picture of the immune response or differentiate infection from vaccination beyond simple diagnosis. ObjectivesHere we use a combination of the Nucleoprotein (NP), the Spike 1 (S1) and Spike 2 (S2) subunits, and the receptor binding domain (RBD) and N-terminal domain (NTD) of the Spike antigens from the Syrius-CoViDiag(R) multiplex IgG assay, to follow the immune response to SARS-CoV-2 infection over a long time period and depending on disease severity. ResultsUsing a panel of 209 sera collected from 61 patients up to eight months after infection, we observed that most patients develop an immune response against multiple viral epitope, but anti-S2 antibodies seemed to last longer. For all the tested IgGs, we have found higher titers for hospitalized patients than for non-hospitalized ones. Moreover the combination of the five different IgG titers increased the correlation to the neutralizing antibody titers than if considered individually. ConclusionMultiplex immunoassays have the potential to improve diagnostic performances, especially for ancient infection or mild form of the disease presenting weaker antibody titers. Also the combined detection of anti-NP and anti-Spike-derived domains can be useful to differentiate vaccination from viral infection and accurately assess the antibody potential to neutralize the virus.

Social Media Terms and Conditions and Informed Consent From Children: Ethical Analysis.

BACKGROUND: Terms and conditions define the relationship between social media companies and users. However, these legal agreements are long and written in a complex language. It remains questionable whether users understand the terms and conditions and are aware of the consequences of joining such a network. With children from a young age interacting with social media, companies are acquiring large amounts of data, resulting in longitudinal data sets that most researchers can only dream of. The use of social media by children is highly relevant to their mental and physical health for 2 reasons: their health can be adversely affected by social media and their data can be used to conduct health research. OBJECTIVE: The aim of this paper is to offer an ethical analysis of how the most common social media apps and services inform users and obtain their consent regarding privacy and other issues and to discuss how lessons from research ethics can lead to trusted partnerships between users and social media companies. Our paper focuses on children, who represent a sensitive group among users of social media platforms. METHODS: A thematic analysis of the terms and conditions of the 20 most popular social media platforms and the 2 predominant mobile phone ecosystems (Android and iOS) was conducted. The results of this analysis served as the basis for scoring these platforms. RESULTS: The analysis showed that most platforms comply with the age requirements issued by legislators. However, the consent process during sign-up was not taken seriously. Terms and conditions are often too long and difficult to understand, especially for younger users. The same applies to age verification, which is not realized proactively but instead relies on other users who report underaged users. CONCLUSIONS: This study reveals that social media networks are still lacking in many respects regarding the adequate protection of children. Consent procedures are flawed because they are too complex, and in some cases, children can create social media accounts without sufficient age verification or parental oversight. Adopting measures based on key ethical principles will safeguard the health and well-being of children. This could mean standardizing the registration process in accordance with modern research ethics procedures: give users the key facts that they need in a format that can be read easily and quickly, rather than forcing them to wade through chapters of legal language that they cannot understand. Improving these processes would help safeguard the mental health of children and other social media users.