Caterina Casari

Jennifer Leung

P D James
email: jamesp@queensu.ca

Dr Paula James Room

New and Emerging Therapies for Women, Girls and People with the Potential to Menstruate (WGPPM) with VWD

Innovation in therapies for von Willebrand disease (VWD) patients has lagged far behind hemophilia, creating inequity in the bleeding disorder community. Although currently existing treatments of antifibrinolytics, desmopressin and plasma-derived von Willebrand factor (pdVWF) replacement are considered effective, multiple studies report poor quality of life in VWD patients, especially those with heavy menstrual bleeding (HMB). This disconnect underscores the need for novel therapies that are safe and effective and that consider a patient's specific contraceptive and reproductive needs. Recombinant VWF (rVWF) is the most recent new therapy for VWD; the data specific to women is reviewed. We also present emerging data on emicizumab for the treatment of VWD, BT200 (rondoraptivon pegol), generalized hemostatic therapies (VGA039 and HMB-011) as well as treatments based on nanotechnology (platelet-inspired nanoparticles and KB-V13A12). We are optimistic as we move towards pivotal clinical trials for these elegant and innovative treatments.

Introduction

In the past decade, the bleeding disorder community has seen an explosion of innovation in therapeutics to treat and prevent bleeds. The majority of this has been focused on hemophilia including extended half-life concentrates, non-factor based therapies, re-balancing agents and gene therapy. [START_REF] Weyand | New therapies for hemophilia[END_REF][START_REF] Batty | Advances and challenges for hemophilia gene therapy[END_REF] In contrast, the development of novel therapies for VWD has lagged far behind, despite it being the most common inherited bleeding disorder. The reasons for this are many and include the heterogeneity of bleeding symptoms experienced by VWD patients, as well as an under-appreciation of the impact of the disease. The latter is strongly rooted in sexist attitudes about the gynecologic and obstetric bleeding symptoms that exclusively affect women, girls and people with the potential to menstruate (WGPPM). [START_REF] Weyand | Sexism in the management of bleeding disorders[END_REF] Additionally, the pathophysiology of bleeding in VWD is complex and involves deficiency of both the multimeric glycoprotein von Willebrand factor (VWF) as well as coagulation Factor (FVIII), in addition to multiple possible specific functional VWF defects (ie: impaired platelet, collagen or FVIII binding). [START_REF] Lavin | New treatment approaches to von Willebrand disease[END_REF][START_REF] Lenting | Towards novel treatment options in von Willebrand disease[END_REF] This makes designing and implementing treatments, as well as performing clinical trials to evaluate novel VWD therapies, challenging.

VWD is currently classified into three types: Type 1 is characterized by reduced levels of functionally normal VWF; Type 2 is divided into 4 subtypes; 2A (decreased VWFplatelet binding because of loss of high molecular weight multimers), 2B (increased affinity of VWF for platelets resulting in loss of high molecular weight multimers and variable thrombocytopenia), 2M (decreased VWF-platelet binding with normal multimers) and 2N (decreased VWF-FVIII binding); and Type 3 which is caused by a virtual absence of VWF with secondary FVIII deficiency. [START_REF] Sadler | Update on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor[END_REF] Patients present with a variety of bleeding symptoms including heavy menstrual bleeding and post-partum hemorrhage, but also non-sex specific symptoms such as hemarthrosis, gastrointestinal bleeds, epistaxis, easy bruising, oral cavity bleeding and bleeding after surgery and dental procedures. Currently existing treatments for VWD, including antifibrinolytics, desmopressin and pdVWF replacement have been around for decades, and are widely considered safe and effective. [START_REF] Mannucci | New therapies for von Willebrand disease[END_REF] However, quality of life in VWD patients is known to be poor, especially for those with HMB, which is the most common presenting symptom. 8- 11 This disconnect underscores the problem facing the VWD community -if current treatments are so good, why are patients still suffering?

Multiple studies highlight the inequity and barriers facing women with VWD, including diagnostic delays, serial dismissal of symptoms and lack of access to appropriate care. [START_REF] Arya | They don ' t really take my bleeds seriously ": Barriers to care for women with inherited bleeding disorders[END_REF][START_REF] Arya | Everything was blood when it comes to me ": Understanding the lived experiences of women with inherited bleeding disorders[END_REF][START_REF] Sanigorska | The lived experience of women with a bleeding disorder: A systematic review[END_REF] Indeed, in a recent publication, focused on the management of heavy menstrual bleeding in a tertiary care multidisciplinary clinic, only 68% could be successfully managed medically and only 17% were satisfied with first line treatment. This means patients sequentially trial multiple therapies alone or in combination, only to end up with surgical management to control their bleeding. [START_REF] Kuthiala | Management of heavy menstrual bleeding in women with bleeding disorders in a tertiary care center[END_REF] Clearly, there is an urgent need for novel therapies and optimized management strategies for VWD patients. In this paper, we summarize new and emerging therapies for WGPPM with VWD including rVWF, emicizumab, BT200, generalized hemostatic therapies, and nanotechnologybased therapies. We conclude with a discussion about the characteristics of an ideal treatment for these patients and a view to future pivotal clinical trials.

New Treatment Options

Recombinant von Willebrand Factor Recombinant VWF (rVWF) is the most recent new therapy to become available for VWD patients. Its pharmacokinetics, safety and efficacy were reported in two prospective trials that enrolled both males and females. [START_REF] Gill | Hemostatic efficacy, safety, and pharmacokinetics of a recombinant von Willebrand factor in severe von Willebrand disease[END_REF][START_REF] Leebeek | Recombinant von Willebrand factor prophylaxis in patients with severe von Willebrand disease: phase 3 study results[END_REF] The first, published in 2015, enrolled 37 patients with severe VWD, including 20 females. Participants received rVWF (+/-rFVIII) for on-demand treatment. Overall, bleed control for all (100%) treated bleeds (n=192 bleeds in 22 subjects) was rated as good or excellent. Thirty two of the mucosal bleeds were menorrhagia, requiring 37 infusions with rVWF. There were a small number of adverse effects (6 nonserious AEs in 4 subjects and 2 concurrent serious AEs in 1 subject; chest discomfort and increased heart rate without cardiac symptomatology) and no thrombotic events or severe allergic reactions. For prophylactic treatment, the safety and efficacy of rVWF was reported in 2022. [START_REF] Leebeek | Recombinant von Willebrand factor prophylaxis in patients with severe von Willebrand disease: phase 3 study results[END_REF] In this prospective, open-label, non-randomized phase 3 study, participants received rVWF prophylaxis at a starting dose of 50 +/-10 VWF:RCo IU/kg intravenously (IV) twice weekly (or based on their prior pdVWF weekly dosing schedule). The results showed a reduction in spontaneous bleeding events of 91.5% in 13 patients previously treated ondemand and of 45.0% in 10 patients previously on pdVWF prophylaxis. Eleven of the 23 included patients were female. Five patients required treatment for spontaneous bleeding events while on study; 3 of these were for menorrhagia. Antifibrinolytics were permitted to treat minor or moderate breakthrough bleeds if additional doses of rVWF were not required, and two patients received this, but it is not clear if these were the patients with menorrhagia. In general, it is not possible to distinguish most of the results from these two studies based on the sex of participants.

In contrast, the VWDMin Study focused exclusively on female patients, and was a phase 3, open-label, randomised crossover trial comparing the use of rVWF to tranexamic acid for heavy menstrual bleeding (HMB) in patients with mild and moderate VWD. [START_REF] Ragni | Recombinant von Willebrand factor and tranexamic acid for heavy menstrual bleeding in patients with mild and moderate von Willebrand disease in the USA (VWDMin): a phase 3, open-label, randomised, crossover trial[END_REF] Patients were excluded if they were on combined hormonal contraceptives and if they were pregnant or lactating. Participants were assigned to two consecutive cycles of rVWF at a dose of VWF:RCo 40 IU/kg IV on day 1 of menses or oral tranexamic acid 1300 mg three times daily on days 1 -5 in random order. Menstrual blood loss was measured by the PBAC (Pictorial Bleeding Assessment Chart) score. Thirty-six patients completed the trial and the results showed a lower median PBAC score for tranexamic acid (146) when compared to rVWF (213); however both remain solidly above 100, the threshold used to define HMB. Importantly, the primary endpoint (a 40-point reduction in PBAC score by day 5 after two cycles of treatment) was not met and the trial was stopped early due to slow recruitment. The fact that all participants received the same single dose of rVWF regardless of baseline VWF levels, and the variability among study participants likely influenced the results. The authors conclude that rVWF is not superior to tranexamic acid in reducing HMB and advocated for shared decision making with patients when discussing treatment options. The fact that all trial participants still had HMB after treatment with both rVWF and tranexamic acid clearly highlights the need for novel therapies.

Emerging Treatment Options

Emicizumab Emicizumab is a bispecific monoclonal antibody that mimics the function of FVIII by bridging activated FIX and FX. It has been demonstrated to be highly effective for the prevention of bleeds in hemophilia patients with and without inhibitors. [START_REF] Callaghan | Long-term outcomes with emicizumab prophylaxis for hemophilia A with or without FVIII inhibitors from the HAVEN 1-4 studies[END_REF] In contrast to standard therapy that involves multiple intravenous infusions of coagulation factor concentrate per week, emicizumab is administered subcutaneously (SC) weekly, biweekly or monthly, dramatically decreasing the burden of care. [START_REF] Callaghan | Long-term outcomes with emicizumab prophylaxis for hemophilia A with or without FVIII inhibitors from the HAVEN 1-4 studies[END_REF] The off label use of emicizumab in Type 3 VWD has been reported in patients with and without anti-VWF antibodies, with an improvement in hemostasis in all cases. [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] Weyand | Efficacy of emicizumab in a pediatric patient with type 3 von Willebrand disease and alloantibodies[END_REF][START_REF] Sigaud | Treatment with emicizumab leads to clinical improvement in a type 3 von Willebrand disease patient with inhibitor suffering from left ankle arthropathy[END_REF][START_REF] Barg | The potential role of emicizumab prophylaxis in severe von Willebrand disease. Blood Cells[END_REF][START_REF] Vo | Prophylactic emicizumab in children and adults with type 3 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] These case reports include the successful treatment of hemarthrosis in males and females, as well as substantial improvement in heavy menstrual bleeding in an 11 year old girl with Type 3 VWD, anemia and hypovolemic shock. She had not responded to hormonal therapy, cryoprecipitate nor tranexamic acid. Following the addition of low dose emicizumab (3mg/kg SC once a month) she had no further menorrhagia or bleeding episodes. [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] Additionally, recent in vitro work shows an improvement in thrombus formation under shear in all types of VWD, potentially expanding its clinical use beyond Type 3 VWD. [START_REF] Yaoi | Emicizumab enhances thrombus formation in vitro under high shear flow conditions in whole blood from patients with type 1 and type 3 von Willebrand disease[END_REF][START_REF] Yaoi | Emicizumab improves thrombus formation of type 2A von willebrand disease under high shear condition[END_REF] BT200 (rondoraptivon pegol) BT200 (rondoraptivon pegol) is a pegylated aptamer that binds to the VWF A1 domain and increases VWF and FVIII by decreasing the clearance of both molecules. [START_REF] Kovacevic | The von Willebrand factor A-1 domain binding aptamer BT200 elevates plasma levels of von Willebrand factor and factor VIII: a first-in-human trial[END_REF] Interestingly, its predecessor, ARC1779, was originally developed as an anti-thrombotic agent due to its ability to inhibit VWF-platelet interactions. 29 BT200 has been studied in Type 2B VWD in a prospective phase 2 trial. Participants initially received loading doses of 3 mg SC on days 1, 4 and 7, followed by 6 -9 mg every week until day 28. [START_REF] Ay | The VWF binding aptamer rondoraptivon pegol increases platelet counts and VWF/FVIII in type 2B von Willebrand disease[END_REF] Five patients, including 2 women, were included and participant-level data is presented. VWF and FVIII levels increased > 2 fold and platelet counts by > 3 fold. In all thrombocytopenic patients, plasma levels of VWF:GPIbM and VWF collagen binding increased, and high molecular weight multimers were restored. Data presented at the 2023 ISTH Congress showed that BT200 prolongs VWF half-life by blocking its interaction with the macrophage scavenger receptor LRP1, providing in vitro support for a role beyond Type 2B VWD. [START_REF] Choin | Aptamer BT200 prolongs VWF half-life by blocking interaction with macrophage scavenger receptor LRP1[END_REF] Further studies evaluating this molecule in larger numbers of VWD patients with bleeding endpoints are necessary.

Generalized Hemostatic Therapies -VGA039 and HMB-001 VGA039 VGA039 is a monoclonal antibody directed against human protein S that inhibits its cofactor activity for TFPI (tissue factor pathway inhibitor) alpha and activated protein C, enhancing thrombin generation through both the initiation and propagation phases of coagulation. It is administered parenterally -either IV or SC and has a relatively long half-life of 21 and 12 days respectively, at doses of 1 mg/kg. In vitro studies showed enhanced thrombin generation in VWF-deficient plasma and ex vivo studies showed concentration dependent increases in thrombin generation as well as dose and concentration dependent increases in D-dimer levels. [START_REF] Leong | Pre-Clinical Characterization of VGA039, an Anti-Protein S Monoclonal Antibody Being Developed As a Universal Hemostatic Agent for Various Bleeding Disorders[END_REF] Future studies in VWD patients are critical to establish safety and efficacy HMB-001 HMB-001 is a bispecific antibody that binds and accumulates endogenous activated Factor VII (FVIIa) in circulation. It promotes activation of Factor X and thrombin generation by placing FVIIa on the surface of activated platelets. [START_REF] Gandhi | HMB-001 -a novel bispecific antibody with potential for subcutaneous prophylactic treatment of multiple bleeding disorders -initial focus on Glanzmann Thrombasthenia[END_REF][START_REF] Ostergaard | HMB-001 -A novel bispecific antibody accumulating and targeting endogenous FVIIa to activated platelets for subcutaneous prophylaxis in multiple bleeding disorders including Glanzmann thrombasthenia[END_REF] To date, studies have been focused on in vivo and ex vivo models of Glanzmann Thrombasthenia but it has the potential to treat other bleeding disorders including VWD.

Nanotechnology -Platelet-inspired nanoparticles and KB-V13A12 Platelet-inspired Hemostatic Nanoparticles Synthetic platelet nanoparticles are small liposomal particles decorated with multiple peptides facilitating concomitant binding to collagen, VWF and activated platelets. In vitro studies using microfluidic assays show an improvement in thrombus formation using blood from mice with Type 2B VWD and deficient of VWF (VWF-KO). In vivo studies using a tail clip assay, showed a reduction in blood loss of 35% in 2B VWD and 68% in VWF-KO mice. [START_REF] Roullet | Efficacy of platelet-inspired hemostatic nanoparticles on bleeding in von Willebrand disease murine models[END_REF] Clearly, much additional work is needed to translate this therapy into humans, and to determine its suitability to treat WGPPM with HMB. However, the customizable nature of synthetic platelet nanoparticles makes them an attractive treatment option for VWD, with its varied and heterogeneous pathophysiology across types/subtypes.

Nanobody-based KB-V13A12

A bifunctional molecule that binds both albumin and VWF, KB-V13A12 prolongs the half-life of endogenous VWF. It has been tested in a mouse model of Type 1 VWD; subcutaneous administration of the molecule was shown to increase plasma VWF levels by ~2 fold for up to 10 days and restore functional hemostasis in a tail-clip assay. [START_REF] Peyron | KB-V13A12, a novel nanobody-based therapeutic molecular for the treatment of von Willebrand disease[END_REF] A single subcutaneous injection with a long half-life is promising for the treatment of HMB, pending translational studies.

Moving Towards the Future

We must consider the unique values and preferences of WGPPM with VWD as we move forward with pivotal clinical trials for these molecules. Of course, safe and effective therapies are required. However, attention must also be paid to the specific issues of fertility, teratogenicity and the changing requirement for contraception over the lifespan of these patients. The monthly nature of menses makes a subcutaneous treatment with a half-life of many days very attractive, and for non-hormonal therapies, the ability to be safely combined with contraception is critical.

As mentioned above, clinical trials in VWD patients are complicated. VWD patients experience a variety of bleeding symptoms and are heterogeneous in terms of the pathobiological manifestations of their disease. This means inclusion/exclusion criteria must be developed thoughtfully. Additionally, clinical trial design must be equitable; we cannot continue to exclude the very patients who need novel treatments the most. In contrast to hemophilia clinical trials, which have the benefit of a fairly straight forward primary outcome of reduction in annual bleeding rate (ABR) focused on musculoskeletal bleeds, an appropriate outcome for the mucocutaneous bleeds seen in VWD is more difficult to define. Therefore, we call on the community to adopt and adhere to a set of core outcomes to be reported by all trials that encompass the burden of disease experienced by VWD patients and expect results to be presented in a way that allows for sex-specific interpretation. We also expect trial endpoints to be feasible and patientfocused and urge regulators to move away from the reliance on arcane measures such as alkaline hematin methods for the evaluation of menstrual blood loss. We understand that access to trial participants is likely to be a challenge in the very near future, however as a community, we managed to navigate this for novel therapies for hemophilia; VWD patients deserve no less.

The innovation and elegance of the molecules discussed above is impressive and inspiring. We are optimistic for the future of care for WGPPM with VWD.

Authorship

All authors contributed to the writing of this manuscript and approve its final version.

VWD pa'ents with poten'al to menstruate suffer from increased bleeding & QoL burdens

	pa#ents with poten#al to menstruate	pa#ents without poten#al to menstruate
	↘↘QoL	sex-specific bleeds	+	non sex-specific bleeds	non sex-specific bleeds	↘QoL
			↗↗bleeds
		Emerging treatment op?ons for VWD
	emicizumab	BT200	VGA039		HMB-001	platelet inspired nanopar?cles	KB-V1316M

Acknowledgements

The visual abstract was created using Biorender.

Conflict of interest disclosures: PJ receives research funding from Bayer, and consultancy fees from Band/Guardian Therapeutics, Star/Vega Therapeutics and Biomarin; CC is co-inventor on a patent regarding KB-V13A12 ORCID profiles: CC: 0000-0002-8271-6795; JL: 0000-0002-2840-7588; PJ: 0000-0003-4649-9014


H Ezzedine
email: houcine.ezzedine@univ-valenciennes.fr

A Trabelsi

C Kolski

Modelling of Agent oriented Interaction using Petri Nets, application to HMI design for transport system supervision

This article presents an original method using high level Petri nets for the specification and design of interactive systems. We suggest an agent oriented architecture based on the classic components of an interactive application (application, dialogue control, interface with the application). Our approach will be validated via the specification and design of a human-machine interface used in the supervision of a land-based transport system (Bus/Tramway).

I. INTRODUCTION

Many applications currently offer Human-Machine interactions which are increasingly flexible, and even adaptive and intelligent [KOL,98;HÖÖ,00;CAL,01]. Indeed, interactive system designers place a far greater degree of importance on the reactivity, the maintainability, the re-use and the adaptation of their system with regard to the context in which it is used. These various properties are facilitated by a modular structuring of the interactive system. In this context, multi-agent systems provide a promising perspective because of their capacities for structuring and organising the different software components in relation to Human-Machine interaction [KEE, 00; KLU, 01; GRI, 01].

However, the majority of models available are mainly concerned with user-controlled applications and do not take into account the specificities found in supervision applications in which the user plays the role of controller and commander of an independent process [MIL,88;SHE,88]. We therefore put forward an agent-oriented specification method for the design of Human-Machine interfaces intended for the control/command of industrial systems. The specialised agents suggested by the method are able to guarantee communication between the process and the user via input/output devices. In the supervision context, each one of these two functions has its own characteristics which are linked to the specificities of the supervision role (such as real time aspects).

The second part describes the basic principles linked to multi-agent systems. The third part concentrates on aspects of interactive system architecture. In the fourth part, we will explain how Petri nets are used for the specification of interactions. The fifth part describes the case study.

II. BASIC PRINCIPLES LINKED TO MULTI-AGENT SYSTEMS

The general principle of Multi-Agent Systems (MAS) is the distribution of the problem, which in our case is Human-Machine interaction, into a certain number of co-operative entities which can communicate and co-ordinate their behaviour in order to reach a common objective. The particularity of each MAS is to be found in the criteria selected for the distribution of capabilities and knowledge (data) between the agents.

The agents have two facets: one facet tends towards the collective aspect, which leads us to define the notion of agent grouping (mechanisms concerning group activities), and another individual facet with the mechanisms and the knowledge regarding the internal functioning rules at the level of the agent, concerning its own individual goals.

Two types of agent can be distinguished in multiagent systems [FER,95;CHA,92;CHA,96]:

-lower level reactive agents (with a low degree of granularity); these agents only have a limited communication language and protocol, their capacities merely obey the law of stimulus/action.

-systems made up of cognitive agents are founded upon the principle of co-operation between agents which are capable of performing complex operations alone. A cognitive system generally includes a small number of agents capable of reasoning using a data base and of processing the various pieces of information linked to the application field.

It should also be noted that hybrid agents are also to be found; these combine the two types of agent mentioned above. The classic models of interactive systems distinguish three essential functions (presentation, control and application). Some models (such as the Seeheim and ARCH models) consider these three functions as being three distinct functional units.

Other approaches using structural components, and in particular those said to be distributed or agent approaches, suggest grouping the three functions together into one unit, the agent. The agents are then organised in a hierarchical manner according to principles of composition or communication (for example PAC [COU,90], MVC (Model-View-Controller de Smalltalk).

Our approach could be considered as being intermediate as it borrows elements for its principles from both types of model given above.

In our architecture, we suggest using a division into three functional components which we have called respectively: interface with the application (connected to the application), dialogue controller, and presentation (this component is directly linked to the user). These three components group together agents:

-the application agents which handle the field concepts and cannot be directly accessed by the user. One of their roles is to ensure the correct functioning of the application and the real time dispatch of the information necessary for the other agents to perform their task.

-the interactive agents (or interface agents), unlike the application agents, are in direct contact with the user (they can be seen by the user). These agents co-ordinate between themselves in order to intercept the user commands and to form a presentation which allows the user to gain an overall understanding of the current state of the application. In this way, a window may be considered as being an interactive agent in its own right; its specification describes its presentation and the services it is to perform.

-the dialogue control agents which are also called mixed agents; these provide services for both the application and the user. They are intended to guarantee coherency in the exchanges emanating from the application towards the user, and vice versa.

IV. USE OF PETRI NETS FOR MULTI-AGENT SPECIFICATION AND DESIGN

Each agent therefore plays a role within its group. This role can be expressed in the form of the services it offers in the interactive system.

A service is defined as being a quadruplet s={E, C a , R, P} with (see Fig. 1):

-E: the event which triggers the service, -C a: the conditions to be met in order to perform this service, -R: the resources necessary for the service to be performed, -P: the property of this service, which can be either an operation concerning the agent alone (with or without a change of state for the interactive agents), or a call for the service of another agent (with a change of state for the reactive agents). The succession of various calls for services gives rise to the succession of windows in the human-machine interface.

The behaviour of the agents is modelled using high level Petri nets; this makes it possible to validate certain properties of the application before it is implemented (vivacity, re-initialisation …). We illustrate the notion of service using two significant examples in section V.4.

V. APPLICATION TO A SUPERVISION INTERFACE FOR A BUS AND TRAMWAY NETWORK

The validation of our approach occurred within the framework of a project involving an industrial partner, the SEMURVAL company which will run the future urban transport network (tramway and bus) in the town of Valenciennes, as well as several research laboratories (LAIL, I3D, LAMIH and INRETS) [MES, 98; CHI, 00; EZZ, 01]. Urban transport networks, and in particular the systems intended to provide information for the passengers will act as an example of the application of our method.

Information in public transport networks has become not only a need but also a requirement. The users of a multi-mode public transport system (trams, buses, underground trains, overground trains) want to be better informed before and during their journeys and to receive reliable information. This information must therefore be regulated (and not simply automatic) and a dynamic interface must be designed to enable the human regulator to perform his/her role of information regulation (control/command of the information displayed).

Each element of information transmitted to the passengers must be truly pertinent and relevant.

The system targeted by our research project will be referred to as the IAS (Information Assistance System) in the rest of this article.

A. Modelling of the interactions between the IAS and the human regulator with interpreted Petri nets

In any interactive application, the use of a formalism describing the system's dynamics is of capital importance. Indeed, modelling is all the more necessary since it provides formal analysis techniques which make it possible to prove properties of the design before its implementation [PAL,97;ABE,01]. We therefore decided to use Petri nets, and more precisely interpreted Petri nets, in order to model the interactions between the system and the human regulator (see Fig. 2). The Petri net is only concerned with the interactive part and therefore only with the relevant information which has to go via the regulator. All other information is dealt with using an automatic mode.

Four main functions performed by the IAS can be distinguished:

-automatic management of information (with no intervention by the regulator). This encompasses times of delayed stops, destinations, following stops, and also information on connections (informing the passengers on the connections possible), -the visualisation function: this makes it possible to inform the regulator, at any time, of the state of the network from the point of view of information intended for the passengers.

One of the roles of this function is to control the state of the display devices (breakdown of display panels, divergence between the information actually displayed and the information sent to the regulation post, …), information being processed (consultation of the information displayed on the mobile units and the network stations at any time) as well as the position of the mobile units and their state, -the message function which allows the regulator to send a message to the network unit of his/her choice at any time.

B. Events linked to the IAS

As shown in Figure 3, the IAS can be affected by two types of event which can considerably change its functioning: "action" events and "network" events:

-The "action" events correspond to orders given by the regulators from the control/command post. They can correspond to:  a modification to the topology of the network (supervision of a stopping point,…),  messages sent by the regulator,  a modification to the running plan.

-The "network" events which come directly from the field:  Disruptions (maintenance work, vehicle breakdown, demonstrations…),  Messages sent by the driver.

C. Agent oriented specification of the humanmachine interface

We suggest the use of an agent oriented architecture which divides the agents into three classes. As a reminder, these classes are called respectively: application agents, dialogue control agents and interface agents (or presentation agents). Specification of application agents: the application agents are intended to manage the passenger information in the vehicles and stations and to calculate the information to be displayed (delays, timetable and route modifications, etc.). According to the traffic context, each agent possesses rules enabling it to act correctly in its environment. Specification of dialogue control agents: the dialogue control agents have a dual role. They make it possible to: (1) Diffuse the modifications observed at the application level on the interactive agents, (2) Diffuse the regulator's command actions towards the application agents. These agents act as an intermediary for interaction between the interface agents and the application agents. Specification of the interface agents: we have identified six types of interface agent responsible for direct interaction with the human regulator. These agents are represented in the form of interactive windows. The regulator can interact with these agents via the various functions possible in the windows, for example: the buttons, the edition zones, the pictures, etc. These agents are: The modelling of the multi-agent system interface with the application using high level Petri nets makes it possible firstly to specify the services between agents and secondly to provide a dynamic simulation of the application with a view to assessing the performance of the whole system.



D. Examples of the use of Petri nets for the definition of an agent's service

Example 1: Table 1 and Figure 4 illustrate an internal operation within an interface agent. Following the event "vehicle delay" indicated by the network support system, the State of the traffic interface agent is notified by an application agent, via a dialogue control agent, of the delay of the vehicle in question. The interface agent analyses this event and checks if the condition "Delay greater than 5 minutes" is fulfilled. The validation of this condition brings about the display of a dialogue box, used as a resource by the interface agent, which will inform the regulator of a considerable disruption in the traffic. The interface agent will use its internal properties to determine the nature and category of the message to be transmitted. As we have stated in the previous section, the interface agents are interactive. Therefore, if the State of the traffic interface agent perceives the event "Click on a vehicle" performed by the regulator (see Fig. 5, Table 2), it calls upon the service of the Vehicle interface agent. In this case, the interface agent will need to use neither resource nor internal properties.

E. Graphic interface

It should be remembered that the aim of the IAS is to enable the regulator to visualise, edit, create and transmit information intended for the passengers in the stations and/or vehicles.

In order to perform his/her task of regulation correctly, the regulator interacts with the six interface agents: the State of the traffic agent, the State of the line agent, the Station agent, the Vehicle agent, the Message agent and the Overall view agent. All of these agents are developed using the C++ language.

The State of the traffic interface agent: the State of the traffic interface agent gives a synthetic representation of all the delays concerning mobile units travelling on the network. Thus, with the help of the network support system, it ensures the real time surveillance of vehicle delays on the network supervised.

The State of the line interface agent: the view of the State of the line interface agent is made up of graphic elements such as stations, route sections, vehicles, etc. (see Fig. 6). A click on a vehicle directly displays the view (window) of the vehicle interface agent which will deal with any further interaction with the regulator. The Message interface agent: the view of the Message interface agent enables the regulator to obtain a synthetic view of all the messages being sent to vehicles and stations. It is made up of four types of panel (see Fig. 8): one panel which contains a list of all the messages which can be sent, one panel making it possible to choose the destination line, with an option to make a multiple selection, the two other panels enable the regulator to select the station and vehicles which are to receive the message selected.

The Overall view interface agent: in order to make the task of supervising the traffic easier for the regulator, we created an overall view interface agent. As its name implies, the view given by this agent provides the regulator with a global view of the traffic on the network. This view encompasses all the lines to be supervised and facilitates access to line, stations and vehicles.

VI. CONCLUSIONS AND PERSPECTIVES

In this article, we have put forward an agent orient specification approach for the design of humanmachine interfaces. This approach was applied during a project involving a bus and tram network, and was intended to provide significant support in the field of passenger information.

The use of an agent oriented interactive system specification approach for the IAS led to a modular breakdown of the interface. These modules were the subject of a specification using high level Petri nets.

Following the specification and design phases, an assessment phase will be performed as regards both the technical and the ergonomic aspects of the interface. This is currently being prepared.

Fig. 1 .

 1 Fig. 1. Definition of an agent's service

Fig. 2 .

 2 Fig. 2. Modelling of interaction-the decision support function, which consists in suggesting messages to be displayed to the regulator in the event of considerable disruptions. These messages can be shortened,

Fig. 3 .

 3 Fig. 3. Events linked to the IAS

  The State of traffic agent,  The State of the line agent,  The Station agent,  The Vehicle agent,  The Message agent,  The Overall view agent.

Fig

  Fig. 4. Agent's internal operation

Fig. 5 .

 5 Fig. 5. Call for another agent's service

The

  Fig. 7.a. View of the Station interface agent

Fig. 6 .

 6 Fig. 6. View of the State of the line interface agent

ACKNOWLEDGEMENTS

The authors would like to thank the FEDER, the GRRT and the Nord-Pas-de Calais regional authorities which have provided support for this research (SART and NIPO projects).


Nathalia Soar

Caio De Oliveira

Pascale Royer

Simon Le Floc'

Chrystelle Po

Nadia Bahlouli

Cédric Mauprivez

Halima Kerdjoudj

Adrien Baldit
email: adrienbaldit@univ-lorraine.fr

BIOMECHANICAL SIMULATION OF HAEMOSTATIC SPONGES USED FOR SINUS LIFT PROCEDURE

Introduction

The objective of this study is to provide a geometrical model that describes the morphological behavior of haemostatic gelatin-based sponges under the effect of hydration following the sinus lift surgery [1]. For this purpose, a finite element model was developed to simulate conditions close to clinical ones and experimentally reproduced by Okley et al. [2]. The final objective is to build a patient specific tool to predict how much initial sponge volume is required to promote the bone formation and support the dental implants once mineralized.

Materials and Methods

Modelling such a shrinking within physiological conditions rises many issues involving hydro-chemo-mechanical couplings.

Aiming a fast answer to the clinical demand, the morphological effects of hydration on haemostatic sponges has been modelled through a thermo-stress problem [3] with Abaqus where the temperature variation represented the sponge hydration. Gelita-spon sponge being cubic (10x10x10mm³), only one fourth has been used with a mesh size of 1mm leading to 250 linear hexahedral temperaturedisplacement coupled elements (C3D8H).

Results

An optimisation loop has been set with Python to reach the set of parameters fitting the experiments presented in Fig 2.

Discussion and Conclusions

The results are satisfactory for an extend to the patient specific geometry, i.e. sinus cavity.

Although, mesh refinement led to convergence issues due to excessive deformations. Besides, the analogy with thermomechanics is far from reality and microstructural analyses are under progress to be coupled with poromechanical simulation.

Figure 1 :

 1 Figure 1: Sinus lift X-ray monitoring with in yellow the defect to fill and in blue the newly formed bone after one-month implantation. b) side view of a dry haemostatic sponge (Gelita-spon) and c) its state after 12 hours within physiological fluid.

Figure 2 :

 2 Figure 2: Simulation results (red line) fitting experimental data (blue dots) in terms of normalized volume as function of time. The associated final geometries (one example in Fig 2) are close to experiments Fig 1c.4. Discussion and ConclusionsThe results are satisfactory for an extend to the patient specific geometry, i.e. sinus cavity.Although, mesh refinement led to convergence issues due to excessive deformations. Besides, the analogy with thermomechanics is far from reality and microstructural analyses are under progress to be coupled with poromechanical simulation.5. References

Acknowledgements:

The authors would like to thank the CNRS MoTiV grant and the FLI IMCOLL grant for providing financial support to this project.


Annie Harroche

Thibaud Sefiane

Maximilien Desvages

Delphine Borgel

Dominique Lasne

Caterina Casari

Ivan Peyron

Laurent Frenzel

Stéphanie Chhun

Peter J Lenting
email: peter.lenting@inserm.fr

Cécile Bally

Non-inhibitory antibodies inducing increased emicizumab clearance in a severe haemophilia A inhibitor patient

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Haemophilia A is a bleeding disorder that results from coagulation factor VIII deficiency, which can be treated via substitution therapy using FVIII concentrates. However, the generation of neutralizing antibodies renders treatment ineffective in up to 30% of the severe patients. [START_REF] Cormier | Advances in knowledge of inhibitor formation in severe haemophilia A[END_REF][START_REF] Scott | Factor VIII: Perspectives on Immunogenicity and Tolerogenic Strategies[END_REF] Emicizumab is a humanized bispecific antibody that binds simultaneously to activated factor IX (FIXa) and factor X (FX), thereby mimicking the cofactor function of activated factor VIII (FVIIIa), even in the presence of FVIII inhibitors. [START_REF] Kitazawa | A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in a hemophilia A model[END_REF][START_REF] Sampei | Identification and multidimensional optimization of an asymmetric bispecific IgG antibody mimicking the function of factor VIII cofactor activity[END_REF] Once-weekly subcutaneous administration of emicizumab markedly decreased the bleeding rate in patients who had haemophilia A with or without factor VIII inhibitors. [START_REF] Oldenburg | Emicizumab Prophylaxis in Hemophilia A with Inhibitors[END_REF][START_REF] Mahlangu | Emicizumab Prophylaxis in Patients Who Have Hemophilia A without Inhibitors[END_REF][START_REF] Young | A multicenter, open-label phase 3 study of emicizumab prophylaxis in children with hemophilia A with inhibitors[END_REF][START_REF] Pipe | Efficacy, safety, and pharmacokinetics of emicizumab prophylaxis given every 4 weeks in people with haemophilia A (HAVEN 4): a multicentre, open-label, non-randomised phase 3 study[END_REF] However, anti-drug antibodies with neutralizing potential can develop in a small number of patients and have been associated with decreased emicizumab plasma concentrations and loss of efficacy. [START_REF] Harkins Druzgal | Neutralizing antidrug antibody to emicizumab in a patient with severe hemophilia A with inhibitors: New case with detailed laboratory evaluation[END_REF][START_REF] Valsecchi | Characterization of the neutralizing anti-emicizumab antibody in a patient with hemophilia A and inhibitor[END_REF] Although the development of neutralizing antiemicizumab antibodies is rare and routinely biological monitoring not recommended in patients treated with emicizumab, it is still important to analyze for the presence of such antibodies in case of bleeding events. In this study, we describe the development of non-inhibitory antiemicizumab antibodies that selectively provoke increased emicizumab clearance in a severe haemophilia A patient with inhibitors.

A 2-year-old boy with severe haemophilia A developed an anti-FVIII inhibitor (1 BU/mL) at 19 exposure days. The patient failed to respond to immune tolerance induction and venous access became extremely complicated. Treatment with emicizumab was therefore initiated with 4 loading doses (3 mg/kg/week) followed by weekly treatment (1.5 mg/kg/week). Clinical outcomes were excellent with no bleeding episodes or bruising. Blood samples were taken as part of routine care, with excess being stored for research (The Hôpital Necker's haemophilia bio-library/Necker Biobank, registration number: DC-2009-955; procedure is in accordance the Helsinki declaration and participants give written informed consent). Analysis revealed emicizumab concentrations in the expected range (66 µg/ml 52 days after emicizumab initiation) [START_REF] Oldenburg | Emicizumab Prophylaxis in Hemophilia A with Inhibitors[END_REF] , and a dramatic decrease in APTT-ratio (0.74, normal range <1.2) was measured (Fig. 1).

A spontaneous hemarthrosis of the ankle occurred 6 months after emicizumab initiation, which was confirmed via clinical examination and ultrasound evaluation. Simultaneously, the APTTratio raised to 2.67, and circulating emicizumab concentrations were below 1 µg/mL (Fig. 1).

Hence, the development of anti-emicizumab antibodies was suspected, and the presence of emicizumab-specific IgGs in patient serum was explored in immunosorbent assays, using normal serum and IgG-depleted patient serum as controls. Binding of IgGs to immobilized emicizumab (5 µg/ml) was determined using isotype-specific peroxidase-labeled monoclonal antibodies. Whereas no specific IgG2 or IgG3 anti-emicizumab antibodies were detected, patient's serum was indeed enriched in anti-emicizumab antibodies of the IgG1 subtype (Fig. 2A-B). We could not test for IgG4 antibodies, since emicizumab is of this subtype. [START_REF] Kitazawa | A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in a hemophilia A model[END_REF] Treatment of the hemarthrosis included rFVIIa and oral corticoids (1 mg/kg/day for 48h to reduce peri-articular inflammation). A minor increase in emicizumab concentrations (1.7 µg/mL) and reduction in APTT-ratio (1.44) was observed (Fig. 1), suggesting a potential corticosensitivity of the anti-emicizumab antibody-producing plasmocytes. Although no bleeds were observed during a 3-week period, emicizumab levels remained undetectable following a short corticosteroid therapy (2 mg/kg/day, conform to the management of children's immunologic thrombocytopenic purpura). Corticosteroid-therapy was therefore halted. Since anti-emicizumab antibodies have been reported to be transient in some patients [START_REF] Paz-Priel | Immunogenicity of Emicizumab in People with Hemophilia A (PwHA): Results from the HAVEN 1-4 Studies[END_REF] , emicizumab therapy (1.5 mg/kg/week) was continued for 3 months. As no improvement was observed, emicizumab therapy was terminated.

To further characterize the anti-emicizumab antibodies, additional tests were performed. We next analyzed eventual inhibition of emicizumab activity using the chromogenic activity assay (Biophen FVIII:C activity assay (ref 221402); Hyphen BioMed, Andresy, France). Surprisingly, no reduction in emicizumab activity was observed, irrespective whether normal or patient serum was tested (Fig. 2C). Similar data were obtained using a one-stage clotting assay, suggesting that the anti-emicizumab antibodies are essentially non-inhibitory. This was further assessed by analyzing binding of biotinylated (bt)-emicizumab to immobilized purified recombinant FIX (Pfizer, Paris, France) or plasma-derived FX (Cryopep; Montpellier, France). Biotinylation was performed using the NHS-PEO4-biotin kit (ref: UPR2027B; Griener Bio-one; Frickenhausen, Germany) without loss of cofactor activity. No inhibition of emicizumab binding to FIX was observed with normal or patient serum (Fig. 2D). However, a modest inhibition of emicizumab binding to FX was observed using patient serum (Fig. 2E). Maximal inhibition was 35±20% (n=4: p=0.0124 compared to normal serum) using 5-fold diluted serum.

The absence of a dominant inhibitory effect on the interactions between emicizumab and FIX/FX is compatible with the non-inhibitory nature of the patient's anti-emicizumab antibodies in the activity assays. Indeed, a modest decrease in emicizumab-FX complex formation would still allow for sufficient ternary complex to be formed (555 pM versus 851 pM in the absence of inhibitor, when calculated according to Kitazawa et al.). [START_REF] Kitazawa | Factor VIIIa-mimetic cofactor activity of a bispecific antibody to factors IX/IXa and X/Xa, emicizumab, depends on its ability to bridge the antigens[END_REF] We previously showed that a threshold of about 300 pM ternary complex is needed to produce sufficient hemostatic activity in vivo. [START_REF] Ferriere | A hemophilia A mouse model for the in vivo assessment of emicizumab function[END_REF] As low emicizumab levels in the patient can be explained by an increased clearance, we determined survival of bt-emicizumab in the absence or presence of serum in immuno-deficient mice. Bt-emicizumab was added to 0.9% NaCl, 100µl control serum or 100µl patient serum (both 5 µg per 100µl), and incubated for 30 min at ambient temperature. Mixtures were then injected via the retro-orbital plexus (at a dose of 0.25 mg/kg bt-emicizumab) to male NOD.CB17-Prkdcscid/NCrHsd-mice (age 9 weeks; ENVIGO, Gannat, France). Housing and experiments of NOD.CB17-Prkdcscid/NCrHsd-mice were performed in accordance with French regulations and the experimental guidelines of the European Community. Experimentation was approved by the local ethical committee of Université Paris-Sud (Comité d'Éthique en Experimentation Animale no. 26, protocol APAFIS#4400-2016021716431023v5). Blood samples were taken at 15min, 6h, 24h and 48h after infusion. Plasma was used to determine residual bt-emicizumab levels in an ELISA in which streptavidin-coated microtiter wells were incubated with plasma samples, and bound bt-emicizumab was probed using peroxidaselabeled anti-human IgG4-Fc antibodies. No difference in the disappearance from the circulation was found between bt-emicizumab alone or the bt-emicizumab/normal serum combination (t1/2=8.5h and 8.3h, respectively; Fig. 2F). In contrast, bt-emicizumab was eliminated significantly faster in the presence of patient serum (t1/2=2.2h; p=0.0013; Fig. 2F), with no btemicizumab detectable at 48h after infusion (compared to 21% for both other conditions). It thus seems that the main effect of the anti-emicizumab antibodies is to accelerate clearance of emicizumab.

Opposite to previously reported inhibitors of emicizumab, we here describe the presence of antibodies that leave emicizumab activity unaffected, but that provoke the rapid elimination of emicizumab in mice, providing an explanation for the low emicizumab levels in the patient.

Although the occurrence of anti-emicizumab antibodies is rare, their presence may severely diminish its clinical efficacy, resulting in the re-appearance of spontaneous bleeds. Clinical monitoring will usually be sufficient for patients receiving emicizumab therapy. However, in case of bleeding in the absence of any compliance concerns, biological monitoring (APTT and emicizumab concentration) is therefore helpful to detect possible anti-drug antibodies.

Legends

Figure 1 :

 1 Figure 1: Evolution of APTT and emicizumab plasma concentration over time. At indicated time-points, blood samples were taken from the patient. Plasma was then analyzed for APTT (left Y-axis, open circles) and emicizumab concentration (right Y-axis; closed squares). Arrow 1 indicates bleeding event; Arrows 2-3 indicate periods of cortico-therapy (2: 1 mg/kg/day for 48 hours during day 187-189; 3: 2 mg/kg/day during 3 weeks during days 194-214, with progressive decrease in dosing).

Figure 2 :

 2 Figure 2: Characterization of anti-emicizumab antibodiesPanel A-B: Emicizumab was immobilized (5 µg/ml) and incubated with control serum (blue), patient serum (red) or IgG-depleted patient serum (black). Bound anti-emicizumab antibodies were probed using peroxidase-labeled IgG-subtype specific antibodies, and detected via 3,3',5,5'-tetramethyl benzidine (TMB) hydrolysis. For panel A, samples were diluted 256-fold, and response was normalized to that of normal plasma, which was arbitrarily set at 1. For panel B, the dose-response for binding of IgG1 antibodies to emicizumab is shown. Panel C: Emicizumab (25 µg/ml) was incubated in the absence or presence of various dilutions of control serum (blue circles) or patient serum (red circles). Presented is the percentage residual emicizumab activity relative to the absence of serum as measured in a chromogenic FVIIIactivity assay using human FIXa and FX. Panel D: Binding of bt-emicizumab (50 µg/ml) to immobilized FIX (5 µg/ml) was performed in the absence or presence of various dilutions of control serum (blue circles) or patient serum (red circles). Bound bt-emicizumab was probed with peroxidase-labeled streptavidin and detected via TMB hydrolysis. Shown is the percentage residual FIX binding relative to the absence of serum. Panel E: Binding of bt-emicizumab (10 µg/ml) to immobilized FX (5 µg/ml) was performed in the absence or presence of various dilutions of control serum (blue circles) or patient serum (red circles). Bound bt-emicizumab was probed with peroxidase-labeled streptavidin and detected via TMB hydrolysis. Shown is the percentage residual FX binding relative to the absence of serum. Statistical assessment was performed using multiple t-test analysis between control and patient serum. Stars indicate p<0.05 as analyzed in a multiple t-test comparing control serum and patient serum. Panel F: Immuno-deficient mice received bt-emicizumab (0.25 mg/kg) alone (orange circles) or in the presence of control serum (100 µl; blue circles) or patient serum (100 µl; red circles) via intravenous injection in the retro-orbital plexus. At indicated time-points, samples were taken and plasma was analyzed for the presence of residual bt-emicizumab. Presented is the percentage of residual bt-emicizumab relative to bt-emicizumab alone at 3 min after injection, which was arbitrarily set at 100%. Lines were generated by fitting the data to an equation describing a single-exponential decay. All panels: Data represent the mean±SD of three experiments.

Disclosure of Conflicts of Interest

AH received fees for consultancy and board expert membership for Roche, Takeda, LFB, CSL-Behring, Sobi and NovoNordisk. DL received consulting fees from Roche (with fees going to Association de Recherche en Hématologie à Necker-Enfants Malades; ARHNEM). PJL received speaker fees from Biotest, Chugai, CSL-Behring, LFB-Biomedicament, NovoNordisk, Roche, Sanofi, Takeda and research support from Sobi, Catalyst Biosciences. All other authors declare no conflict of interest.


Peter J Lenting
email: peter.lenting@inserm.fr

Alexis Texier

Caterina Casari

VON WILLEBRAND FACTOR: FROM FIGURANT TO MAIN CHARACTER IN THE SCENE OF INFLAMMATION

Keywords: von Willebrand factor, LRP1, innate immunity, macrophages

VWF: a multi-purpose protein

Von Willebrand factor (VWF) is a peculiar protein, not only by being the largest protein that travels through our vessels, or by the way hemodynamic forces regulate its platelet-binding functions but also because of its pleiotropic influence on a diverse spectrum of physiological and pathological processes. In addition to the wellestablished mechanisms by which VWF supports primary haemostasis and functions as a chaperone-protein for coagulation factor VIII (FVIII), we now begin to understand in more detail how VWF exerts its activities beyond hemostasis. For instance, the various ways by which VWF may modulate angiogenic processes has been elucidated during the last decade.(1, 2, 3) More recently, it has been shown that VWF promotes smooth muscle cell proliferation via direct interactions with the LRP4-aVb3 receptor complex, thereby inducing signaling pathways that include p38MAP kinase activation.(4).

VWF interacts with macrophages for multiple reasons

Drakeford and colleagues recently reported a new breakthrough in our understanding of VWF function beyond haemostasis, ie its role in innate immunity. [START_REF] Drakeford | von Willebrand factor links primary hemostasis to innate immunity[END_REF] Ample evidence that VWF contributes to inflammatory responses has been reviewed by Kawecki et al.. [START_REF] Kawecki | von Willebrand factor and inflammation[END_REF] VWF is able to recruit leukocytes to sites of inflammation via direct interactions and via the formation of platelet-strings that enable platelet-leukocyte interactions. [START_REF] Pendu | P-selectin glycoprotein ligand 1 and beta 2-integrins cooperate in the adhesion of leukocytes to von Willebrand factor[END_REF][START_REF] Lerolle | von Willebrand factor is a major determinant of ADAMTS-13 decrease during mouse sepsis induced by cecum ligation and puncture[END_REF][START_REF] Petri | von Willebrand factor promotes leukocyte extravasation[END_REF][START_REF] Hillgruber | Blocking von Willebrand factor for treatment of cutaneous inflammation[END_REF][START_REF] Adam | A genetically-engineered von Willebrand disease type 2B mouse model displays defects in hemostasis and inflammation[END_REF][START_REF] Ayme | A Novel Single-Domain Antibody Against von Willebrand Factor A1 Domain Resolves Leukocyte Recruitment and Vascular Leakage During Inflammation-Brief Report[END_REF] However, little was known about the interaction between VWF and another player in the immune system: the macrophage. In the context of VWF biology, macrophages are mainly known as cells that are part of the catabolic pathway that eliminates VWF from the circulation. [START_REF] Van Schooten | Macrophages contribute to the cellular uptake of von Willebrand factor and factor VIII in vivo[END_REF][START_REF] Chion | N-linked glycans within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance[END_REF] In addition, VWF located at the surface of liver macrophages (Kupffer cells) plays a role in the surveillance mechanism that is designed to eliminate platelet/bacteria complexes from the circulation.( 15) Drakeford and colleagues now show that VWF has the potential to drive macrophages into a pro-inflammatory M1-phenotype.(5)

How VWF turns macrophages into pro-inflammatory assets

In a series of elegant experiments, Drakeford et al. demonstrate that VWF binding to macrophages (either THP-1-derived or bloodborne monocyte-derived) induces p38MAP kinase signaling, forcing a change in gene expression pattern with 1334 genes displaying modified expression (Figure 1). Notably, pro-inflammatory cytokines and chemokines are upregulated and increased production of Tumor Necrosis Factor (TNF), interleukin (IL)-6, IL-1b, chemokine C-C ligand (CCL)-2, CCL-3 and CCL-4 was detected. Interestingly, the supernatant of VWF-stimulated macrophages appeared more efficient in recruiting naïve monocytes compared to the supernatant of LPS-stimulated macrophages, illustrating the pro-inflammatory potential of the VWF/macrophage axis.

Perhaps unexpected, VWF not only induced changes in gene associated with the inflammatory response, but also genes involved in metabolic pathways (Fig. 1). These alterations provoked a marked increase in glycolytic activity in combination with changes in the morphology of mitochondria. High glycolytic activity is indeed known to modify the structure of mitochondria, and is characteristic of the inflammatory phenotype of macrophages. [START_REF] O'neill | A guide to immunometabolism for immunologists[END_REF] An important finding during their studies was that VWF-induced macrophage reprogramming is transient, in contrast to changes induced by LPS, which proved long-lasting (Figure 1). Knowing that VWF is one of the socalled acute-phase proteins, it seems possible that transient increases in VWF levels (for instance in response to endothelial stimulation) allow for a temporary, reversible stimulation of macrophages towards the proinflammatory M1-phenotype. This temporary effect may originate from the notion that VWF is efficiently endocytosed and degraded by macrophages.

VWF and LRP1: a flexible interaction

To identify the receptor that mediates the VWF-induced alterations in the gene expression profile and subsequent phenotype of the macrophages, the authors turned to receptors that were previously identified to be involved in the catabolism of VWF. One of these receptors is the scavenger-receptor LDL-receptor related protein-1 (LRP1).( 17) By using dedicated inhibitors of LRP1 (ie. anti-LRP1 antibodies and the universal LRPinhibitor Receptor-Associated Protein), the authors were able to partially neutralize the pro-inflammatory effect of VWF. Signaling via the p38MAP kinase pathway was approximately halved and consequently, macrophages were less willing to turn into the M1-phenotype. This strongly suggests that LRP1 is indeed involved not only in the endocytosis of VWF but also is a key element in VWF-dependent reprogramming of the macrophages. It should be noted that the role of the VWF-LRP1 interaction is perhaps more complex than one would think at first sight. Previous studies have shown that LRP1 can induce both pro-and antiinflammatory outcomes, as illustrated by the findings that a loss of macrophage LRP1 has a dual and opposite effect on atherosclerotic plaque biogenesis, depending on whether the plaque is growing or shrinking. [START_REF] Overton | Deletion of macrophage LDL receptorrelated protein increases atherogenesis in the mouse[END_REF][START_REF] Mueller | Deletion of Macrophage Low-Density Lipoprotein Receptor-Related Protein 1 (LRP1) Accelerates Atherosclerosis Regression and Increases C-C Chemokine Receptor Type 7 (CCR7) Expression in Plaque Macrophages[END_REF] Is it possible that different LRP1-mediated signals into the macrophage are dependent on the ligand that binds to LRP1? In favor of this would be the finding that factor VIII (also a ligand for LRP1) does not modify the phenotype of macrophages. [START_REF] Kis-Toth | Recombinant factor VIII Fc fusion protein drives regulatory macrophage polarization[END_REF] Another possibility is that the multimeric nature of VWF allows for oligomerization of LRP1 receptors at the cellular surface, a condition that perhaps could force LRP1-mediated signaling into the pro-inflammatory direction. Again another option is that there is another yet unidentified receptor involved. LRP1 (and other members of the LRP-family) are known to form heterologous-receptor complexes at the surface of cells, and it is not uncommon that this is done in a cell-specific manner. Examples hereof are complexes of LRP1 and the NMDA-receptor on neuronal cells, of LRP1 and the receptor tyrosine kinase EphA2 on glioblastoma cells, and of LRP1 with the PDGF-receptor on smooth muscle cells. [START_REF] May | Neuronal LRP1 functionally associates with postsynaptic proteins and is required for normal motor function in mice[END_REF][START_REF] Gopal | A novel extracellular Hsp90 mediated co-receptor function for LRP1 regulates EphA2 dependent glioblastoma cell invasion[END_REF][START_REF] Boucher | LRP: role in vascular wall integrity and protection from atherosclerosis[END_REF] Of particular interest in this regard is the capacity of LRP1 to form complexes with b2-integrins at the surface of leukocytes, knowing that these integrins (notably aDb2) are associated with a pro-inflammatory phenotype of macrophages. [START_REF] Spijkers | LDL-receptor-related protein regulates beta 2-integrin-mediated leukocyte adhesion[END_REF][START_REF] Aziz | The Upregulation of Integrin alpha(D)beta(2) (CD11d/CD18) on Inflammatory Macrophages Promotes Macrophage Retention in Vascular Lesions and Development of Atherosclerosis[END_REF] Moreover, b2-integrins are similar to LRP1 in that they can bind to VWF, suggesting that a tertiary complex can be formed between VWF, LRP1 and b2-integrins. [START_REF] Pendu | P-selectin glycoprotein ligand 1 and beta 2-integrins cooperate in the adhesion of leukocytes to von Willebrand factor[END_REF] It would be of interest to investigate whether the formation of this tertiary complex is responsible for the pro-inflammatory reprogramming of macrophages.

In vivo chemotaxis activity of VWF

The in vivo relevance of the VWF-macrophage axis was tested in a mouse model of chemotaxis. Following peritoneal deposition of VWF, the cellular content of the peritoneum was analyzed at 3h and 24h. No differences were observed between vehicle-and VWF-treated mice after 3h. In contrast, after 24h VWFtreated mice were characterized by an increased number of small peritoneal macrophages, a population of macrophages that is known to expand in response to inflammatory stimuli (Fig. 1). Macrophages isolated from the peritoneum also manifested increased expression of pro-inflammatory cytokines, like TNF, IL-6 and IL-1b.

Translational consequences

Having identified VWF as a pro-inflammatory agent, the next question is obviously how this would affect us in daily life? As mentioned above, the link between VWF and inflammation has been fairly well-established in different animal models, including stroke and atherosclerosis. This association is perhaps less clear in the human context, but also less easy to investigate, since confounding variables are often present. However, having more insight into the molecular basis by which VWF exerts its pro-inflammatory activity may help us to further define the conditions in which VWF plays a more or less relevant role in the inflammatory response. In particular, the interplay between lung macrophages and VWF in conditions like systemic inflammatory response syndrome (SIRS) and COVID-19 could be of interest. VWF levels are markedly increased in COVID-19, whereas SIRS is characterized by increased levels of active VWF (a platelet-binding conformation, that also promotes binding to LRP1). [START_REF] Rastegarlari | Macrophage LRP1 contributes to the clearance of von Willebrand factor[END_REF][START_REF] Fogarty | Sustained VWF-ADAMTS-13 axis imbalance and endotheliopathy in long COVID syndrome is related to immune dysfunction[END_REF][START_REF] Rauch | Coagulation biomarkers are independent predictors of increased oxygen requirements in COVID-19[END_REF][START_REF] Hyseni | Active von Willebrand factor predicts 28-day mortality in patients with systemic inflammatory response syndrome[END_REF] Moreover, increased levels of VWF in COVID-19 or active VWF in SIRS are associated with exaggerated inflammatory responses and an increased risk of mortality. [START_REF] Hyseni | Active von Willebrand factor predicts 28-day mortality in patients with systemic inflammatory response syndrome[END_REF][START_REF] Philippe | Von Willebrand factor collagen-binding capacity predicts in-hospital mortality in COVID-19 patients: insight from[END_REF] These findings may provide a rationale for studies aiming to reduce plasma levels of VWF or to interfere with VWF-LRP1 interactions in order to limit the inflammatory response under such conditions.

The other side of the coin would be conditions in which VWF is deficient or dysfunctional, as in von Willebrand disease (VWD). Also here the difficulty is that VWD is a very heterogenous disease, both from a molecular and clinical point of view. It will be complicated to design studies to explore the connection between VWF and innate immunity in such an heterogenous group of patients, especially if these patients receive prophylactic treatment with VWF concentrates. However, the availability of large national healthcare databases may open new avenues to explore this intriguing relationship in more detail.

In conclusion, Drakeford and colleagues have presented an elegant study that further confirms the multifunctional role of VWF beyond hemostasis. Having identified (at least in part) the molecular pathways that contribute to the pro-inflammatory action of VWF towards macrophages. This knowledge will help us to better understand the complex relationship between haemostasis and innate immunity.

Figure 1 VWF

 1 Figure 1 VWF and innate immunity Lenting et al.

Funding

This work received financial support from the National Research Agency (Agence Nationale de la Recherche, ANR-20-CE19-0032-FLAMRING).

Legend Figure 1: VWF turns macrophages into a proinflammatory M1-phenotype

Binding of VWF to LRP1 (and probably a yet unidentified second receptor) at the surface of macrophages induces signaling, resulting in transient changes gene expression, driving these macrophages into a proinflammatory M1-phenotype. Particularly, increased expression and secretion of pro-inflammatory cytokines and chemokines has been observed, promoting chemotaxis in both in vitro and in vivo models. These findings further establish a role of VWF as a pro-inflammatory agent.(5) Figure has been created using Biorender.

Author contribution

All authors contributed to the writing and editing of the manuscript

Conflict of interest

The authors declare no conflict of interest


Franck Nicoud
email: franck.nicoud@umontpellier.fr

Rodrigo Mendez

Alexandre Ranc

Muriel Giansily

Didier Lucor

Simon Mendez

NUMERICAL ASSESSMENT OF DEVICE-RELATED THROMBUS FORMATION TRIGGERED BY THE CONTACT SYSTEM

Keywords: Thrombosis, Contact System, Hemodynamics, Reduced kinetics

Thrombosis is a major concern in blood-coated medical devices. In the present study, pro-coagulant reactions including the contact activation system are coupled with a fluid solver as well as a recently proposed thrombus formation model. The resulting biochemical/fluid model is applied to a backward facing step configuration, a flow configuration that frequently appears in medical devices. Simulations show that the contact system induces a large concentration of thrombin is the recirculation zone without the need of an a priori knowledge of the thrombus location. Platelets are then chemically activated and generate a thrombus whose both position and growth rate are in good agreement with the experimental data available.

INTRODUCTION

One of the main problems of blood-contacting devices is thrombus formation which can lead to device malfunction or thromboembolism. Blood clotting in devices is regulated by a series of intertwined biological processes, such as protein adsorption, platelet activity, complement system and coagulation reactions, as reviewed by Gorbet and Sefton [START_REF] Gorbet | Biomaterial-associated thrombosis: Roles of coagulation factors, complement, platelets and leukocytes[END_REF]. These mechanisms appear due to the presence of the device whose artificial wall lacks the endothelial properties of the vessel. At the material surface, contact activation of factor XII (zymogen of the coagulation cascade) takes place, initiating a cascade of enzymatic reactions [START_REF] Yan | Contact activation by the intrinsic pathway of blood plasma coagulation[END_REF] that produce thrombin, a key coagulation enzyme that activates platelets and forms fibrin. At the same time, platelet adhesion and activation can also occur at the device surface, as explained by Jaffer et al. [START_REF] Jaffer | Medical device-induced thrombosis: what causes it and how can we prevent it?[END_REF]. In the last stage of the thrombus formation process, aggregated platelets and polymerized fibrin form a stable clot that may hinder the device performance.

Computational fluid dynamics (CFD) has been used to study the flow in medical devices and evaluate the risk of thrombosis [START_REF] Yoganathan | Fluid mechanics of heart valves[END_REF]. A straightforward evaluation of thrombosis risk can be performed using specific flow patterns, such as high shear stress yielding to platelet activation [START_REF] Shadden | Potential fluid mechanic pathways of platelet activation[END_REF] or stasis which promotes coagulation [START_REF] De Biasi | Science for surgeons : Understanding pump thrombogenesis in continuous-flow left ventricular assist devices[END_REF]. Approaches focusing only on flow properties do not allow to study the biochemical processes, which play a major role in thrombus formation [START_REF] Fogelson | Fluid Mechanics of Blood Clot Formation[END_REF]. Thus, more exhaustive methods accounting for platelet activity, the coagulation cascade, clot growth and its interaction with the flow have been developed for thrombus triggered by vessel injury [START_REF] Leiderman | Grow with the flow: a spatial-temporal model of platelet deposition and blood coagulation under flow[END_REF], [START_REF] Yazdani | A General Shear-Dependent Model for Thrombus Formation[END_REF]. However, only small computational domains (~100 m) can be considered due to the complexity of these models. Reduced, low-order models accounting for platelet adhesion, activation (by chemical and mechanical pathways) and clot-flow interaction have been developed [START_REF] Taylor | Development of a computational model for macroscopic predictions of device-induced thrombosis[END_REF]Wu et al. 2016). Nevertheless, thrombin produced by the coagulation cascade triggered by the contact system has not been considered in a model of thrombus formation in devices. A model that incorporates the contact activation system and the coagulation reactions until thrombin formation coupled with a realistic representation of flow dynamics is thus needed. This is all the more true as recent clinical studies have pointed at the contact activation phase as a promising therapeutic target providing an anticoagulant strategy without increasing the risk of bleeding [START_REF] Jaffer | Medical device-induced thrombosis: what causes it and how can we prevent it?[END_REF], [START_REF] Larsson | A Factor XIIa Inhibitory Antibody Provides Thromboprotection in Extracorporeal Circulation Without Increasing Bleeding Risk[END_REF].

A first step towards this objective was recently presented by Mendez et al. [START_REF] Mendez | Introducing the pro-coagulant contact system in the numerical assessment of device-related thrombosis[END_REF] who implemented a surface-mediated thrombin production model in a general purpose computational hemodynamics framework. The model accounts for the interaction of the blood flow with the contact activation system and the rest of the blood coagulation cascade. The objective of the present study is then twofold:

1-couple the framework of [START_REF] Mendez | Introducing the pro-coagulant contact system in the numerical assessment of device-related thrombosis[END_REF] to a proper thrombus formation/growth model, 2assess the resulting pipeline by computing a well-controlled configuration relevant to device-induced thrombus formation.

METHODOLOGY

The Navier-Stokes equations are solved along with the Convection-Diffusion-Reaction equations for each chemical species. The set of equations writes:

∇ • 𝒖 = 0 (1) 𝜌 ( 𝜕𝒖 𝜕𝑡 + 𝒖 • ∇𝒖) = -∇p + μ∇ 2 𝒖 -μF(ε)𝒖 (2)
𝜕𝐶 𝑖 𝜕𝑡 + ∇ • (𝐶 𝑖 𝒖) = ∇ • (𝐷 𝑖 ∇𝐶 𝑖 ) + 𝑅 𝑖 ( 3
)
where 𝒖 is the velocity field, p is the pressure field, 𝜌 stands for the density and μ is the dynamic viscosity (both assumed constant-valued). 𝐶 𝑖 represents the concentration of species 𝑖, 𝐷 𝑖 is the diffusion coefficient and 𝑅 𝑖 stands for the reaction source term for each of the coagulation factors, as computed from the kinetic model of Chatterjee et al. [START_REF] Chatterjee | Systems biology of coagulation initiation: Kinetics of thrombin generation in resting and activated human blood[END_REF] (53 species, 63 reactions rates). In order to initiate the coagulation reactions by contact activation of factor XII, the proper boundary condition introduced by Mendez et al. [START_REF] Mendez | Introducing the pro-coagulant contact system in the numerical assessment of device-related thrombosis[END_REF] is used. This condition relates the species diffusive wall flux to the surface reaction rate of factor XII activation. It reads:

𝐷 𝑋𝐼𝐼𝑎 𝜕𝐶 𝑋𝐼𝐼𝑎 𝜕𝑛 = -𝑘 𝑠 𝐶 𝑋𝐼𝐼 ( 4
)
where n is the direction normal to the wall and 𝑘 𝑠 denotes the surface reaction rate of the 𝑋𝐼𝐼 → 𝑋𝐼𝐼 𝑎 reaction. Furthermore, the thrombus evolution is accounted for thanks to the model proposed by Taylor et al. [START_REF] Taylor | Development of a computational model for macroscopic predictions of device-induced thrombosis[END_REF] which considers three biochemical species: non-activated platelets (NP), activated platelets (AP) and Adenosine diphosphate (ADP). Mechanical and chemical activation mechanisms are considered in the model. When a platelet is activated, a certain amount of ADP is released and once a threshold concentration of ADP is reached, chemical activation takes place. As in mechanical activation, chemical activation of platelets also releases ADP and an autoamplification loop is formed. To quantify thrombus growth, the concept of platelet aggregation intensity 𝜀 was introduced in [START_REF] Taylor | Development of a computational model for macroscopic predictions of device-induced thrombosis[END_REF]. The thrombus frontier corresponds to the surface (line in 2D) where the aggregation intensity reaches a pre-defined threshold value. Production/destruction of 𝜀 are also introduced based on the local value of the concentration of activated platelets and viscous shear stress. Finally, the impact of the growing thrombus on the flow is accounted for by considering the thrombus as a porous material and is modeled by adding a Brinkman term in the RHS of the Navier-Stokes equations (see last term of Eq. ( 2)). The details about the evolution equation of 𝜀 and the expression of F(ε) can be found in Taylor et al., 2016 [13]. The coupling between the approaches developed in [START_REF] Mendez | Introducing the pro-coagulant contact system in the numerical assessment of device-related thrombosis[END_REF] and [START_REF] Taylor | Development of a computational model for macroscopic predictions of device-induced thrombosis[END_REF] is done by allowing the platelets to be activated by thrombin instead of only ADP. Moreover, given the simplicity of the thrombus model described above, a reduced order kinetic scheme was used for the coagulation cascade which mimics the scheme of Chatterjee et al. [START_REF] Chatterjee | Systems biology of coagulation initiation: Kinetics of thrombin generation in resting and activated human blood[END_REF] at lower cost (5 species and 9 reaction rates).

The framework described above was applied to predict the thrombus growth in a backward facing step geometry (BFS) inspired from the experimental configuration of Taylor et al. 2014 [START_REF] Taylor | In vitro quantification of time dependent thrombus size using magnetic resonance imaging and computational simulations of thrombus surface shear stresses[END_REF]. The flow equations were solved thanks to the in-house CFD solver YALES2BIO (imag.umontpellier.fr/~yales2bio/) already used by Mendez et al. [START_REF] Mendez | Introducing the pro-coagulant contact system in the numerical assessment of device-related thrombosis[END_REF].

RESULTS

The flow configuration considered is presented in Figure 1 which also displays a typical map of thrombin concentration after convergence. Note that this result corresponds to the case where the thrombus generation/growth model is switched off. This allows illustrating how the coagulation cascade initiated by the contact system is able to produce thrombin behind the BFS, where the characteristic flow time scale is large enough to allow chemistry to dominate. In the other regions of the flow domain, any thrombin production is washed out before accumulation can occur. Note that the boundary condition Eq. ( 4) is applied uniformly over all the walls in Fig. 1. This contrasts with conventional in-vivo models in which the reactions are initialized at user-defined injury sites. When the thrombus generation/growth model is swiched on, the computation predicts blood clotting behind the BFS, as observed in the experimental data of Taylor et al. [START_REF] Taylor | In vitro quantification of time dependent thrombus size using magnetic resonance imaging and computational simulations of thrombus surface shear stresses[END_REF]; this is illustrated in Figure 2. Figure 3 shows that the computed growth of the thrombus is in good agreement with both experimental and computational data. Note however that the evolution of the thrombus is mostly independent (not shown) on the amount of activated platelets injected at the inlet of the flow domain. Indeed, the platelets activation is very efficiently triggered by the thrombin issued by the contact system. This contrasts with the previous study of Taylor et al. 2016 [START_REF] Taylor | Development of a computational model for macroscopic predictions of device-induced thrombosis[END_REF] where the background concentration of activated platelet shows a large influence on the non-normalized results.

Figure 1 :

 1 Figure 1: Schematic of the BFS configuration (left) and concentration of thrombin in absence of thrombus formation modeling (right).

Figure 2 :

 2 Figure 2: Longitudinal velocity component after 5 (left) and 20 (right) minutes. The white line is the boundary of the thrombus.

Figure 3 :

 3 Figure 3: Time evolution of the length (left) and the height (right) of the thrombus behind the BFS.