Delphine Charlotte

La Petite Cécile

Alexandre

Evelyne Yann

Mohamad, Idinath, Julie, Ghassem, Yacine, Bobby, Katie, Ray, but also Fred Ana Maria

Marijke Alex

Cécile L…

Keywords: La maladie de Willebrand: de la pathophysiologie aux nouvelles Facteur de Willebrand, maladie de Willebrand, saignement, modèles de souris, dominants-négatifs von Willebrand factor, von Willebrand disease, bleeding, mouse models, dominant-negative variants

Von Willebrand factor (VWF) is a large glycoprotein best known for its essential role in haemostasis: it supports platelet adhesion and aggregation at sites of vascular injury and carries

I am grateful to Pr. Jacques Young and Mme Hélène Kinnel for their help and guidance through all the numerous steps to obtain this HDR.

I couldn't have undertaken this journey without Cécile, Peter and Olivier, my lifelong mentors and role models, who have welcomed me in their lab as a post-doc and have supported me every step of the way ever since. They are truly inspirational and represent the side of science I admire and I strive to be part of. Thank you for making a place for me in your scientific group and family.

I am deeply indebted to il Prof (Francesco Bernardi) and Mirko, for introducing me to research and to the world of thrombosis and haemostasis; and to Sara, my best friend in life and in science, Matteo e Pierpaolo for having shared the same pipettes, PCR tubes, cell culture dishes and every achievement since our PhD. I am also thankful to Wolfgang, because during my stay in his lab I have learned that science can be full of difficulties but you must find a way to overcome them. I could have not done that without my colleague and friend Dave, too whom I am deeply thankful. Special thanks to Vincent, and Nikolett, because as with Sara and Dave, I spent countless hours with every one of them discussing every small detail of numerous protocols to improve them and achieve better results every day! And sometimes just for the sincere pleasure of chatting with them! Another special thanks to Paulette and Gigi, for all the precious tips and tricks they taught me in the lab! To Valerie, for our interesting exchanges. To Anne-Lise and Florence, for their precious support. I am also grateful to all the professors and colleagues I have worked with in many different labs and projects around the world, Sophie, Antoine, Annabelle, Jenny and the Lillois, Nicolas in Japan, Prs De Cristofaro in Rome, and to all the colleagues I have shared the bench with during the last 15 years.

Von Willebrand factor (VWF) is a large glycoprotein best known for its essential role in haemostasis: it supports platelet adhesion and aggregation at sites of vascular injury and complexes with coagulation FVIII in circulation.

Quantitative/qualitative deficiencies account for an heterogeneous group of bleeding disorders with variable severity and manifestations, historically classified in three major types and further subtypes based on specific molecular/functional defects. Diseases affecting VWF are grouped under the name of von Willebrand disease (VWD), overall representing the most common inherited bleeding disorder. This manuscript summarizes previously published, ongoing and future research projects with the overall aim of a) investigating molecular mechanisms of VWF biology b) untangling pathologic mechanisms c) developing cellular and murine disease models and d) establishing innovative therapeutic options for VWD. Initial postdoctoral studies focused on VWD-type 2B and receptor-mediated VWF clearance, then expanded on the relationship between VWF and platelets and on other thrombocytopenias. The most recent studies have focused on pathogenic mechanisms of dominantnegative genetic variants by developing cellular and murine models to mimic and understand these complex mechanisms, allowing for the development of corrective approaches. Ongoing and future studies will expand on the establishment of new cellular and murine models of VWD & AVWS and progress toward more personalized genetic-and protein-based therapeutic options for VWD.

Le facteur de von Willebrand (FW) est une grande glycoprotéine connue pour son rôle essentiel dans l'hémostase: il favorise l'adhésion et l'agrégation des plaquettes aux sites de lésions vasculaires et transporte le FVIII de la coagulation dans la circulation. Les déficiences quantitatives/qualitatives en VWF représentent un groupe hétérogène de troubles de la coagulation de gravité et manifestations variables, historiquement classés en trois types principaux et plusieurs sous-types basés sur leur défauts moléculaires/fonctionnels. Les maladies affectant le FW sont regroupées sous le nom de maladie de Willebrand (MW), représentant globalement le trouble héréditaire de la coagulation le plus fréquent. Ce manuscrit résume les projets de recherche déjà publiés, en cours et futurs, visant à a) étudier certains mécanismes moléculaires de la biologie du FW, b) décrire les mécanismes pathologiques, c) développer des modèles cellulaires et murines de la maladie et d) proposer des options thérapeutiques innovantes pour la MW. Les premières études postdoctorales ont porté sur la maladie de Willebrand de type 2B et les récepteurs de clairance du FW, puis ont été étendues à la relation entre le FW et les plaquettes et à d'autres thrombocytopénies. Les études les plus récentes se sont concentrées sur les mécanismes pathogéniques des variantes génétiques dominantes-négatives en développant des modèles cellulaires et murins pour imiter et comprendre ces mécanismes afin de proposer des approches correctives. Les études en cours et à venir se concentrent sur le développement de nouveaux modèles murins de la maladie et sur des options thérapeutiques plus personnalisées basées sur des approches génétiques et à l'aide des protéines spécifiques. RESEARCH ACTIVITIES PAST -PRESENT -FUTURE My research studies, which perfectly fit the themes of the unit, are mainly focused on unknown functions and relationships between VWF and its many partners. I am interested in developing in vitro & in vivo models to elucidate molecular mechanisms underlying unsolved clinical aspects of congenital and acquired von Willebrand disease. I am particularly keen to explore new molecular strategies that can be applied as therapeutic approaches for VWFassociated disorders. During my career, I have started very early to be involved in various mentoring activities. As a senior master student I was a tutor for future biology students, later, as senior Ph.D student, post-doc and researcher I supervised several undergraduate & Ph.D. students, post-docs, technicians and engineers. In 2021, I joined the program "Women & Science" at the University Paris-Saclay, for which I am paired to a PhD student for a one-year mentorship. Moreover, I have always been committed to engage and promote the involvement of early career researchers to science at all levels. I have been a member of the International Society of Thrombosis and Haemostasis (ISTH)-Early Career Committee since its creation in 2013, vicechair in 2018-2019 and chair in 2020-2022. Within these roles, I represented early career scientists within the society and organized & moderated several educational in-person and virtual events, with the aim of reaching and engaging scientists from all over the world.

This manuscript does not have the presumption of being exhaustive but includes an overview of von Willebrand factor/von Willebrand disease and briefly summarized relevant studies in which I was primary involved. Some of these studies are currently in progress while the remaining will be the focus of my research efforts in the near future. Present and future studies are unpublished and confidential.

GENERAL INTRO

Von Willebrand factor is a large multimeric glycoprotein essential for its dual role in haemostasis. VWF mediates platelet adhesion and aggregation at sites of vascular injury and carries coagulation factor VIII (FVIII) in circulation. In normal conditions, circulating VWF flows in a globular non-sticky conformation, which is inaccessible to ADAMTS-13, the protease that specifically cleaves VWF and regulate VWF multimers size, and to platelets, however, it binds to FVIII, protecting it by premature clearance and degradation. Vascular damage is associated with local environmental changes highly affecting VWF. Blood flow suddenly change and becomes turbulent, shear stress rapidly increases and subendothelial components are exposed to blood. Meanwhile, VWF unfolds and adopts an open/active conformation. Open VWF adheres to the subendothelium via interaction with exposed collagen (VWF-A1 domain binds collagen I and III, VWF-A3 domain binds collagen IV and VI) and unfolded immobilized VWF recruits platelets to the injury site via reversible interaction with GPIb-IX-V platelet complex (VWF binds to GPIba). Early platelet interaction with the subendothelium results in initial platelet rolling and is followed by firm adhesion sustained by direct platelet interaction with collagen and fibronectin. Upon these early events activated platelets undergoes structural and conformational changes, aIIbb3 integrin at the platelet surface is also activated and intracellular content is released. All these events contribute to further platelet activation and platelet-platelet aggregation (via fibrinogen-and VWF-interactions) supporting coagulation and fibrin generation, thrombus growth and stabilization, and finally arresting the bleeding. Of note, intracellular VWF from endothelial cells (ECs) and platelets also become available at site of vascular injury 1 .

Although a series of relatively new functional properties of VWF have been identified and it is sometimes difficult to completely separate one function from the others, 2,3 large part of my research studies was mostly pertinent to the haemostatic role of VWF, which was summarized in the previous paragraph, however, my on-going and future projects (briefly presented later) also relate to its role in angiogenesis, vascular formation and remodelling.

VWF biosynthesis 4

VWF is synthetized by endothelial cells (ECs) and megakaryocytes (MKs). VWF is present in blood, in the subendothelial matrix and intracellularly, where it is stored in specialized organelles, the Weibel-Palade bodies (WPB) within the ECs and the a-granules in MKs and platelets. WPB formation is driven by VWF synthesis while a-granules are complex compartments storing numerous proteins. Most of circulating VWF originates from ECs and is constitutively secreted 5 , while intracellular stored VWF is locally released in response to EC/platelet activation. VWF biosynthesis, better studied within ECs, is a complex process consisting in a succession of events that follow one another in a precise order. Importantly, VWF is first translated into a pre-pro-peptide (2813 amino acids) with the following revisited domain structure 6 sequence: SP-D1-D2-D'-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK. The signal peptide targets the protein to the endoplasmic reticulum and is cleaved thereof. Within the endoplasmic reticulum, propeptides cotranslationally undergoes tail-to-tail dimerization through covalent binding between two CK domains. Dimerization forms a "bouquet-like" structure with domains aligned in a side-by-side manners and stabilized by the 3 interchain cysteine bridges between two CK domains. Dimers are then transported to the Golgi, where environmental conditions (low pH, low NaCl, high CaCl2) favour head-to-head self-assembly of VWF multimers through covalent interchain bridges between cysteines in the D3 domains. The multimerization process is ensured by the presence of VWF propeptide (D1-D2), which in the meanwhile is cleaved by rest of the protein but remains non-covalently attached to mature VWF. This process generates a wide heterogenous pool of short

Von Willebrand disease 8,9

Due to its essential role in haemostasis, congenital VWF quantitative and/or qualitative deficiency results in von Willebrand disease (VWD), the most common bleeding disorder with estimated prevalence of 0.6-1.3%. However, not all individuals with low VWF present clinically relevant symptoms and those diagnosed with VWD have estimated prevalence around 1/10.000. Congenital VWD in a very heterogenous disease and has been historically classified in three subtypes: VWD-type 1, -type 2 and type 3. Types 1 (partial) & 3 (total) are quantitative defects while type 2 groups all qualitative deficiencies 10,11 . Depending on non-univocal definitions, VWD-type 1 (60-80% of all VWD cases) consists of patients with low VWF antigen levels < 30% and moderate/severe bleeding symptoms. A separate group of patients exhibiting intermediately reduced VWF levels between 30-50% and more modest bleeding phenotypes have been classified as having "low VWF levels" ("low VWF"). 12,13 It is important to mention that recently published and controversial VWD guidelines 14,15 Historically, VWD is a women's disorder, which, in a society that is MEN-centric, has a huge impact on quality of life, 17,[22][23][24][25][26][27] in how the disease is perceived, diagnosed, funded and ultimately in how it is treated. 26,27 Treatment of VWD Briefly, the moderate, tail-vein-transection (TVT) assay 33 consists in a standardized lateral incision applied with a scalpel using customized cutting molds and results in a precise injury to one of the two lateral veins present in the tail. Upon injury, mice are placed on a heating pad and the tail immersed in warm saline for a standardized time (30-60 minutes). This assay can be performed in two ways, with 2-3 challenges at fixed times (at 15-30-45-minute) (more severe) and without challenges (milder). Each challenge consists of swiping twice the injury site with a gauze to physically remove the external part of the clot. Mice are anesthetized with isoflurane for the total duration of the experiment. Every bleeding arrest and spontaneous rebleed are annotated. The amount of blood shed in the first 3 minutes represents "primary haemostasis" and is quantified separately. The amount of blood shed during the rest of the experiment is also quantified. Blood is quantified by measuring haemoglobin-associated absorbance upon red-blood-cell lysis in a fixed amount of water, and comparing unknown values with a reference curve prepared with known blood volumes. The classic tail-clip bleeding assay represent a more severe model, in which the 3-mm distaltip of the tail is amputated in anesthetized mice. In this case venous and arterial blood are collected for the duration of the experiment and quantified. Arrest and restart of the bleeding are annotated but generally, the time to first arrest is reported as read-out, together with the total amount of blood shed during the observation time.

Publications: 34,35 During my thesis, we created an in vitro cellular model for the study of a large in-frame deletion (p.P1127_ C1948delinsR) in the VWF gene (Figure 1-2), found in a patient with a severe manifestation of VWD. Interestingly, the mutant VWF displayed a dominant-negative effect due to the partial deletion of the D3 domain crucial for the multimerization process.

Although dominant-negative variants were well known to affect VWF, using high resolution agarose gels, we were able to visualize, for the first time, heteropolymers of VWF formed by wild-type and mutant molecules. However, these heteropolymers were limited to dimers and tetramers, supporting the hypothesis that the multimerization process is interrupted upon incorporation of the mutant subunit into a growing polymer (Figure 2). Next, we successfully rescued the dominant-negative effect of the deletion by using a silencing approach. siRNAs that specifically targeted the altered junction-sequence between exons 25 and 35, recognized and suppressed the synthesis of mutant-VWF molecules while wild-type-VWF multimers were normally produced (Figure 2). This study was the first published example of siRNA-based corrective strategy applied to VWD.

Publications: 36,37 In this study, we analyzed VWD-type 2B mice transiently expressing two independent variants (p.V1316M-or p.R1306Q-VWF) via the hydrodynamic gene transfer technique, resulting in various disease severities.

• Casari C, Pinotti M, Lancellotti S, Adinolfi E, Casonato A, De Cristofaro R,

Post-doc #1 (France) 3.2.1. Elevated macrophage-dependent platelet clearance contributes to thrombocytopenia in VWD-type 2B

Figure 2. (A) Cellular models of heterozygous VWD and first in vitro mutation-specific siRNA-based correction. (Left) Control cells receiving a wild-type-VWF expression plasmid generated and secreted normal VWF multimers of all sizes. (Centre) Cells co-transfected with wild-type-and mutant-VWF expression plasmids generated heteropolymers (in red) of wild-type and mutant VWF, resulting in the secretion of low molecular weight and ODD multimers. (Right) Co-transfection of siRNAs selectively targeting the mutant-VWF (siDEL) partially restored multimer formation. (B) siRNA treatment effects in cells. (Left) siDEL treatment selectively decreased the amount of mutant-VWF mRNA while the wild-type mRNA was unaffected. (Centre) While untreated cells synthetized higher amount of mutant-VWF, siDEL treatment shifted the equilibrium toward the synthesis of mostly wild-type VWF, which improved the multimer profile of secreted VWF (right). (Panel B adapted by Casari C. et al. Blood 2010.)

I demonstrated that, in VWD-type 2B mice, the thrombocytopenia was associated with decreased platelet half-life and presence of mutant VWF at the surface of circulating single platelets and platelet aggregates. Platelets collected from VWD-type 2B mice and re-injected into mice with the same genotype, were found to accumulate in the liver and the spleen, where they co-localized with Kupffer cells and marginal macrophages, respectively. In addition, chemical depletion of macrophages in VWD-type 2B mice led to near-normalization of their platelet counts, emphasizing the important role of macrophages in VWD-type 2Bassociated thrombocytopenia.

These data indicate that VWF carrying VWD-type 2B variants, binds spontaneously to platelets and this might accelerate macrophage-mediated clearance, which in turn contributes to the thrombocytopenia in VWD-type 2B patients (Figure 3). 38,39 Three experimental models (normal human platelets incubated with recombinant human-VWF, a transient mouse model described before and blood samples from VWD-type 2B patients) have been used to demonstrate that platelet functions are severely dysregulated in severe VWD-type 2B associated with the p.V1316M substitution. Indeed, we observed decreased platelet aggregation, secretion and spreading upon stimulation with all tested agonists (ADP, thrombin, convulxin and collagen) as well as reduced thrombus growth on a collagen matrix. These multiple platelet dysfunctions were associated with impaired activation of the platelet integrin αIIbβ3 and of the small GTPase Rap1. Platelet signaling

Altered platelet functions are a predominant factor in the bleeding tendency in VWDtype 2B patients

VWF-type 2B VWF-type 2B/ Platelet aggregates

Uptake by macrophages

Figure 3. Uptake of VWF/platelet complexes by macrophages. VWF carrying VWD-type 2B variants adsorbs to platelets and these complexes are eliminated by macrophages in the liver and the spleen. This could accelerate platelet clearance and participate in the assessment of the thrombocytopenia in VWD-type 2B.

upstream of Rap1 activation, which is essential for αIIbβ3 activation, was therefore analyzed. Ca 2+ store release as well as Syk phosphorylation and PLCγ2 activation appeared normal upon agonist induced stimulation, suggesting that VWF carrying the p.V1316M variant acts directly on, or downstream of Ca 2+ store release. Based on these data, we concluded that a severe thrombocytopathy contributes to the bleeding tendency in VWD-type 2B patients carrying the p.V1316M substitution (Figure 4).

Creation of a knock-in mouse model of VWD-type 2B 40

During my post-doctoral studies, I conducted several projects on VWD-type 2B using transient mouse models generated via hydrodynamic gene transfer that enables the expression of wildtype and/or mutant VWF. To improve on the transient expression, we next engineered the first knock-in mouse model of VWD-type 2B. These mice closely resemble the human disease showing macrothrombocytopenia, circulating platelet-aggregates and reduced HMWMs. Severe bleeding tendency was demonstrated in a tail-clip assay, while lack of vessel occlusion was observed in a FeCl3-induced injury model. Impaired platelet adhesion was demonstrated by the inability of thrombi to properly form on a collagen surface under venous or arterial shear conditions. Moreover, reduced platelet aggregation in response to both thrombin and collagen was confirmed, further validating the mouse model. We also discover an unexpected issue concerning VWD-type 2B. Indeed, the VWD-type 2B knock-in mice showed increased vascular permeability and increased leukocyte recruitment in an inflammation model (the reverse passive Arthus reaction), indicating that the type 2B phenotype enhances the inflammatory response. Our results demonstrated the knock-in mouse model faithfully reflects VWD-type 2B associated phenotypes. [41][42][43] One of our main research interests is to unravel the clearance mechanisms of VWF and FVIII. In this context, during my post-doctoral fellowship, I was involved in a number of studies aiming to identify clearance receptors for VWF. We have demonstrated that the low-density lipoprotein receptor-related protein-1 (LRP1) expressed on macrophages is a modulator of VWF clearance. Interestingly, binding to LRP1 requires VWF to be in its open conformation, resulting from the unfolding process induced by shear forces. Increased levels and prolonged survival of VWF in mice selectively deficient for the LRP1 in macrophages (macLRP1 --mice) support the idea that macrophage-LRP1 is important for the clearance of VWF in physiological conditions 41 .

VWF clearance

In a second study we demonstrated that, in contrast to wild-type-VWF, LRP1 was able to interact with VWF-type 2B variants (p.V1316M and p.R1306Q-VWF) even in the absence of shear forces. We concluded that enhanced binding of the VWF carrying type 2B variants to LRP1 could explain their increased clearance 44 . In a third study, we identified that the macrophage-receptor Siglec-5, which specifically interacts with sialic acid residues, is able to bind to VWF and FVIII. We demonstrated that overexpression of Siglec-5 in murine hepatocytes by hydrodynamic gene transfer, resulted in decreased levels of VWF (and FVIII) in plasma, suggesting that Siglec-5 may contribute to the regulation of plasma levels of the FVIII/VWF complex 43 .

Publications

Creation of a mouse model of Thrombotic Thrombocytopenic Purpura 45

In the framework of a collaborative study, in May 2013, I worked in the laboratory of Prof. Nicolas Prevost at the Kyoto Gakuen University (Japan) during a short-stay period sponsored by the same University. I collaborated in the creation of a new murine model of Thrombotic Thrombocytopenic Purpura (TTP). TTP is a rare and severe disease, in which patients suffer from quantitative or qualitative defects in ADAMTS13, the protease that processes VWF once secreted in the blood stream. This new mouse model was based on the transient expression via hydrodynamic gene transfer of a VWF variant resistant to ADAMTS13-mediated cleavage, in Vwf-deficient mice. These TTP-mice developed thrombocytopenia, red cell destruction, impaired kidney function and VWF-rich platelet thrombi in the renal microvasculature, all of which are symptoms consistent with TTP.

Publications: As a follow-up on my project on VWD-type 2B, I pursued the investigation of the molecular mechanisms underlying impaired platelet signaling in this pathology. To do so, I expressed the p.V1316M-type 2B VWF variant via hydrodynamic gene transfer in mice with specific platelet defects. Two distinct pathways contribute to Rap1 activation in platelets: rapid activation mediated by the calcium-sensing guanine nucleotide exchange factor (CalDAG-GEFI) and sustained activation dependent on signalling by protein kinase C (PKC) and the ADP receptor, P2Y12. The differential use of the JON/A antibody, which irreversibly binds to the activated form of the murine integrin αIIbβ3, allowed us to determine that the PKC/P2Y12 pathway is altered in VWD-type 2B. Further studies with ADP as an agonist suggested that p.V1316M-VWF engagement of GPIba leads to impaired platelet signalling at the level of PKC, but not P2Y12. Consistently, downstream of PKC we observed impaired α-and dense-granule release and decreased PKC substrate phosphorylation in response to agonist stimulation. Moreover, in platelets from mice and from patients expressing p.V1316M-VWF, we observed, for the first-time, elevated phosphorylation of PKC substrates under resting conditions, suggesting that a PKC-dependent pre-activation prevents normal response to agonist stimulation. In summary, our studies identify altered PKC signalling as the underlying cause of platelet hypofunction in severe VWD-type 2B associated with p.V1316M-VWF expression (Figure 5). VWD-type 2B can be considered an example of acquired platelet disorder.

• Morioka Y*, Casari C*,

Role of Arp2/3 in platelet activation and homeostasis 49

When I was a post-doc at UNC in the lab of Prof. Wolfgang Bergmeier, who is known for his expertise on platelet signaling, I collaborated to some platelet studies, not directly pertaining to VWF. Actin reorganization regulates key processes in platelet activation. We examined the role of the Arp2/3 complex, an essential component in actin filament branching, in platelet functions. The Arpc2 gene, encoding the p34 subunit of the Arp2/3 complex, was deleted in the megakaryocyte lineage (Arpc2 fl/fl PF4-Cre). Deletion of the Arp2/3 complex resulted in marked microthrombocytopenia in mice, caused by premature platelet release into the bone marrow compartment and impaired platelet survival in circulation. Loss of Arp2/3 led to macrophagemediated platelet clearance as demonstrated by significant increase of circulating platelet counts following clodronate liposome treatment. Arp2/3 complex is well known for its role in lamellipodia formation, consistently, Arpc2 fl/fl PF4-Cre platelets and megakaryocytes showed a marked defect in platelet spreading but mild defects in integrin activation and aggregation. However, no significant differences in hemostasis or thrombosis were observed between Arpc2 fl/fl PF4-Cre and control mice. In summary, this study demonstrates that Arp2/3 is critical for platelet homeostasis but, unexpectedly, not vascular hemostasis.

Publications:

•

The RAP1-GAP RASA3 prevents clearance of circulating platelets 50

This is a study I started at UNC, and was recently finalized and published. We used various mouse models and approaches to demonstrate that macrothrombocytopenia associated with Rasa3 deficiency, a negative regulator of the small GTPase Rap1, is predominantly caused by integrin-mediated platelet clearance. Rasa3-mutant megakaryocytes exhibited altered morphology but these abnormalities did not significantly reduce the occurrence of proplatelet formation in vivo. Once released into circulation, Rasa3-mutant platelets are rapidly sequestered in the spleen at the marginal/red pulp interface and subsequently phagocytosed by resident macrophages. Rasa3-mutant platelets are also cleared to a greater extent by the liver, and this clearance mechanism is further enhanced upon splenectomy. Premature clearance of Rasa3-mutant platelets is dependent in part on talin1 and αIIbβ3 integrin, and antibodies against αIIbβ3 caused platelets to be cleared in the spleen in a similar manner to Rasa3-mutant platelets. These findings uncover a potentially novel mechanism of integrindependent platelet clearance.

Researcher (France) 3.4.1. The macrophage receptor SR-A1 contributes to VWF clearance 51

When I came back from the US, I was entrusted with the task of finalizing a project that identified the macrophage-receptor SR-A1 as a potential important contributor of VWF clearance. Several receptors, expressed on different cellular types including macrophages and hepatocytes, were already known as able to participate -at least in part-to VWF clearance. Moreover, clinical and research studies were suggesting that increased VWF clearance was rather common among several VWD subtypes (type 1, type 2 36,44 and type 3). We and others previously demonstrated that binding of wild-type VWF to LRP1 requires shear-mediated unfolding of VWF, whereases shear stress-independent binding of VWF to macrophages in static conditions was also possible when VWF was carrying gain-of-function variants within the A1 domain 36,44 or truncated its N-glycans, [START_REF] O'sullivan | N-linked glycan truncation causes enhanced clearance of plasmaderived von Willebrand factor[END_REF][START_REF] Chion | N-linked glycans within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance[END_REF] or possibly via others receptors. [START_REF] Casari | Clearance of von Willebrand factor[END_REF] Macrophages were responsible for the majority of VWF clearance 55 however, functional inactivation of previously identified receptors was associated only with modest effects on VWF clearance, suggesting that additional receptors had yet to be identified. We studied whether SR-A1 could be one of them. SR-A1 (also known as SCARA1 or CD204) is a trimeric receptor highly expressed on macrophages, is part of the big family of the scavenger receptors and share structural similarities with SCARA5, identified as associated with VWF plasmatic levels in genome wide association studies.

Using in vitro solid-phase assays we demonstrated that VWF efficiently binds to SR-A1 via multiple binding sites on the D'D3, A1 and D4 domains without the need to adopt an open conformation. Moreover, cellular experiments (Figure 6) using 1) cultured HEK293 cells expressing exogenous SR-A1 (left), 2) human THP1-deived macrophages (middle) and 3) primary murine bone marrow-derived macrophages from SR-A1-deficient-and wild-type mice (right), confirmed that VWF binds to macrophages via SR-A1 receptor.

In vivo, we measured reduced propeptide/antigen ratios in SR-A1-deficient mice (Figure 7, left), which is compatible with a slower clearance of VWF in the absence of the receptor. Moreover, we were able to demonstrate that recombinant VWF carrying genetic variants known to be associated with accelerated clearance in VWD patients (p.R1205H and p.S2179F)

Figure 6. SR-A1/hVWF Duolink-PLA analysis in HEK293 cells expressing exogenous SR-A1 (left), THP1-derived human macrophages (middle) and primary bone marrow-derived macrophages from SR-A1-deficient (top right) and wild-type mice (bottom right). Fluorescent red spots indicate complexes between SR-A1 and human-VWF, which was incubated with immobilized cells. (Figure adapted by Wohner N. et. Haematologica 2018)

was actually binding to SR-A1 with increased affinity compared to wild-type VWF in solidphase and cellular experiments. Finally in vivo, accelerated clearance of these mutant-VWF was partially normalized in SR-A1-deficient mice, suggesting that SR-A1-mediated uptake of the so-called "clearance-mutant VWF" (Figure 7, right) could contribute to their reduced circulatory half-life in patients.

Publications: [START_REF] Muczynski | A factor VIII-nanobody fusion protein forming an ultrastable complex with VWF: effect on clearance and antibody formation[END_REF] One of the important roles of VWF in physiologic conditions is to function as carrier of FVIII, which prevents its premature clearance and also modulates anti-FVIII antibody development in haemophilia A patients. Indeed, in circulation, FVIII/VWF complexes co-exist with a small pool of free FVIII (2-5%), which however behaves differently and confuses the study of the complexes in vivo, in animal models. To minimize the amount of free FVIII, we generated a fully functional FVIII-anti-VWF fusion protein, in which the B-domain of FVIII was replaced by a bivalent anti-VWF single domain antibody (sdAb) KB013bv. Because this sdAb efficiently binds to VWF and dissociates slowly, the FVIII-KB013bv fusion protein formed ultra-stable complexes with VWF (Figure 8). Compared to BDD-FVIII, the FVIII-KB013bv had two-fold increased mean residence time (1.6h versus 3h) when infused in F8-deficient mice, which resulted in reduced free FVIII (<0.2%). The prolonged survival was confirmed in a tail-clip experiment. 24 hours post-injection, all mice receiving FVIII-KB013bv shed minimal amount of blood compared to mice receiving BDD-FVIII. Interestingly, mice exposed to repeated infusions of the ultra-stable complex rarely developed anti-FVIII Ab (only 1 out of 8 treated mice) while the great majority (14 out of 16) of mice receiving BDD-FVIII developed anti-FVIII Abs, suggesting that enhanced VWF binding may result in reduced anti-FVIII Ab formation [START_REF] Sorvillo | von Willebrand factor binds to the surface of dendritic cells and modulates peptide presentation of factor VIII[END_REF] . This study demonstrated that increased VWF-binding favors FVIII survival and reduces anti-FVIII antibody formation. 28,[START_REF] Karampini | Correcting dominant-negative von Willebrand disease[END_REF] This study stemmed from my Ph.D. project, but was made possible by the expertise acquired during my post-docs. It was the first study to report correction of dominant-negative VWDassociated defects in vivo. In vivo silencing was first discovered in the 90s and rapidly generated great excitement within the scientific community, which recognized in this discovery the potential to revolutionize medicine. However, despite its rapid and widespread use in research, it took long time for the first RNAi-based drug (patisiran) to be approved, in 2018, with one of the remaining bigger limitation being cell-specific targeting. Indeed, approved or RNAi-based drugs in advanced clinical trial and commercially available products for in vivo research studies, all exclusively targeted hepatocytes, which do not synthetize VWF. To overcome this limitation and test RNAi in VWD models, we applied the hydrodynamic gene transfer technique to Vwf-deficient

• Wohner N, Muczynski V,

An anti-VWF single domain antibody to create a FVIII-VWF ultra stable complex

Modulation of dominant-negative VWD in vivo

. Engineering FVIII to create an ultra-stable complex with VWF: (Left) Study's strategy. FVIII Bdomain was replaced with a bivalent anti-VWF sdAb (KB013bv), resulting in an ultra-stable complex with VWF, mostly due to very slow dissociation rate. FVIII-KB013bv maintained cofactor activity, had prolonged survival and decreased anti-FVIII Abs formation. (Right) According to Sorvillo et al 57 , the VWF-FVIII complex separates at the cellular surface of antigen-presenting cells, with FVIII being endocytosed and most of the VWF molecules remaining outside the cell. Based on this model, we anticipated that FVIII-KB013bv-VWF complex will not dissociate, resulting in reduced uptake of FVIII-KB013bv by antigen-presenting cells. Fewer FVIII-derived peptides will then be presented to T cells, and in turn, less anti-FVIII antibodies will be produced. Figure adapted by Muczynski et al. Blood 2018.

mice and generated transient heterozygous mouse models of VWD-type 2A co-expressing wild-type-VWF and VWF-DEL (p.P1127_ C1948delinsR) (Figure 1). These mice mirrored the severe multimerization defect observed in the patient [START_REF] Bernardi | A de novo and heterozygous gene deletion causing a variant of von Willebrand disease[END_REF][START_REF] Bernardi | In-frame deletion of von Willebrand factor A domains in a dominant type of von Willebrand disease[END_REF] and synthetized VWD within their hepatocytes. Therefore, we were able to use lipid-based nanoparticles to deliver siRNAs specifically targeting the VWF-DEL in vivo. Multimer analysis revealed that higher multimer bands were visible after treatment and the odd bands corresponding to wild-type/DEL heteropolymers were instead decreased (Figure 10). Importantly, the use of negative control siRNAs with sequences not targeting any murine gene transcript did not have any effect on VWF multimer profile, confirming the specificity of the siDEL designed to target the mutant VWF. Moreover, while siNEGs left VWF antigen levels essentially unchanged postadministration, siDEL increased VWF antigen levels of 25% (Figure 10). These results demonstrated that liver-mediated VWF expression can be modulated in vivo by siRNA administration and that highly selective siRNAs can ameliorate severe phenotypes due to genetic variants with dominant-negative effects. Although consistent, phenotypic correction was however modest in siDEL-treated mice, for several reasons: 1) siRNA-treatment is unable to trigger 100% gene knockdown, with 50-80% silencing reported in previous studies using the same lipid nanoparticles; 2) in vitro, we previously demonstrated that VWF-DEL is expressed with high efficiency, suggesting that even small amounts of mutant VWF still can have strong dominant-negative effect, terminating VWF multimerization intracellularly when integrated into a growing multimer and preventing better results; 3) mice subjected to hydrodynamic gene transfer express supra-physiologic VWF levels, making siRNA-mediated silencing more challenging and 4) finally, siRNAs are effective only if internalized by transfected hepatocytes (60-70%), therefore, it is possible that a portion of VWF-synthetizing cells did not uptake siDELs and secreted heteropolymers.

Figure 10. Mutation-selective RNA silencing in vivo in a mouse model of VWD-type 2A. (A) VWF:Ag levels increased 25% upon siDEL administration while remained stable upon siNEG infusion. (B) Multimer profile improved in two ways by siDEL: higher multimers appeared and heteropolymers decreased. siNEG had no effects on VWF multimers. (C) Densitometric analysis of VWF multimers. Arrowheads indicate oddbands/heteropolymers. (Figure adapted by Campioni M. et al. Journal of Thromb Haemost 2021)

To further prove that inhibiting the dominant-negative effect of a VWF variant is a promising approach for VWD, we decided to test a second corrective strategy (Figure 11). The dominant-negative effect of VWF-DEL consists in its ability to terminate the multimerization process of a growing polymer and it is possible because VWF-DEL has a free and functional Cterminal end. Therefore, we hypothesized that preventing VWF-DEL to interact with any other VWF polymer would suppress its dominant-negative nature resulting in the secretion of reduced amount, but only of fully functional, normal VWF multimers. To test this hypothesis, we engineered a pLIVE vector expressing a VWF variant carrying the DEL and the p.C2773R variant, which is well known to prevent CK-mediated, tail-to-tail dimerization.

Heterozygous mice transfected with wild-type and the double-mutant VWF plasmids displayed normal multimer profiles in plasma, with VWF multimers of all sizes and undetectable heteropolymers. Compared to mice expressing wild-type VWF only, these mice expressed around 70% of VWF antigen levels, which was sufficient to correct hemostasis in a classic tail-clip assay (Figure 12).

Figure 11. Effect of heterozygous variants affecting VWF multmerization (top) dimerization (middle) and both polymerization processes (bottom). Dominant-negative variants inhibiting multimerization or dimerization have similar termination effects when aberrant monomers or dimers are incorporated into a growing multimer. VWF monomers with both variants are unable to interact with any other VWF subunit, preventing the termination effect.

Figure 12. DNA-targeting corrective approach. (A) VWF:Ag levels in heterozygous expressing the doublemutant VWF were restored to 70% compared to those of mice expressing VWF/WT only. (B) Multimer profile was essentially normal (B) and the bleeding tendency was similar to that of mice expressing wild-type VWF. (Figure adapted by Campioni M. et al Journal of Thromb Haemost 2021)

Although our strategies were not designed to be applied directly to patients with VWD, we believe that this work has the merit of being the first study to provide in vivo proof-of-concept that corrective approaches aiming to prevent and/or counteract the dominant-negative effect of VWF variants with high efficiency will represent a promising tool toward more personalized treatments for VWD (Figure 13). A commentary highlighting our results was written by Dr Elli Karampini and Prof James O'Donnell, and published in the same issue of The Journal of Thrombosis and Haemostasis.

Schematic overview adapted by Karampini et al. of our corrective approaches to prevent the deleterious effects of a dominant-negative VWF variant. In vivo strategies (left) and intracellular VWF biosynthesis (right). (A) Normal/wild-type VWF multimer synthesis and secretion. (B) Dominant-negative heterozygous variants resulting in the formation of hetropoymers. (C) Mutant-specific siRNA-based corrective approach partially preventing heteropolymer secretion. (D) Alternative corrective approach to prevent the formation of hetropolymer obtained by inserting an additional mutation preventing VWF dimerization to the VWF-DEL variant, which hampers multimerization.

• ITMO-IHP (Instituts Thématiques Multi-organismes Immunologie, Hématologie et Pneumologie) French

Grant on Thrombosis and Hemostasis as principal investigator, in the framework of a French-Italian collaboration. (Bench fees, Obtained as PI) • CSL-Behring (France) "Fonds de dotation" for research in hemostasis (Bench fees, Obtained as PI)

Several years after our first paper about RNAi applied to VWD was published, 34 the group of Prof. Eikenboom in the Netherlands, presented an innovative approach. Instead of targeting specific pathogenic variants, they proposed to target SNPs, aiming to develop siRNAs that could be useful for many patients, carrying various gene mutations, in association with the same SNPs. Short after we first presented our in vivo preliminary studies, we started a collaboration, to test some of their siRNAs in our mouse models of VWD-type 2B. For that project, I mentored Annika De Jong, PhD student in their group, when she was visiting our lab for the in vivo experiments. Despite a lot of optimization efforts and some promising early results, we realized that their siRNAs were not sufficiently selective. (These results have been presented by Prof Eikenboom and by Annika De Jong at several congresses, but they have not been published yet).

VWD variants within the D4 domain 61

In the framework of a collaboration with the group of Prof Remi Neviere and Dr Olivier Nicolas Pierre-Louis (Unit EA7525 Cardiovascular vulnerability, metabolic & endocrine pathologies) at the Martinique's Hospital and University of Antilles, we hosted in our laboratory a Ph.D. student, Marie-Daniela Dubois, for 1-year. My roles was to co-supervise Marie-Daniela in our lab and I continued to mentored her during her thesis writing the following year. The project was focused on the study of VWF variants identified in Afro-Caribbean patients from Martinique. Of particular interest, they identified two patients with multiple gene variants, among which two were located within the VWF-D4 domain. Literature search highlighted that studies untangling the pathologic mechanisms of mutant VWF carrying variants within this domain were limited, therefore, we conducted in vitro studies to investigate how two distinct VWF-D4 variants, affected VWF synthesis/secretion and functions. Briefly, Patient 1, deceased at the time of the publication, was diagnosed with VWD-type 1 (VWF:Ag 35%, VWF:RCo 30%; FVIII:C 33%; VWF:FVIIIB 56%, normal multimer profile/reduced intensity, bleeding score 0; recFVIII requirement before/after surgery). Seven VWF gene alterations have been identified in the propositus, two new variants and five reported as benign. The p.His817Gln substitution within the D' domain (VWD-type 2N-associated variant) was responsible for the decreased FVIII binding but was found with high frequency in African-Americans and classified as non-pathogenic in persons of this ethnicity, [START_REF] Bellissimo | VWF mutations and new sequence variations identified in healthy controls are more frequent in the African-American population[END_REF][START_REF] Wang | Characterizing polymorphisms and allelic diversity of von Willebrand factor gene in the 1000 Genomes[END_REF][START_REF] Johnsen | Common and rare von Willebrand factor (VWF) coding variants, VWF levels, and factor VIII levels in African Americans: the NHLBI Exome Sequencing Project[END_REF] such as Patient 1. The two new heterozygous variants were p.Tyr1086= and the p.Cys2216Phefs*9. Patient 2 was classified as VWD-type 3 (VWF:Ag 1-5%; VWF:RCo 5%; FVIII:C 2+3%; VWFpp 6%; absence of multimers; bleeding score 32). Eight variants have been identified within the VWF, five non-deleterious and three possibly pathogenic, such as heterozygous p.Val510=, p.Cys1149Arg and p.Pro2145Thrfs*5. Importantly, the p.Cys1149Arg and p.Pro2145Thrfs*5 were located in different alleles.

Both patients were carrying heterozygous frameshift variants causing premature stop codons and leading to VWF missing part of the D4-and all the C-terminal end of the molecule -with CK being essential for VWF dimerization-(Figure 14, left). To perform cellular studies, we engineered plasmids for the expression of wild-type and mutant VWF and, to differentiate the two molecules in heterozygous models, we expressed wild-type recombinant VWF followed by a Myc-tag and each of the mutant recVWF followed by an HA-tag (Figure 14). Homozygous (WT-rVWF-Myc; P1-rVWF-HA and P2-rVWF-HA) and heterozygous (WT-rVWF-Myc + P1-rVWF-HA or WT-rVWF-Myc + P2-rVWF-HA) plasmid combinations were expressed in HEK293T cells and VWF analyzed in cellular media and lysates. Cellular media were subsequently concentrated to perform classic VWF functional studies. Total VWF antigen levels were quantified in cellular media and lysates for each condition. Ratios (supernatants over lysates) were also calculated and for each condition including mutant VWF, ratios were lower than those in the control conditions (WT-rVWF-Myc), suggesting some intracellular retention of both mutant recombinant VWF. Taking advantage of the different tags, we also quantified specific expression of wild-type and mutant VWF in the supernatant of heterozygous cellular models. In these conditions, while P2-rVWF-HA was expressed at a similar proportion of WT-rVWF-Myc, the P1-rVWF-HA was instead poorly expressed compared to WT-rVWF-Myc, indicating more intracellular retention for P1-rVWF-HA than for P2-rVWF-HA. In cellular media from heterozygous models, we also evaluated multimer profiles. Not surprisingly, supernatants containing P1-rVWF-HA, poorly secreted, showed normal multimer profiles (Figure 15). Interestingly, after careful analysis of multimer profiles of cellular media containing P2-rVWF-HA, we noticed a faint additional odd-bands (Figure 15, red arrowheads) with intermediate molecular weight, compatible with heteropolymers formed by wild-type & mutant truncated VWF molecules. Extra-and intra-cellular mutant VWF quantifications and multimer analysis of heterozygous models, led us to propose the following pathogenic mechanisms for each frameshift variant (Figure 16). The p.Cys2216Phefs*9 (P1-rVWF-HA) was poorly secreted, probably because of a combination of mRNA degradation and intracellular retention. When co-expressed with WT-rVWF, we measured reduced antigen levels in conditioned media, consistent with VWD-type 1, however, normal/functional multimers of only wild-type subunits were detected, consistent with normal results in all binding tests (with GPIIbIIIa, GPIba, collagen III, FVIII and ADAMTS13). Hence, for this variant, the detrimental effect appears mostly at the expression level. For the p.Pro2145Thrfs*5 (P2-rVWF-HA) variant, the pathogenic mechanism was different. Indeed, mutant VWF subunits lacking the ability to dimerize (because CK-domain is missing) are synthetized and secreted. Therefore, monomeric VWF carrying the p.Pro2145Thrfs*5 variant behaves as a classic dominant-negative so-called "dimerization-variant", terminating further multimer elongation when incorporated into a growing polymer. Lack of a large part of the C-terminal end of VWF, resulted in decreased binding to GPIIbIIIa -as expected-but also in reduced binding to collagen III, ADAMTS13 and GPIba binding. On the contrary, FVIII binding was unaffected. Patient 2 phenotypes however were very severe, much more than expected from the in vitro results. Indeed, in addition to the p.Pro2145Thrfs*5 variant, this patient carried a second dominant-negative variant (p.Cys1149Arg) preventing VWF multimerization, in the other allele. The association of these two deleterious dominant-negative variants was responsible for the severe clinical case consistent with VWD-type 3.

Publications:

• Dubois MD, Peyron I,Pierre-Louis ON, Pierre-Louis S, Rabout J, Boisseau P, De Jong A, Susen S, Goudemand J, Neviere R, Fuseau P, Christophe OD, Lenting

Ongoing projects (unpublished data -confidential)

Unique humanized mouse models of von Willebrand disease to implement the need for innovative therapeutic strategies

Many of the projects I have been involved during my career have benefitted by the use of murine models. Although science is moving fast in the direction of alternative models to replace the use of animals for research purposes, it is undoubted that in the field of thrombosis and haemostasis such animal models are still essential to study the physiopathology of mechanisms where so many factors interplay. VWF has been named the molecular bus and it deploys its functions by interacting with different receptors and cellular types, with components of the extracellular matrix or with soluble proteins, in circulation or within the specialized cellular organelles where they are stored. When using mouse models, it is desirable that the murine actors mimic the human ones and cross-reaction between species is useful, although not always possible. In this context, one of the major limitations of studying VWF in murine models, is the human/mouse incompatibility between VWF and one of its major partners, the platelet receptor GPIba. Human-VWF binds poorly to the murine receptor, preventing the normal recruitment of platelet at site of injury, when human-VWF is express in mice. However, murine-VWF can bind to human-GPIba in vivo, although with a reduced affinity [START_REF] Kanaji | Humanized GPIbα-von Willebrand factor interaction in the mouse[END_REF] .

Our group and others have developed several strategies to overcome this limitation. One of the first attempt developed by Chen et al consisted in engineering a knock-in mouse expressing murine-VWF carrying the p.R1326H substitution, which proved to be sufficient in supporting in vivo thrombus formation in presence of transfused human platelets 66 . One subsequent example was described in our previous study (page 17), for which we set up a chimeric model for transient expression of functional human-VWF in mice, by switching the human-A1 domain with its murine counterpart 46 . In the same study, reverting the p.1326 amino acid to Arginine, as it is in the murine-VWF, was however insufficient to correct in vivo haemostasis. Notably, the group of Zaverio Ruggeri developed few years later a similar chimeric approach by engineering mice that expressed murine-VWF with the human version of exon 28. Then, to assure haemostasis, they crossed these chimeric mice with mice expressing a humanized version of GPIba (mVWF(h-exon28) +/+ /hGPIba). [START_REF] Kanaji | Humanized GPIbα-von Willebrand factor interaction in the mouse[END_REF] Aiming to develop stable rather than transient humanized mouse models to test molecules directed to human-VWF, we decided to engineer double humanized mice expressing fulllength human-VWF and human-GPIba (hVWF +/+ /hGP1BA +/+ mice) (Figure 17). Moreover, one of the VWD subtypes for which we were missing an appropriate knock-in mouse model was group 2 VWD-type 2A. This subtype is due to genetic variants resulting in mutant VWF more susceptible to ADAMTS-13-mediated degradation and is of particular interest because its phenotypes and some of the clinical conditions resemble those observed in patients suffering from acquired von Willebrand Syndrome. Therefore, we decided to also engineered a second double humanized mouse model of VWD-type 2A, expressing human-VWF carrying the p.R1597W variant and human-GPIba (hVWFp.R1597W +/+ /hGP1BA +/+ mice, which will be referred as VWD2A mice) (Figure 17).

While these unique murine models have recently been very helpful tools for several projects within our group and for various collaborations, I was responsible for the generation of the double humanized colonies, which was particularly challenging during the recent pandemic, and I am currently coordinating their characterization.

To evaluate VWF expression in the double humanized mice we used human-pooled normal plasma as reference. First we measured VWF:Ag levels and, unexpectedly, levels were low not only in VWD2A mice but also in mice expressing wild-type human-VWF (3±0.8% in VWD2A mice -red-and 15±4% in hVWF +/+ /hGP1BA +/+ mice -purple-) (Figure 18). Multimer analysis was also performed with murine plasma. Because very low antigen levels can result in low quality gels, multimers were evaluated in normal conditions and 30 minutes post-histamine administration, which resulted in two-fold increase in VWF antigen levels. Normal multimer distribution was observed in mice expressing wild-type human-VWF (Figure 18, purple). On the contrary, the profile was profoundly abnormal in VWD2A mice, with only few sharp bands visible at the low-molecular weights. Moreover, these bands showed unusual intensity and migration patterns, suggesting that indeed, VWF degradation was altered in these mice (Figure 18, red).

Results for the hVWF +/+ /hGP1BA +/+ mice were completely unexpected: multimer profile was normal but antigen levels were low, which is the definition of VWD-type 1. Therefore, we realized that these mice could not represent "control" humanized mice but rather they mimicked VWD-type 1, for which no other murine models were available. For this reason, hVWF +/+ /hGP1BA +/+ mice are referred as VWD1 mice. VWF clearance was estimated by measuring VWF propeptide levels, with VWFpp/VWF:Ag ratios around 1 indicative of normal VWF clearance. Although at different levels, VWFpp/VWF:Ag ratios were elevated in both mice (3±0.2% and 7±2% for VWD1 and VWD2A mice, respectively) suggesting that accelerated VWF clearance could contribute, at least in part, to the low antigen levels. Because VWF is the carrier of FVIII in circulation, we measured FVIII activity in plasma and, in accordance with low VWF antigen levels, FVIII:C was reduced in humanized mice (FVIII:C 44±8% in VWD1 and 7±1% in VWD2A mice). The expression of human GPIba was confirmed by flow cytometry (Figure 19, left) using human and murine platelets and monoclonal antibodies selectively binding to the human (anti-hGPIba/APC clone HIP1) or the murine (anti-mGPIba/PE clone Xia.G5) receptors. These results were also confirmed by immunofluorescence on blood smears prepared from engineered mice (not shown). Next, we also performed basic studies to confirm that platelets were functional in these mice. Platelet count and size were measured with a veterinary blood counter and platelet activation was evaluated by flow cytometry (Figure 19, middle and right).

Platelet count was moderately reduced of 20-25% in VWD1 and VWD2A mice (platelet counts: 471±47, 443±68 and 589±65*10 3 /µl in VWD1-, VWD2A-and control 129Sv mice, respectively) and platelet size was slightly increased (platelet volume: 6±0.3, 6±0.3 and 5±0.3µm 3 in VWD1, VWD2A-and control mice). Although the values differed slightly from those of control mice, neither platelet count nor platelet size were as severe as in macrothombocytopenic mice. Finally, whole blood platelet activation was analyzed by flow cytometry by measuring activated aIIbb3 and P-selectin exposure in response to increasing doses of PAR4-peptide. Data showed that beside a little reduction in counts, when tested in vitro, platelets were functional in humanized VWD1 and VWD2A mice.

To have an overview of the bleeding profile of our new humanized mice, we evaluated haemostasis in a moderate-tail vein transection (TVT) and a severe-tail-clip bleeding assays (Figure 20). (These bleeding assays, were briefly described at page 10) Clinical manifestations of VWD-type 1 patients are very heterogeneous with some patients experiencing severe spontaneous bleedings and the majority having mild symptoms. In VWD1 mice, we did not observe pre-or post-natal spontaneous bleeding episodes nor mortality and when subjected to a moderate TVT assay, these mice were able to form clots that were stopping the bleeding few minutes post-injury and rapidely after each challenge, similar to wild-type control mice. Although some VWD1 mice lost larger amount of blood then control mice, overall, the amount of blood shed was quite variable in VWD1 mice and when compared confidential to control mice, the difference was not statistically significant (84±82µl versus 36±22µl) (Figure 20, top left). The situation was different when these mice were subjected to tail-clip, in which the inflicted injury is more severe. In this assay, VWD1 mice significantly shed more blood compared to control mice (102.0±122.5µl versus 18.24±36.77µl) and the time-to-first arrest was also longer (463±624s versus 88±49s) (Figure 20, top right). In the lab we are currently setting up two additional bleeding models, a tail-artery-transection assay (TAT) and a saphenous-vein bleeding assay, which are both quite severe. Preliminary data suggest that also in these assays, VWD1 mice have a more severe bleeding profile compared to control mice (not shown). These data suggest that VWD1 mice have a variable bleeding profile: in a moderate assay with a minor injury, heamostasis was normal, however, in more severe assays with more serious injuries haemostasis was abnormal and mice displayed a moderate bleeding phenotype. For VWD2A mice, as expected, the situation was different. These mice displayed a severe bleeding phenotype in both assays, although high variability was observed for most of the measurements (Figure 20, bottom). In the TVT, VWD2A mice were rarely able to form occlusive clots and they shed large amounts of blood compared to control mice (662±171µl versus 36±22µl). In the tail-clip assay, none of the VWD2A mice was able to arrest the bleeding after tail amputation and collectively they lost much more blood that control mice (630.5±314.4µl versus 18.24±36.77µl). Although the study and characterization of these mouse models are still ongoing, our data confirm that VWD1 mice have a mild bleeding phenotype, as observed in the majority of patients with the same VWD subtype, while VWD2A mice displayed, as expected, a more severe bleeding profile. We are currently using these mice in several projects aiming to test innovative therapeutic strategies for VWD. For example, we have engineered a nanobody targeting VWF and albumin able to increase circulating VWF levels and to restore haemostasis in VWD1 mice subjected to the tail-clip assay (patent deposited; manuscript in preparation).

Presentations:

•

Investigating the effects of the FVIII-mimetic emicizumab in VWD mouse models

As previously mentioned in this manuscript, one the physiologic roles of VWF is to carry FVIII in circulation. Indeed, because of the existence of the VWF/FVIII complex, it took long time before each of the component was identified as a separate identity. Concentration of VWF in plasma is 10µg/ml (35nM) while FVIII concentration is 200ng/ml (0.8nM) therefore, in physiologic conditions there is a 50-molar excess of VWF. Although FVIII binds to VWF with high affinity (kd ~ 0.2-0.5nmol/l), the interaction is reversible. It is estimated that in normal individuals the majority of FVIII (95-97%) circulates in complex with VWF, while a small proportion of FVIII (3-5%) always circulate as free protein. [START_REF] Pipe | Life in the shadow of a dominant partner: the FVIII-VWF association and its clinical implications for hemophilia A[END_REF][START_REF] Turecek | the iPATH study group. Biological mechanisms underlying inter-individual variation in factor VIII clearance in haemophilia[END_REF] VWF clearance is not affected by FVIII, on the contrary, in regard to FVIII, interaction with VWF is essential to prevent premature FVIII degradation and clearance, with FVIII half-life reduced about six-fold in the absence of VWF. [START_REF] Morfini | Pharmacokinetics of monoclonally-purified and recombinant factor VIII in patients with severe von Willebrand disease[END_REF] Therefore, in pathologic conditions characterized by very low VWF levels or when VWF/FVIII interaction is prevented, a secondary FVIII-deficiency is observed. For example, it is the case in VWD-type 3, in which VWF is absent or undetectable and in VWDtype 2N, in which genetic variants on VWF within the FVIII-binding region prevent or highly reduce the formation of VWF/FVIII complexes. Moreover, FVIII levels are variably reduced in some cases of VWD-type 1 and VWD-type 2, other than type 2N. These complex situations in which VWF and FVIII dual deficiency coexists, manifest mixed bleeding phenotypes. Management can vary but often rely on the administration of VWF/FVIII concentrates, ondemand or on prophylaxis for the more severe cases. Albeit rare, inhibitory autoantibody against VWF can develop (~10% of VWD-type 3), suddenly limiting the treatment options for these patients. A few clinical cases have been reported about VWD-type 3 patients with inhibitors, who successfully received FVIII concentrates for the management of major surgeries, suggesting that FVIII was indeed sufficient to support haemostasis. [START_REF] Bergamaschini | Posttransfusion anaphylactic reactions in a patient with severe von Willebrand disease: role of complement and alloantibodies to von Willebrand factor[END_REF][START_REF] Boyer-Neumann | Multi-therapeutic approach to manage delivery in an alloimmunized patient with type 3 von Willebrand disease: Letters to the editor[END_REF][START_REF] Pergantou | The challenging management of a child with type 3 von Willebrand disease and antibodies to von Willebrand factor: LETTER TO THE EDITORS[END_REF][START_REF] Sakurai | Successful use of recombinant factor VIII devoid of von Willebrand factor during multiple teeth extractions in a patient with type 3 von Willebrand disease[END_REF][START_REF] Sottilotta | Efficacy of Octocog Alfa (Advate) in a Child with Type 3 von Willebrand Disease and Alloantibodies[END_REF] However, a part from these specific cases, whether or not FVIII or VWF contribute equally to the hemostatic response, or, in other words, should FVIII be considered as treatment option for VWD patients, has long been debated, and remains an open question. One major con is the short half-life of FVIII concentrates in these patients, due to the absence of VWF. Since its initial approval in 2017, the use of emicizumab, a bispecific antibody that mimics FVIII activity, with long half-life independent from VWF, has greatly expanded in patients with Haemophilia with remarkable improvement of their quality of life. Recently, a few clinical cases have been reported about severe VWD-type 3 patients, with/without inhibitors, who were successfully treated with emicizumab, [START_REF] Weyand | Efficacy of emicizumab in a pediatric patient with type 3 von Willebrand disease and alloantibodies[END_REF][START_REF] Sigaud | Posters Abstracts[END_REF][START_REF] Cefalo | Effectiveness of emicizumab in preventing life-threatening bleeding complications in type 3 von Willebrand disease with inhibitors: A paediatric report[END_REF][START_REF] Barg | The potential role of emicizumab prophylaxis in severe von Willebrand disease[END_REF][START_REF] Shanmukhaiah | Efficacy of emicizumab in von Willebrand disease (VWD) patients with and without alloantibodies to von Willebrand factor (VWF): Report of two cases and review of literature[END_REF] reinitiating the debate, whether or not this treatment option should be considered as standard of care.

Notwithstanding the large use of emicizumab, pre-clinical studies have been largely limited to non-human primate because of the non-cross reactivity of the antibody for rodent FIX and FX. This barrier, has substantially limited the number of in-vivo studies in small animal models and, despite the clinical efficacy of emicizumab is undoubted, some mechanistic aspects of its mode of action are still unidentified. To overcome this limitation, our group has recently optimized an administration protocol for its use in murine models [START_REF] Ferriere | A hemophilia A mouse model for the in vivo assessment of emicizumab function[END_REF] , therefore, we decided to investigate the use of emicizumab in mouse models of VWD. The first model we thought to test was VWD-type 2N, which is a very heterogeneous VWD subtype with VWF and FVIII levels wildly varying between patients. Because we did not have a knock-in mouse model available in the laboratory, we decided to create transient murine models of VWD-type 2N with low VWF and FVIII, using the hydrodynamic gene transfer technique. Despite big optimization efforts, due to the intrinsic variability of the technique, only a small number of mice subjected to hydrodynamic injection were displaying the expected phenotypes, such as VWF levels high enough to induce a VWD but low enough to generate the secondary FVIII-deficiency. Because of ethical concerns on the number of mice that would have been necessary to pursue the study using transient VWD-type 2N mice, we decided to abandon this model. Instead, we proposed to test emicizumab in our knock-in mice that were meeting the characteristics of low VWF levels and low FVIII levels, such as Vwf-knockout mice (mimicking VWD-type 3, VWD3 mice thereafter) and our new humanized mouse model of VWD-type 2A (VWD2A). First, we tested the effects of emicizumab on murine plasma, in vitro, in a global thrombin generation assay (Figure 21). Similar to rFVIII, emicizumab restored the hemostatic potential of a VWD3 murine plasma, (ETP increased 52%, thrombin peak increased 48%) (left) while rVWF had no effect, suggesting that emicizumab could indeed be beneficial in these mice. For the VWD2A plasma, the results were different, although FVIII activity was very low, the hemostatic potential was only moderately reduced compared to normal murine plasma and was not modified in presence of rFVIII/rVWF nor emicizumab (right). These data suggest that, for an unknown reason, thrombin generation in plasma from mice with a 129Sv genetic background (VWD2A mice and littermate controls) was less sensitive, nevertheless, FVIII or FVIII-mimetic seemed not to modify the profiles. Subsequently, to test the hemostatic efficacy of emicizumab in vivo, we applied the bispecific antibody (5mg/kg), in conjunction with human factors IX and X or recFVIII (or recVWF) as control, and performed the tail-vein-transection assay, in the milder (without challenges) or more severe settings (with challenges).

As for VWD3 mice, upon injury, they were unable to arrest the bleeding (Fugure 23).

Emicizumab administration improved the bleeding tendency in a mild or severe assay, as suggested by a significant decrease of the time to first arrest. In the mild assay, 5/7 emicizumab-receiving mice formed an occlusive clot within 5 min post-injury, suggesting that primary haemostasis is restored, with similar effects observed in mice receiving low-or highdose FVIII. When a challenge was performed, primary haemostasis was again restored in 10/18 mice, however, only half of the emicizumab-treated mice were able to arrest the bleeding, suggesting that clots were somehow unstable. Despite results about bleeding times were statistically significant and solid, results on the amount of blood shed during the bleeding assays were much more heterogenous, suggesting that emicizumab improved the bleeding profile of these assays, but did not completely restored haemostasis. Interestingly, in the TVT assays, rFVIII administration did not correct the bleeding phenotypes of VWD3 mice (confirming previous data independently generated in the lab).

As for VWD2A mice, results were different (Figure 24). As shown in the previous paragraph, these mice displayed a severe bleeding phenotype, nevertheless, some variability was observed. Most of the mice bled profusely upon injury and ~ one third (3/10 and 5/14) were able to arrest the bleeding within few minutes. If challenged however, these clots appeared unstable. Because of this quite high variability, we had to increase the number of mice per group. When treatments were tested, consistent with in vitro results, not rFVIII neither emicizumab displayed any clear significant effect on the bleeding tendency of these mice. Because previous data were not available, we also tested whether rVWF administration was able to restore haemostasis in these mice, similarly to VWD3 mice. Indeed, all VWD2A mice receiving rVWF arrested the bleeding within few minutes and the bleeding phenotype was completely corrected. Experiments are still ongoing for this project. The effects of emicizumab will be evaluated in a third VWD model, which mimic VWD-type 2B; in vitro, because of the lack of sensitivity of the thrombin generation in 129Sv murine plasma, we also decided to further test emicizumab in perfusion assays. Moreover, due to the variability of the results obtained upon FVIIIadministration in VWF-deficient mice, the question was raised whether or not that could be due to consumption of exogenous FVIII during the experiment. Therefore, we will test a new recombinant FVIII molecule, with a much longer half-life (BIVV001). Nevertheless, the data obtained so far, indicated that as observed in patients, emicizumab improved haemostasis in a mouse model of VWD-type 3, suggesting that the bispecific antibody assists in restoring primary clot formation in the absence of VWF. However, no such corrective effects were observed in VWD-type 2A mice, suggesting that, degraded VWF, even at low concentrations, can have a negative effect on clot formation in a TVT bleeding model.

Mentoring:

• Post-doc fellow (Genevieve Mc Cluskey). These preliminary data were presented by Genevieve at the 2022 XXX ISTH Congress, London, UK.

Clot structure in FVIII-deficient mice receiving FVIII-or emicizumab

Since 2020, I am co-supervising a Ph.D. student, Thibaud Sephiane, who's major project aims to evaluate the composition of clots formed upon a standardized injury, in FVIII-deficient mice, treated with recFVIII/FVIII-Fc or emicizumab, and their hemostatic efficacy in moderate bleeding models (TVT with/without challenge). Indeed, while the use of emicizumab expand and benefits for patients are undoubted, in-depth studies untangling specific effects of this antibody to overall clot formation mechanisms are lacking. In our study, mice were treated with recFVIII/FVIII-Fc or emicizumab and subjected to tail-vein-transection without challenges, to evaluate the hemostatic response and clot stability (Figure 25). In these conditions, no significant differences were observed between recFVIII-and emicizumabreceiving mice. Time bleeding profiles were similar, all mice stopped bleeding within 2 minutes post-injury and never rebled, except one mouse receiving emicizumab. The amount of blood collected from treated mice was also similar (28±17µl versus 59±29µl, in recFVIIIand emicizumab-receiving mice, respectively) and profoundly reduced compared to the amount of blood shed by vehicle-treated mice (760±146µl). (Results in FVIII-Fc receiving mice are similar to those observed in mice infused with recFVIII, thus, only data for recFVIII-infused mice are presented in this manuscript.) In this model, all treatments displayed similar hemostatic efficacy.

To perform microscopic analysis of the clots, the tails were excised 10-minute post-injury and a small portion surrounding the injury was further isolated and prepared for longitudinal sectioning, before undergoing microscopic studies (Figure 26). HES staining (Figure 26) revealed a similar general architecture of the clots with recFVIII and emicizumab, however, careful observation highlighted the presence of dense structures in the internal portion of clots formed by recFVIII, which were absent in clots formed by emicizumab. Next, scanning electron microscopy (SEM) was performed (Figure 26). In recFVIII-treated mice, the external, superficial portion of the clot appeared covered by compact and dense structures, in which components were fused together and no individual cells, nor fibrillar structures were visible.

In contrast, clot induced by emicizumab appeared very different, with an unexpected fractured surface and individual RBCs distinguishable at higher magnifications. Moreover, truncated fibers seemed to stem from RBC-rich structures into the empty spaces left by the cracks. These qualitative data indicated that although recFVIII and emicizumab had a similar efficacy in the mild TVT bleeding model without challenges, they were supporting generation of clots with different architecture. Therefore, we decided to focus on the study of fibrin as essential element of clots. Post-injury tail tissue sections were then immunostained for fibrin and quantitative analysis was performed by measuring surface area covered by fibrin and mean fluorescence intensity (Figure 26). In clots generated by mice receiving emicizumab, we observed a larger amount of fibrin with both measurements, compared to mice receiving recFVIII (or FVIII-Fc) at low or high doses. Area covered by fibrin 10±3% versus 216±6% and mean fluorescence intensity 0.20±0.11 versus 0.56±0.29, in recFVIII-receiving versus emicizumab-receiving mice. Fibrin formation and degradation were then studied in vitro in human FVIII-deficient plasma (not shown). In presence of emicizumab, fibrin formation appeared more efficient, as indicated by higher amount of fibrin generated in shorter times. Moreover, clots formed by emicizumab appeared more resistant to lysis and degradation as suggested by slower fibrinolysis in vitro (not shown). In accordance with previous experiments, SEM performed on different preparations allowing finely visualization of fibrinfibers architecture (Figure 26), confirmed that emicizumab-supported clots produced fibrin networks with different morphology compared to those observed in recFVIII-generated clots. Emicizumab infusion into mice generated thicker fibers (3.9 increase in fiber diameter) which were covering more surface area, resulting in fibrin networks with less intersections and 2fold less pores (Figure 26). Therefore, we hypothesized that the different morphology of emicizumab-supported clots could be somehow more fragile, and decided to perform TVT with challenges, to evaluate hemostatic responses to clot removal (Figure 27). In these conditions, all but one recFVIII/FVIII-Fc treated mice were able to efficiently arrest bleeding after each challenge within 3 minutes, resulting in low amount of total blood lost during the experiment. In contrast, emicizumab treated mice, were also able to arrest the bleeding after the first challenge, however, spontaneous bleeding occurred after few minutes, resulting in larger amount of blood shed at the end of the experiment. Although compared to recFVIII-, emicizumab-infusion was associated with more blood loss, it is important to mention that the amount of blood was less compared to that measured in vehicle-treated mice. Blood loss 164±33µl, 568±96µl and 867±98µl with recFVIII, emicizumab and vehicle, respectively.

Although still ongoing, these results showed that FVIII & emicizumab equally arrest bleedings upon injury in a TVT bleeding model, however, emicizumab-induced clots are less stable upon challenge. In-depth analysis of clot architecture highlighted structural differences between FVIII-and emicizumab-sustained clots. While FVIII clots were dense and smooth, Emicizumab clots were less dense and displayed a fractured surface, which appeared to result from abnormal fibrin formation and fibrin degradation. Indeed, enhanced fibrin generation and decreased fibrinolysis were associated with thicker fibrin fibers and abnormal fibrin network, suggesting that these odd structures could perhaps be less elastic and responsive to (shear) stress, and providing a potential explanation for the instability of emicizumab-supported clots upon challenge.

  Bernardi F. "The dominant-negative von Willebrand factor gene deletion p.P1127_C1948delinsR: molecular mechanism and modulation" Blood. 2010 Dec 9;116(24):5371-6 • Marchetti G, Pinotti M, Lunghi B, Casari C, Bernardi F. "Functional genetics" Thromb Res. 2012 Mar;129(3):336-40 Presentations: • Oral Presentation July 2009: XXII Congress of the International Society on Thrombosis and Haemostasis (ISTH), Boston (MA) USA • Poster Presentation June 2008: 21 th Biochemical PhD Students National Meeting A. Castellani, University of Pavia, Brallo di Pregola, Italy

Figure 1 .

 1 Figure 1. Wild-type and deleted-VWF domain structure. At the protein level, VWF-DEL lacks all A domains, part of the D3 domain (important for multimer formation) and part of the D4 domain (red). Because the CK domain is intact, the VWF-DEL can form dimers either with normal or with another VWF-DEL monomer.However, because part of the D3 domain is missing, the N-terminal head of a DEL subunit cannot sustain Nterminal (head-to-head) multimerization thus displaying a dominant-negative effect.

  Du V, Wu Y-P, Kauskot A, de Groot PG, Christophe OD, Denis CV, de Laat B, Lenting PJ "Accelerated uptake of VWF/platelet complexes in macrophages contribute to VWD-type 2B associated thrombocytopenia" Blood. 2013 Oct 17;122(16):2893-902 • Comment in: Castaman G. "2B or not 2B: when VWF is not a good platelet friend" Blood. 2013 Oct 17;122(16):2772-3. Presentations: • Oral Presentation April 2014: Thrombosis & Hemostasis Summit of North America (THSNA), Chicago (IL), USA • Oral Presentation July 2013: XXIV ISTH Congress, Amsterdam, The Netherlands Recognitions: • Trainee Travel Award, THSNA, Chicago (IL), USA (2014) Mentoring: • Principal supervisor of a License student, 2 months internship (Sophie Paskarajesudasan)

  Publications: • Casari C*, Berrou E*, Lebret M, Adam F, Kauskot A, Bobe R, Desconclois C, Fressinaud E, Christophe OD, Lenting PJ, Rosa J-P, Denis CV, Bryckaert M. "An unexpected thrombocytopathy in von Willebrand disease type 2B" J Clin Invest. 2013 Dec 2;123(12):5071-8 (*C.C. and E.B. equally contributed to this work) • Comment in: Ware J. "Thrombocytopathy and type 2B von Willebrand disease" J Clin Invest. 2013 Dec;123(12):5004-6. Presentations: • Oral Presentation & Poster Presentation July 2014: Gordon Research Seminar & Conference on Hemostasis, Waterville Valley (NH), USA • Oral Presentation July 2013: XXIV ISTH Congress, Amsterdam, The Netherlands Recognitions: • Best oral presentation at the GRS in Hemostasis, Waterville Valley (NH), USA (2014) • Young Investigator Award, XXIV ISTH, Amsterdam, The Netherlands (2013)

Figure 4 :

 4 Figure 4: Platelet signaling pathway downstream ADP, Thrombin and Convulxin/Collagen stimulation. Normal platelet activation was prevented in VWD-type 2B platelets, particularly, Rap1 and consequently αIIbβ3 responses were inhibited. The signalling events upstream calcium store release appeared normal, suggesting that VWF carrying type 2B mutations acts downstream this event.

  : • Rastegarlari G, Pegon JN, Casari C, Odouard S, Navarrete AM, Saint-Lu N, van Vlijmen BJ, Legendre P, Christophe OD, Denis CV, Lenting PJ. "Macrophage LRP1 contributes to the clearance of von Willebrand factor" Blood. 2012 Mar 1;119(9):2126-34. • Wohner N, Legendre P, Casari C, Christophe OD, Lenting PJ, Denis CV. "Shear stress-independent binding of von Willebrand factor-type 2B mutants p.R1306Q & p.V1316M to LRP1 explains their increased clearance" J Thromb Haemost. 2015 May;13(5):815-20 • Pegon JN, Kurdi M, Casari C, Odouard S, Denis CV, Christophe OD, Lenting PJ. "Factor VIII and von Willebrand factor are ligands for the carbohydrate-receptor Siglec-5" Haematologica. 2012 Dec;97(12):1855-63 Mentoring: • Co-supervisor of one post-doc (Nikolett Wohner)

48

 48

  Publications: • Casari C, Paul DS, Susen S, Lavenu-Bombled C, Harroche A, Piatt R, Poe KO, Lee RH, Bryckaert M, Christophe OD, Lenting PJ, Denis CV, Bergmeier W. "Protein kinase C signaling dysfunction in von Willebrand disease (p.V1316M) type 2B platelets". Blood Adv. 2018 Jun 26;2(12):1417-1428 • Casari C & Bergmeier W. "Acquired platelet disorders" Thromb Res. 2016 May;141 Suppl 2:S73-5 Presentations: • Oral Presentation November 2017: 5 th French Club on Platelets and Megakaryocytes, Paris, France • Oral Presentation October 2017: GFHT/CoMET Congress, Caen, France • Oral Presentation & Poster Presentation July 2016: Gordon Research Seminar & Conference on Hemostasis, Stoweflake (VT), USA • Poster Presentation March 2016: 8 th Symposium on Hemostasis, Chapel Hill (NC), USA • Poster Presentation March 2016: McAllister Heart Institute/Integrative Vascular Biology Annual Research Symposium, University of North Carolina, Chapel Hill (NC), USA • Oral Presentation June 2017: XXVI ISTH, Berlin, Germany • Poster Presentations April 2015: Gordon Research Seminar & Conference on Cell Biology of Megakaryocytes & Platelets, Barga (LU), Italy Mentoring: • Co-supervisor of two research technicians (Raymond Piatt and Kathryn Poe) Funding: • EHA (European Hematology Association) 3-year Non-Clinical Junior Research Fellowship. (Salary and bench fees, Obtained as PI)

Figure 5 :

 5 Figure 5: Rap1 activation in platelets and VWD-type 2B defects. The rapid and transient response CalDAG-GEFImediated appeared normal in VWD-type 2B platelets, whereas the late and sustained response dependent on PKC/P2Y12 was affected. Pre-activation of PKC-and not P2Y12-signaling resulted in impaired agonist response.

•

  Lee RH, Ghalloussi D, Harousseau GL, Kenny JP, Kramer PA, Proamer F, Nieswandt B, Flick MJ, Gachet C, Casari C, Eckly A, Bergmeier W. "Rasa3 deficiency minimally affects thrombopoiesis but promotes severe thrombocytopenia due to integrin-dependent platelet clearance" JCI Insight 2022 Apr 22;7(8):e155676.

  Mohamadi A, Aymé G, Christophe OD, Lenting PJ, Denis CV, Casari C. "Macrophage scavenger-receptor SR-AI contributes to the clearance of von Willebrand factor" Haematologica. 2018 Apr;103(4):728-737 Mentoring: • Co-supervisor of one post-doc (Nikolett Wohner)

Figure 7 .

 7 Figure 7. Clearance of VWF in engineered murine models. (Left) Wild-type human VWF was expressed by hydrodynamic gene transfer in macrophage-specific LRP1-deficient and SR-A1-deficient mice and clearance was evaluated by measuring VWF propeptide to antigen ratio. Apparently, VWF was cleared less rapidly in SR-A1-deficient mice suggesting that SR-A1 contributes more than LRP1 to basal VWF clearance. (Right) Human VWF wild-type or carrying the clearance-variants (p.R1205H and p.S2179F) was overexpressed in SR-A1-deficient or control mice and VWF clearance analyzed. In the absence of SR-A1, mutant VWF clearance was almost normalized, suggesting that SR-A1 contribute to the rapid clearance of these VWF mutants. (Figure adapted by Wohner N. et. Haematologica 2018)

•

  Muczynski V, Casari C, Moreau F, Aymé G, Kawecki C, Legendre P, Proulle V, Christophe OD, Denis CV, Lenting PJ. "A factor VIII-nanobody fusion protein forming an ultra-stable complex with VWF: effect on clearance & antibody formation". Blood 2018 Sep 13;132(11):1193-1197 Mentoring: • Co-supervisor of a research engineer (François Moreau)

Figure 9

 9 Figure 9. Engineering FVIII to create an ultra-stable complex with VWF: (Left) Study's strategy. FVIII Bdomain was replaced with a bivalent anti-VWF sdAb (KB013bv), resulting in an ultra-stable complex with VWF, mostly due to very slow dissociation rate. FVIII-KB013bv maintained cofactor activity, had prolonged survival and decreased anti-FVIII Abs formation. (Right) According to Sorvillo et al[START_REF] Sorvillo | von Willebrand factor binds to the surface of dendritic cells and modulates peptide presentation of factor VIII[END_REF] , the VWF-FVIII complex separates at the cellular surface of antigen-presenting cells, with FVIII being endocytosed and most of the VWF molecules remaining outside the cell. Based on this model, we anticipated that FVIII-KB013bv-VWF complex will not dissociate, resulting in reduced uptake of FVIII-KB013bv by antigen-presenting cells. Fewer FVIII-derived peptides will then be presented to T cells, and in turn, less anti-FVIII antibodies will be produced. Figure adapted by Muczynski et al. Blood 2018.

58

 58

  Publications: • Campioni M, Legendre P, Loubiere C, Lunghi B, Pinotti M, Christophe OD, Lenting PJ, Denis CV, Bernardi F, Casari C "In vivo modulation of a dominant-negative variant in mouse models of von Willebrand disease type 2A" J Thromb Haemost. 2021 Jan;19(1):139-14 • Comment in: Karampini E, O'Donnell JS. Correcting dominant-negative von Willebrand disease. J Thromb Haemost. 2021 Jan;19(1):55-57 Presentations: • Invited Presentation October 2021: European Haemophilia Consortium (EHC) (virtual) • Invited Presentation September 2021 11 th Bari International Conference (BIC), Venice, Italy • Invited Presentation July 2021: XXIX ISTH Congress, Philadelphia, USA (virtual), SSC VWD session • Oral Presentation October 2019: 3 nd European Congress of Thrombosis and Hemostasis (ECTH), Glasgow, United Kingdom (Ted-style selected plenary presentation) • Poster Presentation September 2019: 10 th Bari International Conference (BIC), Genova, Italy • Oral Presentation June 2015: XXV ISTH Congress, Toronto, Canada Recognitions: • Young Investigator Award, XXV ISTH, Toronto, Canada (2015) Mentoring: • Principal supervisor in France for one visiting PhD student (Matteo Campioni) and two research engineers (Legendre Paulette and Cécile Loubiere) Funding:

Figure 13 .

 13 Figure 13. Schematic overview adapted by Karampini et al. of our corrective approaches to prevent the deleterious effects of a dominant-negative VWF variant. In vivo strategies (left) and intracellular VWF biosynthesis (right). (A) Normal/wild-type VWF multimer synthesis and secretion. (B) Dominant-negative heterozygous variants resulting in the formation of hetropoymers. (C) Mutant-specific siRNA-based corrective approach partially preventing heteropolymer secretion. (D) Alternative corrective approach to prevent the formation of hetropolymer obtained by inserting an additional mutation preventing VWF dimerization to the VWF-DEL variant, which hampers multimerization.

Figure 14 .

 14 Figure 14. Frameshift variants identified in Afro-Caribbean patients. (Top-left) VWF domain structure. Both frameshift variants were located in the D4 domain and led to VWF molecules truncated within the same domain (missing part of D4 and all C-domains). (Bottom-left) Nucleotidic and aminoacidic sequences for wild-type and aberrant VWF in patients 1 & 2 (P1-P2) with formation of premature stop codons. (Right) pcDNA3.1 expression vectors for cellular studies were engineered to express VWF-wild-type with a Myc tag (top), VWF carrying the p.Pro2145Thrfs*5 variant with an HA tag (middle) and VWF carrying the p.Cys2261PheThrfs*9 variant with an HA tag (bottom). Abbreviated names for these vectors are indicated in brackets.

Figure 16 .

 16 Figure 16. Pathogenic mechanisms of frameshift variants within the D4 domain in cellular models of VWD. Lack of expression of the P1-mutant VWF is less deleterious than residual expression of dominantnegative P2-mutant-VWF lacking the ability to dimerize, especially if expressed (in P2) in association with a "complementary" dominant-negative mutant-VWF unable to multimerize. Although the two frameshift variants were both affecting the same domain leading to similar truncated VWF, they are associated with very different pathologic mechanisms and resulted in completely different phenotypes.

Figure 17 .

 17 Figure 17. Engineering double-humanized mice for VWF and GPIba.Three heterozygous transgenic mice (129Sv genetic background) were generated by homologous recombination (outsourced to GeneOway). Both human proteins were expressed under the control of endogenous, murine regulatory sequences. For expression of the human-VWF, human-cDNA was inserted within murine exons 5 and 6, inactivating the expression of the murine gene. Within the human-VWF cDNA we included the p.T789A polymorphism, which is associated with increased VWF:Ag. For human-GPIba, the murine gene was replaced by the human-cDNA, thus preventing expression of the murine receptor. The 129Sv background was selected because mice with this background express fully functional ADAMTS13. Heterozygous mice were intercrossed to generate three homozygous colonies. Finally, homozygous mice for each of the human-VWF (wild-type or p.R1597W variant) were crossed with homozygous mice for human-GPIba to obtain mice expressing human-VWF and human platelet receptor. Double humanized mouse colonies were bred and maintained in our animal facility.

Figure 18 .

 18 Figure 18. VWF analysis in humanized mice. VWF antigen, clearance and multimer profiles were evaluated in murine plasma and compared to human-pooled-normal-plasma (hPNP). Multimers were analyzed in normal conditions or 30-minute post-histamine administration (13nM/g IP).Densitometry of multimer gels was also performed. FVIII:C was measured and compared to murine-pooled-normal-plasma.

  confidential

Figure 19 .

 19 Figure 19. Platelet analysis in humanized mice. (Left) Human/murine GPIba expression in human/murine platelets by flow cytometry. (Middle) Platelet counts and volume measured by a veterinary counter (Scil Vet abc Plus from Scil animal care). (Right) PAR4p-induced platelet activation measured by quantification of activated-aIIbb3 and P-selectin exposure in VWD1, VWD2A and control mice.

Figure 20 .

 20 Figure 20. Bleeding profiles of humanized mice. (Left) Moderate, tail-vein-transection (TVT) assay. Representative time plots illustrate time of active bleeding (filled bars) and times of no-bleeding (empty bars). Dotted lines indicate primary haemostasis (3min) or eventual challenges (15-30-45min). The graphs on the right show the total amount of blood shed by each mouse for the duration of the experiment (0-60 min). (Right)Severe tail-clip model. Graphs represent the amount of blood lost by each individual mouse over 30-minute and the time to fist arrest.

  confidential

Figure 21 .

 21 Figure 21. Thrombin generation in murine plasma. (Left) TGA was performed on VWF-deficient murinepooled plasma, mean of n=4 experiments in duplicates. (Right) TGA was performed on VWD2A plasma from individual mice, mean of n=8 mice in duplicates.

Figure 23 .Figure 24 .

 2324 Figure 23. TVT without (top) and with (bottom) challenges in VWD3 mice. The graphs on the left show time to first arrest and quantification of the total amount of blood shed in 30-min. Bleeding time profiles are reported. Active bleeding times, filled bars; no bleeding times, empty bars.

  confidential

  Figure 25. TVT without challenges. Representative bleeding time profiles. Active bleeding times, filled bars; no bleeding times, empty bars. The graph on the right shows total amount of blood shed in 45-min.

Figure 26 .

 26 Figure 26. Microscopic analysis of in-vivo clots generated in FVIII-deficient mice. (A) Cartoon representation of tissue preparation. (B) HES staining of FVIII-and emicizumab-generated clots. Bottom pictures were acquired at higher magnifications. (C) SEM showing the surface of clots in non-dehydrated preparations. Bottom pictures show higher magnifications. (D) Fibrin immunostainig (left) and quantification (right). (E) SEM of clots showing the architecture of fibrin-fibers network (top) and quantitative analysis (bottom graphs).

  confidential

Figure 27 .

 27 Figure 27. TVT with challenges. Representative bleeding time profiles. Active bleeding times, filled bars; no bleeding times, empty bars. The graph on the right shows total amount of blood shed in 45-min.

  I was born in Ferrara (Italy) in 1981, and I was first introduced to the field of thrombosis and haemostasis during my Master's degree studies at the University of Ferrara (Italy), where I did my training internships in the laboratory of Profs. Francesco Bernardi and Mirko Pinotti. Under their mentorship, in 2010, I obtained my Ph.D. in Biochemistry, Molecular Biology and Biotechnology with a thesis focused on von Willebrand Factor (VWF). Fascinated by the biochemical features and by the many pathophysiological roles of VWF, I had the opportunity to move to Paris for my first post-doctoral experience (2010-2013). There, I joined the team of Drs. Cécile Denis, Peter Lenting and Olivier Christophe where I strengthened my knowledge on VWF and acquired experience with in vivo mouse models. While investigating several molecular pathways resulting in von Willebrand Disease (VWD)type 2B, a paradoxical bleeding disorder associated with thrombocytopenia, I extended my curiosity and research efforts to platelet biology and platelet-associated disorders. In 2014, I was awarded a three-year Non-clinical Junior Research Fellowship by the European Hematology Association to join the group of Prof. Wolfgang Bergmeier at the McAllister Heart Institute (University of North Carolina in Chapel Hill, USA). Then, I returned to INSERM in Paris,

	3.5.2 Investigating the effects of the FVIII-mimetic emicizumab in VWD 1. CANDIDATE PRESENTATION	35-40
		mouse models
	3.5.3 Clot structure in FVIII-deficient mice receiving FVIII-or emicizumab	41-44
	3.6	FUTURE PROJECTS
	3.6.1 The role of von Willebrand factor in bleedings from angiodysplasias	44-46
		(confidential)
	3.6.2 Genetic approaches to correct the deleterious effects of dominant-	46-47
		negative variants in models of von Willebrand disease (confidential)
		REFERENCES	47-51
		CV	52-55
		ANNEXES
	6.1	LIST OF PUBLICATIONS	56-58
	3.1 6.2	PhD (Italy) LIST OF PRESENATATIONS	59-60
	3.1.1 In vitro study & correction of a large in-frame deletion in the von Willebrand factor gene 6.3 SELECTED PAPERS	13-14 61-159
	3.2	POST-DOC #1 (France)
	3.2.1 Elevated macrophage-dependent platelet clearance contributes to	14-15
		thrombocytopenia in VWD-type 2B
	3.2.2 Altered platelet functions are a predominant factor in the bleeding	15-16
		tendency in VWD-type 2B patients
	3.2.3 Creation of a knock-in mouse model of VWD-type 2B	16-17
	3.2.4 VWF clearance	17
	3.2.5 Creation of a mouse model of Thrombotic Thrombocytopenic	17-18
		Purpura
	3.2.6 Mouse models expressing engineered human VWF to test	18
		antithrombotic molecules
	3.3	POST-DOC #2 (USA)
	3.3.1 Protein kinase C (PKC) signaling dysfunction in VWD-type 2B	18-19
		(p.V1316M) platelets
	3.3.2 Role of Arp2/3 in platelet activation and homeostasis	19-20
	3.3.3 The RAP1-GAP RASA3 prevents clearance of circulating platelets	20
	3.4	RESEARCHER
	3.4.1 The macrophage receptor SR-A1 contributes to VWF clearance	21-22
	3.4.2 An anti-VWF single domain antibody to create a FVIII-VWF ultra	22-23
		stable complex
	3.4.3 Modulation of dominant-negative VWD in vivo	23-27
	3.4.4 VWD variants within the D4 domain	27-30
	3.5	ONGOING PROJECTS (unpublished data/confidential)
			30-35

3.5.1 Unique humanized mouse models of von Willebrand disease to

implement the need for innovative therapeutic strategies where I was selected as tenured researcher (Chargé de recherche de classe normale) in 2018, within the unit 1176, Hemostasis, Inflammation, Thrombosis (HITh).

  -, intermediate-and large VWF multimers formed by 2 to > 60-mers. VWF is incredibly rich in cysteines, which are abundantly clustered at the N-and C-terminal domains of the mature subunit but rare within the A domains. Whether or not all cysteines are involved in disulfide bridges is a matter of debate, however, cysteine-bridges are undoubtedly essential for appropriate dimerization and multimerization processes 7 .

Importantly, during its biosynthesis, VWF is also subjected to glycosylation, which starts early in the endoplasmic reticulum (N-glycosylation) and continue throughout protein maturation. Mature VWF contains 13 Nand 10 O-linked carbohydrates with various structures. The majority of VWF glycans are sialylated (capped with sialic acid); five glycans are sulphated and both N-and O-glycans can harbor ABO(H) blood group determinants. Of note, glycosylation patterns are different in EC-and platelet-derived VWF 4 .

  Most mutations are heterozygous missense variants and the associated defects result from abnormal VWF synthesis, secretion and clearance. Of note, VWF clearance varies significantly between individuals and this variability pertains not only to endogenous basal VWF half-life but also to endogenous VWF survival in circulation following desmopressin (DDAVP) administration and to exogenous VWF concentrates. Moreover, because abnormal clearance has significant therapeutic implications, VWD-type 1 patients with increased VWF clearance have been named VWD-type 1C and were historically identified by an elevated VWF propeptide to antigen ratio (VWFpp/VWF:Ag). More recently, it has been suggested that identification of VWD-type 1C should be based on VWF survival following DDAVP. To add some complexity, increased VWF clearance is not only associated with (some) VWD-type 1 but also to some VWD-type 2.

	FVIII (low VWF:FVIIIB). As a consequence, FVIII half-life is variably decreased and patients
	suffer from secondary FVIII-deficiency (low FVIII:C).
	Finally, VWD-type 3, is rare (less than 5% of all VWD) and severe as identifies patients with
	absent/undetectable VWF in circulation. Mutations are often homozygous/compound
	heterozygous nonsense, frameshift or splice site mutations that can be associated with
	nonsense-mediated decay resulting in null alleles, large deletion or even
	homozygous/compound heterozygous missense variants. These variants impair synthesis of
	VWF, but can also prevent VWF release or accelerate VWF clearance.
	VWD can be acquired (acquired von Willebrand syndrome (AVWS)) in patients with aortic
	stenosis, in patients with mechanical circulatory assist devices (left ventricular assist devices
	(LVAD) or extracorporeal membrane oxygenation (ECMO)) due to heart failure and
	respiratory-or heart&respiratory-failure 16 , but also in patients with lymphoproliferative,
	myeloproliferative or immunologic disorders. Pathogenic mechanisms include loss of
	HMWMs and increased VWF degradation in situation of high shear stress, autoantibody-
	binding/immune complexes formation and elimination, adsorption of VWF by malignant cells.
	The prevalence of AVWS is unknown but the use of mechanical assist devices has
	exponentially increased over the last years. Abnormal high shear stress associated AVWS
	resembles congenital VWD-type 2A (group 2).
	VWD symptoms vary but generally consist of mucosal bleedings. Bruising and epistaxis are
	frequent in children, hematomas, menorrhagia and bleeding from minor wounds are frequent
	in adults with severe bleeding associated with surgery, especially dental surgery/extraction.
	Major complication is gastrointestinal (GI) bleeding frequent in VWD-type 2A (group 2), VWF-
	type 3 and AVWS. Severe VWD patients with very low VWF antigen levels and VWD-type 2N
	that virtually any heterozygous patients suffer secondary FVIII-deficiency and manifest haemophilia A-like phenotypes such
	mutation affecting VWF is dominant-negative. Group 2 variants mostly localize within or close as joint bleedings/hemarthrosis/arthropathy. Although VWD should equally affect patients of
	to ADAMTS13 cleavage site (Tyr1605/Met1606 within the A2 domain) and show increased both gender, women are more frequently diagnosed and more often manifest bleeding
	satellite bands in multimer gels of plasmatic VWF. symptoms, because women are subjected to more physiologic haemostatic challenges during
	VWD-type 2B variants (increased platelet binding) are ~ 40 gain-of-function mutations menstruations and childbirth. Heavy-menstrual-bleeding is very difficult to diagnose mostly
	clustered in the A1 domain, within/close to the binding site for platelet receptor GPIba. These because definition of normal menstrual bleeding is scarce, nevertheless, heavy-menstrual-
	variants force VWF in an open/active conformation, without the need of shear stress-induced bleeding in women with VWD has been reported as greater than 50% (100% in some studies).
	VWF elongation. Phenotypes are heterogeneous but are associated with decreased VWF On the other hand, taking into account women with heavy-menstrual-bleeding, around 13%
	activity, normal or reduced multimer profiles (low HMWMs), normal platelet count or of adults have VWD or even more if adolescents are considered. Abnormal bleedings during
	fluctuating macrothrombocytopenia, normal or often increased VWF clearance. periods are often associated with anaemia/iron deficiency. Of note, women are also generally
	Macrothrombocytopenia is associated with impaired platelet functions. diagnosed at later age than men. It has been reported that 76% of men are correctly
	VWD-type 2M (decreased VWF activity, reduced platelet binding or collagen binding; normal diagnosed by the age of 10-year while 50% of women, are not-diagnosed by the age of 12-
	year. Undiagnosed VWD in young women translates into difficulties in the management of multimers) groups ~ 80 variants that interfere with GPIba binding (often located in the A1 domain) or with collagen-binding (with most identified mutation in this category affecting menstrual bleedings. 17-21
	binding to collagen type I and III which interact with VWF A1 domain).
	VWD-type 2N (decreased FVIII-binding) is due to rare (~ 30) homozygous/compound
	heterozygous variants mostly clustered within the D' domain and preventing VWF binding to

recommended to label individuals with VWF levels between 30-50% but abnormal bleeding profiles as having VWD-type 1. Around 250 genetic variants spread throughout the whole VWF have been associated with VWD-type 1 but only for ~ one quarter the pathologic mechanism has been investigated/identified. VWD-type 2 is less common than VWD-type 1 and accounts for ~ 20% of all VWD cases. VWDtype 2 groups all qualitative/functional defects and is further subdivided in four subtypes, 2A, 2B, 2M and 2N. Briefly, VWD-type 2A (decreased VWF activity, decreased platelet binding) is characterized by abnormal VWF multimer profile (reduced/absence of high-molecular-weight multimers (HMWMs)) as a result of intracellular multimerization defects (group 1 mutations), increased VWF susceptibility to ADAMTS13-mediated cleavage (group 2 mutations) or both. Of note and relevant for this manuscripts, group 1, includes variants (mostly affecting cysteines) preventing/altering VWF dimerization (clustered in the CK domain and resulting in odd bands in multimer gels) or multimerization (localized within VWF propeptide (D1-D2) or in the D3 domain). Heterozygous variants affecting dimerization or multimerization are the quintessential example of VWF variants with dominant-negative effects, as they clearly have a negative impact on the wild-type counterpart (see Figure

11

and page 24). On the other hand, due to its biosynthetic process, it has been proposed

2.3. Bleeding models to evaluate haemostasis in mice

	8,15,22 has remained essentially unchanged for decades and essentially
	consists in replacing missing/dysfunctional VWF by the use of plasma-derived concentrates
	or recombinant VWF (available since 2015). Desmopressin (DDAVP)-induced release of
	intracellular VWF is also a widely used and standard care treatment option for patients with
	quantitative VWF deficiency. Prophylaxis is less common in VWD patients compared to
	haemophilia patients and reserved for severe cases. Adjuvant therapies often used in VWD
	patients are anti-fibrinolytics (i.e. tranexamic acid). Treatment of gynaecologic bleedings in
	women with VWD includes oral contraceptives and tranexamic acid to control heavy
	menstrual bleedings; DDAVP/VWF concentrates /tranexamic acid for pre-postpartum
	management. Of note, during surgery or in case of acute heavy bleedings, when rapid
	increase of FVIII levels is also required, FVIII concentrates can be infused.
	It is nowadays evident that there is an unmet clinical need for new treatment options for
	patients with VWD aiming to achieve better efficacy and to improve quality of life. Our group,
	and others have long been engaged in the development of such alternative options, including
	genetic-and protein-based approaches. Our own studies have investigated RNA-silencing,
	gene therapy, antibody-based option (emicizumab, Mab508, nanobodies). 27-32

(relevant for this manuscript)

3. RESEARCH ACTIVITIES: PAST -PRESENT -FUTURE 3.1. PhD (Italy) 3

  .1.1. In vitro study and correction of a large in-frame deletion in the von Willebrand factor gene

	• Peyron I, Kizlik-Masson C, Dubois M, Atsou S, Ferrière S, Denis CV, Lenting PJ, Casari C, Christophe OD
	"Camelid-derived single chain antibodies in hemostasis: mechanistic, diagnostic and therapeutic
	applications" Res Pract Thromb Haemost. 2020 Sep 9;4(7):1087-1110
	• Van Belle E*, Vincent F*, Rauch A*, Casari C, Jeanpierre E, Loobuyck V, Rosa M, Delhaye C, Spillemaeker H,
	Paris C, Debry N, Verdier B, Vincentelli A, Dupont A, Lenting PJ*, Susen S*. "Von Willebrand Factor and
	Management of Heart Valve Disease: Ready for Prime Time?" J Am Coll Cardiol 2019 Mar 12;73(9):1078-
	1088
	• Casari C, Lenting PJ, Christophe OD, Denis CV. "Von Willebrand factor abnormalities studied in the mouse
	model: what we learned about VWF functions" Mediterr J Hematol Infect Dis. 2013 Jul 10;5(1):e2013047
	• Casari C, Lenting PJ, Wohner N, Christophe OD, Denis CV. "Clearance of von Willebrand factor" J Thromb
	Haemost. 2013 Jun 11; Suppl 1:202-11
	• Lenting PJ, Casari C, Christophe OD, Denis CV. "von Willebrand factor: the old, the new and the unknown" J
	Thromb Haemost. 2012 Dec;10(12):2428-37
	• Lenting PJ, Kizlik-Manson C, Casari C. "Towards novel treatment options in von Willebrand Disease"
	Haemophilia 2022 May; 28 Suppl 4:5-10

  Prévost N, Kauskot A, Loubière C, Legendre P, Repérant C, Baruch D, Rosa JP, Bryckaert M, de Groot PG, Christophe OD, Lenting PJ, Denis CV. "A genetically-engineered von Willebrand disease type 2B mouse model displays defects in hemostasis and inflammation" Sci. Rep. 2016 May;23(6):26306 (*A.F. and C.C. equally contributed to this work)

	Publications:
	• 7.	Adam F*, Casari C*,

  Wohner N, Cho S, Kurata S, Kitano A, Christophe OD, Lenting PJ, Li R, Denis CV, Prévost N. "Expression of a structurally constrained von Willebrand factor variant triggers acute thrombotic thrombocytopenic purpura in mice" Blood. 2014 May 22:123(21): 3344-53 (*Y.M. and C.C. contributed equally to this work)

3.2.6. Mouse models expressing engineered human VWF to test antithrombotic molecules

46

In search for new target molecules for the treatment of cardiovascular and cerebrovascular disease, interfering with the first step of platelet adhesion to the subendothelium remains an attractive option. In this context, inhibition of the VWF-GPIba axis would target this very early phase of platelet adhesion. However, most inhibitors target human VWF and due to species incompatibility, human VWF is not efficient in mice since it does not bind to murine GPIba. Therefore, to overcome this incompatibility, we engineered a functional human-mouse chimeric VWF and transiently expressed this molecule in Vwf-deficient mice. Using this new mouse model, we confirmed the antithrombotic potential of interfering with the VWF-GPIba interaction and validated our murine model as a simple in vivo tool to evaluate anti-human VWF agents. Publications: • Navarrete AM, Casari C, Legendre P, Marx I, Hu JR, Lenting PJ, Christophe OD, Denis CV. "A murine model to characterize the antithrombotic effect of molecules targeting human von Willebrand factor" Blood. 2012 Sep 27;120(13):2723-32 3.3. Post-doc #2 (USA) 3.3.1. Protein kinase C (PKC) signaling dysfunction in VWD-type 2B (p.V1316M) platelets

47

  6.2. List of presentations (Congresses, Lectures & Scientific Meetings) (OP: oral presentation; PP: poster presentation; IP: invited presentation) • OP July 2022: XXX Congress of the International Society on Thrombosis and Haemostasis (ISTH), London, UK "Unique humanized mouse models of von Willebrand disease to Chair of the session "The Future of Gene Therapy" • IP October 2021: European Haemophilia Consortium (EHC) (virtual) "Tailored treatments for VWD" • IP October 2021: 3 rd French Research day MHEMO "(Mal)Formations Vasculaires : ce que nous avons appris des modèles murins" • IP September 2021 11 th Bari International Conference (BIC), Venice, Italy "Future therapies in von Willebrand disease: time for a tailored fit?" • IP July 2021: XXIX Congress of the International Society on Thrombosis and Haemostasis (ISTH), Philadelphia, USA (virtual) "New Treatment Options for VWD" • IP (plenary lecture) November 2020: XXVI SISET (Italian Society of Thrombosis and Haemostasis) Congress (virtual). "VWF beyond haemostasis" ("Vecchi e nuovi percorsi del bus molecolare: il fattore di von Willebrand") • October 2019: 3 nd European Congress of Thrombosis and Hemostasis (ECTH), Glasgow, United Kingdom. OP "A cure for VWD? The long and winding road" (Ted-style selected plenary presentation) • September 2019: GFHT (Groupe Français d'études sur l'Hémostase et la Thrombose) and CoMET (Coordination médicale pour l'étude et le traitement des maladies hémorragiques constitutionnelles) Congress, Montpellier, France IP "Dégradation du facteur Willebrand et syndrome de Willebrand acquis: Utilisation des dispositifs de support mécanique" (in French) • September 2019: 10 th Bari International Conference (BIC), Genova, Italy PP "In vitro and in vivo modulation of von Willebrand factor gene mutations with dominant-negative effect" • July 2019: XXVII Congress of the International Society on Thrombosis and Haemostasis (ISTH), Melbourne, Australia Chair of the session "VWF Biology" • October 2018: 2 nd European Congress of Thrombosis and Hemostasis (ECTH), Marseille, France • October 2018: 6 th French Club on Platelets and Megakaryocytes, Marseille, France • November 2017: 5 th French Club on Platelets and Megakaryocytes, Paris, France OP "Dysfonction de la voie de signalisation PKC dans la maladie de Willebrand de type 2B" • October 2017: GFHT (Groupe Français d'études sur l'Hémostase et la Thrombose) and CoMET (Coordination médicale pour l'étude et le traitement des maladies hémorragiques constitutionnelles) Congress, Caen, France OP "Protein Kinase C Signaling Dysfunction in von Willebrand Disease Type 2B Platelets" • September 2017: 9 th Bari International Conference (BIC), Rome, Italy • July 2017: XXVI Congress of the International Society on Thrombosis and Haemostasis (ISTH), Berlin, Germany OP "Protein Kinase C Signaling Dysfunction in von Willebrand Disease Type 2B Platelets" Chair of the session "VWF: Functional Implications in Health and Disease" • 12 th May 2017: University of Sussex, School of life; Science Summer Seminar Series, Brighton, UK IP "Von Willebrand Disease type 2B" • October 2016: Topics in Haemathology and Blood Coagulation, "The Development of a Curriculum and Establishment of a Regional Training Platform for Haematology in Life Sciences and Medicine [DECERPH]" within the framework of the EU Tempus project 544282-TEMPUS-2013-JPCR, Ferrara, Italy IP "von Willebrand Disease: genotype phenotype correlation" • July 2016: Gordon Research Seminar & Conference on Hemostasis, Stoweflake (VT), USA OP at GRS and PP at GRC "Protein Kinase C signalling dysfunction in VWD-type 2B platelets" • March 2016: 8 th Symposium on Hemostasis, Chapel Hill (NC), USA PP "Protein Kinase C signalling dysfunction in von Willebrand disease type 2B platelets" • March 2016: McAllister Heart Institute/Integrative Vascular Biology Annual Research Symposium, University of North Carolina, Chapel Hill (NC), USA PP "Von Willebrand disease type 2B: does GPIbalpha-mediated activation contribute to in increased platelet clearance?" • June 2015: XXV Congress of the International Society on Thrombosis and Haemostasis (ISTH), Toronto, Canada OP "New in vitro and in vivo strategies to modulate von Willebrand factor gene mutations with dominantnegative effect" PP "The RAP1-GAP RASA3 prevents clearance of circulating platelets" • April 2015: Gordon Research Seminar & Conference on Cell Biology of Megakaryocytes & Platelets, Barga (LU), Italy PP "Von Willebrand disease type 2B: does GPIbalpha-mediated activation contribute to an increased platelet clearance?" • March 2015: McAllister Heart Institute/Integrative Vascular Biology Annual Research Symposium, University of North Carolina, Chapel Hill (NC), USA PP "The RAP1-GAP RASA3 prevents clearance of circulating platelets" • July 2014: Gordon Research Seminar & Conference on Hemostasis, Waterville Valley (NH), USA OP at GRS and PP at GRC "von Willebrand disease type 2B: a platelet point of view" • May 2014: 7 th Symposium on Hemostasis, Chapel Hill (NC), USA • April 2014: Thrombosis & Hemostasis Summit of North America (THSNA), Chicago (IL), USA OP "New insights into von Willebrand disease type 2B" • June-July 2013: XXIV Congress of the International Society on Thrombosis and Haemostasis (ISTH), Amsterdam, The Netherlands OP "The von Willebrand disease type 2B p.V1316M mutation inhibits platelet functions by interfering with Ca2+ mobilization and integrin aIIbß3 activation pathways" OP: "Accelerated uptake of VWF/platelet complexes in macrophages contribute to VWD-type 2B associated thrombocytopenia" • July 2011: XXIII Congress of the International Society on Thrombosis and Haemostasis (ISTH), Kyoto, Japan • September 2009: Nano and Biotechnology for diagnostic and therapies, University of Urbino, Italy • July 2009: XXII Congress of the International Society on Thrombosis and Haemostasis (ISTH), Boston (MA) USA OP "Cellular model of a dominant-negative VWF gene deletion: a breakpoint specific RNAi restores secretion of functional VWF" • June 2008: 21 th Biochemical PhD Students National Meeting A. Castellani, University of Pavia, Brallo di Pregola, Italy PP "Cellular models support the dominant-negative effects of a deleted VWF" • May 2008: 6 th Bari International Conference on Hemophilia, von Willebrand and ADAMTS-13 (BIC), Pugnochiuso, Vieste del Gargano -Foggia-Italy PP "Cellular models support the dominant-negative effects of a deleted VWF" • March 2007: XIV Telethon Scientific Convention, Salsomaggiore Terme -Parma-Italy • Dubois MD et al. Res Pract Thromb Haemost. 2022 Jun 15;6(4):e12737 • Campioni M et al. J Thromb Haemost. 2021 Jan;19(1):139-14 • Muczynski V et al. Blood 2018 Sep 13;132(11):1193-1197 • Casari C et al. Blood Adv. 2018 Jun 26;2(12):1417-1428 • Wohner N et al. Haematologica. 2018 Apr;103(4):728-737 • Adam F*, Casari C* et al. Sci. Rep. 2016 May;23(6):26306 • Morioka Y*, Casari C* et al. Blood. 2014 May 22:123(21): 3344-53 • Casari C*, Berrou E* et al. J Clin Invest. 2013 Dec 2;123(12):5071-8 • Casari C et al. Blood. 2013 Oct 17;122(16):2893-902 • Casari C et al. Blood. 2010 Dec 9;116(24):5371-6

	6.3.	Selected publications (attached)

ACKNOWLEDGEMENTS

First, I would like to express my deepest gratitude to Dr. Pierre Mangin, Pr. Agnés Veyradier, Pr. Yves Gruel, Pr. Delphine Borgel, Dr. Christophe Guignabert and Dr. Elisa Rossi for having kindly accepted to be part of this committee.

Mentoring:

• PhD student (Thibaud Sefiane). This • M2 student (Antoine Babuti), post-doc fellow (Marco Heestermans)

Genetic approaches to correct the deleterious effects of dominant-negative variants in models of von Willebrand disease

Confidential

Curriculum Vitae et Studiorum