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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.

BACKGROUND: Protein S (PS) is a natural anticoagulant acting as a cofactor for activated protein C (APC) in the proteolytic inactivation of activated factor 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 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 VWD patients. 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 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 SP to improve thrombus formation in VWF-deficient condition (with human platelets) or using blood from VWD-2B mice and VWF-deficient mice (VWF-KO, i.e., type 3 VWD). In vivo, using a tail clip assay, SP treatment reduced blood loss by 35% in VWD-2B mice and 68% in VWF-KO mice. Additional studies using nanoparticles decorated with various combinations of peptides demonstrated that the collagen binding peptide, although not sufficient by itself, was absolutely crucial for SP efficacy in VWD-2B while all three peptides appeared necessary for VWF-KO mice. Clot imaging by immunofluorescence and scanning electron microscopy revealed that SP treatment of VWF-KO mice 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.

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.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.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.

The opportunistic fungal pathogenCryptococcus neoformanscauses lethal infections in immunocompromised patients. Macrophages are central to the host response to cryptococci; however, it is unclear howC. neoformansis recognized and phagocytosed by macrophages. Here we investigate the role of TLR4 in the nonopsonic phagocytosis ofC. neoformans. We find that loss of TLR4 function unexpectedly increases phagocytosis of nonopsonized cryptococci. The increased phagocytosis observed inTlr4-/-cells was dampened by pre-treatment of macrophages with either a TLR3 inhibitor or 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 inTlr4-/-macrophages and there was a 75% decrease in phagocytosis of nonopsonized cryptococci byMsr1-/-macrophages. Furthermore, immunofluorescence imaging revealed colocalization of MSR1 and internalised cryptococci. Together, these results identify MSR1 as a key receptor for the phagocytosis of nonopsonizedC. neoformansand demonstrate TLR4/MSR1 crosstalk in the phagocytosis ofC. neoformans.

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. While 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 of congenital 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, p0.0001) 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.

Aims Von Willebrand factor (VWF) is a plasma glycoprotein involved in primary hemostasis, while also having additional roles beyond hemostasis 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 αvβ3 signaling abolished proliferation. However, VWF did not bind to αvβ3 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 αvβ3 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 significant reduced, if any, 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. Conclusions VWF mediates VSMC proliferation through a mechanism involving A2 domain binding to the LRP4 receptor and integrin αvβ3 signaling. 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. Translational perspective The mechanisms that drive physiological repair and pathological hyperplasia of the arterial vessel wall are complex and only partially understood. Specifically, the role of subendothelial-matrix proteins remains unclear. Here, we show that the hemostatic protein von Willebrand factor (VWF) accumulates in the vascular wall of atherosclerotic lesions and localizes to areas of vascular smooth muscle cell (VSMC) proliferation. VWF was found to use its A2-domain for binding to the VSMC-receptor LRP4, which in turn triggered outside-in signaling via integrin αVβ3, thereby inducing VSMC proliferation. Interfering with A2-domain/LRP4 interactions might offer innovative and additional therapeutical approaches to limit pathological hyperplasia.

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.

Background Activated factor VII (FVIIa) is pertinent to the initiation of blood coagulation. Proteolytic and amidolytic activity of FVIIa are greatly enhanced by its cofactor, tissue factor (TF). Objective We aimed to generate a single-domain antibody (sdAb) that recognizes free FVIIa rather than TF-bound FVIIa. Methods A llama-derived phage library was used to screen for anti-FVIIa sdAbs. Results One sdAb, KB-FVIIa-004, bound to FVIIa, but not to its precursor FVII or to homologous proteins (prothrombin, factor X, or their activated derivatives). FVIIa amidolytic activity was inhibited by KB-FVIIa-004 (Ki = 28-45 nM) in a competitive manner. KB-FVIIa-004 also inhibited FVIIa-mediated FX activation (Ki = 26 nM). In contrast, KB-FVIIa-004 was inefficient in prolonging the clotting time of the prothrombin time-test, which was prolonged by a maximum of 10 s at high sdAb concentrations (10 μM). Furthermore, FVIIa/TF amidolytic activity or FVIIa/TF-mediated FX activation remained unaffected up to a 50-fold to 1000-fold molar excess of KB-FVIIa-004. These data suggest that KB-FVIIa-004 loses its inhibitory activity in the presence of TF. A KB-FVIIa-004/albumin fusion-protein (004-HSA) was generated for in vivo testing. By using 004-HSA, we observed that this sdAb blocked the therapeutic capacity of FVIIa to correct bleeding in FVIII-deficient mice. Discussion This observation is compatible with the view that FVIIa functions independently of TF under these conditions. In conclusion, we have generated a sdAb that specifically blocks TF-independent activity of FVIIa. This antibody can be used to gain insight into the roles of TF-bound and TF-free FVIIa.

Objective— Platelet secretion is crucial for many physiological platelet responses. Even though several regulators of the fusion machinery for secretory granule exocytosis have been identified in platelets, the underlying mechanisms are not yet fully characterized. Approach and Results— By studying a mouse model (cKO [conditional knockout] Kif5b ) lacking Kif5b (kinesin-1 heavy chain) in its megakaryocytes and platelets, we evidenced unstable hemostasis characterized by an increase of blood loss associated to a marked tendency to rebleed in a tail-clip assay and thrombus instability in an in vivo thrombosis model. This instability was confirmed in vitro in a whole-blood perfusion assay under blood flow conditions. Aggregations induced by thrombin and collagen were also impaired in cKO Kif5b platelets. Furthermore, P-selectin exposure, PF4 (platelet factor 4) secretion, and ATP release after thrombin stimulation were impaired in cKO Kif5b platelets, highlighting the role of kinesin-1 in α-granule and dense granule secretion. Importantly, exogenous ADP rescued normal thrombin induced-aggregation in cKO Kif5b platelets, which indicates that impaired aggregation was because of defective release of ADP and dense granules. Last, we demonstrated that kinesin-1 interacts with the molecular machinery comprising the granule-associated Rab27 (Ras-related protein Rab-27) protein and the Slp4 (synaptotagmin-like protein 4/SYTL4) adaptor protein. Conclusions— Our results indicate that a kinesin-1–dependent process plays a role for platelet function by acting into the mechanism underlying α-granule and dense granule secretion.

The development of therapeutic strategies for the treatment of hemophilia A has been a long-lasting issue since the initial identification of the disease in the early 19th century and has involved several major steps to reach contemporary treatment. The current replacement therapy involving intravenous infusion of plasmatic or recombinant factor VIII (FVIII) has been efficient to control bleeding episodes of patients with hemophilia A. Nonetheless, replacement therapy requires frequent infusions to maintain values of FVIII above the threshold of efficacy and represents a serious burden that significantly impacts the patient's quality of life. The recent half-life extension technologies have permitted the development of a first generation of long-acting FVIII variants with improved circulating survival. These molecules, which are now in clinical phase III studies or have already received food and drug administration (FDA) approval for therapeutic use will allow a reduction in the frequency of infusion and lighten the treatment of hemophilia A. However, the half-life extension of FVIII was not as efficient as initially expected, particularly with regard to the results obtained with other therapeutic molecules. To overcome these limitations inherent to the nature of FVIII biology, several bypassing therapies independent of the FVIII molecule are currently in development. These emerging approaches exploit the modulation of the coagulation/anticoagulation balance taking place in the hemostatic process to compensate for the FVIII deficiency. In this review, we recount the history of hemophilia A treatment up to the recent emerging therapies, with particular focus on infusion-based strategies.

Essentials Treatment options for von Willebrand disease (VWD) patients are limited. The p.P1127_C1948delinsR deletion/variant is a useful model to study VWD in vitro and in vivo. Counteracting dominant-negative effects restores von Willebrand factor multimerization in mice. This is the first siRNA-based treatment applied to a mouse model of VWD-type 2A. ABSTRACT: Background Treatment options for patients suffering from von Willebrand disease (VWD) are limited. Von Willebrand factor (VWF) is a polymeric protein that undergoes regulated dimerization and subsequent multimerization during its biosynthesis. Numerous heterozygous variants within the VWF gene display a dominant-negative effect and result in severe VWD. Previous studies have suggested that preventing the assembly of wild-type and mutant heteropolymers using siRNAs may have beneficial effects on VWF phenotypes in vitro. Objectives To study heterozygous dominant-negative variants in vivo, we developed a mouse model of VWD-type 2A and tested two independent strategies to modulate its detrimental effect. Methods The p.P1127_C1948delinsR deletion/variant, causing defective VWF multimerization, was expressed in mice as a model of VWD-type 2A variant. Two corrective strategies were applied. For the first time in a mouse model of VWD, we applied siRNAs selectively inhibiting translation of the mutant transcripts and we combined the VWD-type 2A deletion with the Cys to Arg substitution at position 2773, which is known to prevent dimerization. Results The RNA silencing approach induced a modest but consistent improvement of the VWF multimer profile. However, due to incomplete efficiency, the dominant-negative effect of the original variant could not be completely prevented. In contrast, the DNA approach resulted in increased antigen levels and restoration of a normal multimer profile. Conclusions Our data showed that preventing the detrimental impact of dominant-negative VWF variants by independent molecular mechanisms has beneficial consequences in vivo, in mouse models of dominant VWD.

Engineered recombinant factor X (FX) variants represent a promising strategy to bypass the tenase complex and restore hemostasis in hemophilia patients. Previously, a thrombin-activatable FX variant with fibrinopeptide-A replacing the activation peptide (FX-delAP/FpA) has been described in this regard. Here we show that FX-delAP/FpA is characterized by a sixfold shorter circulatory half-life compared with wild-type FX, limiting its therapeutical applicability. We therefore designed a variant in which the FpA sequence is inserted C-terminal to the FX activation peptide (FX/FpA). FX/FpA displayed a similar survival to wt-FX in clearance experiments and could be converted into FX by thrombin and other activating agents. In in vitro assays, FX/FpA efficiently restored thrombin generation in hemophilia A and hemophilia B plasmas, even in the presence of inhibitory antibodies. Expression following hydrodynamic gene transfer of FX/FpA restored thrombus formation in FVIII-deficient mice in a laser-induced injury model as well as hemostasis in a tail-clip bleeding model. Hemostasis after tail transection in FVIII-deficient mice was also corrected at 5 and 90 minutes after injection of purified FX/FpA. Our data indicate that FX/FpA represents a potential tenase-bypassing agent for the treatment of hemophilia patients with or without inhibitors.

The bispecific antibody emicizumab is increasingly used for hemophilia A treatment. However, its specificity for human factors IX and X (FIX and FX) has limited its in vivo functional analysis to primate models of acquired hemophilia. Here, we describe a novel mouse model that allows emicizumab function to be examined. Briefly, FVIII-deficient mice received IV emicizumab 24 hours before tail-clip bleeding was performed. A second infusion with human FIX and FX, administered 5 minutes before bleeding, generated consistent levels of emicizumab (0.7-19 mg/dL for 0.5-10 mg/kg doses) and of both FIX and FX (85 and 101 U/dL, respectively, after dosing at 100 U/kg). Plasma from these mice display FVIII-like activity in assays (diluted activated partial thromboplastin time and thrombin generation), similar to human samples containing emicizumab. Emicizumab doses of 1.5 mg/kg and higher significantly reduced blood loss in a tail-clip–bleeding model using FVIII-deficient mice. However, reduction was incomplete compared with mice treated with human FVIII concentrate, and no difference in efficacy between doses was observed. From this model, we deducted FVIII-like activity from emicizumab that corresponded to a dose of 4.5 U of FVIII per kilogram (ie, 9.0 U/dL). Interestingly, combined with a low FVIII dose (5 U/kg), emicizumab provided enough additive activity to allow complete bleeding arrest. This model could be useful for further in vivo analysis of emicizumab.

The development of an immune response against therapeutic factor VIII is the major complication in hemophilia A patients. Oligomannose carbohydrates at N239 and/or N2118 on factor VIII allow its binding to the macrophage mannose receptor expressed on human dendritic cells, thereby leading to factor VIII endocytosis and presentation to CD4+ T lymphocytes. Here, we investigated whether altering the interaction of factor VIII with mannose-sensitive receptors on antigen-presenting cells may be a strategy to reduce factor VIII immunogenicity. Gene transfer experiments in factor VIII-deficient mice indicated that N239Q and/or N2118Q factor VIII mutants have similar specific activities as compared to non-mutated factor VIII; N239Q/N2118Q mutant corrected blood loss upon tail clip. Production of the corresponding recombinant FVIII mutants or light chains indicated that removal of the N-linked glycosylation site at N2118 is sufficient to abrogate in vitro the activation of FVIII-specific CD4+ T cells by human monocyte-derived dendritic cells. However, removal of mannose-ending glycans at N2118 did not alter factor VIII endocytosis and presentation to CD4+ T cells by mouse antigen-presenting cells. In agreement with this, the N2118Q mutation did not reduce factor VIII immunogenicity in factor VIII-deficient mice. Our results highlight differences in the endocytic pathways between human and mouse dendritic cell subsets, and dissimilarities in tissue distribution and function of endocytic receptors such as CD206 in both species. Further investigations in preclinical models of hemophilia A closer to humans are needed to decipher the exact role of mannose-ending glycans in factor VIII immunogenicity.

International audience; Hemophilia A and B, diseases caused by the lack of factor VIII (FVIII) and factor IX (FIX) respectively, lead to insufficient thrombin production, and therefore to bleeding. New therapeutic strategies for hemophilia treatment that do not rely on clotting factor replacement, but imply the neutralization of natural anticoagulant proteins, have recently emerged. We propose an innovative approach consisting of targeting a natural and potent thrombin inhibitor, expressed by platelets, called protease nexin-1 (PN-1). By using the calibrated automated thrombin generation assay, we showed that a PN-1-neutralizing antibody could significantly shorten the thrombin burst in response to tissue factor in platelet-rich plasma (PRP) from patients with mild or moderate hemophilia. In contrast, in PRP from patients with severe hemophilia, PN-1 neutralization did not improve thrombin generation. However, after collagen-induced platelet activation, PN-1 deficiency in F8-/-mice or PN-1 blocking in patients with severe disease led to a significantly improved thrombin production in PRP, underlining the regulatory role of PN-1 released from platelet granules. In various bleeding models, F8-/-/PN-1-/- mice displayed significantly reduced blood loss and bleeding time compared with F8-/-mice. Moreover, platelet recruitment and fibrin(ogen) accumulation were significantly higher in F8-/-/PN-1-/- mice than in F8-/-mice in the ferric chloride-induced mesenteric vessel injury model. Thromboelastometry studies showed enhanced clot stability and lengthened clot lysis time in blood from F8-/-/PN-1-/- and from patients with hemophilia A incubated with a PN-1-neutralizing antibody compared with their respective controls. Our study thus provides proof of concept that PN-1 neutralization can be a novel approach for future clinical care in hemophilia.

Summary Essentials Factor VIIa is cleared principally as a complex with antithrombin. Enzyme/serpin complexes are preferred ligands for the scavenger-receptor LRP1. Factor VIIa/antithrombin but not factor VIIa alone is a ligand for LRP1. Macrophage-expressed LRP1 contributes to the clearance of factor VIIa/antithrombin. SummaryBackground Recent findings point to activated factor VII (FVIIa) being cleared predominantly (± 65% of the injected protein) as part of a complex with the serpin antithrombin. FVIIa–antithrombin complexes are targeted to hepatocytes and liver macrophages. Both cells lines abundantly express LDL receptor-related protein 1 (LRP1), a scavenger receptor mediating the clearance of protease–serpin complexes. Objectives To investigate whether FVIIa–antithrombin is a ligand for LRP1. Methods Binding of FVIIa and pre-formed FVIIa–antithrombin to purified LRP1 Fc-tagged cluster IV (rLRP1-cIV/Fc) and to human and murine macrophages was analyzed. FVIIa clearance was determined in macrophage LRP1 (macLRP1)-deficient mice. Results Solid-phase binding assays showed that FVIIa–antithrombin bound in a specific, dose-dependent and saturable manner to rLRP1-cIV/Fc. Competition experiments with human THP1 macrophages indicated that binding of FVIIa but not of FVIIa–antithrombin was reduced in the presence of annexin-V or anti-tissue factor antibodies, whereas binding of FVIIa–antithrombin but not FVIIa was inhibited by the LRP1-antagonist GST-RAP. Additional experiments revealed binding of both FVIIa and FVIIa–antithrombin to murine control macrophages. In contrast, no binding of FVIIa–antithrombin to macrophages derived from macLRP1-deficient mice could be detected. Clearance of FVIIa–antithrombin but not of active site-blocked FVIIa was delayed 1.5-fold (mean residence time of 3.3 ± 0.1 h versus 2.4 ± 0.2 h) in macLRP1-deficient mice. The circulatory presence of FVIIa was prolonged to a similar extent in macLRP1-deficient mice and in control mice. Conclusions Our data show that FVIIa–antithrombin but not FVIIa is a ligand for LRP1, and that LRP1 contributes to the clearance of FVIIa–antithrombin in vivo.

International audience; Von Willebrand factor (VWF) modulates factor VIII (FVIII) clearance and the anti-FVIII immune response. Despite the high affinity that defines the FVIII/VWF interaction, association/dissociation kinetics dictates 2% to 5% FVIII being present as free protein. To avoid free FVIII when studying the FVIII-VWF complex in vivo, we designed a FVIII-nanobody fusion protein, with the nanobody part being directed against VWF. This fusion protein, designated FVIII-KB013bv, had a 25-fold higher affinity compared with B-domainless FVIII (BDD-FVIII) for VWF. In vitro analysis revealed full cofactor activity in 1-stage clotting and chromogenic assays (activity/antigen ratio 1.0 ± 0.3 and 1.1 ± 0.3, respectively). In vivo, FVIII-013bv displayed a twofold increased mean residence time compared with BDD-FVIII (3.0 hours vs 1.6 hours). In a tail clip–bleeding assay performed 24 hours after FVIII infusion, blood loss was significantly reduced in mice receiving FVIII-KB013bv vs BDD-FVIII (15 ± 7 μL vs 194 ± 146 μL; P = .0043). Unexpectedly, when examining anti-FVIII antibody formation in FVIII-deficient mice, the immune-response toward FVIII-KB013bv was significantly reduced compared with BDD-FVIII (1/8 vs 14/16 mice produced anti-FVIII antibodies after treatment with FVIII-KB013bv and BDD-FVIII, respectively). Our data show that a stabilized interaction between FVIII and VWF is associated with a prolonged survival of FVIII and a reduced immune response against FVIII.

Gene therapy mediated by recombinant adeno-associated virus (AAV) vectors is a promising treatment for systemic monogenic diseases. However, vector immunogenicity represents a major limitation to gene transfer with AAV vectors, particularly for vector re-administration. Here, we demonstrate that synthetic vaccine particles encapsulating rapamycin (SVP[Rapa]), co-administered with AAV vectors, prevents the induction of anti-capsid humoral and cell-mediated responses. This allows successful vector re-administration in mice and nonhuman primates. SVP[Rapa] dosed with AAV vectors reduces B and T cell activation in an antigen-selective manner, inhibits CD8+ T cell infiltration in the liver, and efficiently blocks memory T cell responses. SVP[Rapa] immunomodulatory effects can be transferred from treated to naive mice by adoptive transfer of splenocytes, and is inhibited by depletion of CD25+ T cells, suggesting a role for regulatory T cells. Co-administration of SVP[Rapa] with AAV vector represents a powerful strategy to modulate vector immunogenicity and enable effective vector re-administration., Immunogenicity of AAV vectors renders repeated AAV dosing ineffective. Here the authors show that coadministration of nanoparticle-encapsulated rapamycin overcomes AAV immunogenicity through Treg induction, enabling efficient AAV redosing in mice and nonhuman primates.

Results from clinical trials of liver gene transfer for hemophilia demonstrate the potential of the adeno-associated virus (AAV) vector platform. However, to achieve therapeutic transgene expression, in some cases high vector doses are required, which are associated with a higher risk of triggering anti-capsid cytotoxic T-cell responses. Additionally, anti-AAV preexisting immunity can prevent liver transduction even at low neutralizing antibody (NAb) titers. Here, we describe the use of exosome-associated AAV (exo-AAV) vectors as a robust liver gene delivery system that allows the therapeutic vector dose to be decreased while protecting from preexisting humoral immunity to the capsid. The in vivo efficiency of liver targeting of standard AAV8 or AAV5 and exo-AAV8 or exo-AAV5 vectors expressing human coagulation factor IX (hF.IX) was evaluated. A significant enhancement of transduction efficiency was observed, and in hemophilia B mice treated with 4 × 1010 vector genomes per kilogram of exo-AAV8 vectors, a staggering ∼1 log increase in hF.IX transgene expression was observed, leading to superior correction of clotting time. Enhanced liver expression was also associated with an increase in the frequency of regulatory T cells in lymph nodes. The efficiency of exo- and standard AAV8 vectors in evading preexisting NAbs to the capsid was then evaluated in a passive immunization mouse model and in human sera. Exo-AAV8 gene delivery allowed for efficient transduction even in the presence of moderate NAb titers, thus potentially extending the proportion of subjects eligible for liver gene transfer. Exo-AAV vectors therefore represent a platform to improve the safety and efficacy of liver-directed gene transfer.

Objective— von Willebrand factor (VWF) is crucial to hemostasis, but also plays a role in inflammatory processes. Unfortunately, no proper monoclonal antibodies to study VWF function in mice are currently available. We therefore aimed to generate single-domain antibodies (sdAbs) recognizing murine VWF and blocking its function in vivo. Approach and Results— Llama-derived sdAbs recognizing both human and murine VWF were isolated via phage display technology. One of them (designated KB-VWF-006) recognized the VWF A1 domain with picomolar affinity. This sdAb avidity was strongly enhanced via dimerization using a triple Ala linker (KB-VWF-006bi). When administered in vivo to wild-type mice, KB-VWF-006bi dose dependently induced bleeding in a tail clip model. In 2 distinct models of inflammation, KB-VWF-006bi efficiently interfered with leukocyte recruitment and vascular leakage. Conclusions— KB-VWF-006bi is an sdAb recognizing the A1 domain of human VWF and murine VWF that interferes with VWF–platelet interactions in vivo. By using this sdAb, we now also show that the A1 domain is pertinent to the participation of VWF in the inflammatory response.

During the last decade, the development of improved and novel approaches for the treatment of hemophilia A has expanded tremendously. These approaches include factor VIII (FVIII) with extended half-life (eg, FVIII-Fc and PEGylated FVIII), monoclonal antibodies targeting tissue factor pathway inhibitor, small interfering RNA to reduce antithrombin expression and the bispecific antibody ACE910/emicizumab. Emicizumab is a bispecific antibody recognizing both the enzyme factor IXa and the substrate factor X. By simultaneously binding enzyme and substrate, emicizumab mimics some part of the function exerted by the original cofactor, FVIII, in that it promotes colocalization of the enzyme–substrate complex. However, FVIII and the bispecific antibody are fundamentally different proteins and subject to different modes of regulation. Here, we will provide an overview of the similarities and dissimilarities between FVIII and emicizumab from a biochemical and mechanistical perspective. Such insight might be useful in the clinical decision making for those who apply emicizumab in their practice now or in the future, particularly in view of the thrombotic complications that have been reported when emicizumab is used in combination with FVIII-bypassing agents.

International audience; Recently, we have identified scavenger receptor class A member I (SR-AI) as a receptor for coagulation factor X (FX), mediating the formation of an FX reservoir at the macrophage surface. Here, we demonstrate that the FX/SR-AI-complex comprises a third protein, pentraxin-2 (PTX2). The presence of PTX2 is essential to prevent internalization of FX by SR-AI, and the presence of FX is needed to interfere with internalization of PTX2. Binding studies showed that FX, SR-AI, and PTX2 independently bind to each other (KD,app: 0.2-0.7 μM). Surprisingly, immunoprecipitation experiments revealed that FX and PTX2 circulate as a complex in plasma, and complex formation involves the FX activation peptide. No binding of PTX2 to other vitamin K–dependent proteins was observed. Short hairpin RNA–mediated inhibition of PTX2 levels in mice resulted not only in reduced levels of PTX2, but also in similarly reduced FX levels. Moreover, PTX2 and FX levels were correspondingly reduced in SR-AI–deficient mice. Analysis of 71 human plasma samples uncovered a strong correlation between FX and PTX2 plasma levels. Furthermore, plasma samples of patients with reduced FX levels (congenital/acquired FX deficiency or after anti–vitamin K treatment) were characterized by concomitantly decreased PTX2 levels. In conclusion, we identified PTX2 as a novel partner for FX, and both proteins cooperate to prevent their SR-AI–mediated uptake by macrophages. Interestingly, their respective plasma levels are interdependent. These findings seem of relevance in perspective of ongoing clinical trials, in which plasma depletion of PTX2 is used as a therapeutical approach in the management of systemic amyloidosis.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease that presents with thrombocytopenia, disseminated thrombosis, hemolytic anemia, and organ dysfunction. The etiology of TTP has revealed that patients share a deficiency in plasma protease a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), the enzyme responsible for cleaving ultra-large von Willebrand factor (VWF) multimers into nonthrombogenic fragments. Therefore, existing TTP mouse models were developed by targeted disruption of the ADAMTS13 gene. ADAMTS13(-/-) mice are mostly asymptomatic in the absence of a trigger, as redundant proteases appear to take on VWF processing. As an alternative approach to creating one such model, we devised a strategy based on the expression of a cleavage-resistant VWF mutant in mice. The creation of a disulfide bond within the A2 domain of VWF was found to render VWF multimers resistant to proteolysis by plasma proteases under flow. Furthermore, mice expressing the murine VWF/p.S1494C-p.A1534C mutant present with symptoms characteristics of acute TTP such as thrombocytopenia, red cell shredding, accumulation of VWF-rich thrombi in the microvasculature, and advanced TTP symptoms such as renal dysfunction and splenomegaly. Because this model appears to faithfully emulate the pathophysiology of TTP, it should prove most useful in the study of microangiopathic diseases and their treatment.

Background: Several first-generation gene therapies are currently in clinical trials for Haemophilia A. These trials have to date exhibited varying results including issues with large patient to patient variation in FVIII levels and lack of durability. It is hypothesized that these challenges may, in part, be caused by the large size of the F8 transgenes used, resulting in genome sizes surpassing the optimal packaging capacity of AAV (~4.7kbp). We hypothesised that the oversize AAV vectors contain truncated genomes which cause batch to batch variations and decrease the quality of the resulting drug product. Aim: Armed with clinical success with our proprietary AAVS3 capsid in Haemophilia B (Nathwani et al, 2018), we designed novel, next generation potent AAV-F8 vectors with genomes of less than 4.9kbp to increase the quality of the resulting product and the predictability of FVIII expression. Methods: We deployed several methods to achieve our goal, including optimisations to the 'F8-SQ' coding sequence and optimisations of components of the expression cassette from the perspective of balancing size and potency of the resulting vectors. Over 20 optimized gene sequences, 10 different signal peptides, more than 60 short liver-specific promoters with a size of less than 270bp, and several shorter FVIII coding sequences were designed. We evaluated the resulting constructs for potency in human liver cell line Huh7 cells as well as in C57BI/6 mice. In addition, a range of different deletion lengths encompassing the B-domain were assessed for their in vitro and in vivo efficiency. Selected potent constructs were further evaluated in haemophilia A mice, and their haemostatic ability was demonstrated in the classical tail clip model. Results: All elements of the expression cassette were optimized to generate a construct with more than 70-fold higher expression than the wild-type FVIII construct as evaluated in the human liver cell line Huh7. Importantly, we were able to develop a series of short promoters ( Conclusion: We have developed several highly potent lead candidates with sizes of less than 4.9kbp, some being less than 4.75kbp in length. These candidates all have vector genomes markedly shorter in length than AAV-FVIII candidates currently in development and amongst these, we have selected the top best candidate, FLT210, for further development. Disclosures Kia: Freeline Therapeutics: Employment, Equity Ownership. Comper:Freeline: Employment, Equity Ownership. Correia:Freeline: Employment, Equity Ownership. Casari:Freeline: Research Funding. Negash:Freeline: Employment, Equity Ownership. Portillo:Freeline: Employment, Equity Ownership. Pandya:Freeline: Employment, Equity Ownership. Dodev:Freeline: Employment, Equity Ownership. Schulze:Freeline: Employment, Equity Ownership. Sonntag:Freeline: Employment, Equity Ownership. Kober:Freeline: Employment, Equity Ownership. Cocita:Freeline Therapeutics: Employment, Equity Ownership. Chisari:Freeline Therapeutics: Employment, Equity Ownership. Alade:Freeline: Employment, Equity Ownership. Shehu:Freeline: Employment, Equity Ownership. Jeyakumar:Freeline Therapeutics: Employment, Equity Ownership. Khinder:Freeline: Employment, Equity Ownership. Foley:Freeline: Employment, Equity Ownership. Dane:Freeline: Employment, Equity Ownership. McIntosh:Freeline Therapeutics: Consultancy, Equity Ownership. Ollerton:Freeline: Employment. Christophe:Freeline: Research Funding. Denis:Freeline: Research Funding. Corbau:Freeline: Employment, Equity Ownership.

SummaryHaemorrhagic episodes in patients carrying circulatory assist devices represent a severe life-threatening clinical complication. These bleeding episodes may originate from a reduced functionality of von Willebrand factor (VWF), a multimeric protein pertinent to the formation of a haemostatic plug. It has been reported that the reduced functionality is due to increased proteolytic degradation by the enzyme ADAMTS13, a phenomenon that is facilitated by device-induced increases in shear stress to which VWF is exposed. Here, we have tested a series of VWF-derived protein fragments and monoclonal murine anti-VWF antibodies for their capacity to reduce shear stress-dependent degradation of VWF. Via direct binding experiments, we identified an anti-VWF antibody that partially blocked VWF-ADAMTS13 interactions (46 ± 14%). Epitope mapping experiments revealed that the antibody, designated mAb508, is directed against the distal portion of the VWF D4-domain (residues 2134–2301) and recognises a synthetic peptide encompassing residues 2158–2169. Consistent with its partial inhibition of VWF-ADAMTS13 interactions in binding assays, mAb508 reduced ADAMTS13-mediated VWF degradation in a vortex-based degradation assay by 48 ± 10%. In a HeartMateII-based whole bloodperfusion system, mAb508 was able to reduce degradation of highmolecular- weight (HMW)-VWF-multimers dose-dependently, with a maximal inhibition (83 ± 8%) being reached at concentrations of 10 μg/ml or higher. In conclusion, we report that partial inhibition of VWF-ADAMTS13 interactions using an anti-VWF antibody can prevent excessive degradation of HMW-VWF multimers. This strategy may be used for the development of therapeutic options to treat bleeding episodes due to shear stress-dependent VWF degradation, for instance in patients carrying circulatory assist devices.

Quantitative deficiencies in von Willebrand factor (VWF) are associated with abnormal hemostasis that can manifest in bleeding or thrombotic complications. Consequently, many studies have endeavored to elucidate the mechanisms underlying the regulation of VWF plasma levels. This review focuses on the role of VWF clearance pathways. A summary of recent developments are provided, including results from genetic studies, the relationship between glycosylation and VWF clearance, the contribution of increased VWF clearance to the pathogenesis of von Willebrand disease and the identification of VWF clearance receptors. These different studies converge in their conclusion that VWF clearance is a complex phenomenon that involves multiple mechanisms. Deciphering how such different mechanisms coordinate their role in this process is but one of the remaining challenges. Nevertheless, a better insight into the complex clearance pathways of VWF may help us to better understand the clinical implications of aberrant clearance in the pathogenesis of von Willebrand disease and perhaps other disorders as well as aid in developing alternative therapeutic approaches.

International audience; von Willebrand disease type 2B (VWD-type 2B) is characterized by gain-of-function mutations of von Willebrand factor (vWF) that enhance its binding to platelet glycoprotein Ibα and alter the protein's multimeric structure. Patients with VWD-type 2B display variable extents of bleeding associated with macrothrombocytopenia and sometimes with thrombopathy. Here, we addressed the molecular mechanism underlying the severe macrothrombocytopenia both in a knockin murine model for VWD-type 2B by introducing the p.V1316M mutation in the murine Vwf gene and in a patient bearing this mutation. We provide evidence of a profound defect in megakaryocyte (MK) function since: (a) the extent of proplatelet formation was drastically decreased in 2B MKs, with thick proplatelet extensions and large swellings; and (b) 2B MKs presented actin disorganization that was controlled by upregulation of the RhoA/LIM kinase (LIMK)/cofilin pathway. In vitro and in vivo inhibition of the LIMK/cofilin signaling pathway rescued actin turnover and restored normal proplatelet formation, platelet count, and platelet size. These data indicate, to our knowledge for the first time, that the severe macrothrombocytopenia in VWD-type 2B p.V1316M is due to an MK dysfunction that originates from a constitutive activation of the RhoA/LIMK/cofilin pathway and actin disorganization. This suggests a potentially new function of vWF during platelet formation that involves regulation of actin dynamics.

von Willebrand disease (VWD)-type 2B is characterized by gain-of-function mutations in the von Willebrand factor (VWF) A1-domain, leading to increased affinity for its platelet-receptor, glycoprotein Ibα. We engineered the first knock-in (KI) murine model for VWD-type 2B by introducing the p.V1316M mutation in murine VWF. Homozygous KI-mice replicated human VWD-type 2B with macrothrombocytopenia (platelet counts reduced by 55%, platelet volume increased by 44%), circulating platelet-aggregates and a severe bleeding tendency. Also, vessel occlusion was deficient in the FeCl3-induced thrombosis model. Platelet aggregation induced by thrombin or collagen was defective for KI-mice at all doses. KI-mice manifested a loss of high molecular weight multimers and increased multimer degradation. In a model of VWF-string formation, the number of platelets/string and string-lifetime were surprisingly enhanced in KI-mice, suggesting that proteolysis of VWF/p.V1316M is differentially regulated in the circulation versus the endothelial surface. Furthermore, we observed increased leukocyte recruitment during an inflammatory response induced by the reverse passive Arthus reaction. This points to an active role of VWF/p.V1316M in the exfiltration of leukocytes under inflammatory conditions. In conclusion, our genetically-engineered VWD-type 2B mice represent an original model to study the consequences of spontaneous VWF-platelet interactions and the physiopathology of this human disease.

von Willebrand factor (VWF) is a protein best known from its critical role in hemostasis. Indeed, any dysfunction of VWF is associated with a severe bleeding tendency known as von Willebrand disease (VWD). Since the first description of the disease by Erich von Willebrand in 1926, remarkable progress has been made with regard to our understanding of the pathogenesis of this disease. The cloning of the gene encoding VWF has allowed numerous breakthroughs, and our knowledge of the epidemiology, genetics and molecular basis of VWD has been rapidly expanding since then. These studies have taught us that VWF is rather unique in terms of its multimeric structure and the unusual mechanisms regulating its participation in the hemostatic process. Moreover, it has become increasingly clear that VWF is a more all-round protein than originally thought, given its involvement in several pathologic processes beyond hemostasis. These include angiogenesis, cell proliferation, inflammation, and tumor cell survival. In the present article, an overview of advances concerning the various structural and functional aspects of VWF will be provided.

von Willebrand factor (VWF) is a promising target for developing antithrombotic drugs. The absence of accessible animal models impedes the study of specific human VWF (huVWF) targeting molecules in thrombosis. huVWF is not functional in the mouse because of a lack of interaction between huVWF and murine glycoprotein Ib. Using site-directed mutagenesis, we have replaced single or multiple amino acids in huVWF with their murine counterparts to eliminate species incompatibility. Using hydrodynamic injection, we have expressed the different chimeric VWF constructs into VWF−/− mice. Only huVWF with a complete murine A1 domain insertion was able to correct bleeding in vivo and form occlusive thrombi in mesenteric vessels after FeCl3 treatment. Using this model, we tested the antithrombotic effect of monoclonal antibodies against huVWF, blocking its interaction with collagens (mAbs 203 and 505) or with glycoprotein IIbIIIa (mAb 9). The 3 mAbs inhibited the thrombotic process in arterioles of VWF−/− mice expressing huVWFmuA1. Inhibiting VWF-interaction with collagens was more potent, emphasizing the potential of such a target as an antithrombotic tool. Our results validate our murine model as a simple in vivo tool to evaluate anti-huVWF agents.

Objective— Although von Willebrand factor (VWF) is a heavily glycosylated protein, its potential to associate with glycan-binding proteins is poorly investigated. Here, we explored its interaction with the glycan-binding proteins galectin-1 and galectin-3. Methods and Results— Immunofluorescence analysis using Duolink proximity ligation assays revealed that VWF colocalizes with galectin-1 and galectin-3 in endothelial cells, both before and after stimulation of endothelial cells. Moreover, galectin-1 was found along the typical VWF bundles that are released by endothelial cells. Galectin-1 and galectin-3 could be coprecipitated with VWF from plasma in immunoprecipitation assays, whereas plasma levels of galectin-1 and galectin-3 were significantly reduced in VWF-deficient mice. Binding studies using purified proteins confirmed that VWF could directly interact with both galectins, predominantly via its N -linked glycans. In search of the physiological relevance of the VWF-galectin interaction, we found that inhibition of galectins in in vitro perfusion assays was associated with increased VWF-platelet string formation, a phenomenon that was reproduced in galectin-1/galectin-3 double-deficient mice. These mice were also characterized by a more rapid formation of initial thrombi following ferric chloride–induced injury. Conclusion— We have identified galectin-1 and galectin-3 as novel partners for VWF, and these proteins may modulate VWF-mediated thrombus formation.

The relationship between low-density lipoprotein receptor–related protein-1 (LRP1) and von Willebrand factor (VWF) has remained elusive for years. Indeed, despite a reported absence of interaction between both proteins, liver-specific deletion of LRP1 results in increased VWF levels. To investigate this discrepancy, we used mice with a macrophage-specific deficiency of LRP1 (macLRP1−) because we previously found that macrophages dominate VWF clearance. Basal VWF levels were increased in macLRP1− mice compared with control mice (1.6 ± 0.4 vs 1.0 ± 0.4 U/mL). Clearance experiments revealed that half-life of human VWF was significantly increased in macLRP1− mice. Ubiquitous blocking of LRP1 or additional lipoprotein receptors by overexpressing receptor-associated protein in macLRP1− mice did not result in further rise of VWF levels (0.1 ± 0.2 U/mL), in contrast to macLRP1+ mice (rise in VWF, 0.8 ± 0.4 U/mL). This points to macLRP1 being the only lipoprotein receptor regulating VWF levels. When testing the mechanism(s) involved, we observed that VWF-coated beads adhered efficiently to LRP1 but only when exposed to shear forces exceeding 2.5 dyne/cm2, implying the existence of shear stress-dependent interactions. Furthermore, a mechanism involving β2-integrins that binds both VWF and LRP1 also is implicated because inhibition of β2-integrins led to increased VWF levels in control (rise, 0.19 ± 0.16 U/mL) but not in macLRP1− mice (0.08 ± 0.15 U/mL).

The development of inhibitory anti-factor VIII (FVIII) antibodies in patients with haemophilia A following replacement therapy is associated with several types of risk factors. Among these, the purity of FVIII concentrates, and in particular the presence of von Willebrand factor (VWF), was controversially proposed to influence the immunogenicity of exogenous FVIII. We re-assessed in vivo and in vitro the immuno-protective effect of VWF towards FVIII. The immuno-protective effect of VWF towards FVIII was investigated in vivo, in a model of haemophilia A. We studied the endocytosis of FVIII by murine bone marrow-derived dendritic cells and evaluated the capacity of VWF to block the internalization of FVIII. We characterized the relevance of VWF for the accumulation of FVIII in the marginal zone of the spleen, a secondary lymphoid organ where the immune response to therapeutically administered FVIII initiates. Our results confirm that VWF reduces the immunogenicity of FVIII in FVIII-deficient mice. Paradoxically, VWF is important for the accumulation of FVIII in the marginal zone of the spleen. We propose that VWF exerts at least two non-mutually exclusive immunoprotective roles towards FVIII in haemophilic mice: VWF prevents the endocytosis of FVIII by professional antigen-presenting cells by blocking the interaction of FVIII with as yet unidentified endocytic receptor(s). Hypothetically, VWF, by virtue of increasing the half-life of FVIII in the circulation, may allow an increased contact time with tolerogenic marginal zone B cells in the spleen.

Summary. Although factor VIII (FVIII) and von Willebrand factor (VWF) are products of two distinct genes, they circulate in plasma as a tight non-covalent complex. Moreover, they both play a critical role in the haemostatic process, a fact that is illustrated by the severe bleeding tendency associated with the functional absence of either protein. FVIII is an essential cofactor for coagulation factor IX, while VWF is pertinent to the recruitment of platelets to the injured vessel wall under conditions of rapid flow. FVIII and VWF have in common that they are heavily glycosylated: full-length FVIII contains 20 N-linked and at least seven O-linked glycans, while VWF contains 12 N-linked and 10 O-linked glycans. Three decades of research have revealed that the carbohydrate structures of FVIII and VWF contribute to many of the steps that can be distinguished in the life-cycle of these proteins, including biosynthesis/secretion, function and clearance. In this review, several of these aspects will be discussed. In addition, the interaction of the FVIII/VWF complex with two families of carbohydrate-binding proteins, i.e. Galectins and Siglecs, and their potential physiological relevance will be discussed.

Von Willebrand disease (VWD)–type 2B originates from a gain-of-function mutation in von Willebrand factor (VWF), resulting in enhanced platelet binding. Clinical manifestations include increased bleeding tendency, loss of large multimers, thrombocytopenia, and circulating platelet aggregates. We developed a mouse model to study phenotypic consequences of VWD-type 2B mutations in murine VWF: mVWF/R1306Q and mVWF/V1316M. Both mutations allow normal multimerization but are associated with enhanced ristocetin-induced platelet aggregation, typical for VWD-type 2B. In vivo expression resulted in thrombocytopenia and circulating aggregates, both of which were more pronounced for mVWF/V1316M. Furthermore, both mutants did not support correction of bleeding time or arterial vessel occlusion in a thrombosis model. They further displayed a 2- to 3-fold reduced half-life and induced a 3- to 6-fold increase in number of giant platelets compared with wild-type VWF. Loss of large multimers was observed in 50% of the mice. The role of ADAMTS13 was investigated by expressing both mutants in VWF/ADAMTS13 double-deficient mice. ADAMTS13 deficiency resulted in more and larger circulating platelet aggregates for both mutants, whereas the full multimer range remained present in all mice. Thus, we established a mouse model for VWD-type 2B and found that phenotype depends on mutation and ADAMTS13.

von Willebrand disease (VWD), caused by quantitative or qualitative abnormalities in von Willebrand factor (VWF) is considered the most common inherited bleeding disorder in humans. Mild and severe quantitative defects in VWF cause VWD types 1 and 3 respectively, whereas qualitative abnormalities induce VWD type 2. VWD has also been diagnosed in a number of animal species such as dogs, pigs, cats and horses, as a result of naturally occurring mutations. More recently, murine models have drawn a great deal of attention. Their small size along with their well-defined genetic background makes them ideal tools to study the in vivo function of VWF. The most commonly used model is the VWF-deficient mouse engineered through homologous recombination. However, models resulting from changes in modifier genes indirectly affecting VWF have also been described. These various models have proven very useful in elucidating some aspects of VWF biology not easily addressed through in vitro approaches.

SummaryVon Willebrand factor (VWF) structure-function relationship has been studied only through in vitro approaches. The VWF-deficient mouse model has been extremely useful to examine the in vivo function of VWF but does not allow a more subtle analysis of the relative importance of its different domains. However, considering the large size of VWF and its capacity to interact with various ligands in order to support platelet adhesion and aggregation, the necessity to evaluate independently these interactions appeared increasingly crucial. A recently developed technique, known as hydrodynamic injection, which allows transient expression of a transgene by mouse hepatocytes, proved very useful in this regard. Indeed, transient expression of various VWF mutants in VWF-deficient mice contributed to improve our knowledge about the role of VWF interaction with subendothelial collagens and with platelets receptors in VWF roles in haemostasis and thrombosis. These findings can provide new leads in the development of anti-thrombotic therapies.