Anna Nozdracheva

Svetlana Ishenko

Galina Shipareva

Simon Shnoll

Anastasia Sveshnikova

Ekaterina Simonenko

Larisa Menshinina

Vsevolod Tverdislov

Enno Ruuge

Alexander Tikhonov

Valentin Lobyshev

Leonid Yakovenko

Yuruj Nechipurenko

Mariya Popzova

Sergey Yakovenko

Dmitry N Galya

Andrey U Taya

Sergey O Fedya

Sasha R Soslan

Nastya I Polina

Lesha Kirill

Emily Janus-Bell

Alexandra Yakusheva

Cyril Scandola

Nicolas Receveur

Usman Muhammad Ahmed

Clarisse Mouriaux

Catherine Bourdon

Cécile Loubière

Anita Eckly

Yotis A Senis

Mikhail A Panteleev

Christian Gachet

Pierre H Mangin

The role of integrin α5β1 in thrombus formation during hemostasis and arterial thrombosis

Keywords: shear rate, rheology, hemostasis, integrins, platelet, fibrin, arterial thrombosis Le rôle des plaquettes, de

Thanks to my partners in crime

works, helping in realizing my ideas and explaining how present them to the World in a right way. Thank you for tolerating my horrible English and French, my harsh assessments in each sphere of life, my infinity rides home and the millions of my emails each minute of your life.

You helped me to integrate into a grown-up life and to see the wider world. Thank you for helping with all my stuff during relocations, for family diners, for all tickets to football games, for your advices and chatting and for all this care during my time in France! I would like to express my special thanks of gratitude to my teacher and boss Mikhail Panteleev who gave me the opportunity to work in real science. You sparked my interest in science and supported it during long period of time. You believed in me and entrusted the project which has changed my life. You have even been believing in me when I didn't believe in myself. Thank you for allowing me to make my own decisions and for giving me a lot of freedom.

I would like to earnestly acknowledge the sincere efforts and valuable time given by our scientific "grandpa" of Russian team Fazly Ataullakhanov. Thank you for giving me a chance, for all your criticisms and comments, for confidence in me and for forgiveness of all my stunts. Platelets are small discoid-shaped anucleated cells derived from megakaryocytes in the bone marrow and circulating freely in the bloodstream. Their main role is to ensure hemostasis, which represents the physiological process leading to the arrest of bleeding. After vessel lesion, under high flow conditions, von Willebrand factor (vWF) bound to subendothelial collagen recruits platelets through its interaction with the glycoprotein complex (GP) Ib-IX-V. Stable adhesion of platelets is then ensured by the interaction of integrins α2β1, α5β1 and α6β1 with their respective ligands namely collagen, fibronectin and laminins. Integrin αIIbβ3 is also involved in stable platelet adhesion through the binding to vWF and fibronectin. This adhesion step leads to the interaction between subendothelial collagen and GPVI, which initiates strong platelet activation, notably through the release of ADP and TxA2. These soluble agonists, via their receptors, amplify platelet activation by increasing the affinity of integrin αIIbβ3 for its main plasma ligand, soluble fibrinogen, which ensures aggregation and the formation of the hemostatic plug. Some activated platelets expose negatively charged phospholipids on their surface allowing the recruitment of coagulation factors and the generation of thrombin. This serine protease is then able to cleave fibrinogen within the clot into an insoluble fibrin network which stabilizes the aggregate and stops bleeding under physiological conditions.

My thanks to

Table of contents

List of figures

List of tables

A similar process can occur in a pathological context, after erosion or rupture of an evolved atherosclerotic plaque in a diseased artery. This leads to the exposure of tissue factor and highly reactive proteins such as fibrillar collagen, initiating the coagulation cascade and the recruitment and aggregation of flowing platelets. The forming thrombus can become occlusive and block the bloodstream which leads to necrosis of surrounding tissue responsible for a high rate of mortality.

Rheology plays a central role in the regulation of hemostasis and arterial thrombosis.

First of all, it regulates receptor-ligand bond formation during the initial step of platelet adhesion at the site of injury. Once platelets have adhered, hemodynamic forces can stimulate their mechano-receptors to activate them. In addition, the amplification of platelet activation by soluble agonists is finely tuned by the flow which carries them away from the place of injury.

While the importance of blood flow in the regulation of platelet activation, thrombus growth and stability has been defined, the precise rheological conditions occurring after lesion of a healthy vessel remain undetermined. Moreover, while the molecular mechanism that occurs during hemostasis and arterial thrombosis is well established, and the role of the main adhesion receptors has been characterized, the importance of integrin α5β1 remains elusive. Finally, the process by which the platelet plug stops his growth to limit unwanted vessel occlusion at site of lesion of healthy vessels is also unclear.

The main objective of my thesis project consists in: i) the characterization of flow conditions occurring after lesion of a healthy vessel; ii), define the importance of platelet integrin α5β1 in hemostasis and arterial thrombosis; iii) evaluate the role of fibrin in the termination of the hemostatic response.

Review of Literature

I. Hemostasis

Hemostasis is the physiological process leading to the arrest of bleeding [START_REF] Sang | Interplay between platelets and coagulation[END_REF]. It includes primary hemostasis, coagulation and fibrinolysis. Primary hemostasis is mainly ensured by platelets which adhere, activate and aggregate at the injured site (Broos et al., 2011). Coagulation is a cascade of enzymatic reactions leading to generation of thrombin which cleaves fibrinogen within the clot to form an insoluble fibrin network leading to stabilization of the platelet aggregate (Palta et al., 2014). Finally, at the end of the process, fibrinolysis is initiated and generates locally plasmin, which degrades fibrin to dissolve the clot and restore normal blood flow (Gale, 2011).

A. Primary hemostasis: the major role of platelets Primary hemostasis relies on the ability of platelets to form the hemostatic plug at the site of vascular injury to limit bleeding.

Platelet morphology and ultrastructure

Platelets are anucleate fragments derived from the cortical cytoplasm of bone marrow megakaryocytes (Figure 1) [START_REF] Gremmel | Platelet Physiology[END_REF]. In man they have a mean diameter of 3.1 ± 0.3 μm, an average thickness of 1.0 ± 0.2 μm and a volume of approximately 7 fm 3 (David-Ferreira, 1964). After being released from the megakaryocytes, platelets circulate in the blood in a discoid shape for about 7 to 10 days. A normal human platelet count is ranging from 150,000 up to 450,000 platelets per microliter. The platelet cytoskeleton is responsible for the discoid shape of platelets. It is mainly composed of microtubules forming a circumferential network under the plasma membrane and acto-myosin filaments located under the plasma membrane and in the cytosol [START_REF] White | The submembrane filaments of blood platelets[END_REF].

Upon activation, platelets change their shape which is regulated by rapid and dramatic actin filament de-and re-polymerization [START_REF] Bearer | Actin dynamics in platelets[END_REF]. Platelets contain on average seven times more actin by cell volume than non-muscle cells [START_REF] Hartwig | The elegant platelet: signals controlling actin assembly[END_REF]. Inside platelets, actin exists in a dynamic equilibrium between the monomeric or globular form (G-actin) and the polymeric filamentous form (F-actin). The actin dynamics underlying shape changes of platelets depend on a large number of actin-binding proteins. For example, VASP inhibits filament disassembly and Arp2/3 is required to polymerize new filaments [START_REF] Bearer | Actin dynamics in platelets[END_REF].

Platelets have a complete set of organelles: mitochondria, endoplasmic reticulum, glycogen grains, dense tubular system, and granules. Platelets contain three types of granules-

Marginal band

Dense granule Glycogen Alpha granule

Lysosome

Open canalicular system Mitochondria α-granules, dense granules and lysosomeswhich carry distinct cargos and vary in biogenesis, trafficking, and exocytosis [START_REF] Heijnen | Platelet secretory behaviour: as diverse as the granules ... or not?[END_REF].

The α-granules count is 40-80 per platelet with a diameter of 200-500 nm and they account for about 10-16% of the platelet volume [START_REF] Eckly | Respective contributions of single and compound granule fusion to secretion by activated platelets[END_REF][START_REF] Frojmovic | Human platelet size, shape, and related functions in health and disease[END_REF][START_REF] White | Use of the electron microscope for diagnosis of platelet disorders[END_REF]. The content of -granules comes either from its biosynthesis in megakaryocytes or endocytosis from the plasma (Table 1) [START_REF] Fitch-Tewfik | Platelet granule exocytosis: a comparison with chromaffin cells[END_REF][START_REF] Mumford | A review of platelet secretion assays for the diagnosis of inherited platelet secretion disorders[END_REF]. Defects of α-granule formation lead to a severe bleeding disorder named gray platelet syndrome, which highlights the importance of these organelles in platelet function [START_REF] Buchanan | Platelet function in the Chediak-Higashi syndrome[END_REF][START_REF] Costa | A platelet abnormality in the Chediak-Higashi syndrome of man[END_REF].

The dense granules count is 3-8 per platelet and their diameter is about 150 nm [START_REF] White | Use of the electron microscope for diagnosis of platelet disorders[END_REF]. These granules contain small non-protein molecules such as ADP, ATP, serotonin, pyrophosphates, calcium and polyphosphates (Table 1) [START_REF] Holmsen | Physiological functions of platelets[END_REF][START_REF] Smith | Polyphosphate enhances fibrin clot structure[END_REF]. The role of dense granules in hemostasis is evidenced by the bleeding tendency in patients with inherited dense granule deficiency, such as Hermansky-Pudlak syndrome [START_REF] Ambrosio | Storage pool diseases illuminate platelet dense granule biogenesis[END_REF][START_REF] Toro | Chediak-Higashi Syndrome[END_REF] and by increasing bleeding times in mice with dense granule defects [START_REF] Ren | Munc13-4 is a limiting factor in the pathway required for platelet granule release and hemostasis[END_REF].

The lysosomes count is lower than 3 per platelet and their diameter is about 200-250 nm. Platelet lysosomes contain β-hexosaminidase, acid glycohydrolases, membrane bound proteins such as LAMP-1, LAMP-2 and LAMP-3 (Table 1) [START_REF] Holmsen | Measurement of secretion of lysosomal acid glycosidases[END_REF].

Secretion of the lysosomal content has important extracellular functions, such as supporting receptor cleavage, fibrinolysis and degradation of extracellular matrix components, and remodeling of the vasculature [START_REF] Heijnen | Platelet secretory behaviour: as diverse as the granules ... or not?[END_REF].

The molecular mechanisms of primary hemostasis

The molecular mechanisms of primary hemostasis have been widely described. After vessel lesion, under high flow conditions, von Willebrand factor (vWF) bound to subendothelial collagen recruits platelets through its interaction with the glycoprotein complex Ib-IX-V (Savage et al., 1996). Stable adhesion of platelets is then ensured by the interaction of integrins notably, α2β1, α5β1 and α6β1 which bind to their respective ligands namely collagen, fibronectin and laminins (Bergmeier and Hynes, 2012). Integrin αIIbβ3 is also involved in stable platelet adhesion through the binding to vWF and fibronectin [START_REF] Giuliano | Bidirectional integrin alphaIIbbeta3 signalling regulating platelet adhesion under flow: contribution of protein kinase C[END_REF][START_REF] Maurer | Fibrillar cellular fibronectin supports efficient platelet aggregation and procoagulant activity[END_REF]. This adhesion step leads to the interaction of collagen with GPVI, which initiates strong platelet activation, that is amplified through the release of the soluble agonists, ADP, ATP and TxA2 [START_REF] Zahid | The future of glycoprotein VI as an antithrombotic target[END_REF]. These soluble agonists via their receptors, P2Y1 and P2Y12 for ADP, P2X1 for ATP and the thromboxane receptor (TP) for TxA2, amplify platelet activation by increasing the affinity of integrin αIIbβ3 for its main plasma ligand, soluble fibrinogen, which ensures aggregation and the formation of the hemostatic plug (Figure 2) [START_REF] Versteeg | New fundamentals in hemostasis[END_REF].

Figure 2:

The molecular mechanisms of primary hemostasis. ADP, adenosine diphosphate; ATP, adenosine triphosphate; Fgn, fibrinogen; FN, fibronectin; vWF, von Willebrand factor; GP, glycoprotein complex; LM, laminin; PAR, protease-activated receptor; TP, thromboxane receptor; TxA2, thromboxane A2.

Platelet receptors involved in primary hemostasis

Primary hemostasis is mediated by receptors that allow platelet adhesion, activation and aggregation.

Activation and adhesion receptors

The GPIb-IX-V Complex

The GPIb-IX-V complex is expressed only in megakaryocytes and platelets. It belongs to the leucine-rich repeat protein family [START_REF] Hickey | Human platelet glycoprotein IX: an adhesive prototype of leucine-rich glycoproteins with flank-center-flank structures[END_REF][START_REF] Lopez | The alpha and beta chains of human platelet glycoprotein Ib are both transmembrane proteins containing a leucine-rich amino acid sequence[END_REF]. GPIb-IX-V contains 4 types of subunits: 1 GPIbα molecule linked to 2 GPIbβ subunits via disulfide bridges to form the GPIb; the latter is non-covalently associated with 1 GPIX subunit to form the GPIb-IX complex. GPIb-IX complex is weakly linked to one GPV subunit [START_REF] Luo | Glycoprotein Ibalpha forms disulfide bonds with 2 glycoprotein Ibbeta subunits in the resting platelet[END_REF][START_REF] Mo | Transmembrane domains are critical to the interaction between platelet glycoprotein V and glycoprotein Ib-IX complex[END_REF][START_REF] Modderman | Glycoproteins V and Ib-IX form a noncovalent complex in the platelet membrane[END_REF][START_REF] Phillips | Platelet plasma membrane glycoproteins. Evidence for the presence of nonequivalent disulfide bonds using nonreduced-reduced two-dimensional gel electrophoresis[END_REF]. There are about 25,000 copies of GPIb-IX and 12,500 copies of GPV per platelet which was measured by antibody binding [START_REF] Modderman | Glycoproteins V and Ib-IX form a noncovalent complex in the platelet membrane[END_REF].

The main ligand of GPIb is vWF, which becomes instrumental in platelet recruitment at elevated shear (>900 s -1 ) at site of vessel injury or to a growing thrombus. In vitro, this interaction induces platelet rolling on immobilized vWF which is explained by the fact that GPIb-vWF bonds have fast association and dissociation rates [START_REF] Fressinaud | von Willebrand factor-mediated platelet adhesion to collagen involves platelet membrane glycoprotein IIb-IIIa as well as glycoprotein Ib[END_REF]. GPIb also binds to additional ligands including thrombin, P-selectin, thrombospondin-1, factor XI, factor XII and high-molecular weight kininogen, which all bind to the 45-kDa globular Nterminal extracellular domain of the receptor [START_REF] Andrews | Glycoprotein Ib-IX-V[END_REF][START_REF] Bradford | Human kininogens regulate thrombin binding to platelets through the glycoprotein Ib-IX-V complex[END_REF][START_REF] Bradford | Human factor XII binding to the glycoprotein Ib-IX-V complex inhibits thrombin-induced platelet aggregation[END_REF][START_REF] Jurk | Thrombospondin-1 mediates platelet adhesion at high shear via glycoprotein Ib (GPIb): an alternative/backup mechanism to von Willebrand factor[END_REF][START_REF] Simon | Platelet glycoprotein ibalpha is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/CD18)[END_REF]. At the intracytoplasmic level, GPIbα interacts with the actin binding protein filamin A, phosphoinositide 3-kinase and the adapter 14-3-3ζ through its cytoplasmic tail domain [START_REF] Bryckaert | Of von Willebrand factor and platelets[END_REF].

Ligation of vWF to the GPIbα subunit induces an intracellular signal involving a member of the Src-kinase family, which leads to phospholipase Cγ2 activation, and subsequent Ca 2+ release from internal stores generating oscillations [START_REF] Ozaki | Platelet GPIb-IX-Vdependent signaling[END_REF]. This signal is relatively weak, but is nevertheless proposed to contribute to αIIbβ3 activation and filopodia extension (Figure 3) [START_REF] Fredrickson | Shear-dependent rolling on von Willebrand factor of mammalian cells expressing the platelet glycoprotein Ib-IX-V complex[END_REF][START_REF] Mangin | Signaling role for phospholipase C gamma 2 in platelet glycoprotein Ib alpha calcium flux and cytoskeletal reorganization. Involvement of a pathway distinct from FcR gamma chain and Fc gamma RIIA[END_REF].

Figure 3:

The signaling pathway triggered by the GPIb-IX-V complex. Akt, protein kinase B alpha; DAG, diacylglycerol; GP, glycoprotein; IP3, inositol 1,4,5-triphosphate; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PKG, protein kinase G; PLCγ, phospholipase Cγ; Syk, spleen tyrosine kinase (Adapted from [START_REF] Andrews | Glycoprotein Ib-IX-V[END_REF]).

Defects of one of the members of the GPIb-IX complex leads to a rare bleeding disorder named Bernard-Soulier syndrome, which is characterized by a macrothrombocytopenia, highlighting the importance of this receptor in hemostasis [START_REF] Buchanan | Platelet function in the Chediak-Higashi syndrome[END_REF][START_REF] Costa | A platelet abnormality in the Chediak-Higashi syndrome of man[END_REF][START_REF] Liang | A common ancestral glycoprotein (GP) 9 1828A>G (Asn45Ser) gene mutation occurring in European families from Australia and Northern Europe with Bernard-Soulier Syndrome (BSS)[END_REF][START_REF] Strassel | Intrinsic impaired proplatelet formation and microtubule coil assembly of megakaryocytes in a mouse model of Bernard-Soulier syndrome[END_REF]. Numerous in vitro and in vivo studies have shown that absence of the GPIb-IX complex or blockade of the vWF-GPIb interaction reduces experimental thrombosis, indicating the importance of the GPIb-IX complex in thrombus formation [START_REF] Bergmeier | The role of platelet adhesion receptor GPIbalpha far exceeds that of its main ligand, von Willebrand factor, in arterial thrombosis[END_REF][START_REF] Konstantinides | Distinct antithrombotic consequences of platelet glycoprotein Ibalpha and VI deficiency in a mouse model of arterial thrombosis[END_REF][START_REF] Maurer | Targeting platelet GPIbbeta reduces platelet adhesion, GPIb signaling and thrombin generation and prevents arterial thrombosis[END_REF]. In contrast, this receptor does not appear to play a major role in thrombus stability [START_REF] Ni | Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen[END_REF].

Targeting GPIb-IX complex is recognized as a potential efficient antithrombotic strategy, however, the function blocking antibodies could have an impact on the bleeding risk and need therefore to be further investigated.

Glycoprotein VI

GPVI is a 62 kDa glycoprotein expressed only on platelets and megakaryocytes [START_REF] Nieswandt | Platelet-collagen interaction: is GPVI the central receptor?[END_REF][START_REF] Zahid | The future of glycoprotein VI as an antithrombotic target[END_REF]. It is best known as the main platelet activating receptor for collagen. It belongs to the Ig receptor superfamily and has: i) two extracellular Ig domains (D1 and D2), to which ligands are binding, ii) a mucin-like stalk rich in serine and threonine with sites of O-glycosylation, iii) a single transmembrane helix and iv) a cytoplasmic tail. There are 3,000 to 4,000 copies of GPVI per platelet [START_REF] Best | GPVI levels in platelets: relationship to platelet function at high shear[END_REF].

GPVI is mainly expressed as a monomer on resting platelets, but 30% of it is expressed as a dimer and this proportion increases upon activation up to 44% [START_REF] Jung | Constitutive dimerization of glycoprotein VI (GPVI) in resting platelets is essential for binding to collagen and activation in flowing blood[END_REF]. GPVI is associated with two FcRγ-chains through a salt bridge [START_REF] Feng | Convergence on a distinctive assembly mechanism by unrelated families of activating immune receptors[END_REF]. The FcRγ-chain contains an immunoreceptor tyrosine-based activation motif (ITAM) which initiates a signaling cascade following GPVIligand interaction. The coupling to the FcRγ-chain with GPVI is necessary for the surface expression of GPVI, and to initiate its signaling [START_REF] Zheng | Expression of the platelet receptor GPVI confers signaling via the Fc receptor gamma -chain in response to the snake venom convulxin but not to collagen[END_REF].

Ligand binding to GPVI promotes its clustering and brings into close proximity the Src family kinases, Fyn and Lyn, bound to the proline-rich region of the cytoplasmic tail of GPVI and the ITAM domain of the FcRγ-chain, promoting its phosphorylation [START_REF] Ezumi | Physical and functional association of the Src family kinases Fyn and Lyn with the collagen receptor glycoprotein VI-Fc receptor gamma chain complex on human platelets[END_REF][START_REF] Quek | Fyn and Lyn phosphorylate the Fc receptor gamma chain downstream of glycoprotein VI in murine platelets, and Lyn regulates a novel feedback pathway[END_REF][START_REF] Suzuki-Inoue | Association of Fyn and Lyn with the proline-rich domain of glycoprotein VI regulates intracellular signaling[END_REF]. This leads to the recruitment and phosphorylation of the tyrosine kinase Syk which initiates a downstream signaling cascade leading to the formation of a LAT-based signaling complex located at the cell membrane [START_REF] Pasquet | LAT is required for tyrosine phosphorylation of phospholipase cgamma2 and platelet activation by the collagen receptor GPVI[END_REF]. Formation of this complex allows effector proteins such as the tyrosine kinases Btk and Tec to come into contact with their substrate resulting in the activation of phospholipase Cγ2 [START_REF] Watson | GPVI and integrin alphaIIb beta3 signaling in platelets[END_REF]. Phospholipase Cγ2 hydrolyzes phospho-inositol-4,5-bisphosphate into DAG and inositol-1,4,5-triphosphate, which leads to the release of Ca 2+ from internal stores into the cytosol. Post-calcium events are then initiated which ultimately increase the affinity of αIIbβ3 for its main ligand, fibrinogen, promoting platelet aggregation. The GPVI signaling pathway is particularly efficient in releasing soluble agonists such as ADP and TxA2, which enhance platelet activation (Figure 4) [START_REF] Ahmed | Pharmacological Blockade of Glycoprotein VI Promotes Thrombus Disaggregation in the Absence of Thrombin[END_REF].

Fibrillar collagen is the first identified, best known and most potent ligand of GPVI [START_REF] Nieswandt | Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen[END_REF]. The glycine-proline-hydroxyproline (GPO) sequences of type I and type III collagens are instrumental to bind GPVI [START_REF] Jarvis | Identification of a major GpVI-binding locus in human type III collagen[END_REF]. Additional ligands have been identified including the adhesive proteins fibrinogen [START_REF] Induruwa | Platelet collagen receptor Glycoprotein VI-dimer recognizes fibrinogen and fibrin through their D-domains, contributing to platelet adhesion and activation during thrombus formation[END_REF][START_REF] Mangin | Immobilized fibrinogen activates human platelets through glycoprotein VI[END_REF], fibrin [START_REF] Alshehri | Fibrin activates GPVI in human and mouse platelets[END_REF][START_REF] Mammadova-Bach | Platelet glycoprotein VI binds to polymerized fibrin and promotes thrombin generation[END_REF] and laminins [START_REF] Inoue | Laminin stimulates spreading of platelets through integrin alpha6beta1-dependent activation of GPVI[END_REF]. A family of snake venom toxinsthe C-type lectins (convulxin, ophioluxin, alboagregin-A and alboluxin)have been shown to activate GPVI [START_REF] Dormann | Alboaggregin A activates platelets by a mechanism involving glycoprotein VI as well as glycoprotein Ib[END_REF]Du et al., 2002a;Du et al., 2002b;[START_REF] Murakami | Crystal structure of the platelet activator convulxin, a disulfide-linked alpha4beta4 cyclic tetramer from the venom of Crotalus durissus terrificus[END_REF]. GPVI is not considered as a key platelet receptor for hemostasis as patients presenting a defect in GPVI only present minor bleedings such as purpura or epistaxis [START_REF] Jandrot-Perrus | Platelet glycoprotein VI genetic quantitative and qualitative defects[END_REF]. In addition, mice deficient or immuno-depleted for GPVI do not present a prolongation in tail-bleeding time [START_REF] Kato | The contribution of glycoprotein VI to stable platelet adhesion and thrombus formation illustrated by targeted gene deletion[END_REF][START_REF] Lockyer | GPVI-deficient mice lack collagen responses and are protected against experimentally induced pulmonary thromboembolism[END_REF][START_REF] Mangin | A humanized glycoprotein VI (GPVI) mouse model to assess the antithrombotic efficacies of anti-GPVI agents[END_REF].

In contrast, numerous studies based on in vitro, ex vivo and in vivo models have identified a role of these receptor in some experimental models of thrombosis [START_REF] Andrews | Targeting GPVI as a novel antithrombotic strategy[END_REF][START_REF] Bender | Genetic and antibody-induced glycoprotein VI deficiency equally protects mice from mechanically and FeCl(3) -induced thrombosis[END_REF]Hechler et al., 2010;[START_REF] Munnix | The glycoprotein VIphospholipase Cgamma2 signaling pathway controls thrombus formation induced by collagen and tissue factor in vitro and in vivo[END_REF]. Ultrasound or mechanical injuries of atherosclerotic plaque in aged ApoE -/-mice, which are proposed to better mimic pathological conditions of atherothrombosis, further highlighted an important role of GPVI in arterial thrombosis [START_REF] Cosemans | Contribution of platelet glycoprotein VI to the thrombogenic effect of collagens in fibrous atherosclerotic lesions[END_REF]Hechler and Gachet, 2011a;[START_REF] Kuijpers | Complementary roles of platelets and coagulation in thrombus formation on plaques acutely ruptured by targeted ultrasound treatment: a novel intravital model[END_REF][START_REF] Mangin | Thrombin overcomes the thrombosis defect associated with platelet GPVI/FcRgamma deficiency[END_REF][START_REF] Massberg | A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo[END_REF]. Together, these observations suggest that GPVI represents an attractive anti-thrombotic target with a potential minor effect on bleedings.

3.1.3. C-type lectin-like type II transmembrane receptor C-type lectin-like type II transmembrane receptor (CLEC-2) is a 30 kDa protein expressed on platelets and megakaryocytes, and at low levels on dendritic cells and myeloid cells [START_REF] Lowe | The expression of mouse CLEC-2 on leucocyte subsets varies according to their anatomical location and inflammatory state[END_REF][START_REF] Suzuki-Inoue | A novel Syk-dependent mechanism of platelet activation by the C-type lectin receptor CLEC-2[END_REF]. Its extracellular domain consists of a stem and a carbohydrate-recognizing domain (CTLD) [START_REF] Watson | The platelet receptor CLEC-2 is active as a dimer[END_REF] and its cytoplasmic domain is composed of a short cytosolic tail harboring a single YxxL sequence termed hemITAM [START_REF] Watson | The crystal structure and mutational binding analysis of the extracellular domain of the plateletactivating receptor CLEC-2[END_REF][START_REF] Watson | GPVI and CLEC-2 in hemostasis and vascular integrity[END_REF]. There are 2,000 to 4,000 copies of CLEC-2 per platelet [START_REF] Gitz | CLEC-2 expression is maintained on activated platelets and on platelet microparticles[END_REF]. On non-activated platelets, CLEC-2 is mainly expressed as a monomer with only about a third of the receptors being in a dimerized state, but after ligand binding with CLEC-2, most molecules pass into an oligomerized state, which contributes to the receptor clustering to induce signals [START_REF] Hughes | CLEC-2 activates Syk through dimerization[END_REF][START_REF] Martin | The structure of CLEC-2: mechanisms of dimerization and higher-order clustering[END_REF][START_REF] Martyanov | Control of Platelet CLEC-2-Mediated Activation by Receptor Clustering and Tyrosine Kinase Signaling[END_REF][START_REF] Martyanov | CLEC-2 induced signalling in blood platelets[END_REF].

CLEC-2 was identified by affinity chromatography as a receptor for rhodocytin, the snake C type lectin toxin, which activates platelets through a Src kinase-regulated pathway (Suzuki- [START_REF] Inoue | Laminin stimulates spreading of platelets through integrin alpha6beta1-dependent activation of GPVI[END_REF]. In contrast to the GPVI pathway, CLEC-2 activates Syk through a dimerization mechanism via a hemITAM, while downstream signaling is similar to the one induced by GPVI, with phosphorylation of Syk, LAT, SLP-76, Btk and PLCγ2 [START_REF] Fuller | The C-type lectin receptors CLEC-2 and Dectin-1, but not DC-SIGN, signal via a novel YXXL-dependent signaling cascade[END_REF][START_REF] Spalton | The novel Syk inhibitor R406 reveals mechanistic differences in the initiation of GPVI and CLEC-2 signaling in platelets[END_REF][START_REF] Suzuki-Inoue | A novel Syk-dependent mechanism of platelet activation by the C-type lectin receptor CLEC-2[END_REF] ultimately leading to Ca 2+ release and platelet activation events (Figure 5) [START_REF] Lombard | Mouse podoplanin supports adhesion and aggregation of platelets under arterial shear: A novel mechanism of haemostasis[END_REF]. [START_REF] Astarita | Podoplanin: emerging functions in development, the immune system, and cancer[END_REF]. Under pathological conditions it was found on the surface of malignant tumors (squamous cell carcinomas, melanomas, gliomas) [START_REF] Sekiguchi | Targeting a novel domain in podoplanin for inhibiting platelet-mediated tumor metastasis[END_REF][START_REF] Shirai | C-type lectin-like receptor 2 promotes hematogenous tumor metastasis and prothrombotic state in tumor-bearing mice[END_REF], macrophages during macro-inflammation process [START_REF] Hitchcock | Inflammation drives thrombosis after Salmonella infection via CLEC-2 on platelets[END_REF] and inside atherosclerotic plaques [START_REF] Inoue | Vascular Smooth Muscle Cells Stimulate Platelets and Facilitate Thrombus Formation through Platelet CLEC-2: Implications in Atherothrombosis[END_REF].

CLEC-2 does not appear to play a key role in hemostasis, since bleeding time in mice with CLEC-2 deficiency is not prolonged [START_REF] Shirai | C-type lectin-like receptor 2 promotes hematogenous tumor metastasis and prothrombotic state in tumor-bearing mice[END_REF]. However, mice deficient in CLEC-2 or treated with a depleting anti-podoplanin antibody have been reported to experience reduced experimental thrombosis [START_REF] Bender | Combined in vivo depletion of glycoprotein VI and Ctype lectin-like receptor 2 severely compromises hemostasis and abrogates arterial thrombosis in mice[END_REF][START_REF] Inoue | Vascular Smooth Muscle Cells Stimulate Platelets and Facilitate Thrombus Formation through Platelet CLEC-2: Implications in Atherothrombosis[END_REF][START_REF] May | CLEC-2 is an essential platelet-activating receptor in hemostasis and thrombosis[END_REF][START_REF] Suzuki-Inoue | Essential in vivo roles of the C-type lectin receptor CLEC-2: embryonic/neonatal lethality of CLEC-2-deficient mice by blood/lymphatic misconnections and impaired thrombus formation of CLEC-2-deficient platelets[END_REF]. Podoplanin is unlikely to be the ligand explaining the potential role of CLEC-2 in thrombosis models as it is not found on the healthy vessel wall. Of note, the role of CLEC-2 in experimental thrombosis could not be confirmed in our laboratory with GPIbCre-CLEC-2-deficent mice which presented a normal thrombus formation after mechanical injury of the aorta and FeCl3 of the carotid artery (unpublished data). CLEC-2 has also been shown to be involved in arterial thrombosis independently of hemITAM signaling. Indeed, mice presenting the CLEC-2 receptor with a mutation impairing hemITAM signaling, have defects in thrombus formation only with an anti-CLEC-2 antibody [START_REF] Haining | CLEC-2 contributes to hemostasis independently of classical hemITAM signaling in mice[END_REF].

CLEC-2/podoplanin interaction has been proposed to play a role beyond hemostasis. As podoplanin is present on the surface of endothelial cells of lympho-capillaries, CLEC-2mediated platelet activation appears important during vessel development to block blood effusion and to prevent blood filling of the lymphatic system, a process potentially restricted to chronic vascular remodeling [START_REF] Haining | Lymphatic blood filling in CLEC-2-deficient mouse models[END_REF][START_REF] Suzuki-Inoue | Essential in vivo roles of the C-type lectin receptor CLEC-2: embryonic/neonatal lethality of CLEC-2-deficient mice by blood/lymphatic misconnections and impaired thrombus formation of CLEC-2-deficient platelets[END_REF][START_REF] Zhang | Transient loss of venous integrity during developmental vascular remodeling leads to red blood cell extravasation and clearance by lymphatic vessels[END_REF]. In addition, a role for the CLEC-2-podoplanin interaction has been shown to contribute to tumor progression notably through the ability of podoplanin expressed on tumor cells to promote platelet aggregation [START_REF] Christofori | Cancer: division of labour[END_REF]. In agreement with this study, anti-podoplanin antibody has been reported to suppress lung colonization of colon adenocarcinoma intestines revealing that CLEC-2-mediated platelet activation is a potential trigger of tumor metastasis [START_REF] Sugimoto | Suppression of experimental lung colonization of a metastatic variant of murine colon adenocarcinoma 26 by a monoclonal antibody 8F11 inhibiting tumor cell-induced platelet aggregation[END_REF][START_REF] Suzuki-Inoue | Platelets and cancer-associated thrombosis: focusing on the platelet activation receptor CLEC-2 and podoplanin[END_REF]).

Integrins

Integrins are a family of transmembrane glycoproteins functioning as cell adhesion and signaling molecules. They are heterodimers composed of α and β subunits which associate noncovalently [START_REF] Burke | Invertebrate integrins: structure, function, and evolution[END_REF]. Each subunit consists of 3 domains: a large extracellular domain that is responsible for ligand binding, a single-pass transmembrane domain and a smaller cytoplasmic tail [START_REF] Hynes | Integrins: bidirectional, allosteric signaling machines[END_REF]. In mammals, 24 integrins have been described which consist of the association of 18 different α-chains with 8 β subunits [START_REF] Humphries | Integrin ligands at a glance[END_REF][START_REF] Kinashi | Intracellular signalling controlling integrin activation in lymphocytes[END_REF]Luo et al., 2007a). Integrins are regulating many biological functions, such as cell migration, proliferation, differentiation, survival and apoptosis [START_REF] Nieswandt | Integrins in platelet activation[END_REF]. Two subgroups of integrins are present on human platelets: β1 and β3 families, which account for a total of five human platelet integrins: α2β1, α5β1, α6β1, αvβ3 and αIIbβ3 [START_REF] Piotrowicz | Glycoprotein Ic-IIa functions as an activation-independent fibronectin receptor on human platelets[END_REF][START_REF] Sonnenberg | Laminin receptor on platelets is the integrin VLA-6[END_REF][START_REF] Staatz | The membrane glycoprotein Ia-IIa (VLA-2) complex mediates the Mg++-dependent adhesion of platelets to collagen[END_REF].

Integrins can adopt three different conformational states as identified by electronic microscopy [START_REF] Takagi | Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling[END_REF]: i) on resting platelets, integrins are mostly inactive with low affinity for extracellular ligands. The integrin ectodomain is folded forming an inverted V which presents a closed binding side; ii) after platelet activation which generates an inside-out signal, integrins switch to an intermediate state in which the molecule expands, but contains a closed globular head. In this conformation, integrins have an intermediate affinity for their ligands [START_REF] Huang | Platelet integrin alphaIIbbeta3: signal transduction, regulation, and its therapeutic targeting[END_REF]; and iii) in case of stronger signals integrins expand and the globular head opens allowing a high affinity for ligands [START_REF] Chen | An integrin alphaIIbbeta3 intermediate affinity state mediates biomechanical platelet aggregation[END_REF]. Besides these conformational changes, integrins can cluster into oligomers to increase avidity for their ligands [START_REF] Carman | Integrin avidity regulation: are changes in affinity and conformation underemphasized?[END_REF].

Inside-out signaling of integrins, which has been mainly studied for IIb3, is initiated upon ligand binding of agonists such as collagen, ADP, TxA2 or thrombin to their receptors (Figures 6). respectively. This promotes activation of a small GTPase Rap1 which is a common downstream activator in this signaling pathway [START_REF] Boettner | Control of cell adhesion dynamics by Rap1 signaling[END_REF][START_REF] Bos | Linking Rap to cell adhesion[END_REF][START_REF] Katagiri | Rap1 and integrin inside-out signaling[END_REF]. Activated Rap1b forms a complex with the integrin activator talin through Rap1-GTP-interacting adaptor molecule [START_REF] Han | Reconstructing and deconstructing agonist-induced activation of integrin alphaIIbbeta3[END_REF] and thus facilitates talin interaction with the cytoplasmic tail of the β subunit [START_REF] Bromberger | Direct Rap1/Talin1 interaction regulates platelet and neutrophil integrin activity in mice[END_REF] resulting in conformational change and increase in the affinity of integrins for their ligands [START_REF] Lefort | Distinct roles for talin-1 and kindlin-3 in LFA-1 extension and affinity regulation[END_REF]. Kindlin-3

cooperates with talin to facilitate talin binding to the β subunit [START_REF] Haydari | Kindlin Assists Talin to Promote Integrin Activation[END_REF]. CalDAG-GEFI, calcium and DAG-regulated guanine-nucleotide-exchange factor; CLEC- [START_REF] Stefanini | RASA3 is a critical inhibitor of RAP1dependent platelet activation[END_REF]).

The ligand binding to integrins initiates an outside-in signal, which has also been mainly studied for IIb3 (Luo et al., 2007a). This process initiates focal adhesion kinase (FAK) and Src recruitment to the β integrin cytoplasmic tail. They phosphorylate paxillin and P130C, leading to Rac activation. This Rac pathway promotes formation of lamellipodial protrusion [START_REF] Kurokawa | Mechanism and role of localized activation of Rho-family GTPases in growth factor-stimulated fibroblasts and neuronal cells[END_REF][START_REF] Machacek | Coordination of Rho GTPase activities during cell protrusion[END_REF]. In parallel, integrin linked kinase (ILK) is recruited to the β integrin cytoplasmic domain and together with Src promote Akt activity. This

Akt pathway regulates sensitivity to thrombin receptor activating peptide (TRAP)-dependent fibrinogen binding and secretion of dense and α-granule contents [START_REF] Woulfe | Defects in secretion, aggregation, and thrombus formation in platelets from mice lacking Akt2[END_REF]. Activated Src also initiates Ras-Erk pathway which regulates store-mediated Ca 2+ entry in human platelets (Figure 7) [START_REF] Hu | Integrin bi-directional signaling across the plasma membrane[END_REF][START_REF] Rosado | Role of the ERK pathway in the activation of storemediated calcium entry in human platelets[END_REF].

Integrin α2β1

Integrin α2β1 is expressed on the surface of platelets, fibroblasts, epithelial, and endothelial cells [START_REF] Ojalill | Integrin alpha2beta1 decelerates proliferation, but promotes survival and invasion of prostate cancer cells[END_REF][START_REF] Zutter | Widespread histologic distribution of the alpha 2 beta 1 integrin cell-surface collagen receptor[END_REF]. Two polymorphisms in the 2 gene are associated with the cell-surface density of integrin 21 [START_REF] Corral | The number of platelet glycoprotein Ia molecules is associated with the genetically linked 807 C/T and HPA-5 polymorphisms[END_REF][START_REF] Kritzik | Nucleotide polymorphisms in the alpha2 gene define multiple alleles that are associated with differences in platelet alpha2 beta1 density[END_REF]. The expression of this receptor varies between 2,000 and 8,000 copies per platelet [START_REF] Di Paola | Platelet glycoprotein I(b)alpha and integrin alpha2 beta1 polymorphisms: gene frequencies and linkage disequilibrium in a population diversity panel[END_REF]. The α2β1 integrin is the first collagen receptor identified on platelets [START_REF] Santoro | Isolation and characterization of a platelet surface collagen binding complex related to VLA-2[END_REF] and mainly supports stable adhesion [START_REF] Sarratt | GPVI and alpha2beta1 play independent critical roles during platelet adhesion and aggregate formation to collagen under flow[END_REF]. It recognizes type I fibrillar collagen with high affinity [START_REF] Knight | The collagen-binding A-domains of integrins alpha(1)beta(1) and alpha(2)beta(1) recognize the same specific amino acid sequence, GFOGER, in native (triple-helical) collagens[END_REF], but can also bind type IV subendothelial collagen [START_REF] Vandenberg | Characterization of a type IV collagen major cell binding site with affinity to the alpha 1 beta 1 and the alpha 2 beta 1 integrins[END_REF]. Integrin α2β1 has also a large number of additional ligands, such as tenascin C, laminins, proteoglycans endorepellin/ perlecan and decorin [START_REF] Bix | Endorepellin causes endothelial cell disassembly of actin cytoskeleton and focal adhesions through alpha2beta1 integrin[END_REF][START_REF] Chan | Multiple functional forms of the integrin VLA-2 can be derived from a single alpha 2 cDNA clone: interconversion of forms induced by an anti-beta 1 antibody[END_REF][START_REF] Guidetti | The small proteoglycan decorin supports adhesion and activation of human platelets[END_REF][START_REF] Sriramarao | Endothelial cell attachment and spreading on human tenascin is mediated by alpha 2 beta 1 and alpha v beta 3 integrins[END_REF].

The outside-in signaling pathway of this receptor results in shape change, filopodia extension, lamellipodia formation and platelet spreading [START_REF] Inoue | Integrin alpha2beta1 mediates outside-in regulation of platelet spreading on collagen through activation of Src kinases and PLCgamma2[END_REF].

Integrin α2β1 does not seem to play a critical role in hemostasis. Two patients with α2β1 genetic defects presented only a moderate bleeding phenotype [START_REF] Kehrel | Deficiency of intact thrombospondin and membrane glycoprotein Ia in platelets with defective collagen-induced aggregation and spontaneous loss of disorder[END_REF][START_REF] Nieuwenhuis | Human blood platelets showing no response to collagen fail to express surface glycoprotein Ia[END_REF]. This result is in agreement with the normal tail-bleeding time in mice with a deficiency of the α2 chain [START_REF] Chen | The alpha(2) integrin subunit-deficient mouse: a multifaceted phenotype including defects of branching morphogenesis and hemostasis[END_REF][START_REF] Habart | Conditional knockout of integrin alpha2beta1 in murine megakaryocytes leads to reduced mean platelet volume[END_REF][START_REF] Holtkotter | Integrin alpha 2-deficient mice develop normally, are fertile, but display partially defective platelet interaction with collagen[END_REF][START_REF] Nieswandt | Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen[END_REF]. In humans with overexpression of α2β1, an increased risk of myocardial infarction, diabetic retinopathy and stroke has been reported, pointing out to a role of this integrin in arterial thrombosis [START_REF] Matsubara | Association between diabetic retinopathy and genetic variations in alpha2beta1 integrin, a platelet receptor for collagen[END_REF][START_REF] Santoso | Association of the platelet glycoprotein Ia C807T gene polymorphism with nonfatal myocardial infarction in younger patients[END_REF]. The role of integrin α2β1 in thrombus formation and stability has been demonstrated in a flow-based assay consisting in blood perfusion over collagen [START_REF] He | The contributions of the alpha 2 beta 1 integrin to vascular thrombosis in vivo[END_REF][START_REF] Kuijpers | Role of murine integrin alpha2beta1 in thrombus stabilization and embolization: contribution of thromboxane A2[END_REF]. The role of this integrin has also been shown in experimental thrombosis in in vivo mouse models based on chemical injuries (FeCl3; Rose Bengal) but appears dispensable after intravascular injection of collagen, a model mimicking thromboembolism [START_REF] He | The contributions of the alpha 2 beta 1 integrin to vascular thrombosis in vivo[END_REF][START_REF] Kuijpers | Role of murine integrin alpha2beta1 in thrombus stabilization and embolization: contribution of thromboxane A2[END_REF].

and adult tissues, including vascular endothelial and smooth muscle cells [START_REF] Magnusson | Fibronectin: structure, assembly, and cardiovascular implications[END_REF]. Integrin α6β1 can also interact with TSP-1 as proposed after observation of a reduction in adhesion to TSP-1 of platelets deficient for α6 [START_REF] Schaff | Integrin alpha6beta1 is the main receptor for vascular laminins and plays a role in platelet adhesion, activation, and arterial thrombosis[END_REF]. The integrin binding to its ligands supports platelet adhesion under static and flow conditions [START_REF] Geberhiwot | Blood platelets contain and secrete laminin-8 (alpha4beta1gamma1) and adhere to laminin-8 via alpha6beta1 integrin[END_REF][START_REF] Hindriks | Platelet adhesion to laminin: role of Ca2+ and Mg2+ ions, shear rate, and platelet membrane glycoproteins[END_REF][START_REF] Inoue | Laminin stimulates spreading of platelets through integrin alpha6beta1-dependent activation of GPVI[END_REF][START_REF] Nigatu | Megakaryocytic cells synthesize and platelets secrete alpha5-laminins, and the endothelial laminin isoform laminin 10 (alpha5beta1gamma1) strongly promotes adhesion but not activation of platelets[END_REF][START_REF] Schaff | Integrin alpha6beta1 is the main receptor for vascular laminins and plays a role in platelet adhesion, activation, and arterial thrombosis[END_REF][START_REF] Sonnenberg | Laminin receptor on platelets is the integrin VLA-6[END_REF] and promotes outside-in signaling resulting in an increase in intracellular Ca 2+ concentrations, shape change and filopodia extension [START_REF] Inoue | Laminin stimulates spreading of platelets through integrin alpha6beta1-dependent activation of GPVI[END_REF][START_REF] Schaff | Integrin alpha6beta1 is the main receptor for vascular laminins and plays a role in platelet adhesion, activation, and arterial thrombosis[END_REF].

Integrin α6β1 does not seem to play a critical role in hemostasis as bleeding time in mice with an α6 deficiency is not increased [START_REF] Schaff | Integrin alpha6beta1 is the main receptor for vascular laminins and plays a role in platelet adhesion, activation, and arterial thrombosis[END_REF]. In contrast, this integrin plays a role in thrombus formation both in vitro under shear flow and in vivo in 3 experimental thrombosis animal models based on mechanical-injury of the aorta, laser-injury of the mesenteric arteriole and guide wire-injury of the carotid artery [START_REF] Schaff | Integrin alpha6beta1 is the main receptor for vascular laminins and plays a role in platelet adhesion, activation, and arterial thrombosis[END_REF].

Integrin αIIbβ3

Integrin αIIbβ3 is a heterodimer of 230 kDa composed of an αIIb subunit and a β3

subunit [START_REF] Jennings | Purification of glycoproteins IIb and III from human platelet plasma membranes and characterization of a calcium-dependent glycoprotein IIb-III complex[END_REF]. Integrin αIIbβ3 is specifically expressed on platelets, even though it has been proposed to also be expressed by tumor cells [START_REF] Boukerche | Platelet-melanoma cell interaction is mediated by the glycoprotein IIb-IIIa complex[END_REF][START_REF] Grossi | Role of tumor cell glycoproteins immunologically related to glycoproteins Ib and IIb/IIIa in tumor cell-platelet and tumor cell-matrix interactions[END_REF]Honn et al., 1992a;[START_REF] Timar | Expression and function of the high affinity alphaIIbbeta3 integrin in murine melanoma cells[END_REF]. This receptor is present at 80,000 copies on the surface of a resting platelet and 30,000 additional copies are found on the membrane of the open canalicular system and α granules which are exposed after platelet activation [START_REF] Wagner | Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding to human platelets[END_REF]. Fibrinogen is the main ligand of integrin αIIbβ3. This receptor recognizes both the RGD peptide-binding sequence of the α chain of fibrinogen and the KQAGDV sequence of the γ chain. This interaction allows the bridging of adjacent platelets resulting in their aggregation, which represents the main function of αIIbβ3 (Marguerie et al., an RGD peptide-binding sequence, and its ligands were proposed to be vitronectin, vWF, osteopontin, fibrinogen and fibronectin [START_REF] Adair | Three-dimensional EM structure of the ectodomain of integrin {alpha}V{beta}3 in a complex with fibronectin[END_REF][START_REF] Bennett | Agonist-activated alphavbeta3 on platelets and lymphocytes binds to the matrix protein osteopontin[END_REF]. At the functional level, αvβ3 supports platelet adhesion to fibronectin, vitronectin and osteopontin which are present in the vessel wall [START_REF] Mccarty | Evaluation of the role of platelet integrins in fibronectin-dependent spreading and adhesion[END_REF][START_REF] Paul | Concurrent signaling from Galphaq-and Galphai-coupled pathways is essential for agonist-induced alphavbeta3 activation on human platelets[END_REF]. This integrin has also been proposed to participate in clot retraction [START_REF] Mor-Cohen | Disulfide Bonds as Regulators of Integrin Function in Thrombosis and Hemostasis[END_REF].

The importance of the αvβ3 integrin in hemostasis and arterial thrombosis has not yet been reported. In humans, no αv integrin mutation has been described to induce hemorrhagic disease. Unpublished data of our group indicate that mice deficient for αv specifically in the platelet lineage have a normal tail bleeding time and an unaltered response in models of experimental thrombosis (unpublished, Mangin P. and Léon C. 2016).

Receptors for soluble agonists

Soluble agonists such as ADP, ATP, thrombin or TxA2 play an instrumental role in amplifying platelet activation to ensure thrombus growth and stability. They act through ion channel receptors (P2X1 receptor) and G protein-coupled receptors (P2Y1, P2Y12, TP and PARs receptors) (Figure 8) [START_REF] Gurbel | G-protein-coupled receptors signaling pathways in new antiplatelet drug development[END_REF]. 3.2.1. The P2X1 receptor P2X1 belongs to the ion channel family that is activated by ATP and inhibited by ADP [START_REF] Sun | P2X1 purinoceptor in human platelets. Molecular cloning and functional characterization after heterologous expression[END_REF]. This receptor is expressed on the surface of platelets, smooth muscle cells, neutrophils, macrophages [START_REF] Hinze | P2X1 receptor-mediated inhibition of the proliferation of human coronary smooth muscle cells involving the transcription factor NR4A1[END_REF][START_REF] Lecut | P2X1 ion channels promote neutrophil chemotaxis through Rho kinase activation[END_REF][START_REF] Sim | P2X1 and P2X4 receptor currents in mouse macrophages[END_REF]. The expression of this receptor ranges from 100 to 150 copies per platelet [START_REF] Mackenzie | Activation of receptor-operated cation channels via P2X1 not P2T purinoceptors in human platelets[END_REF][START_REF] Vial | Presence of P2X1 purinoceptors in human platelets and megakaryoblastic cell lines[END_REF]. The activation of P2X1 results in a rapid intracellular calcium influx into platelets and, further, myosin light chain phosphorylation that leads to platelet activation evidenced by platelet shape change, pseudopodia formation and degranulation [START_REF] Mahaut-Smith | The unique contribution of ion channels to platelet and megakaryocyte function[END_REF][START_REF] Rolf | Platelet shape change evoked by selective activation of P2X1 purinoceptors with alpha,beta-methylene ATP[END_REF][START_REF] Toth-Zsamboki | P2X1-mediated ERK2 activation amplifies the collagen-induced platelet secretion by enhancing myosin light chain kinase activation[END_REF]. Stimulation of the P2X1 receptor does not induce platelet aggregation, but it amplifies aggregation under high shear conditions or in response to various agonists [START_REF] Erhardt | P2X1 stimulation promotes thrombin receptor-mediated platelet aggregation[END_REF][START_REF] Fung | Primary and secondary agonists can use P2X(1) receptors as a major pathway to increase intracellular Ca(2+) in the human platelet[END_REF][START_REF] Gachet | The platelet P2 receptors in arterial thrombosis[END_REF][START_REF] Ilkan | P2X1 Receptors Amplify FcgammaRIIa-Induced Ca2+ Increases and Functional Responses in Human Platelets[END_REF][START_REF] Jones | Ca2+ influx through P2X1 receptors amplifies P2Y1 receptor-evoked Ca2+ signaling and ADP-evoked platelet aggregation[END_REF][START_REF] Oury | ATP augments von Willebrand factor-dependent shear-induced platelet aggregation through Ca2+-calmodulin and myosin light chain kinase activation[END_REF][START_REF] Oury | The ATP-gated P2X1 ion channel acts as a positive regulator of platelet responses to collagen[END_REF][START_REF] Vial | A study of P2X1 receptor function in murine megakaryocytes and human platelets reveals synergy with P2Y receptors[END_REF].

The P2X1 receptor is rapidly desensitized after interaction with ATP, which has complicated the in vitro study of its role in platelet function [START_REF] Gachet | The platelet P2 receptors in arterial thrombosis[END_REF]. Experiments with P2X1-deficient mice identified a reduced ability of platelets to adhere and form aggregates on collagen, particularly under high shear stress [START_REF] Hechler | A role of the fast ATP-gated P2X1 cation channel in thrombosis of small arteries in vivo[END_REF]. Deletion or inhibition of P2X1 in mice does not modify bleeding time, but reduces thrombus size in a laser-induced thrombosis model [START_REF] Erhardt | P2X1 stimulation promotes thrombin receptor-mediated platelet aggregation[END_REF][START_REF] Hechler | A role of the fast ATP-gated P2X1 cation channel in thrombosis of small arteries in vivo[END_REF][START_REF] Hechler | Inhibition of platelet functions and thrombosis through selective or nonselective inhibition of the platelet P2 receptors with increasing doses of NF449 [4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis-(carbonylimino)))tetrakis -benzene-1,3disulfonic acid octasodium salt][END_REF]. In agreement, genetically modified mice overexpressing P2X1 have a prothrombotic phenotype [START_REF] Oury | Overexpression of the platelet P2X1 ion channel in transgenic mice generates a novel prothrombotic phenotype[END_REF].

G protein-coupled receptors

G protein-coupled receptors (GPCRs) account for the largest family of proteins in the human genome [START_REF] Fredriksson | The G-proteincoupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints[END_REF][START_REF] Pierce | Seven-transmembrane receptors[END_REF][START_REF] Vassilatis | The G protein-coupled receptor repertoires of human and mouse[END_REF]. GPCRs consist of 7 transmembrane α-helices connected to 3 extracellular loops containing ligand binding sites and to 3 intracellular loops associated with guanine nucleotide binding proteins (G proteins). A GPCR can be associated with different G proteins which determine its specific intracellular responses to agonists. G proteins are heterodimers with α, β, and γ subunits. In their inactive state Gα subunit is bound to GDP and tightly associated with Gβ-Gγ unit. After agonist binding, GDP becomes phosphorylated and induces Gα subunit dissociation allowing interaction with downstream effectors [START_REF] Offermanns | Activation of platelet function through G protein-coupled receptors[END_REF][START_REF] Smyth | G-protein-coupled receptors as signaling targets for antiplatelet therapy[END_REF].

The P2Y1 receptor

The P2Y1 receptor is a GPCRs coupled to the Gαq protein. This receptor is widely expressed in human tissues and is found in particular on endothelial cells, smooth muscle cells, immune cells including macrophages, eosinophils and lymphocytes [START_REF] Abbracchio | International Union of Pharmacology LVIII: update on the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy[END_REF].

The expression of this receptor is estimated to be around 150 copies per platelet [START_REF] Gurbel | G-protein-coupled receptors signaling pathways in new antiplatelet drug development[END_REF][START_REF] Leon | The P2Y1 receptor is an ADP receptor antagonized by ATP and expressed in platelets and megakaryoblastic cells[END_REF]. The binding of ADP to P2Y1 results in the interaction of Gαq subunit with PLCβ leading to the generation of IP3 and subsequent mobilization of intracellular Ca 2+ stores [START_REF] Cattaneo | Platelet P2 receptors: old and new targets for antithrombotic drugs[END_REF]Hechler and Gachet, 2011b;[START_REF] Offermanns | Activation of platelet function through G protein-coupled receptors[END_REF]. P2Y1 signaling results in platelet shape change, integrin activation and subsequent platelet aggregation [START_REF] Fabre | Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice[END_REF][START_REF] Gachet | P2 receptors, platelet function and pharmacological implications[END_REF][START_REF] Hechler | The P2Y1 receptor is necessary for adenosine 5'-diphosphate-induced platelet aggregation[END_REF][START_REF] Leon | Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice[END_REF]. P2Y1 also participates in the procoagulant function of platelets [START_REF] Leon | Platelet ADP receptors contribute to the initiation of intravascular coagulation[END_REF][START_REF] Leon | Differential involvement of the P2Y1 and P2Y12 receptors in platelet procoagulant activity[END_REF].

Bleeding time in mice deficient in P2Y1 or treated by its inhibitor MRS 2500 is slightly increased suggesting a minor role of this receptor in hemostasis [START_REF] Fabre | Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice[END_REF]Hechler et al., 2006;[START_REF] Leon | Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice[END_REF][START_REF] Wong | The P2Y1 receptor antagonist MRS2500 prevents carotid artery thrombosis in cynomolgus monkeys[END_REF]. In contrast, deletion or inhibition of P2Y1 in mice leads to protection from collagen-, ADP-or TF-induced thromboembolism [START_REF] Fabre | Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice[END_REF][START_REF] Leon | Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice[END_REF] and reduction of thrombus formation after laser-induced injury of the mesenteric arterioles or FeCl3 injury of the carotid artery [START_REF] Gachet | Regulation of platelet functions by P2 receptors[END_REF][START_REF] Hechler | MRS2500 [2-iodo-N6-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'bisphosphate], a potent, selective, and stable antagonist of the platelet P2Y1 receptor with strong antithrombotic activity in mice[END_REF][START_REF] Lenain | Inhibition of localized thrombosis in P2Y1-deficient mice and rodents treated with MRS2179, a P2Y1 receptor antagonist[END_REF][START_REF] Leon | Key role of the P2Y(1) receptor in tissue factor-induced thrombin-dependent acute thromboembolism: studies in P2Y(1)-knockout mice and mice treated with a P2Y(1) antagonist[END_REF]. These results indicate an important role of P2Y1 in arterial thrombosis.

The P2Y12 receptor

The P2Y12 receptor is a GPCR coupled to the Gαi protein. This receptor is mainly expressed in platelets [START_REF] Gurbel | G-protein-coupled receptors signaling pathways in new antiplatelet drug development[END_REF], but is also found in particular in brain tissue, smooth muscle cells, dendritic cells and some leukocytes [START_REF] Ben Addi | Role of the P2Y12 receptor in the modulation of murine dendritic cell function by ADP[END_REF][START_REF] Wang | P2 receptor mRNA expression profiles in human lymphocytes, monocytes and CD34+ stem and progenitor cells[END_REF][START_REF] Wihlborg | ADP receptor P2Y12 is expressed in vascular smooth muscle cells and stimulates contraction in human blood vessels[END_REF]. The expression of this receptor is estimated to be around 600 copies per platelet [START_REF] Gurbel | G-protein-coupled receptors signaling pathways in new antiplatelet drug development[END_REF][START_REF] Ohlmann | The platelet P2Y(12) receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [(3)H]PSB-0413[END_REF]. The binding of ADP to P2Y12 results in the Gαi-associated inhibition of adenylyl cyclase leading to the reduction of cAMP [START_REF] Cattaneo | Platelet P2 receptors: old and new targets for antithrombotic drugs[END_REF]Hechler and Gachet, 2011b;[START_REF] Hollopeter | Identification of the platelet ADP receptor targeted by antithrombotic drugs[END_REF]. Since cAMP levels control protein kinase A activation and by consequence the inhibition of IP3 receptor, which itself mediates Ca 2+ levels, a decrease in cAMP leads to an increase of intracellular Ca 2+ [START_REF] Quinton | Cyclic AMP-dependent phosphorylation of the inositol-1,4,5-trisphosphate receptor inhibits Ca2+ release from platelet membranes[END_REF][START_REF] Tertyshnikova | Inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ release by cAMP-dependent protein kinase in a living cell[END_REF]. In parallel, ADP-binding to the P2Y12 receptor results in a downstream signaling through the Gβγ which activates PI3K, and then Akt and Rap1b, resulting in activation of αIIbβ3 and subsequent platelet aggregation [START_REF] Cosemans | Continuous signaling via PI3K isoforms beta and gamma is required for platelet ADP receptor function in dynamic thrombus stabilization[END_REF][START_REF] Guidetti | The Gi-coupled P2Y12 receptor regulates diacylglycerol-mediated signaling in human platelets[END_REF][START_REF] Kim | Akt activation in platelets depends on Gi signaling pathways[END_REF][START_REF] Schoenwaelder | Identification of a unique co-operative phosphoinositide 3-kinase signaling mechanism regulating integrin alpha IIb beta 3 adhesive function in platelets[END_REF][START_REF] Stefanini | Negative regulators of platelet activation and adhesion[END_REF][START_REF] Woulfe | Activation of Rap1B by G(i) family members in platelets[END_REF]. P2Y12 also participates in the procoagulant function of platelets [START_REF] Leon | Differential involvement of the P2Y1 and P2Y12 receptors in platelet procoagulant activity[END_REF].

Absence of P2Y12 in humans results in an increased bleeding time and mild signs of hemorrhages [START_REF] Cattaneo | Molecular bases of defective signal transduction in the platelet P2Y12 receptor of a patient with congenital bleeding[END_REF][START_REF] Conley | Scientific and therapeutic insights into the role of the platelet P2Y12 receptor in thrombosis[END_REF][START_REF] Lecchi | Identification of a new dysfunctional platelet P2Y12 receptor variant associated with bleeding diathesis[END_REF][START_REF] Nurden | An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. Its influence on glycoprotein IIb-IIIa complex function[END_REF][START_REF] Remijn | Novel molecular defect in the platelet ADP receptor P2Y12 of a patient with haemorrhagic diathesis[END_REF][START_REF] Shiraga | Impaired platelet function in a patient with P2Y12 deficiency caused by a mutation in the translation initiation codon[END_REF]. In agreement, bleeding times in mice deficient in P2Y12 were shown to be increased [START_REF] Andre | P2Y12 regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries[END_REF]. The importance of P2Y12

receptor in arterial thrombosis is attested by the clinical use of antithrombotic drugs targeting this receptor. One finds irreversible antagonists of P2Y12, such as clopidogrel, prasugrel and ticlopidine, and reversible antagonists, such as ticagrelor and cangrelor [START_REF] Gachet | Regulation of platelet functions by P2 receptors[END_REF]. In agreement, deletion or inhibition of P2Y12 in mice leads to reduction of thrombus formation after FeCl3 injury of the mesenteric arterioles or after photochemical injury of the carotid artery [START_REF] Andre | P2Y12 regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries[END_REF][START_REF] Conley | Scientific and therapeutic insights into the role of the platelet P2Y12 receptor in thrombosis[END_REF][START_REF] Reiner | Ticagrelor, but not clopidogrel, reduces arterial thrombosis via endothelial tissue factor suppression[END_REF].

The TP receptor

The thromboxane A2 receptor is a GPCR coupled to Gαq and Gα12/13 proteins. This receptor is mainly expressed in platelets [START_REF] Gurbel | G-protein-coupled receptors signaling pathways in new antiplatelet drug development[END_REF], but is also found on endothelial cells, smooth muscle cells, monocytes and macrophages [START_REF] Davi | Thromboxane receptors antagonists and/or synthase inhibitors[END_REF]. The expression of this receptor is around 1,500 copies per platelet [START_REF] Halushka | Binding of a thromboxane A2/prostaglandin H2 receptor antagonist to guinea-pig platelets[END_REF]. The main ligand of this receptor is TxA2 whose action is locally restricted because of its short half-life [START_REF] Hamberg | Thromboxanes: a new group of biologically active compounds derived from prostaglandin endoperoxides[END_REF][START_REF] Offermanns | Activation of platelet function through G protein-coupled receptors[END_REF]. As mentioned above, platelet activation leads to production of TxA2 through synthesis of arachidonic acid which is further metabolized to unstable PGH2 by cyclooxygenase-1 and then to TxA2 by TxA2 synthase [START_REF] O'donnell | Platelet lipidomics: modern day perspective on lipid discovery and characterization in platelets[END_REF]. The binding of TxA2 to its receptor results in the interaction of Gαq subunit with PLCβ leading to the generation of IP3 and subsequent mobilization of intracellular Ca 2+ stores [START_REF] Cattaneo | Platelet P2 receptors: old and new targets for antithrombotic drugs[END_REF]Hechler and Gachet, 2011b;[START_REF] Offermanns | Activation of platelet function through G protein-coupled receptors[END_REF]. In parallel, ligand binding to the TP receptor results in a downstream signaling through the Gα12 subunit that activates Rho-associated protein kinase pathway leading to phosphorylation of the myosin light chain [START_REF] Klages | Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets[END_REF]. TP signaling results in platelet shape change, granule secretion, integrin activation and subsequent platelet aggregation [START_REF] Bauer | Dichotomous regulation of myosin phosphorylation and shape change by Rho-kinase and calcium in intact human platelets[END_REF][START_REF] Getz | Differential phosphorylation of myosin light chain (Thr)18 and (Ser)19 and functional implications in platelets[END_REF].

Genetic defects of TP in humans result in a modest bleeding, highlighting the role of this receptor in hemostasis [START_REF] Defreyn | Familial bleeding tendency with partial platelet thromboxane synthetase deficiency: reorientation of cyclic endoperoxide metabolism[END_REF][START_REF] Lages | Impaired platelet response to thromboxane-A2 and defective calcium mobilization in a patient with a bleeding disorder[END_REF][START_REF] Samama | Constitutional thrombocytopathy with subnormal response to thromboxane A2[END_REF][START_REF] Wu | Abnormal platelet response to thromboxane A2[END_REF]. This is further supported by the increased bleeding time in mice deficient in TP receptor [START_REF] Cathcart | Cyclooxygenase-2-linked attenuation of hypoxiainduced pulmonary hypertension and intravascular thrombosis[END_REF][START_REF] Thomas | Coagulation defects and altered hemodynamic responses in mice lacking receptors for thromboxane A2[END_REF][START_REF] Yu | TXAS-deleted mice exhibit normal thrombopoiesis, defective hemostasis, and resistance to arachidonate-induced death[END_REF]. Moreover, deletion or inhibition of TP receptor in mice leads to a reduction of thrombus formation after catheterinduced injury of the carotid artery or after photochemical injury of the femoral artery indicating an important role of this receptor in arterial thrombosis [START_REF] Capra | Impact of vascular thromboxane prostanoid receptor activation on hemostasis, thrombosis, oxidative stress, and inflammation[END_REF][START_REF] Cheng | Role of prostacyclin in the cardiovascular response to thromboxane A2[END_REF][START_REF] Grad | Role of thromboxane receptor in C-reactive protein-induced thrombosis[END_REF]. The importance of TP receptor in arterial thrombosis is further attested by the clinical trials of antithrombotic drugs targeting this receptor: terutroban, terbogrel [START_REF] Capra | Impact of vascular thromboxane prostanoid receptor activation on hemostasis, thrombosis, oxidative stress, and inflammation[END_REF][START_REF] Gurbel | G-protein-coupled receptors signaling pathways in new antiplatelet drug development[END_REF].

The protease-activated receptors

The protease-activated receptors (PARs) are GPCRs coupled to Gαq, Gα12/13 and Gαi [START_REF] Klages | Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets[END_REF][START_REF] Offermanns | G proteins of the G12 family are activated via thromboxane A2 and thrombin receptors in human platelets[END_REF]. These receptors are expressed in platelets [START_REF] Gurbel | G-protein-coupled receptors signaling pathways in new antiplatelet drug development[END_REF], but are also found in endothelial cells, smooth muscle cells, monocytes and astrocytes [START_REF] Ossovskaya | Protease-activated receptors: contribution to physiology and disease[END_REF]. Human platelets express PAR1 and PAR4 at around 1,000 to 2,000 copies per platelet, while mouse platelets express PAR3 and PAR4 at 1,500 to 5,000 copies per platelet [START_REF] Kahn | Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin[END_REF][START_REF] Zeiler | Copy number analysis of the murine platelet proteome spanning the complete abundance range[END_REF]. The main ligand of these receptors is thrombin [START_REF] Coughlin | Protease-activated receptors in hemostasis, thrombosis and vascular biology[END_REF] which cleaves the extracellular N-terminal end of the receptor between residues Arg41 and Ser42, in order to expose a new N-terminal end which can then bind and activate the PAR receptors [START_REF] Vu | Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation[END_REF]. PAR1 and PAR3 have additionally a hirudin-like sequence close to the C-terminal thrombin cleavage site which facilitates the binding to thrombin and is not present on PAR4 [START_REF] Kahn | A dual thrombin receptor system for platelet activation[END_REF][START_REF] Xu | Cloning and characterization of human proteaseactivated receptor 4[END_REF]. After ligand binding to the PARs, a signaling is initiated through Gαq which activates

PLCβ leading to mobilization of intracellular Ca 2+ stores, while Gα12-mediated activation of the Rho-associated protein kinase pathway leads to phosphorylation of the myosin light chain and actin remodeling [START_REF] Voss | PAR1, but not PAR4, activates human platelets through a Gi/o/phosphoinositide-3 kinase signaling axis[END_REF][START_REF] Woulfe | Platelet G protein-coupled receptors in hemostasis and thrombosis[END_REF]. The Gαi subunit-mediated signaling leads to the inhibition of adenylyl cyclase inducing the reduction of cAMP [START_REF] Kim | Protease-activated receptors 1 and 4 do not stimulate G(i) signaling pathways in the absence of secreted ADP and cause human platelet aggregation independently of G(i) signaling[END_REF].

PAR1 and PAR3 are activated by a low concentration of thrombin (order of 1 nM), while PAR4 is activated by higher concentrations (order of 50 nM) [START_REF] Kahn | Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin[END_REF]. Due to this unique mechanism of action, these two receptors signaling pathways complete each other: the signal from PAR1 is fast, but it is quickly switched off, while, PAR4 signals are slow but prolonged [START_REF] Leger | Blocking the protease-activated receptor 1-4 heterodimer in platelet-mediated thrombosis[END_REF]. PAR signaling results in platelet shape change, TxA2 release, granule secretion, integrin activation and subsequent platelet aggregation [START_REF] Bauer | Dichotomous regulation of myosin phosphorylation and shape change by Rho-kinase and calcium in intact human platelets[END_REF][START_REF] Henriksen | PAR-4 agonist AYPGKF stimulates thromboxane production by human platelets[END_REF][START_REF] Kahn | Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin[END_REF]. These receptors are also involved in the procoagulant activity of platelets (Figure 9) [START_REF] Andersen | Protease-activated receptor 1 is the primary mediator of thrombin-stimulated platelet procoagulant activity[END_REF]. The importance of PAR receptors in arterial thrombosis is attested by the clinical use of the vorapaxar which inhibits PAR1 and is recommended in the prevention of ischemic events [START_REF] Gupta | Protease-Activated Receptor Antagonist for Reducing Cardiovascular Events -A Review on Vorapaxar[END_REF][START_REF] Kraft | Effect of Vorapaxar Alone and in Combination with Aspirin on Bleeding Time and Platelet Aggregation in Healthy Adult Subjects[END_REF]. These results were in line with pre-clinical data indicating that the deletion of PAR3 or PAR4 in mice leads to a reduction in thrombus formation after FeCl3 injury of the mesenteric arteriole or the carotid artery, after laser injury of the cremaster arteriole or after thromboplastin-induced pulmonary embolism [START_REF] Cornelissen | Roles and interactions among protease-activated receptors and P2ry12 in hemostasis and thrombosis[END_REF][START_REF] Hamilton | Impaired hemostasis and protection against thrombosis in protease-activated receptor 4-deficient mice is due to lack of thrombin signaling in platelets[END_REF][START_REF] Sambrano | Role of thrombin signalling in platelets in haemostasis and thrombosis[END_REF][START_REF] Vandendries | Par4 is required for platelet thrombus propagation but not fibrin generation in a mouse model of thrombosis[END_REF][START_REF] Weiss | Protection against thrombosis in mice lacking PAR3[END_REF]. Interestingly, vorapaxar does not prolong bleeding time suggesting that PAR1 does not play a major role in hemostasis [START_REF] Kraft | Effect of Vorapaxar Alone and in Combination with Aspirin on Bleeding Time and Platelet Aggregation in Healthy Adult Subjects[END_REF]. In agreement, bleeding time in cynomolgus monkeys inhibited of PAR1 receptor was not modified [START_REF] Chintala | SCH 602539, a proteaseactivated receptor-1 antagonist, inhibits thrombosis alone and in combination with cangrelor in a Folts model of arterial thrombosis in cynomolgus monkeys[END_REF][START_REF] Coughlin | Protease-activated receptors in hemostasis, thrombosis and vascular biology[END_REF].

In contrast, mice deficient in PAR3 or PAR4 receptors have a prolonged bleeding time [START_REF] Hamilton | Impaired hemostasis and protection against thrombosis in protease-activated receptor 4-deficient mice is due to lack of thrombin signaling in platelets[END_REF][START_REF] Sambrano | Role of thrombin signalling in platelets in haemostasis and thrombosis[END_REF][START_REF] Weiss | Protection against thrombosis in mice lacking PAR3[END_REF].

B. Coagulation

La coagulation sanguine est une cascade de réactions enzymatiques aboutissant à la génération de thrombine qui clive le fibrinogène plasmatique en fibrine pour former un réseau insoluble consolidant l'agrégat plaquettaire (Norris, 2003). Ce processus est subdivisé en deux voies : la voie extrinsèque et la voie intrinsèque, qui conduisent toutes deux à la génération de facteur X (Figure 10). L'activation du facteur X via ces deux voies marque le début de la voie commune, menant à la génération de thrombine (Palta et al., 2014). 1. La voie extrinsèque L'activateur principal de la voie extrinsèque est le facteur tissulaire, qui est une glycoprotéine transmembranaire (TF) [START_REF] Williams | Tissue factor in health and disease[END_REF]. Cette glycoprotéine de 47kD contient un domaine extracellulaire composé de 2 fibronectine de type III qui se lient à deux endroits différents du facteur VII : l'un à sa partie transmembranaire, l'autre à une courte partie intracellulaire. [START_REF] Banner | The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor[END_REF][START_REF] Butenas | Tissue factor structure and function[END_REF]. TF est exprimé à la surface des cellules périvasculaires et des cellules épithéliales entourant les vaisseaux sanguins et dans le placenta, le coeur, les poumons et le cerveau [START_REF] Drake | Selective cellular expression of tissue factor in human tissues. Implications for disorders of hemostasis and thrombosis[END_REF][START_REF] Fleck | Localization of human tissue factor antigen by immunostaining with monospecific, polyclonal anti-human tissue factor antibody[END_REF][START_REF] Grover | Tissue Factor: An Essential Mediator of Hemostasis and Trigger of Thrombosis[END_REF][START_REF] Hoffman | Tissue factor around dermal vessels has bound factor VII in the absence of injury[END_REF]. Lors d'une lésion vasculaire, le facteur tissulaire est exposé au flux sanguin, ce qui permet la liaison du facteur VII et conduit à son activation (VIIa) [START_REF] Owens | Tissue factor and thrombosis: The clot starts here[END_REF]. Le complexe TF-VIIa permet alors la liaison calcium-dépendante des facteurs de coagulation plasmatiques (F), à savoir le facteur IX et le facteur X [START_REF] Zeerleder | Factor VII-Activating Protease: Hemostatic Protein or Immune Regulator?[END_REF].

La voie intrinsèque

La voie intrinsèque est initiée par le contact du facteur XII avec une surface chargée négativement, entraînant son activation (van der [START_REF] Van Der Graaf | The contact activation mechanism in human plasma: activation induced by dextran sulfate[END_REF]. Le FXIIa active la prékallikréine en α-kallikréine qui va alors activer le FXII 30 fois plus efficacement qu'une surface chargée négativement et sert de rétroaction positive [START_REF] Wiggins | The autoactivation of rabbit Hageman factor[END_REF]. Le FXIIa conduit à l'activation ultérieure du facteur XI et du facteur IX [START_REF] Mackman | Role of the extrinsic pathway of blood coagulation in hemostasis and thrombosis[END_REF].

Le facteur IX agit avec son cofacteur VIII pour former un complexe ténase pour transformer/cliver les facteurs X en facteur Xa [START_REF] Chaudhry | ))[END_REF].

La voie commune

Le facteur Xa produit par les voies extrinsèques et intrinsèques clive la prothrombine pour générer de la thrombine [START_REF] Smith | How it all starts: Initiation of the clotting cascade[END_REF]. Quelques secondes après le début du processus de coagulation, seules de très petites quantités de thrombine (<<1 nM) sont générées, mais elles sont suffisantes pour activer les facteurs V et VIII, ces deux derniers étant des cofacteurs de protéases actives [START_REF] Wolberg | Thrombin generation and fibrin clot structure[END_REF]. Le facteur Va se lie au facteur Xa et à la prothrombine sur la membrane chargée négativement des microparticules ou des plaquettes activées, pour former le complexe prothrombinase qui accélère la réaction de formation de thrombine de 10 5 fois [START_REF] Mann | Surfacedependent reactions of the vitamin K-dependent enzyme complexes[END_REF]Podoplevova et al., 2016). À la suite de cette réaction, la thrombine est générée à des concentrations très élevées explosives atteignant de l'ordre 100 nM [START_REF] Cawthern | Blood coagulation in hemophilia A and hemophilia C[END_REF]. La thrombine catalyse la conversion du fibrinogène en filaments de fibrine et active également le facteur XIII, qui réticule et renforce les polymères de fibrine conduisant à la formation d'un caillot de fibrine qui stabilise les agrégats plaquettaires [START_REF] Wolberg | Thrombin generation and fibrin clot structure[END_REF]. ce stade, la voie extrinsèque a un fort impact car elle permet de produire localement de la thrombine (Palta et al., 2014). Cependant, la voie extrinsèque ne peut pas assurer la propagation de la génération de thrombine conduisant à la croissance linéaire d'un caillot de fibrine dans l'espace car la distance de diffusion de la thrombine et du FXa est limitée : ils sont rapidement et irréversiblement inactivés par des inhibiteurs plasmatiques [START_REF] Travis | Human plasma proteinase inhibitors[END_REF]. Par conséquent, l'étape de la voie intrinsèque concernant l'activation du facteur IX par la thrombine a un impact important sur l'étape de propagation (Figure 11). Le taux d'inactivation du facteur IXa est d'un ordre de grandeur inférieur à celui du facteur Xa et de la thrombine [START_REF] Afosah | Factor IX(a) inhibitors: an updated patent review (2003-present)[END_REF]. Cette caractéristique permet au facteur IXa de diffuser efficacement, assurant la polymérisation de la fibrine à de plus grandes distances du site initial d'activation de la coagulation [START_REF] Dashkevich | Thrombin activity propagates in space during blood coagulation as an excitation wave[END_REF]. La propagation par diffusion du facteur IXa peut entraîner une propagation d'onde auto-entretenue de la thrombine dans l'espace et une augmentation linéaire de la taille du caillot de fibrine à une vitesse d'environ 1 μm/sec [START_REF] Ataullakhanov | Spatial aspects of the dynamics of blood coagulation. I. Hypothesis[END_REF].

Régulation négative de la coagulation

Pour éviter l'activation incontrôlée du processus telle que la coagulation intravasculaire disséminée, plusieurs régulateurs négatifs de la cascade de coagulation existent, notamment la protéine C, l'antithrombine, la α2-macroglobuline et d'autres [START_REF] Travis | Human plasma proteinase inhibitors[END_REF]. Ces inhibiteurs régulent l'activité des protéases de coagulation et peuvent réduire à zéro la concentration locale de thrombine au site de la lésion en un temps caractéristique d'environ 100 s. La protéine C est activée par la thrombine et dégrade les facteurs Va et VIIIa [START_REF] Panteleev | Spatial propagation and localization of blood coagulation are regulated by intrinsic and protein C pathways, respectively[END_REF]. L'activation de la protéine C par la thrombine est accélérée par un cofacteur de la thrombine, la thrombomoduline, présente à la surface de l'endothélium vasculaire intact [START_REF] Esmon | Protein C and the endothelium[END_REF]. L'antithrombine est un inhibiteur de sérine protéase, qui inactive la thrombine et les facteurs IXa, Xa, XIa et XIIa. Son activité est renforcée en présence d'héparine [START_REF] Opal | Antithrombin, heparin, and heparan sulfate[END_REF].

Défauts de la cascade de coagulation

Les défauts génétiques du système de coagulation chez l'homme peuvent entraîner des saignements massifs [START_REF] Bolton-Maggs | Factor XI deficiency[END_REF][START_REF] Martin-Salces | Review: Factor XI deficiency: review and management in pregnant women. Clinical and applied thrombosis/hemostasis[END_REF][START_REF] Perry | Factor VII deficiency. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis[END_REF]. L'une des maladies les plus courantes de la coagulation sanguine est l'hémophilie A et B associée à un déficit en facteurs de coagulation sanguine VIII et IX, respectivement [START_REF] Kurian | Successful liver transplant from a hemophilia A donor with no development of hemophilia A in recipient[END_REF] [START_REF] Drury | The Liver as the Source of Fibrinogen[END_REF]. Sa concentration dans le plasma varie entre 2 et 4 mg/mL [START_REF] Lowe | Blood viscosity and risk of cardiovascular events: the Edinburgh Artery Study[END_REF]. Le fibrinogène est également stocké dans les granules plaquettaires α [START_REF] Harrison | Improved outcome of paracetamol-induced fulminant hepatic failure by late administration of acetylcysteine[END_REF][START_REF] Wencel-Drake | Ultrastructural localization of human platelet thrombospondin, fibrinogen, fibronectin, and von Willebrand factor in frozen thin section[END_REF]. Cette glycoprotéine est composée de 2 ensembles de 3 chaînes polypeptidiques, Aα, Bβ et γ, délimitées par des ponts disulfure [START_REF] Kattula | Fibrinogen and Fibrin in Hemostasis and Thrombosis[END_REF]. La partie centrale du fibrinogène est constituée des extrémités N-terminales des six chaînes présentant les sites de clivage des fibrinopeptides A et B [START_REF] Mosesson | Fibrinogen and fibrin structure and functions[END_REF]. Le clivage par la thrombine du fibrinopeptide A entraîne la formation de monomérique de fibrine, ce qui, ensuite, initie la polymérisation de la fibrine en présence d'une transglutaminase (facteur XIIIa), entraînant la formation d'un caillot de fibrine stable (Figure 12) [START_REF] Litvinov | Polymerization of fibrin: specificity, strength, and stability of knob-hole interactions studied at the singlemolecule level[END_REF][START_REF] Weisel | Mechanisms of fibrin polymerization and clinical implications[END_REF]. (Adapté de [START_REF] Kohler | The Internal Dynamics of Fibrinogen and Its Implications for Coagulation and Adsorption[END_REF][START_REF] Slater | Does fibrin(ogen) bind to monomeric or dimeric GPVI, or not at all?[END_REF]).

Le fibrinogène et la fibrine participent tous les deux à la formation d'un thrombus stable.

La liaison du fibrinogène à αIIbβ3 permet l'agrégation plaquettaire [START_REF] Isenberg | The platelet fibrinogen receptor: an immunogold-surface replica study of agonist-induced ligand binding and receptor clustering[END_REF][START_REF] Mangin | Immobilized fibrinogen activates human platelets through glycoprotein VI[END_REF]. La fibrine forme une structure insoluble. Cela facilite à son tour la liaison des facteurs de coagulation ce qui conduit à une stimulation supplémentaire de la formation de thrombus et de sa stabilisation [START_REF] Sang | Interplay between platelets and coagulation[END_REF][START_REF] Van Geffen | Platelets and coagulation in thrombus formation: aberrations in the Scott syndrome[END_REF]. Les patients afibrinogénémiques ont des complications avec des événements thromboemboliques artériels et veineux qui démontrent l'importance de la fibrine et du fibrinogène dans la formation de thrombus [START_REF] De Moerloose | Fibrinogen and the risk of thrombosis[END_REF][START_REF] Dupuy | Embolized ischemic lesions of toes in an afibrinogenemic patient: possible relevance to in vivo circulating thrombin[END_REF][START_REF] Girolami | Arterial and venous thrombosis in rare congenital bleeding disorders: a critical review[END_REF][START_REF] Lak | Bleeding and thrombosis in 55 patients with inherited afibrinogenaemia[END_REF].

La structure du caillot de fibrine

La structure du caillot de fibrine peut avoir différentes architectures en fonction des variations génétiques des chaînes polypeptidiques, de la concentration locale de thrombine et du débit à travers le thrombus nécessaire pour un accès optimal aux régulateurs négatifs de la coagulation [START_REF] Stalker | A systems approach to hemostasis: 3. Thrombus consolidation regulates intrathrombus solute transport and local thrombin activity[END_REF][START_REF] Weisel | Structure of fibrin: impact on clot stability[END_REF]. Le caillot peut être composé de fibres de fibrine épaisses et lâches conduisant à une porosité élevée ou de fibres fines et serrées induisant à une faible porosité [START_REF] Gu | Fibrin films: overlooked hemostatic barriers against microbial infiltration[END_REF]. Dans des études cliniques, il a été démontré que le plasma des patients hémophiles forme des caillots de fibrine lâches anormaux, tandis que le plasma des patients atteints d'anévrisme de l'aorte abdominale, d'accident vasculaire cérébral ischémique, de thromboembolie veineuse ou chez les fumeurs forme des caillots de fibrine denses [START_REF] Brummel-Ziedins | Discordant fibrin formation in hemophilia[END_REF][START_REF] Laurens | Fibrin structure and wound healing[END_REF][START_REF] Parastatidis | Fibrinogen beta-chain tyrosine nitration is a prothrombotic risk factor[END_REF][START_REF] Scott | Clot architecture is altered in abdominal aortic aneurysms and correlates with aneurysm size[END_REF]Undas et al., 2009a ;Undas et al., 2009c). Étant donné que le réseau / maillage de fibrine est reconnu comme une caractéristique spécifique de la formation stable du thrombus, le ciblage de la polymérisation de la fibrine avec des peptides synthétiques qui bloquent les interactions entre les monomères de fibrine tels que les peptides avec des séquences de boutons de fibrine A ou B attachés à l'albumine, a été proposé comme une stratégie innovante pour bloquer la thrombose [START_REF] Risser | Engineered Molecular Therapeutics Targeting Fibrin and the Coagulation System: a Biophysical Perspective[END_REF][START_REF] Stabenfeldt | Engineering fibrin polymers through engagement of alternative polymerization mechanisms[END_REF][START_REF] Watson | Peptide-derivatized albumins that inhibit fibrin polymerization[END_REF].

Le rôle respectif du fibrinogène et de la fibrine dans la formation du thrombus

Malgré des études de longue date sur la contribution de la fibrine et du fibrinogène dans la formation du thrombus, il est encore difficile de distinguer leur rôle respectif car ce sont les ligands des récepteurs plaquettaires αIIbβ3 et GPVI, et ils dérivent l'un de l'autre [START_REF] Inoue | Laminin stimulates spreading of platelets through integrin alpha6beta1-dependent activation of GPVI[END_REF][START_REF] Mammadova-Bach | Platelet glycoprotein VI binds to polymerized fibrin and promotes thrombin generation[END_REF]. Pour ces raisons, le groupe de M. Flick a généré des souris exprimant le gène FibAEK n'ayant pas la capacité de former un réseau de fibrine, mais avec des quantités normales de fibrinogène (Prasad et al., 2015). Dans les modèles hémostatiques, certaines de ces souris ont pu arrêter la perte de sang après la coupe du bout de la queue contrairement aux souris déficientes en fibrinogène qui ne parviennent pas à arrêter le saignement, confirmant le rôle clé du fibrinogène. Dans un modèle de thrombose après application de FeCl3, les souris FibAEK forment un thrombus, mais la formation de fibrine était nécessaire pour une occlusion complète du vaisseau. Ces résultats laissent une certaine incertitude sur le rôle du réseau de fibrine par rapport au fibrinogène soluble dans la formation du thrombus. Au cours de mes études doctorales, nous avons utilisé ces souris FibAEK dans des modèles d'hémostase et de thrombose pour étudier l'importance de la formation de fibrine dans la dynamique de croissance du thrombus et dans la limitation de la thrombose, au-delà de son rôle dans la stabilisation du caillot (voir Publication 3).

La fibrinolyse est le processus physiologique conduisant à la dégradation de la fibrine [START_REF] Chapin | Fibrinolysis and the control of blood coagulation[END_REF]Gale, 2011 ;[START_REF] Mackie | Normal haemostasis and its regulation[END_REF]. Les principaux activateurs de ce processus sont les activateurs du plasminogène tissulaire (tPA) et de l'urokinase (uPA). Le tPA est sécrété dans la circulation sanguine par l'endothélium vasculaire au cours d'un processus d'inflammation, d'une stase sanguine et d'états pathologiques supplémentaires. L'uPA est sécrétée par les monocytes, les macrophages et l'épithélium urinaire [START_REF] Chapin | Fibrinolysis and the control of blood coagulation[END_REF].

Les deux activateurs du plasminogène ont de courtes demi-vies de 4 à 8 minutes dans la circulation en raison d'une concentration élevée d'inhibiteurs présents dans le plasma, notamment la α2-antiplasmine, la α1-antitrypsine et l'inhibiteur de l'activateur du plasminogène-1 [START_REF] Rijken | New insights into the molecular mechanisms of the fibrinolytic system[END_REF]. Le tPA et uPA forment un complexe avec le plasminogène à la surface de la fibrine [START_REF] Ilich | Global assays of fibrinolysis[END_REF] induisant son clivage pour générer la plasmine, qui est l'acteur majeur de la fibrinolyse [START_REF] Cesarman-Maus | Molecular mechanisms of fibrinolysis[END_REF]. Le plasminogène est une protéine de 92 kDa qui circule dans le sang à une concentration de 200 mg/L (2 μM) [START_REF] Keragala | Plasminogen: an enigmatic zymogen[END_REF]). Le plasminogène s'associe au fibrinogène, par conséquent, lorsqu'un caillot riche en fibrine se forme, le plasminogène est déjà présent à l'intérieur du caillot où il est nécessaire pour former la plasmine [START_REF] Mosesson | Fibrinogen and fibrin structure and functions[END_REF]. La plasmine est régulée positivement par le FXIIa de la voie de coagulation par contact et régulée négativement par l'inhibiteur de fibrinolyse activé par la thrombine (TAFI) (Figure 13). TAFI est une protéase qui est activée par la thrombine et élimine les résidus de lysine et d'arginine Cterminaux sur la fibrine, entraînant une diminution du nombre de sites de liaison au plasminogène [START_REF] Sillen | Thrombin Activatable Fibrinolysis Inhibitor (TAFI): An Updated Narrative Review[END_REF]. La plasmine clive les polymères de fibrine aux deux extrémités de la chaîne α, libérant ainsi des fragments αC qui forment le fragment X -un produit de dégradation de poids moléculaire élevé [START_REF] Mutch | Thrombus lysis by uPA, scuPA and tPA is regulated by plasma TAFI[END_REF]. Ce fragment peut être à nouveau polymérisé par la thrombine ou dégradé en fragments D et E qui sont inhibiteurs de la polymérisation du monomère de fibrine [START_REF] Lane | Characterization of serum fibrinogen and fibrin fragments produced during disseminated intravascular coagulation[END_REF]. Le fragment D de deux molécules de fibrine liées l'une à l'autre est appelé D-dimères et est un indicateur de la coagulation intravasculaire en cours [START_REF] Bailey | Action of thrombin in the clotting of fibrinogen[END_REF][START_REF] Tripodi | D-dimer testing in laboratory practice[END_REF]. activateur du plasminogène de l'urokinase (Adapté de [START_REF] Meltzer | The impact of the fibrinolytic system on the risk of venous and arterial thrombosis[END_REF]).

II. Arterial thrombosis

Arterial thrombosis is a pathological process taking place in a diseased vessel after erosion or rupture of an evolved atherosclerotic plaque [START_REF] Lippi | Arterial thrombus formation in cardiovascular disease[END_REF]. The thrombus forming on the injured plaque is mainly composed of platelets and fibrin and can lead to the obstruction of a vessel resulting in a reduction in blood supply for downstream tissues [START_REF] Kurihara | Degree of luminal narrowing and composition of thrombus in plaque erosion[END_REF], and as a consequence tissue necrosis and a high mortality rate [START_REF] Leadley | Contribution of in vivo models of thrombosis to the discovery and development of novel antithrombotic agents[END_REF]. According to statistics, Cardiovascular Diseases are the most common underlying cause of death in the world. In 2018, they accounted for an estimated 30.9% (95% uncertainty interval, 30.3%-32.9%) of all global deaths [START_REF] Virani | Heart Disease and Stroke Statistics-2021 Update: A Report From the American Heart Association[END_REF].

Arterial thrombosis is the final complication of a chronic vascular disease named atherosclerosis (Figure 14). vWF, von Willebrand factor; GP, glycoprotein complex; TF, tissue factor; TxA2, thromboxane A2.

From atherosclerosis to atherothrombosis

Atherosclerosis is a progressive vascular inflammatory disease resulting in the accumulation of lipids in the arterial vessel wall and the formation of an atherosclerotic plaque narrowing the blood vessel lumen [START_REF] Mackman | Triggers, targets and treatments for thrombosis[END_REF]. In humans, atherosclerotic plaques are usually found in the aorta, coronary, carotid and cerebral arteries (Lusis, 2000). An evolved plaque can be stable for a long time, and suddenly rupture under the effect of blood flow forces thereby precipitating thrombus formation (Emini [START_REF] Veseli | Animal models of atherosclerosis[END_REF]. Ruptured plaque exposes to the blood stream a highly thrombogenic surface containing TF and collagen that initiates thrombus growth [START_REF] Toschi | Tissue factor modulates the thrombogenicity of human atherosclerotic plaques[END_REF][START_REF] Van Zanten | Increased platelet deposition on atherosclerotic coronary arteries[END_REF].

1.2.The process of atherosclerosis

The process of atherosclerotic plaque formation is initiated by high plasma levels of low-density lipoprotein (LDL) [START_REF] Gimbrone | Endothelial Cell Dysfunction and the Pathobiology of Atherosclerosis[END_REF]. LDLs accumulate in the sub-endothelial space of the arterial wall through endocytosis [START_REF] Libby | Progress and challenges in translating the biology of atherosclerosis[END_REF]. There, they are oxidized and initiate an inflammatory response of the endothelial cells which starts to express chemotactic proteins such as monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin and P-selectin [START_REF] Fuster | Animal models of atherosclerosis[END_REF][START_REF] Tabas | Recent insights into the cellular biology of atherosclerosis[END_REF]. These proteins recruit circulating immune and pro-inflammatory cells, especially monocytes into arterial vessel wall [START_REF] Galkina | Vascular adhesion molecules in atherosclerosis[END_REF][START_REF] Sakakura | Pathophysiology of atherosclerosis plaque progression[END_REF]. Within the vessel wall, the monocytes differentiate into macrophages capable of cholesterol phagocytosis leading to formation of foam cells secreting inflammatory mediators [START_REF] Libby | Progress and challenges in translating the biology of atherosclerosis[END_REF] (SMCs) from the tunica media to the tunica intima where they proliferate and synthesize components of the extracellular matrix such as collagen and elastin that form the fibrous cap covering and stabilizing the plaque [START_REF] Fuster | Animal models of atherosclerosis[END_REF]. Foam cells and SMCs in the center of the plaque undergo apoptosis and necrosis inducing the release of oxidized LDL and generating a cholesterol-rich area called the lipid core or necrotic core plaque [START_REF] Libby | Progress and challenges in translating the biology of atherosclerosis[END_REF]. If the plaque contains a limited amount of lipids and is covered by a thick fibrous cap, it represents a "stable plaque" and its probability to rupture is low [START_REF] Finn | Concept of vulnerable/unstable plaque[END_REF]. In contrast, if the plaque has a lipid-reach core covered by a weakened fibrous cap, it is considered as a "vulnerable plaque" and its probability to erode or rupture is very high (Figure 15) [START_REF] Tomaniak | Vulnerable plaques and patients: state-of-the-art[END_REF].

Treatment of arterial thrombosis

The management of arterial thrombosis depends on its severity and the affected artery.

The therapy usually consists in pharmacological treatment which can be combined to an endovascular approach with the aim to restore normal circulation and prevent thrombus regrowth. The endovascular approach is usually an angioplasty consisting in the introduction of a catheter in an artery of the arm or a leg which is guided to the blocked artery [START_REF] Kerzmann | Acute mesenteric ischemia][END_REF][START_REF] Sorini Dini | Contemporary Approach to Heavily Calcified Coronary Lesions[END_REF]. This catheter has a small balloon on its tip whose inflation dilates the artery, crushing the atherosclerotic plaque. This procedure can be accompanied by the placement of a stent that keeps the vessel open and helps restore normal blood flow [START_REF] Mehta | Acute Myocardial Infarction in Women: A Scientific Statement From the American Heart Association[END_REF].

Concerning the pharmacological treatment, it mainly relies on antiplatelet agents, anticoagulants or fibrinolytic agents (Figure 16). The first anti-platelet agent identified and still widely used in clinics is aspirin. It irreversibly inhibits cyclooxygenase-1 (COX-1) in platelets by acetylation of a serine residue within its catalytic pocket [START_REF] Finamore | A high glucose level is associated with decreased aspirin-mediated acetylation of platelet cyclooxygenase (COX)-1 at serine 529: A pilot study[END_REF]. This leads to prevention of the arachidonic acid transformation to prostaglandins impairing/limiting TxA2 formation and the subsequent platelet activation through TP receptors (Awtry and Loscalzo, 2000). Aspirin is generally used orally at a dose of 75-100 mg per day in the treatment and secondary prevention of thrombotic cardiovascular diseases such as myocardial infarction and stroke [START_REF] Johnston | Ticagrelor and Aspirin or Aspirin Alone in Acute Ischemic Stroke or TIA[END_REF]. The half-life of aspirin in the bloodstream is 13-19 min after its single oral administration [START_REF] Ornelas | Beyond COX-1: the effects of aspirin on platelet biology and potential mechanisms of chemoprevention[END_REF]. Aspirin treatment is accompanied by adverse effects including bleeding, which occurs notably at the gastrointestinal level [START_REF] Lanas | Non-variceal upper gastrointestinal bleeding[END_REF].

P2Y12 receptor antagonists

A second class of antiplatelet drugs widely used in arterial thrombosis targets the P2Y12 receptor [START_REF] Gachet | Antiplatelet drugs: which targets for which treatments?[END_REF]. Antagonists of this receptor can be divided into two groups based on their mechanism of action: i) prodrugs whose active metabolites inhibit irreversibly the P2Y12 receptor, such as ticlopidine, clopidogrel and prasugrel; ii) direct and reversible inhibitors of the P2Y12 receptor, such as ticagrelor and cangrelor [START_REF] Secco | P2Y12 inhibitors: pharmacologic mechanism and clinical relevance[END_REF] (Table 2). These antithrombotics are used in the prevention and treatment of thrombotic events such as acute coronary syndrome, stent thrombosis and ischemic stroke [START_REF] Baqi | Antithrombotic P2Y12 receptor antagonists: recent developments in drug discovery[END_REF][START_REF] Lasica | Use of Anticoagulant Therapy in Patients with Acute Myocardial Infarction and Atrial Fibrillation[END_REF][START_REF] Verheugt | P2Y12 blocker monotherapy after percutaneous coronary intervention[END_REF]. The main limitation of targeting P2Y12 is the risk of bleeding, which increases with the degree of inhibition of the receptor, explaining that the doses used in the clinic only promote 50-60% inhibition of ADP-induced platelet aggregation [START_REF] Gachet | Regulation of platelet functions by P2 receptors[END_REF][START_REF] Wallentin | P2Y(12) inhibitors: differences in properties and mechanisms of action and potential consequences for clinical use[END_REF]. The treatment recommended for secondary prevention of arterial thrombosis and stent thrombosis is based on dual-antiplatelet therapy combining aspirin and a P2Y12 inhibitor [START_REF] Sharma | Dual Antiplatelet Therapy in Coronary Artery Disease[END_REF][START_REF] Sinnaeve | Dual Antiplatelet Therapy De-escalation Strategies[END_REF]. This dual therapy consisting of aspirin and clopidogrel at 12 months has been reported in the CURE study to reduce cardiovascular events (cardiovascular death, non-fatal myocardial infarction or stroke) more than aspirin alone.

It is however, accompanied with an increased risk of major bleeding in patients with acute coronary syndrome [START_REF] Roberto | Dual antiplatelet therapy is under-prescribed in patients with surgically treated acute myocardial infarction[END_REF][START_REF] Sharma | Dual Antiplatelet Therapy in Coronary Artery Disease[END_REF]. In the CHARISMA trial the combination of aspirin with clopidogrel is also more effective than clopidogrel alone by reducing the risk of cardiovascular events [START_REF] Bhatt | International prevalence, recognition, and treatment of cardiovascular risk factors in outpatients with atherothrombosis[END_REF].

Current recommendations suggest to use the newest P2Y12 inhibitors, prasugrel and ticagrelor. These inhibitors act faster than clopidogrel (30 min vs 2 hours) and lead to lesser high on-treatment residual platelet reactivity (HTPR) (3% vs 30%) [START_REF] Kamran | Oral Antiplatelet Therapy After Acute Coronary Syndrome: A Review[END_REF]. In patients with risk of acute coronary syndrome after coronary intervention, the combination of aspirin with prasugrel has been reported to reduce cardiovascular risk further than clopidogrel, with, nevertheless, an increased risk of bleeding [START_REF] Wiviott | Prasugrel versus clopidogrel in patients with acute coronary syndromes[END_REF]. The same result was observed in a study comparing the rate of cardiovascular risk in patients with acute coronary syndrome after a therapy combining ticagrelor and aspirin or clopidogrel and aspirin [START_REF] Wallentin | Ticagrelor versus clopidogrel in patients with acute coronary syndromes[END_REF].

Dual-antiplatelet therapy is not recommended following an ischemic stroke because of the high risk of bleeding [START_REF] Kamran | Oral Antiplatelet Therapy After Acute Coronary Syndrome: A Review[END_REF]. In this situation, the combination of platelet aggregation inhibitors amplifies the risk of hemorrhagic transformation which can be fatal for the patient.

Integrin αIIbβ3 blockers

A third class of antiplatelet drugs used in arterial thrombosis targets integrin αIIbβ3 [START_REF] Huang | Platelet integrin alphaIIbbeta3: signal transduction, regulation, and its therapeutic targeting[END_REF]. There are only three agents inhibiting this integrin approved for clinical use: abciximab, eptifibatide and tirofiban [START_REF] Giordano | Effects Of Glycoprotein IIb/IIIa Antagonists: Anti Platelet Aggregation And Beyond[END_REF]. Abciximab (ReoPro) is a Fab fragment of a chimeric monoclonal antibody inhibiting the interaction of αIIbβ3 with fibrinogen [START_REF] Giordano | Effects Of Glycoprotein IIb/IIIa Antagonists: Anti Platelet Aggregation And Beyond[END_REF]. Eptifibatide (Integrilin) is a cyclic heptapeptide containing a KGD sequence [START_REF] Scarborough | Design of potent and specific integrin antagonists. Peptide antagonists with high specificity for glycoprotein IIb-IIIa[END_REF]. Finally, tirofiban (Aggrastat) is a non-peptide antagonist structurally mimicking a RGD sequence [START_REF] Hartman | Non-peptide fibrinogen receptor antagonists. 1. Discovery and design of exosite inhibitors[END_REF]. These therapeutic agents are administered intravenously in emergency situations such as myocardial infarction or during percutaneous coronary interventions [START_REF] Jamasbi | Platelet receptors as therapeutic targets: Past, present and future[END_REF]. The main limitation of anti-αIIbβ3 agents is the risk of bleeding which is higher than for P2Y12 receptor antagonists [START_REF] Gammie | Abciximab and excessive bleeding in patients undergoing emergency cardiac operations[END_REF][START_REF] Junghans | Bleeding risk of tirofiban, a nonpeptide GPIIb/IIIa platelet receptor antagonist in progressive stroke: an open pilot study[END_REF][START_REF] Rasty | Bleeding associated with eptifibatide targeting higher risk patients with acute coronary syndromes: incidence and multivariate risk factors[END_REF][START_REF] Tigen | Bleeding risk with concomitant use of tirofiban and thirdgeneration P2Y12 receptor antagonists in patients with acute myocardial infarction: A real-life data[END_REF]. A new strategy to inhibit αIIbβ3 with a potential lower risk of bleeding is to only target the activated form of αIIbβ3.

These agents are promising in pre-clinical studies but have not yet been evaluated in clinic [START_REF] Hohmann | Delayed targeting of CD39 to activated platelet GPIIb/IIIa via a single-chain antibody: breaking the link between antithrombotic potency and bleeding?[END_REF][START_REF] Huang | Evaluation of targeting c-Src by the RGT-containing peptide as a novel antithrombotic strategy[END_REF][START_REF] Li | RUC-4: a novel alphaIIbbeta3 antagonist for prehospital therapy of myocardial infarction[END_REF].

Anticoagulant therapy

Another class of agents targeting arterial thrombosis are anticoagulants which can be divided on their mode of action in: i) heparin and low molecular weight heparins (LMWHs); ii) vitamin K antagonists; iii) direct factor Xa inhibitors and iv) direct thrombin inhibitors [START_REF] Dewald | Anticoagulants: Pharmacokinetics, Mechanisms of Action, and Indications[END_REF].

Heparin and LMWHs interact with antithrombin (AT) and catalyze AT-mediated inhibition of thrombin, and factors IXa, Xa, XIa, and XIIa [START_REF] Garcia | Parenteral anticoagulants: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines[END_REF]. At high doses, heparin catalyzes thrombin inactivation by heparin cofactor II and binds to IXa leading to inhibition of Xa [START_REF] Hirsh | Heparin: mechanism of action, pharmacokinetics, dosing considerations, monitoring, efficacy, and safety[END_REF]. Heparin equally inhibits the activity of thrombin and Xa while LMWHs efficiently inhibits the activity of Xa compared to thrombin whose inhibition requires the presence of the high affinity pentasaccharide sequence and an oligosaccharide chain with at least 18 units length that is absent in LMWH due to its shortened chain length [START_REF] Garcia | Parenteral anticoagulants: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines[END_REF][START_REF] Lam | The separation of active and inactive forms of heparin[END_REF][START_REF] Petitou | Synthesis of thrombin-inhibiting heparin mimetics without side effects[END_REF]. A potential major side effect of heparin treatment is heparin-induced thrombocytopenia (HIT) which is triggered by the ability of heparin to bind platelet factor 4 (PF4) which is released after platelets activation [START_REF] Onishi | Heparin and anticoagulation[END_REF]. The heparin/PF4 complex can activate the immune system producing antibodies against the complex activating platelets and monocytes. This leads to TF and procoagulant microparticles releases resulting in facilitating of platelets activation and formation of aggregates [START_REF] Marcucci | Heparin induced thrombocytopenia: position paper from the Italian Society on Thrombosis and Haemostasis (SISET)[END_REF]. LMWHs have weaker protein binding capacities and therefore led to a lower rate of HIT [START_REF] Cosmi | Effect of nonspecific binding to plasma proteins on the antithrombin activities of unfractionated heparin, low-molecular-weight heparin, and dermatan sulfate[END_REF]. Heparin and LMWHs are used to treat pulmonary embolism, but can also be combined to aspirin in the setting of complications of acute coronary syndrome (myocardial infarction and unstable angina) [START_REF] Amane | A comparative study of dalteparin and unfractionated heparin in patients with unstable angina pectoris[END_REF][START_REF] Cohen | Managing pulmonary embolism from presentation to extended treatment[END_REF][START_REF] Undas | Systemic blood coagulation activation in acute coronary syndromes[END_REF].

Another anticoagulant family comprises the vitamin K antagonists. Vitamin K participates in the biosynthesis of several key coagulation factors as a cofactor for their carboxylation in the liver [START_REF] Dowd | The mechanism of action of vitamin K[END_REF]. Warfarin, is one of the member of the vitamin K antagonists, and inhibits the C1 subunit of vitamin K leading to distortions of its function in the synthesis of thrombin and factors VII, IX, and X [START_REF] Stirling | Warfarin-induced changes in procoagulant and anticoagulant proteins[END_REF]. Therefore, warfarin blocks the generation of coagulation factors but has no impact on circulating factors or pre-existing thrombi. This anticoagulant is used to reduce the risk of recurrent myocardial infarction, systemic embolism after it and stroke [START_REF] Jones | The use of novel oral anticoagulants compared to vitamin K antagonists (warfarin) in patients with left ventricular thrombus after acute myocardial infarction[END_REF][START_REF] Mant | Warfarin versus aspirin for stroke prevention in an elderly community population with atrial fibrillation (the Birmingham Atrial Fibrillation Treatment of the Aged Study, BAFTA): a randomised controlled trial[END_REF]. The main disadvantage of this agent is its interaction with numerous drugs and food which influences its anticoagulant response [START_REF] Wells | Interactions of warfarin with drugs and food[END_REF].

Another type of anticoagulants is inhibitors of factor Xa. Apixaban is a direct reversible inhibitor that binds to free and clot-bound factor Xa leading to decrease of thrombin generation [START_REF] Byon | Apixaban: A Clinical Pharmacokinetic and Pharmacodynamic Review[END_REF]. This anticoagulant is used in the prevention and treatment of thrombotic events such as pulmonary embolism and stroke in patients with non-valvular atrial fibrillation [START_REF] Cirincione | Population Pharmacokinetics of Apixaban in Subjects With Nonvalvular Atrial Fibrillation[END_REF][START_REF] Halvorsen | Efficacy and safety of apixaban compared with warfarin according to age for stroke prevention in atrial fibrillation: observations from the ARISTOTLE trial[END_REF]. Its main limitation is the risk of hemorrhage and thrombocytopenia [START_REF] Gresham | Case files of the Medical Toxicology Fellowship at Banner Good Samaritan Medical Center in Phoenix, AZ: a non-warfarin anticoagulant overdose[END_REF][START_REF] Harter | Anticoagulation drug therapy: a review[END_REF].

Finally, one of the member of anticoagulants which directly inhibit thrombin is dabigatran. It binds to free and fibrin-bound thrombin [START_REF] Antonijevic | Dabigatran -Metabolism, Pharmacologic Properties and Drug Interactions[END_REF]. This anticoagulant is used in the prevention of embolic events in patients with non-valvular atrial fibrillation and with non-hemorrhagic stroke [START_REF] Connolly | Dabigatran versus warfarin in patients with atrial fibrillation[END_REF][START_REF] Gomez-Outes | Dabigatran, Rivaroxaban, or Apixaban versus Warfarin in Patients with Nonvalvular Atrial Fibrillation: A Systematic Review and Meta-Analysis of Subgroups[END_REF].

Possible side effects of dabigatran are gastrointestinal bleeding and intracranial hemorrhage [START_REF] Eikelboom | Risk of bleeding with 2 doses of dabigatran compared with warfarin in older and younger patients with atrial fibrillation: an analysis of the randomized evaluation of long-term anticoagulant therapy (RE-LY) trial[END_REF].

Thrombolytic therapy

The thrombolytic therapy consists in transforming plasminogen into plasmin to promote the degradation of the fibrin clot and restore vessel patency. The clinically used thrombolytic agents includes recombinant forms of tPA (rtPA) and urokinase [START_REF] Simpson | Reteplase: a review of its use in the management of thrombotic occlusive disorders[END_REF]. rtPA is the most widely used pharmacologically approved treatment for acute ischemic stroke [START_REF] Tsivgoulis | Timing of Recanalization and Functional Recovery in Acute Ischemic Stroke[END_REF]. It is used alone or combined with thrombectomy to promote the dissolution of a clot which occludes a cerebral artery [START_REF] Alberts | Endovascular Therapy for Acute Ischemic Stroke: Dawn of a New Era[END_REF][START_REF] Anfray | Single-and two-chain tissue type plasminogen activator treatments differentially influence cerebral recovery after stroke[END_REF][START_REF] Frey | Recombinant tissue plasminogen activator (rtPA) for stroke. The perspective at 8 years[END_REF]. Due to the risk of intracerebral haemorrhage associated with rtPA-mediated thrombolysis, a strict treatment window of up to 4.5 hours post-stroke onset has been applied as a threshold for administration of rtPA [START_REF] Shobha | Thrombolysis at 3-4.5 hours after acute ischemic stroke onset--evidence from the Canadian Alteplase for Stroke Effectiveness Study (CASES) registry[END_REF][START_REF] Su | Activation of PDGF-CC by tissue plasminogen activator impairs blood-brain barrier integrity during ischemic stroke[END_REF][START_REF] Yepes | Tissue-type plasminogen activator and neuroserpin: a well-balanced act in the nervous system?[END_REF]. rtPA-treated patients have a low recanalization rate of 17% and 38%

depending on the vessel [START_REF] Nichols | Intravenous recombinant tissue plasminogen activator improves arterial recanalization rates and reduces infarct volumes in patients with hyperdense artery sign on baseline computed tomography[END_REF][START_REF] Rohan | Length of occlusion predicts recanalization and outcome after intravenous thrombolysis in middle cerebral artery stroke[END_REF]. Side effect rtPA is its ability to induce seizure and excitotoxicity through promoting the activation of N-Methyl-D-Aspartate receptors (NMDAR) [START_REF] Alvarez | Acute seizures in acute ischemic stroke: does thrombolysis have a role to play?[END_REF][START_REF] Nicole | The proteolytic activity of tissue-plasminogen activator enhances NMDA receptor-mediated signaling[END_REF][START_REF] Tsirka | Excitotoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator[END_REF]. Taken together, only a small fraction of stroke patients benefit from thrombolysis with no side effects (National Institute of Neurological and Stroke rt, 1995; Vivien, 2017).

Murine models of experimental thrombosis

Models of experimental thrombosis represent unique tools to study the mechanism of thrombus formation in vivo, allowing to dissect cellular and molecular events of this complex process. This section is focusing on the in vivo models which were used during my PhD studies.

Mouse models are extensively used because of numerous advantages including their high fertility, low cost of breeding and small size [START_REF] Kohnken | Overview of the Use of Murine Models in Leukemia and Lymphoma Research[END_REF]. The use of mouse models also allows to study thrombus formation in transgenic mice which were instrumental to better characterize the function of numerous receptors or signaling molecules [START_REF] Whinna | Overview of murine thrombosis models[END_REF]. A very common approach to induce thrombosis in murine healthy vessels consists in damaging the vessel wall chemically, mechanically, with electricity or a laser [START_REF] Cooley | Murine models of thrombosis[END_REF][START_REF] Westrick | Murine models of vascular thrombosis (Eitzman series)[END_REF]. Besides their advantages, mouse models have limitations, notably the absence of correlation to any clinical situation as the lesion are performed on healthy vessels in young mice which do not mimic a clinical setting in patients.

The FeCl3-induced injury model

FeCl3-induced injury is realized by putting a drop or the application of the filter paper with this chemical to the external part of the mouse vessel (Figure 17) [START_REF] Grambow | The slow releasing hydrogen sulfide donor GYY4137 reduces neointima formation upon FeCl3 injury of the carotid artery in mice[END_REF][START_REF] Zhou | The C-terminal CGHC motif of protein disulfide isomerase supports thrombosis[END_REF]. FeCl3 in small vesicles has been shown to cross the endothelium by an endocytic-exocytic pathway and to generate reactive oxygen species which initiates thrombus formation [START_REF] Kurz | Rat model of arterial thrombosis induced by ferric chloride[END_REF][START_REF] Tseng | Transendothelial migration of ferric ion in FeCl3 injured murine common carotid artery[END_REF]. Although this thrombosis model is widely used the exact mechanism by which it triggers thrombosis is still not fully understood. In some studies, the generation of reactive oxygen species triggers the denudation of endothelial cells and the exposure of the subendothelium matrix [START_REF] Dubois | Glycoprotein VI-dependent and -independent pathways of thrombus formation in vivo[END_REF][START_REF] Westrick | Murine models of vascular thrombosis (Eitzman series)[END_REF][START_REF] Woollard | Erythrocyte hemolysis and hemoglobin oxidation promote ferric chloride-induced vascular injury[END_REF]. In other studies, FeCl3 damages the vessel wall but the endothelial denudation is absent [START_REF] Barr | Red blood cells mediate the onset of thrombosis in the ferric chloride murine model[END_REF]Eckly et al., 2011). In this case the mechanism initiating the thrombus formation is unclear. One hypothesis is that RBC-derived structures recruit platelets and this process initiates thrombus formation. Another hypothesis is that FeCl3 bodies exposed on the injured vessel wall to the blood flow contain large amounts of tissue factor on their surface that could support thrombus formation by generating thrombin (Eckly et al., 2011).

These different outcomes probably depend on the vessel type to which FeCl3 is applied [START_REF] Chauhan | von Willebrand factor and factor VIII are independently required to form stable occlusive thrombi in injured veins[END_REF][START_REF] Konstantinides | Distinct antithrombotic consequences of platelet glycoprotein Ibalpha and VI deficiency in a mouse model of arterial thrombosis[END_REF].

The technical simplicity of this model allows to target any mouse vessel (especially, the mesenteric, cremaster or carotid vessels), and resulted in a broad use in our research field. The level of thrombus formation depends on the concentration of FeCl3, application time and the injury size which could result in the formation of occlusive or non-occlusive thrombi (Eckly et al., 2011;[START_REF] Kurz | Rat model of arterial thrombosis induced by ferric chloride[END_REF]. The recording of thrombus formation can be done in real time by visualizing platelet accumulation with fluorescent microscopy (and another thrombus components if it is necessary) or by measuring the blood flow with a Doppler probe [START_REF] Denis | A mouse model of severe von Willebrand disease: defects in hemostasis and thrombosis[END_REF][START_REF] Fay | Vitronectin inhibits the thrombotic response to arterial injury in mice[END_REF].

The laser-induced injury model

Laser-induced injury is realized with the pulsed, high power laser focused on a small tissue volume with minimum damaging of surrounding tissue due to the microscope optics (Figure 18) [START_REF] Stalker | Mouse laser injury models: variations on a theme[END_REF]. This type of injury includes thermal and mechanical damages. The laser beam superheats the tissue or cells which are further destroyed by mechanical forces created by cavitation bubble formation [START_REF] Dubois | Thrombininitiated platelet activation in vivo is vWF independent during thrombus formation in a laser injury model[END_REF][START_REF] Falati | Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse[END_REF][START_REF] Rau | Pulsed laser microbeam-induced cell lysis: time-resolved imaging and analysis of hydrodynamic effects[END_REF][START_REF] Vogel | Mechanisms of pulsed laser ablation of biological tissues[END_REF]. As a result, laser injury is a mechanical injury, but subsequent physical rupture of cells or tissue has additional biochemical effects. The level of laser injury depends on its wavelength, pulse duration, pulse energy and beam diameter [START_REF] Ando | Comparison of therapeutic effects between pulsed and continuous wave 810-nm wavelength laser irradiation for traumatic brain injury in mice[END_REF][START_REF] Larsson | Scanning laser-induced endothelial injury: a standardized and reproducible thrombosis model for intravital microscopy[END_REF]. In our laboratory we showed that minimal laser ablation induces endothelial cell denudation, while strong laser ablation leads to the disruption of all vessel wall layers (Hechler et al., 2010). However, depending on the laser intensity and studied vessel different levels of injury could be induced: endothelial cell denudation when thrombus formation is induced by subendothelium matrix and activation of endothelial cells without denudation [START_REF] Atkinson | Laserinduced endothelial cell activation supports fibrin formation[END_REF]. Because of the thickness of many vessel walls and the presence of fat in surrounding tissue which absorbed laser energy, a main limitation of this murine thrombosis model is that not every vessel can be targeted. This model is currently mainly used in small murine vessels: mesenteric arterioles, ear microcirculation and the cremaster muscle microcirculation [START_REF] Dubois | Glycoprotein VI-dependent and -independent pathways of thrombus formation in vivo[END_REF][START_REF] Falati | Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse[END_REF][START_REF] Falati | In vivo models of platelet function and thrombosis: study of real-time thrombus formation[END_REF][START_REF] Hechler | A role of the fast ATP-gated P2X1 cation channel in thrombosis of small arteries in vivo[END_REF]Nonne et al., 2005;[START_REF] Stalker | Mouse laser injury models: variations on a theme[END_REF]. The thrombus formation in laserinduced model is studied in real time by using brightfield and/or fluorescence imaging [START_REF] Dubois | Thrombininitiated platelet activation in vivo is vWF independent during thrombus formation in a laser injury model[END_REF][START_REF] Stalker | Hierarchical organization in the hemostatic response and its relationship to the platelet-signaling network[END_REF].

The electrolytic injury model

Electrolytic injury is generated by an electrical current delivery through a probe to the vessel that is often also subjected to a stasis mediated with a hemostatic clamp (Figure 19) [START_REF] Sturgeon | Adaptation of the Folts and electrolytic methods of arterial thrombosis for the study of anti-thrombotic molecules in small animals[END_REF]. This type of injury leads to a damage of the intima, the extent of which appears to be controlled by regulation of the voltage of the stimulation current and by application time. This model is currently used in carotid artery and results mostly in occlusive thrombus formation [START_REF] Aleman | Elevated prothrombin promotes venous, but not arterial, thrombosis in mice[END_REF][START_REF] Hughan | Dok-2 adaptor protein regulates the shear-dependent adhesive function of platelet integrin alphaIIbbeta3 in mice[END_REF][START_REF] Schoenwaelder | Corrigendum: 14-3-3zeta regulates the mitochondrial respiratory reserve linked to platelet phosphatidylserine exposure and procoagulant function[END_REF].

The electrolytic injury model is not widely used to study the mechanism of arterial thrombosis because the level of damage is extremely high. Thrombus formation in electrolytic injury models is studied in real time by using brightfield imaging or by measuring the flow with a Doppler probe [START_REF] Kusada | Electric injury model of murine arterial thrombosis[END_REF][START_REF] Mangin | Thrombin overcomes the thrombosis defect associated with platelet GPVI/FcRgamma deficiency[END_REF].

The mechanical injury model

Mechanical injury is generated by forceps compression of the vessel which can sometimes be coupled to concentric vessel stenosis (Folts-like method) (Figure 20) [START_REF] Westrick | Murine models of vascular thrombosis (Eitzman series)[END_REF]. This type of injury leads to the deformation or detachment of the endothelium depending on compression by the forceps jaws or application time (Tang et al., 2016). Two levels of injuries were reported with a moderate injury corresponding to the deformation of the endothelium but not a complete detachment, while the severe injury promotes denudation and breakage of the internal elastic lamina (Tang et al., 2016). This model is used in the carotid artery, aorta and femoral vein [START_REF] Gruner | Relative antithrombotic effect of soluble GPVI dimer compared with anti-GPVI antibodies in mice[END_REF][START_REF] Pierangeli | Induction of thrombosis in a mouse model by IgG, IgM and IgA immunoglobulins from patients with the antiphospholipid syndrome[END_REF][START_REF] Pozgajova | Reduced thrombus stability in mice lacking the alpha2A-adrenergic receptor[END_REF]. Thrombus formation in mechanical injury models is studied in real time by using fluorescent imaging or by measuring the flow with a Doppler probe [START_REF] Mangin | Thrombin overcomes the thrombosis defect associated with platelet GPVI/FcRgamma deficiency[END_REF];

III. The role of blood flow in hemostasis and arterial thrombosis

In the 1,800s the physician Rudolf Virchow described three key features of intravascular venous thrombosis, which later became known as Virchow's Triad: stagnant flow, hypercoagulability and endothelial injury [START_REF] Bagot | Virchow and his triad: a question of attribution[END_REF]. Later, this concept was expanded to recognize the importance of blood flow in the regulation of hemostasis and arterial thrombosis [START_REF] Sakariassen | The impact of blood shear rate on arterial thrombus formation[END_REF][START_REF] Storch | Oscillatory shear stress induces hemostatic imbalance in healthy men[END_REF]. The importance of flow in thrombosis is notably evidenced by the impact of the flow regime on thrombus composition. Indeed, arterial thrombi formed under high flow velocities are rich in platelets and fibrin, while venous thrombi formed under lower flow velocities and are rich in RBC and fibrin [START_REF] Baumgartner | The role of blood flow in platelet adhesion, fibrin deposition, and formation of mural thrombi[END_REF].

Rheology applicable to blood flow

Blood is a viscous fluid which consists of a liquid fractionplasmaand a cellular fractioncells: platelets, red blood cells (RBCs) and leukocytes [START_REF] Sweeney | The blood bank physician as a hemostasis consultant[END_REF]. Blood cells tend to move towards the streamlines of higher velocities, i.e. towards the center of the lumen with gravity having only a negligible effect on their motion in bloodstream. RBC are the dominant part of blood cellular mass and are usually localized in the center of bloodstream where they push other cells towards the walls, a process called margination [START_REF] Basmadjian | The effect of flow and mass transport in thrombogenesis[END_REF][START_REF] Goldsmith | Rheological aspects of thrombosis and haemostasis: basic principles and applications. ICTH-Report--Subcommittee on Rheology of the International Committee on Thrombosis and Haemostasis[END_REF]. This lateral migration creates a layer of platelets and white blood cells (WBC) near the vessel wall in the so-called cell-free layer [START_REF] Aarts | Blood platelets are concentrated near the wall and red blood cells, in the center in flowing blood[END_REF][START_REF] Czaja | The influence of red blood cell deformability on hematocrit profiles and platelet margination[END_REF][START_REF] Eckstein | Conditions for the occurrence of large near-wall excesses of small particles during blood flow[END_REF].

Concept of blood flow motion

For simplification blood is usually considered as an incompressible fluid with a constant viscosity, named Newtonian fluid. The blood movement through a cylindrical tube is considered as a Poiseuille flow, which means that it relies on a pressure difference created by the heart. Blood flowing through a cylindrical tube has a parabolic velocity profile with a maximal velocity in the center that decreases towards the wall (Figure 21) [START_REF] Ruggeri | Platelet adhesion under flow[END_REF].

Therefore, blood flow is modelled as a series of adjacent layers sliding smoothly one over another, which creates a friction called shear. Due to the parabolic profile of velocities the flow near the vessel wall being close to zero, it facilitates the initiation of coagulation reaction as well as platelet adhesion [START_REF] Hathcock | Flow effects on coagulation and thrombosis[END_REF]. Although, blood is usually described as a Newtonian fluid, in reality, it is non-Newtonian because under low flow its viscosity depends on the hematocrit which decreases exponentially with increasing blood flow [START_REF] Goldsmith | Rheological aspects of thrombosis and haemostasis: basic principles and applications. ICTH-Report--Subcommittee on Rheology of the International Committee on Thrombosis and Haemostasis[END_REF][START_REF] Schmid-Schonbein | Rheology of thrombotic processes in flow: the interaction of erythrocytes and thrombocytes subjected to high flow forces[END_REF]. As a consequence, blood exhibits non-Newtonian behaviors such as shear thinning, yield stress and viscoelasticity [START_REF] Fisher | Effect of non-newtonian behavior on hemodynamics of cerebral aneurysms[END_REF][START_REF] Gijsen | The influence of the non-Newtonian properties of blood on the flow in large arteries: unsteady flow in a 90 degrees curved tube[END_REF][START_REF] Merrill | Non-Newtonian Rheology of Human Blood--Effect of Fibrinogen Deduced by "Subtraction[END_REF][START_REF] Thurston | Viscoelasticity of human blood[END_REF]. These properties affect the blood movement inside the vessel, fluid transport and blood force acting on vessel walls and surrounding tissues especially in paths with irregular lumen geometry or stenosed arteries [START_REF] Liu | Influence of non-Newtonian properties of blood on the wall shear stress in human atherosclerotic right coronary arteries[END_REF][START_REF] Lou | A computer simulation of the non-Newtonian blood flow at the aortic bifurcation[END_REF].

Another important parameter to consider is the pulsatile nature of arterial flow which ranges from 0 (or even reversed flow) up to two times the average velocity during each cyclic period [START_REF] Rhode | Novel approaches to the measurement of arterial blood flow from dynamic digital X-ray images[END_REF]. Taking into account the pulsatility, the peak blood flow parameters exceed the values of blood flow regimes calculated for non-Newtonian fluid [START_REF] Lutz | Comparison of steady and pulsatile flow in a double branching arterial model[END_REF].

1.2.Shear rate and shear stress as major hemodynamic parameters

A key parameter to define blood flowing in different vessels is the shear rate (). It is used to characterize the rate at which one fluid layer passes over another. For a Poiseuille flow, the wall shear rate increases linearly with a volume velocity (Q) and decreases as the inverse cube of tube radius (R) [START_REF] Sierra | The physiology of hemostasis[END_REF]:

𝛾 = 4𝑄 𝜋𝑅 3 (1)
For healthy human vessels the mean wall shear rates are 450-2,000 s -1 in the microcirculation and in arterioles, 300-800 s -1 in the large arteries and 15-200 s -1 in veins (Table 3) [START_REF] Goldsmith | Rheological aspects of thrombosis and haemostasis: basic principles and applications. ICTH-Report--Subcommittee on Rheology of the International Committee on Thrombosis and Haemostasis[END_REF][START_REF] Nader | Blood Rheology: Key Parameters, Impact on Blood Flow, Role in Sickle Cell Disease and Effects of Exercise[END_REF][START_REF] Panteleev | Wall shear rates in human and mouse arteries: Standardization of hemodynamics for in vitro blood flow assays: Communication from the ISTH SSC subcommittee on biorheology[END_REF]. Of note, these values are mean wall shear rates and they can vary in arteries due to pulsatile flow. which linearly depends on shear rate with a proportionality constant called viscosity (η) (2) [START_REF] Benis | Determination of the shear stress-shear rate relation for blood by Couette viscometry[END_REF]:

𝜏 = 𝜂 • 𝛾 (2)
Shear stress and shear rate regulate receptor-ligand bond formation during the initial step of platelet adhesion at the site of injury.

Role of blood flow in thrombus formation

The process of platelet aggregate formation in thrombosis and hemostasis shows a high degree of similarity. Following vascular injury, platelets carried by flow adhere, become activated and aggregate. In parallel, the coagulation cascade becomes activated leading to the generation of thrombin and to the formation of fibrin insoluble network. Blood flow affects every step of this process [START_REF] Savage | Specific synergy of multiple substrate-receptor interactions in platelet thrombus formation under flow[END_REF][START_REF] Weiss | Effect of shear rate on platelet interaction with subendothelium in citrated and native blood. I. Shear rate--dependent decrease of adhesion in von Willebrand's disease and the Bernard-Soulier syndrome[END_REF]. First, the flow has an impact on the ability of platelet receptors to engage their ligands immobilized at site of injury. While under relatively low shear (< 900 s -1 ) almost all the adhesive receptors can bind their ligands to ensure the capture of flowing platelets, under high shear only the GPIb-IX complex has the potential to sustain platelet attachment through its interaction with vWF [START_REF] Maxwell | Identification of a 2-stage platelet aggregation process mediating shear-dependent thrombus formation[END_REF][START_REF] Turitto | Platelet interaction with rabbit subendothelium in von Willebrand's disease: altered thrombus formation distinct from defective platelet adhesion[END_REF]. Once platelets have adhered, hemodynamic forces can stimulate mechanosensors, such as the GPIb-IX complex and integrin αIIbβ3, mechano-receptor Piezo1 and mechano-sensitive ion channels TRPV2 and TRPV4, to initiate signal transduction and subsequent platelet activation [START_REF] Abbonante | A new path to platelet production through matrix sensing[END_REF][START_REF] Chen | An integrin alphaIIbbeta3 intermediate affinity state mediates biomechanical platelet aggregation[END_REF][START_REF] Li | Platelet-derived microvesicles induce calcium oscillations and promote VSMC migration via TRPV4[END_REF][START_REF] Mazzucato | Sequential cytoplasmic calcium signals in a 2-stage platelet activation process induced by the glycoprotein Ibalpha mechanoreceptor[END_REF][START_REF] Shen | A directional switch of integrin signalling and a new anti-thrombotic strategy[END_REF][START_REF] Zhao | Piezo1 initiates platelet hyperreactivity and accelerates thrombosis in hypertension[END_REF]. Moreover, once the aggregate is formed, the shear forces tend to disrupt the receptor/ligand and/or receptor/receptor bonds and thereby promote thrombus instability and platelet disaggregation. Finally, the amplification of platelet activation by soluble agonists, such as ADP, TxA2 or thrombin, is finely tuned by the flow which carries away those released by activated platelets at site of thrombus formation to avoid excessive thrombus growth at site of vascular injury.

vWF, a shear sensitive molecule

vWF is a multimeric plasma glycoprotein which is synthesized by endothelial cells and megakaryocytes [START_REF] Jaffe | Synthesis of von Willebrand factor by cultured human endothelial cells[END_REF][START_REF] Sporn | Biosynthesis of von Willebrand protein by human megakaryocytes[END_REF]. It is also stored in the platelet α granules and becomes released after platelet activation [START_REF] Mumford | A review of platelet secretion assays for the diagnosis of inherited platelet secretion disorders[END_REF]. vWF is composed of several domains: i) the A1 domain which binds GPIbα and type IV collagen, ii) the A2 domain, site of cleavage of the vWF by A Desintegrin and Metalloproteinase with ThromboSpondin type 1 repeat 13 (ADAMTS13) which reduces the size and reactivity of circulating multimers, iii) the A3 domain binding site of type I and III subendothelial collagen, iv) the C4 domain which binds the integrin αIIbβ3 and v) the D3 domain which binds FVIII [START_REF] Brehm | Von Willebrand factor processing[END_REF].

Under low shear rates, this protein has a closed globular conformation hiding a cryptic binding site in the A1 domain, and therefore preventing its accessibility to the GPIb-IX complex [START_REF] De Luca | Structure and function of the von Willebrand factor A1 domain: analysis with monoclonal antibodies reveals distinct binding sites involved in recognition of the platelet membrane glycoprotein Ib-IX-V complex and ristocetin-dependent activation[END_REF][START_REF] Di Stasio | The effect of shear stress on protein conformation: Physical forces operating on biochemical systems: The case of von Willebrand factor[END_REF][START_REF] Ulrichts | The von Willebrand factor self-association is modulated by a multiple domain interaction[END_REF]. Under high shear rates (> 5,000 s -1 ) or when immobilized on a surface in the presence of low shear, vWF stretches due to the hydrodynamic force and exposes the A1 domain allowing platelet adhesion through the GPIb-IX complex [START_REF] Alexander-Katz | Shear-flow-induced unfolding of polymeric globules[END_REF]Li et al., 2008;[START_REF] Shankaran | Aspects of hydrodynamic shear regulating shear-induced platelet activation and self-association of von Willebrand factor in suspension[END_REF][START_REF] Springer | von Willebrand factor, Jedi knight of the bloodstream[END_REF].

Platelet aggregation under pathological conditions

In diseased arteries with a thickening of the wall due to an evolved atherosclerotic plaque or a pre-existing thrombus, the irregular vessel geometry leads to generation of flow perturbations (Figure 22) [START_REF] Glagov | Hemodynamics and atherosclerosis. Insights and perspectives gained from studies of human arteries[END_REF][START_REF] Young | Flow characteristics in models of arterial stenoses. II. Unsteady flow[END_REF]. For example, blood flowing through a stenosed vessel generates i) flow acceleration in the pre-stenotic area, ii) high shear exceeding 45,000 s -1 in the stenosis throat and iii) regions of flow recirculation in the post-stenotic zone [START_REF] Bark | Wall shear over high degree stenoses pertinent to atherothrombosis[END_REF]. In the pre-stenotic region, acceleration of the blood flow generates elongational flows and shear gradients which facilitate vWF unfolding that exposes the A1 domain and can promote platelet aggregation [START_REF] Sing | Elongational flow induces the unfolding of von Willebrand factor at physiological flow rates[END_REF].

Concerning the apex of the stenosis, the shear exceeds threshold values of 5,000 s -1 and can also unfold circulating vWF [START_REF] Kroll | Platelets and shear stress[END_REF]. Finally, in the post-stenotic zone, the recirculation flows have been shown to be highly prothrombotic in in vitro and in vivo models.

This effect is likely explained by the accumulation of soluble activators and platelets which are not washed away and can more easily accumulate [START_REF] Jackson | Dynamics of platelet thrombus formation[END_REF]. (Figure from [START_REF] Gorog | Platelet function tests: why they fail to guide personalized antithrombotic medication[END_REF]).

Shear-selective therapy

Shear-selective anti-platelet therapies have been proposed as an innovative treatment to prevent pathological thrombus formation while only modestly increasing the risk of bleeding.

Two approaches to block high shear-mediated thrombosis were proposed. One is focusing on inhibition of the shear-driven interaction between the vWF A1 domain and GPIbα (a singlechain antibody scFv-A1) [START_REF] Hoefer | Targeting shear gradient activated von Willebrand factor by the novel single-chain antibody A1 reduces occlusive thrombus formation in vitro[END_REF]. The second approach is based on shear-sensitive vehicles or nanoparticles aggregates with anti-thrombotic agents which release their contents only under high shear rates [START_REF] Holme | Shear-stress sensitive lenticular vesicles for targeted drug delivery[END_REF][START_REF] Korin | Shear-activated nanotherapeutics for drug targeting to obstructed blood vessels[END_REF][START_REF] Marosfoi | Shear-Activated Nanoparticle Aggregates Combined With Temporary Endovascular Bypass to Treat Large Vessel Occlusion[END_REF][START_REF] Molloy | Shear-sensitive nanocapsule drug release for site-specific inhibition of occlusive thrombus formation[END_REF].

Results

Blood flow conditions taking place at the edge of the wound after traumatic injury of the vessel

Publication 1:

« Traumatic vessel injuries initiating hemostasis generate high shear conditions»

Alexandra Yakusheva 1-3 , Kirill Butov 2-4 , Georgii Bykov 2 , Gábor Závodszky 6 , Anita Eckly 1 , Fazly Ataullakhanov 2,3,5 , Christian Gachet 1 , Mikhail Panteleev #2,3,5 , Pierre H Mangin #1

Introduction

The parameter most commonly used to characterize blood flow is the shear rate, which describes the rate at which one fluid layer passes over another and estimates the velocity change in the direction perpendicular to the flow. The current view is that low and intermediate flows (50 s -1 , 500 s -1 ) occur in intact healthy vessels, while high shear levels (>2,000 s -1 ) are reached in stenosed arteries, notably during thrombosis [START_REF] Goldsmith | Rheological aspects of thrombosis and haemostasis: basic principles and applications. ICTH-Report--Subcommittee on Rheology of the International Committee on Thrombosis and Haemostasis[END_REF][START_REF] Panteleev | Wall shear rates in human and mouse arteries: Standardization of hemodynamics for in vitro blood flow assays: Communication from the ISTH SSC subcommittee on biorheology[END_REF]. High shear found at the apex of a plaque believed to be a specific feature of thrombosis.

It has been proposed that targeting high shear through the inhibition of the shear gradient specific conformation of VWF or by shear-sensitive vehicles of anti-thrombotic agents represents an innovative strategy to selectively block thrombosis with a minor impact on hemostasis, thereby potentially avoiding bleeding complications [START_REF] Hoefer | Targeting shear gradient activated von Willebrand factor by the novel single-chain antibody A1 reduces occlusive thrombus formation in vitro[END_REF][START_REF] Korin | Shear-activated nanotherapeutics for drug targeting to obstructed blood vessels[END_REF]. While the range of physiological wall shear rates values is well established in intact vessels, the shear rates occurring at the edge of a lesion in a healthy vessel remain unknown.

The objective of my first project was to measure the blood flow occurring in wounds after different types of vessel injuries in a context relevant to hemostasis in human and mice.

For this purpose, we developed two novel mouse models of hemostasis in distinct vessels (carotid artery, aorta, saphenous vein and spermatic artery) which represent the two basic scenarios of traumatic injury, i.e. vessel puncture or vessel transection. The lesions and the plugs forming in these models were characterized by fluorescence and scanning electron microscopies. Combining Doppler probe measurements and computations, we determined the variation of blood flow over time after vessel damage. On the basis of the flows and the size of the injury measured experimentally, the shear rate at the edge of the wound was calculated using Navier-Stokes equations and ComSol Multiphysics software. An original model was also developed in humans, based on measurement of the blood loss after injury of the median cubital vein. A puncture was created by inserting a catheter into the cubital vein of healthy human volunteers and the blood loss was measured every minute after injury to calculate the shear levels by applying Poiseuille's equation with volumetric rates of blood loss. This work has been published in Blood Advances in June 2022.

Conclusion

My study shows that after vessel puncture of the murine carotid artery, the shear rate values appear very elevated with medians of 22,000 s -1 at the edge of the hole where platelets start to adhere. The shear rates were increased up to 3 times as compared to a carotid with a stenosis and 20 times when compared to an intact carotid. Similar results were obtained after puncture of the aorta and saphenous vein with medians of shear rates reaching 25,000 s -1 and 7,000 s -1 , respectively. In all models, the high shear conditions were accompanied by elevated levels of elongational flow exceeding the threshold levels required for unfolding of vWF.

Similar shear and elongational levels were observed after transection of the mouse spermatic artery with medians of 14,500 s -1 and 1,000 s -1 , respectively. In humans, the level of shear at the edge of a wound was highly increased after vessel damage with a catheter and reached 2,000-27,000 s -1 . Another interesting observation made in the puncture models in human and mice, was that the shear rates decreased steeply with increasing injury size. This phenomenon was explained by the low hydrodynamic resistance of the injuries as compared to that of the downstream vessel network.

All of these results indicate that various types of lesions in small and large mouse and human vessels can result in extremely high shear rates, which was unexpected. We propose that elevated shear rates are not specific to pathological conditions and can be equally relevant to the physiological process of hemostasis after vessel damage.

Introduction

Platelets express at their surface five different integrins of the β1 and β3 families: The β1 integrins comprising α2β1, α5β1 and α6β1, and the β3 integrins including αvβ3 and αIIbβ3.

Among the β1 integrin family, α2β1 and α6β1 are involved in the initial step of platelet adhesion and activation to adhesive proteins of the subendothelium, and their roles in hemostasis and thrombosis have also been well studied [START_REF] He | The contributions of the alpha 2 beta 1 integrin to vascular thrombosis in vivo[END_REF][START_REF] Schaff | Integrin alpha6beta1 is the main receptor for vascular laminins and plays a role in platelet adhesion, activation, and arterial thrombosis[END_REF]. While the role of integrin α5β1 in platelet adhesion, activation and aggregation on fibrillar fibronectin has been identified [START_REF] Maurer | Fibrillar cellular fibronectin supports efficient platelet aggregation and procoagulant activity[END_REF], the importance of this integrin in hemostasis and in experimental thrombosis remained unknown when we started this work.

The objective of this project was to evaluate the role of integrin α5β1 in hemostasis and experimental thrombosis. For this purpose, mice invalidated for the α5β1 integrin in the platelet lineage (PF4Cre-α5-/-) were generated. We first characterized these mice and their platelets by evaluating the expression of surface receptors, in order to ensure the absence of major abnormalities. Then, we studied in vitro platelet functions by performing aggregometry, determining platelet activation state in response to soluble agonists by flow cytometry and by perfusing murine whole blood on different adhesive proteins. Further, we evaluated the role of integrin α5β1 in arterial thrombosis in vivo in three different experimental animal models of arterial thrombosis for which the rheological conditions as well as the exposed adhesive proteins were different. Finally, we examined the role of integrin α5β1 in hemostasis by using a tail bleeding time model. This work has been published in Thrombosis and Haemostasis in October 2021.

Conclusion

The results of this work show that the platelets of mice deficient for α5β1 do not show any particular defect. PF4Cre-α5 -/-platelets have a normal count and volume, as well as a normal expression of the main surface receptors (αIIbβ3, α2, α6, β1, GPIbα, GPV, GPIX, GPVI), apart for the α5 integrin which is absent. Aggregation of PF4Cre-α5 -/-platelets was also normal in response to series of agonists such as collagen, ADP, U46619, a thromboxane A2 analog, and thrombin. Absence of α5 integrin on platelets has no major impact on integrin αIIbβ3 activation and granule secretion. Platelets of these mice adhere normally to a surface of vWF, fibrinogen or laminins under shear flow. However, they present a major defect in adhesion, activation, and aggregation on a fibrillar fibronectin surface. In three experimental models of arterial thrombosis, mice invalidated for the α5β1 presented a normal profile of thrombus formation and disaggregation as compared to controls. Finally, the normal tailbleeding time favors no major role of this integrin in hemostasis.

Together, the results gathered in this study suggest that α5β1 integrin plays an instrumental role in the adhesion, activation and aggregation of platelets to fibrillar fibronectin, and might also participate in thrombus growth on collagen. However, our results indicate that integrin α5β1 does not play a major role in hemostasis or arterial thrombosis in mice. It remains possible that integrin α5β1 could play a more important role in a lesion exposing a large amount of fibronectin, but this remains to be demonstrated. We conclude that it is unlikely that integrin α5β1 represents an interesting anti-thrombotic target.

Introduction

Following an injury of a healthy vessel, tissue factor and adhesive proteins of the subendothelium are exposed to the flowing blood and initiate the hemostatic response. Thanks to the use of intravital microscopy in various experimental models of vessel injury in mice, the dynamic process of platelet aggregation has been unraveled. First, platelets accumulate to form a large, sometimes subocclusive aggregate, which then disaggregates to leave a platelet rich plug to cover the site of injury. This plug is stabilized by fibrin which forms as the result of fibrinogen cleavage by thrombin generated locally upon tissue factor exposure. The bi-phasic nature of plug formation is essential to avoid unwanted occlusion and ischemia in surrounding tissues. While the mechanism allowing platelet aggregation is very well characterized, the one limiting this process and switching off the hemostatic response is still unclear.

The objective of my third project was to evaluate the role of fibrin in the dynamics of the formation of a hemostatic plug, beyond its stabilizing role. For this purpose, we characterized the dynamic of thrombus and fibrin formation and the plug composition in FeCl3 injury and needle puncture models by using histology, fluorescence microscopy, and electron microscopy approaches. Then, we studied in vitro the ability of platelets to form aggregates on fibrin-poor and fibrin-rich aggregates by using a blood flow assay. Further, we evaluated in vivo the role of fibrin in thrombus formation by mechanically or pharmacologically removing the aggregate before fibrin was predominant in the clot. Finally, we examined in vivo the dynamic of thrombus growth using FeCl3-induced model, in transgenic FibAEK mice presenting a mutation of the Aa chain of fibrinogen which cannot form a fibrin network. A manuscript related to this work is in preparation.

Fibrin covering the thrombus surface turns off the hemostatic response

Alexandra Yakusheva

INTRODUCTION

Hemostasis is a finely tuned physiological process initiated after vessel injury and which aims at repairing the vessel to rapidly stop blood loss and to restore a normal blood intake to supply downstream tissues and organs 1 . It is composed of three interconnected steps and starts with primary hemostasis, which is initiated thanks to the unique ability of blood platelets to be recruited to thrombogenic subendothelial proteins exposed at sites of injury 2 . Platelets then activate and form an aggregate through their ability to bind plasma proteins such as von Willebrand factor (vWF) and fibrinogen, allowing platelet aggregation to form the hemostatic plug 3,4 . In parallel, the tissue factor (TF) exposed in the vessel wall initiates secondary hemostasis or coagulation, which is a cascade of enzymatic reactions amplified at the surface of negatively charged surfaces of platelets and endothelial cells 5 . This leads to the generation of thrombin, a key serine protease, which cleaves fibrinogen into an insoluble fibrin network which stabilizes the clot 6 . At later stages, fibrinolysis is activated and removes the remaining part of the clot to restore normal blood flow and full vessel wall repair 7 .

Platelet plug formation is central for a relevant hemostatic response and to ensure the arrest of bleeding. One of its unique feature is that it is very rapidly initiated following vessel injury to form the aggregate as quickly as possible, thereby limiting blood loss.

Plug formation requires to be finely regulated to allow a suitable response, i.e.

occlusive clot formation in case of vessel rupture or a non-occlusive plug for weaker injuries 8 . This is essential to avoid an excessive response which could result in unnecessary occlusion in vessels presenting modest injuries, thereby avoiding reduction of blood supply to downstream tissues. While the process of platelet adhesion, activation and aggregation at site of injury is very well understood, the process limiting extensive platelet aggregation and switching off the hemostatic response remains fully unknown. In this manuscript, we studied the dynamics of platelet plug formation using in vivo mouse models to investigate the regulation of the hemostatic process. We identified a central role of fibrin, which in addition to its very well-established role in stabilizing the clot, is instrumental in regulating the hemostatic response by turning off the physiological response of plug formation.

MATERIALS AND METHODS

FibAEK mice

Mice carrying a mutant form of fibrinogen were generated by backcrossing FibAEK progeny mice (Matthew J. Flick Laboratory) with C57Bl/6J (Jackson Labs) as was described 9 . In all animal studies, gender and age matched (6-8 weeks) mice were used

Mouse model of hemostasis based on vessel puncture

WT mice with a pure C57BL/6 background were maintained in the animal facilities of the EFS Grand-Est. Ethical approval for the experiments was obtained from the French Ministry of Research. Fluorescent agents (3,3'-dihexyloxacarbocyanine iodide (DIOC6) (Thermo Fisher Scientific, MA, USA); DyLight650-coupled anti-fibrin antibody 59d8

(Inserm UMR_S11076, Marseille, France)) were injected into the jugular vein of 7-28 week-old mice, 5 min before the experiment, to label platelets. The common carotid artery was exposed surgically and was then punctured with a 25-gauge needle.

Thrombus formation was monitored in real time with fluorescent microscope coupled to a CCD camera. At the end of the experiment, the thrombus was fixed and processed for histology or electron microscopy 10 .

In vivo models of arterial thrombosis

Fluorescent agents were injected into the jugular vein 5 min before initiation of thrombus formation. The abdominal aorta of anesthetized WT mice (8-10 weeks old)

was exposed and mechanically injured by pinching with forceps (type 11063-07, F.S.T., Heidelberg, Germany), as previously described 11 . FeCl3-injury was performed as in 10 . Briefly, the common carotid arteries of anesthetized WT mice (7 to 10 weeks old) were exposed and the left carotid artery was injured by applying a 1 mm long patch of filter paper (1M Whatmann) saturated with 7.5% FeCl3 for 2.5 min.

Drug administration

Mice were infused through the jugular vein with either saline (control), rt-PA (10 mg/kg) or hirudin (20 mg/kg) at the indicated times.

Histology of the carotid artery

Before injury, the ligature was prepared in both ends of the carotid artery. At the end of experiment, the vessel was fixed with 300 ul 4% paraformaldehyde was placed on the vessel as previously described 12 . After 15 minutes, the ligature was put and the carotid artery was excised, placed in 4% paraformaldehyde overnight and rinsed with phosphate buffer saline. Then it was placed in increasing concentrations of saccharose and was frozen in optimal cutting temperature compound. The sample was sectioned at 8-um thickness by microtome-cryostat. The sample was labelled with DAPI. Imaging was performed with a Leica confocal microsystem SP5 DMI 6000.

Scanning electron microscopy

Before injury, the ligature was prepared in both ends of the carotid artery. At the end of experiment, the vessel was fixed with 2% glutaraldehyde and 2% paraformaldehyde by transcardiac perfusion. The carotid artery was then excised and post-fixed in 2% glutaraldehyde and 2% paraformaldehyde overnight. The isolated vessel segment was sectioned in the middle by razor and it was dehydrated in increasing concentrations of ethanol followed by hexamethyldisilazine and glued onto a cover slip with the lumen uppermost. The cover slips were sputtered with gold prior to observation at 5 kV under an FEG Sirion SEM (FEI). The chemical distribution of elements was studied by energy dispersive X-ray emission microanalysis (EDX) of selected sections using an ESEM (FEI) at 20 kV, spot size 4.

In vitro flow-based adhesion assay

Microfluidic flow chambers were prepared as previously described 13 . Briefly, the chambers were coated with a solution of fibrillary Horm collagen (200 µg/mL) overnight at 4℃ and blocked with phosphate buffered saline (PBS) 10 mg/mL human serum albumin for 30 minutes at room temperature. For fibrin-poor thrombus formation, hirudinated (100 U/mL) whole blood from healthy human volunteers was labelled with DiOC6 (3,3'-dihexyloxacarbocyanine iodide;1 µmol/L) and was perfused through the coated microfluidic flow chambers with a syringe pump (Harvard Apparatus, Holliston, MA, USA) at 37℃ at 1500 s-1. For fibrin-reach thrombus formation, citrated (3.2%)

human whole blood was labelled with a DyLight 647-coupled anti-fibrin antibody (10 µg/mL) and DiOC6 (3,3'-dihexyloxacarbocyanine iodide;1 µmol/L) and was recalcified by adding CaCl2 (12.5 mmol/L) and MgCl2 (3.5 mmol/L) just before perfusing it through microfluidic flow chambers at 1500 s-1. Once platelet rich thrombi were formed, a brief washing step was performed to rinse the thrombi before perfusing hirudinated blood stained with RAM.1-A567 (2 µg/mL) from the same donor. Fluorescence emission was measured using a confocal Leica SP8 inverted microscope with a resonant scanner and a 40× oil objective. Series of optical sections in xyz were taken from the base to the peak of the thrombi (Leica LAS X software). Images were then stacked and the volume of the thrombi was determined with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Statistical analyses

All statistical analyses were performed using a GraphPad Prism program, version 6.0 (Prism, GraphPad, La Jolla, CA, USA). All values are reported as the mean ± standard error of the mean for a normal distribution, or median for a non-normal distribution. The data of two groups were compared by the two-tailed paired test.

RESULTS

Insight into the dynamics of the formation of a hemostatic plug. To gain insight into the process stopping the hemostatic response after a localized vessel injury, we used in vivo mouse models of vascular injury. We first observed that puncturing the carotid artery of adult mice with a needle resulted in bleeding followed by the formation of a platelet and fibrin-rich plug which stops blood loss. Real-time video microscopy showed that the plug formation is reversible presenting a bell-shaped curve with a phase of growth, embolization to finally stabilize. The thrombus embolization occurs at 250 ± 50 sec after thrombus onset, after which no platelets accumulated anymore (Figure 1A). Fibrin appeared with a delay of 60 ± 20 sec after the first platelets adhered at site of injury, and grew progressively occupying 40 ± 20 % of thrombus area at the time of embolization. By 450 sec after thrombosis initiation fibrin occupied 60 ± 15 % of thrombus area coinciding with no more platelet accumulation (Figure 1B).

Interestingly, when FeCl3 was applied to the carotid, a similar kinetic of platelet aggregation and fibrin formation was evidenced, with the only distinction of the formation of a mural thrombus (Figure 1C). In this model, there was no more platelets accumulation after thrombus embolization, which starts at 1,030 ± 80 sec, when fibrin occupied 52 ± 9 % of its maximum area and its amount was 70 ± 9% of fibrin maximum area 300 sec after embolization (Figure 1D). Similar observations were also obtained after a mechanical injury with forceps of the aorta, even though the overall process was more rapid compared to the two other models (Figure 1E-F). Together, a common feature of three distinct models of vascular injury indicate that the arrest of thrombus growth, i.e. of the hemostatic response, appeared after thrombus embolization and coincides with the time at which fibrin occupied most of the area of the plug.

Fibrin covers the hemostatic plug. To better appreciate the spatiotemporal localization of fibrin within a plug, the carotid artery of adult mice was injured with FeCl3 and fibrin distribution in the thrombus was studied by different approaches.

Fluorescence images using a macroscope to have a large field of view, indicated that after 1500 sec, when the most part of the thrombus was embolized, fibrin occupied 32 ± 8 % of the area of the residual platelet rich plug (Figure 2A). Histological analysis of these thrombi revealed that fibrin staining reached the top of the thrombus, suggesting its exposure to the flowing blood (Figure 2B). This result was confirmed using scanning electron microscopy (SEM) which showed that a large area of the plug contained fibrin on its top that covers the thrombus surface (Figure 2C). Similar observations were made for plugs forming after puncture of the carotid artery and pinching of aorta.

for puncture (Figure 2D-E) and pinching models (Figure 2F-G), respectively.

Together, these imaging approaches indicate that the residual platelet plug is very rich in fibrin, which reaches its top to form a layer. This observation questions whether such a layer exhibits a protective function and could stop further platelet adhesion and aggregation to stop the hemostatic response.

A fibrin layer prevents thrombus growth in vitro. To test the hypothesis of fibrin being involved in arresting the hemostatic response, we used an in vitro approach.

Using a flow-based assay, we formed fibrin-poor and fibrin-rich thrombi by perfusing hirudinated and recalcified citrated blood over collagen at 1,500 s -1 , respectively (Figure 3A). The mean thrombus volumes of fibrin-poor and fibrin-rich thrombi were 10±2 x 10 4 m 3 and 9±3 x 10 4 m 3 (mean ± SEM, n=5), respectively (Figure 3B,C).

We next perfused human hirudinated whole blood over the pre-formed thrombi to evaluate their thrombogenic potential. We observed thrombus build-up on fibrin poor aggregates (3±1 x 10 4 m 3 ), while this process was reduced by more than 10 times on fibrin-rich thrombi (0.3±0.1 x 10 4 m 3 (mean ± SEM, n=5) (Figure 3B,D). This in vitro observation indicates a negative role of fibrin in thrombus growth and allows to hypothesize that a fibrin layer could exhibit a protective function and efficiently limit platelet adhesion and aggregation.

Fibrin stops the process of platelet recruitment and aggregation in vivo. To test the hypothesis that fibrin limits thrombus growth in vivo after thrombus embolization, we injured the carotid artery of an adult mouse with a solution of FeCl3, and once the thrombus reached its maximal size and fibrin only started to appear in the core (fibrin poor thrombus), we mechanically removed the thrombus by flicking the vessel.

Interestingly, under such conditions where fibrin is largely absent from the aggregate, the thrombus was able to repeatedly form again and stopped only when fibrin reached its maximal level i.e., the thrombus surface (Figure 4A-B). This result was confirmed by using a pharmacological approach based on rtPA injection to lyse the fibrin content within the aggregate when the thrombus reached its maximal size (Figure 4C). As expected rtPA in contrast to saline and even hirudin efficiently reduced the level of fibrin within the thrombus and destabilized the aggregate to induce embolization that was not observed to other solutions (Figure 4D, E; Sup. Figure 1A). Interestingly, this did not lead to the arrest of the hemostatic process but led to a regrowth process which far exceeded the time at which aggregation stops in the control and with hirudin (Figure 4F). In addition, this regrowth could be repeated several times, further highlighting a role of fibrin in the arrest of the hemostatic response (Figure 4F).

A genetically modified mouse defective in fibrin formation a marked impairment in the arrest of the hemostatic response. To confirm the hypothesis that fibrin limits thrombus growth and exclude side effects of mechanical and pharmacological approaches, we studied the thrombus formation after FeCl3-induced thrombosis in transgenic FibAEK mice presenting a mutation of the Aa chain of fibrinogen. These mice present a normal level of circulating fibrinogen, but do not have the ability to form an insoluble fibrin network. The dynamic of thrombus formation in FibAEK mice consists of a repeated series of growths and embolizations during all experimental time (Figure 5A,B). In addition, the amount of regrowth processes during thrombus formation was significantly higher in FibAEK mice compared to WT mice (Figure 5C).

More importantly, we could not detect a time at which the process stopped and had to terminate the experiments after 60 min, which far exceeds the time at which aggregation stops in controls (Figure 5D). In conclusion, these findings identified a central role of fibrin, which in addition to its very well-established role in stabilizing the clot, is also instrumental in regulating the hemostatic response by turning off the physiological response of plug formation and preventing further platelet incorporation.

DISCUSSION

The results of this work show that the process of thrombus growth leads to the formation of a stable platelet rich plug which ended up covered by fibrin in three distinct experimental models of vascular injury in large vessels. Using a flow-based assay, we observed that the presence of a fibrin layer on top of an aggregate prevents its growth.

In agreement, we observed that mechanical removal of a thrombus formed in vivo, after vessel flicking, resulted in continuous regrowth which stopped when fibrin reached the top of the thrombus. Moreover, in vivo experiments with pharmacological agents removing the fibrin during the thrombus growth phase, leads to thrombus embolization, which allowed the aggregate to grow again. Interestingly this regrowth process could be repeated many times. Finally, the dynamic of thrombus formation in FibAEK mice was profoundly altered with an expected defect in thrombus stabilization, and an additional prolongation of the hemostatic process composed of successive growth and embolization phases. This study identified a novel instrumental role for fibrin in turning off the physiological response of plug formation by limiting further incorporation of circulating platelets.

Vessel injury in various mouse models of localized vascular injury results in a very similar dynamic of thrombosis, with an initial growth of a very large thrombus, followed by a stage of embolization leaving a patch covering the lesion. The main focus of our study was to understand why following embolization of the large aggregate, the patch composed of highly activated platelets remained inert and did not grow again. This study is purely focused on understanding the physiological process of hemostasis, and is not considering a situation of arterial thrombosis. It is also important to highlight that we did not study the process of embolization per se, neither the process of occlusive thrombus formation. Our study was clearly focused on the first couple of minutes after vessel injury and the profile of the thrombus that forms is common to various vessels and mode of injury including a mechanical injury of the aorta, photochemical injury of the carotid, electrolytic injury of the femoral vein, laser-injury of the cremaster vessels and mesenteric arterioles, puncture injury of femoral vein and aorta and FeCl3-induced injury of the aorta and mesenteric arterioles 10,11,14-19 .

We observed that the surface of the residual patch formed in three different experimental models was covered by a layer of fibrin. These results correlate with the thrombus structure obtained after laser-induced injury in mouse mesenteric vessels which was reported to be mainly composed of platelets and covered by fibrin 20 . Such a fibrin biofilm was also detected on human intracoronary thrombi which were aspirated in patients with acute myocardial infarction 21 . Similar observations were made on thrombi responsible for large vessel occlusion in the setting of acute ischemic stroke 22 . Together, these results indicate that various thrombi from animal models or from patients share in common the presence of a fibrin layer on the luminal side. While the group of Robert Ariëns proposed that such fibrin biofilms play an antimicrobial function, its role in hemostasis had never been addressed 23 . We propose here that the fibrin layer plays an important role in the passivation of the thrombus and promotes thereby the arrest of the hemostatic response.

While the main result of this study indicates that the fibrin layer covering the thrombus switches off the process of thrombus growth, the precise mechanism remains to be determined. An obvious explanation of the arrest of thrombus growth is that the fibrin layer is poorly adhesive as shown in in vitro based experiments with a fibrin layer not efficiently promoting platelet activation and aggregation, and do therefore not represent a nucleation site for further growth 24 . In addition, it is well established that fibrin traps thrombin limiting its diffusion and pro-thrombotic effects 25,26 . This is best illustrated when we compared the effect rtPA versus rtPA combined to the direct thrombin inhibitor hirudin. While rtPA started to lyse fibrin and free thrombin a regrowth was observed which was due to thrombin as this effect disappeared in the presence of hirudin (Figure 4, Sup. Figure 1). Whether there are additional ways by which fibrin arrests thrombus growth cannot be excluded. One potential explanation could be that fibrin decreases the permeability-porosity of thrombus, thereby limits the transport velocity of soluble agonists released by platelets in thrombus core 27,28 . It is also possible that the fibrin layer on the residual thrombus surface restricts the distribution of granule content, for example, the exposure of vWF from α-granules as this molecule could remained trapped in fibrin fibers and not reach the thrombus surface to allow platelet recruitment 29,30 .

From a hemostatic standpoint, the platelet plug should be formed as quickly as possible to limit blood loss after vessel injury. While an occlusive aggregate is required after rupture of a vessel wall, a non-occlusive plug should be formed in case of nonpenetrating injuries. This is essential to avoid an unnecessary vessel occlusion, which would impair blood supply to downstream tissues. Our study proposes that the arrest of the hemostatic process is finely tuned by fibrin. Based on this data, it could be assumed that fibrin would be present in elevated quantities to form a non-occlusive thrombus and low quantities to allow the formation of an occlusive clot when a dramatically injured vessel has to be closed to avoid excessive blood loss. This hypothesis is in agreement with the literature, as murine thrombi formed after perforating laser injury have lower mounts of fibrin compared to thrombi forming after non-penetrating injury induced by laser ablation 31,32 . Because of a lack of sufficient amount of experimental evidence, the difference in occlusive and non-occlusive thrombi composition and the role of fibrin in it, requires further investigations.

In conclusion, our findings identify a central role of fibrin in the arrest of the hemostatic response, by passivating the surface of the thrombus and thereby limiting platelet adhesion to the plug. We propose that, in addition to its very well-established role in stabilizing the clot, fibrin is also instrumental in regulating the hemostatic response by turning off the physiological response of plug formation and preventing further platelet incorporation.

Conclusion

The results of this work show that the process of thrombus growth leads to the formation of a stable platelet rich plug which was covered by fibrin in three distinct experimental models of vascular injury in large vessels. Using a flow-based assay, we observed that the presence of a fibrin layer on top of an aggregate prevents its growth. Mechanical removal of the thrombus after vessel flicking resulted in continuous regrowth which stopped only when fibrin reached its maximal level within the thrombus surface and covered it. In vivo experiments with pharmacological agents removing the fibrin during thrombus growth lead to thrombus embolization, which allowed the aggregate to grow again, and this regrowth process was repeated many times. Finally, the dynamic of thrombus formation in FibAEK mice was profoundly altered with as expected a lack of thrombus stabilization, but additionally we also observed that the hemostatic process was prolonged compared to a control with successive growth and embolization phases.

In conclusion, these findings identified a central role of fibrin in the arrest of the hemostatic response, by limiting platelet adhesion to the plug. We propose that, in addition to its very well-established role in stabilizing the clot, fibrin is also instrumental in regulating the hemostatic response by turning off the physiological response of plug formation and preventing further platelet incorporation.

After vascular injury, platelets adhere, activate and aggregate at the injury site to form a hemostatic plug that limits bleeding. They are also involved in arterial thrombosis, which is the cause of serious pathologies such as myocardial infarction or ischemic stroke. Arterial thrombosis is treated by antiplatelet drugs which have shown their effectiveness in markedly reducing the mortality rates of thrombotic pathologies. However, the main limitation of these treatments is the increased risk of bleeding limiting their use in some clinical situations, such as ischemic stroke, for which the risk of hemorrhagic transformation is high and can have deleterious consequences for the patient. Identifying the specific features of hemostasis and arterial thrombosis in terms of molecular mechanisms or rheological conditions could provide valuable information to reduce the risk of bleeding while maintaining the antithrombotic effect.

To address this question, the objective of my thesis project consisted in: i) the characterization of flow conditions occurring after lesion of a healthy vessel; ii) the identification of the importance of platelet integrin α5β1 in hemostasis and arterial thrombosis;

iii) the evaluation of the role of fibrin in the termination of the hemostatic response.

In the 1,800s the physician Rudolf Virchow described three key features of intravascular venous thrombosis, i.e, endothelial injury, stasis of blood flow and hypercoagulability, which later were expanded to recognize the importance of blood flow in the regulation of hemostasis and arterial thrombosis. The parameter most commonly used to characterize blood flow is the shear rate. In intact vessels, the wall shear rate (WSR), which is the velocity gradient near the vessel wall, ranging from about 100 s -1 in veins up to 2,000 s -1 in small arterioles. In diseased vessels, in which an atherosclerotic plaque forms a severe stenosis, the WSR can reach several 10,000 s -1 . Since the high shear found at the apex of a plaque is recognized as a specific feature of thrombosis, targeting high shear has been proposed as an innovative strategy to selectively block thrombosis with a minor impact on hemostasis, thereby potentially avoiding bleeding complications. A major caveat of this paradigm and the anti-thrombotic strategy derived from it, is that the current knowledge of blood flow parameters occurring in vivo, are based on intact vessels in homeostatic conditions, and the rheological conditions taking place in the wound after vessel wall rupture have never been measured experimentally. The main finding of my first project is that various types of lesions in small and large mouse and human blood vessels result in extremely high shear rates at the edge of the wound where hemostasis occurs. The data demonstrate that shear rates reached during hemostasis are much greater than those occurring in intact vessels and currently proposed as being relevant for hemostasis. These results challenge the belief in the field that hemostasis is a process that depends on low and intermediate shears (< 2,000 s -1 ), while occlusive thrombosis in regions of plaque occurs under high shear. This paradigm shift in the field questions the postulate that targeting thrombus formation at high shear is a specific anti-thrombotic approach.

Hemostasis is taking place in many distinct settings including after injuries resulting in internal bleeding and resulting in bruising, or after open injuries responsible for external bleeding with blood leaving into the external environment. Any type of bleeding is induced by a pressure drop between two points of flow, i.e. inside the circulation and outside the vessel.

My first project was focused on traumatic injuries resulting in external bleeding. In this situation the bleeding is induced by pressure drop occurring between the blood circulation system and the atmosphere, with a drop evaluated at about 600 mmHg (Δ P≈600 mmHg). By comparison, internal bleeding is induced by a pressure drop appearing between the surrounding tissues and the circulation system that should be much lower due to tissue compression (Δ P≈10-74 mmHg) [START_REF] Ashton | The effect of increased tissue pressure on blood flow[END_REF]. Our study reports the occurrence of high shear rates in the case of external bleeding, while blood flow levels taking place during internal bleeding are still unknown and require further investigation. It would have been very interesting to obtain results by using, for example, a laser-induced model adapted to induce a perforating injury of small vessels resulting in internal bleeding and study the effect of active closure of small arterioles under vasomotor tone and passive collapse of soft-walled capillaries.

In our study, we proposed that external bleeding occurs under two different flow regimes: "constant pressure" for small injuries (< 30 μm) and "constant flow" for big injuries (> 150 μm). The occurrence of a "constant flow" regime was determined experimentally as it was possible to generate a large wound in the vessel wall by using a needle. However, a limitation of my study is that it was not possible to provide experimental evidence that for small injuries the shear rate relies on pressure drop -the "constant pressure" regime. We could unfortunately not perform smaller injuries due to technical limitations and generate such data.

Other studies faced similar issues, notably a publication using an experimental hemostasis model based on laser injury of the saphenous vein where the injury diameter was 48 μm, demonstrating the high challenge to perform injuries smaller than 30 µm in diameter which is necessary to switch to another flow regime for this vessel (Getz et al., 2015). A detailed analysis of fluid dynamics for tiny injuries would be definitely interesting and important but requires development of new technical approaches.

Based on observation of the presence of elevated shear at the edge of a wound in healthy vessels, we can now hypothesize that targeting high shear will probably not be devoid of bleeding complications after traumatic injuries (falls, trauma, surgery, etc) and, therefore, would not represent a specific antithrombotic strategy. However, whether there is a rheological parameter which could be used as a specific antithrombotic target remains an open and attractive question. One of them could be an excessive elongation flow which is known to be thrombogenic due to its efficiency in unfolding vWF and promoting platelet adhesion and activation. In our study, we also observed occurrence of increased elongational flows in injured healthy vessels when compared to values in intact vessels. Upcoming studies should identify the levels of elongational flows occurring in hemostatic and thrombotic settings and define the threshold of elongational flows precipitating vWF unfolding to determine whether this parameter can discriminate such situations. Another potential flow parameter discriminating hemostasis from thrombosis is turbulence which does not occur in the setting of hemostasis but could take place in the post-stenotic areas in diseased arteries. A detailed analysis of fluid dynamics in the setting of thrombosis would be definitely interesting and important to define turbulent conditions an elongational flow conditions found in regions where thrombosis is exacerbated.

Antiplatelet agents used in the clinic mainly target the platelet activation and aggregation stages, which is the case of aspirin, P2Y12 inhibitors and integrin αIIbβ3 blockers.

Another option of antiplatelet treatment would be to develop drugs targeting the interaction of platelets with the extracellular matrix in order to interrupt the initial step of the thrombotic process. These platelet-extracellular matrix interactions notably involve integrins of the β1 family (Bergmeier and Hynes, 2012). Among these are the integrins α2β1 and α6β1 which have already been proposed to participate in experimental thrombosis, and whose involvement in hemostasis appears modest, making them potentially interesting new antiplatelet targets [START_REF] He | The contributions of the alpha 2 beta 1 integrin to vascular thrombosis in vivo[END_REF][START_REF] Schaff | Integrin alpha6beta1 is the main receptor for vascular laminins and plays a role in platelet adhesion, activation, and arterial thrombosis[END_REF]. At the beginning of my PhD, the importance of integrin α5β1 in hemostasis and experimental thrombosis had never been evaluated, and became the aim of my second project.

The bleeding time of mice not expressing the α5β1 integrin specifically at the platelet level was not modified. The same results were published for mice deficient for α2β1 and α6β1 in which bleeding times were normal [START_REF] He | The contributions of the alpha 2 beta 1 integrin to vascular thrombosis in vivo[END_REF][START_REF] Schaff | Integrin alpha6beta1 is the main receptor for vascular laminins and plays a role in platelet adhesion, activation, and arterial thrombosis[END_REF]. Interestingly, the mice deficient for the three β1 integrins were described to present an increased bleeding time which suggested that these three receptors are important to ensure hemostasis, but present an important functional compensation. It also suggested that blocking only one of these receptors in the setting of a pharmacological antithrombotic approach might reduce arterial thrombosis for some of them and potentially be devoid of bleeding risk [START_REF] Petzold | beta1 integrin-mediated signals are required for platelet granule secretion and hemostasis in mouse[END_REF].

Our findings indicate that the platelet integrin α5β1 plays a key role in the adhesion, activation and aggregation of platelets in vitro on a surface of fibrillar cellular fibronectin.

However, our study did not show any major role of this integrin in models of experimental thrombosis in vivo. A potential explanation for this apparent discrepancy between the results obtained in vitro and in vivo, could rely in different rheological conditions. Indeed, while the blood perfusion in vitro over fibronectin was performed at 300 s -1 , it is well known that WSR found in mouse arteries are 5 to 10-times higher [START_REF] Panteleev | Wall shear rates in human and mouse arteries: Standardization of hemodynamics for in vitro blood flow assays: Communication from the ISTH SSC subcommittee on biorheology[END_REF]. Unfortunately, using such elevated WSR in vitro was not achievable as adhesion no longer occurred onto fibrillar fibronectin above 1,000 s -1 , which is in agreement with previously published studies [START_REF] Maurer | Fibrillar cellular fibronectin supports efficient platelet aggregation and procoagulant activity[END_REF]. At the molecular level, this lack of adhesion is most likely explained by the fact that immobilized fibronectin very inefficiently adsorbs plasma vWF compared to collagen. It would have been very interesting to obtain results at high wall shear rates in vitro by using, for example, a mixed matrix of vWF and cellular fibrillar fibronectin or by covering the flow chamber surface with cell-derived extracellular matrix. This matrix could be produced by immortalized fibroblasts and it is rich in fibrillar fibronectin which supports platelet adhesion even under high shear rates [START_REF] Maurer | Fibrillar cellular fibronectin supports efficient platelet aggregation and procoagulant activity[END_REF].

Our results indicate that α5β1 alone does not play a central role in experimental thrombosis, and one could be tempted to conclude that α5β1 is less important than the two other β1 integrins, α2β1 and α6β1, which have both been shown to contribute to experimental thrombosis [START_REF] He | The contributions of the alpha 2 beta 1 integrin to vascular thrombosis in vivo[END_REF][START_REF] Schaff | Integrin alpha6beta1 is the main receptor for vascular laminins and plays a role in platelet adhesion, activation, and arterial thrombosis[END_REF]. This hypothesis is also supported by the fact that we have studied PF4Cre-6-deficient mice in the same experimental thrombosis models as PF4Cre-5-deficient mice and only observed a defect in the former strain [START_REF] Schaff | Integrin alpha6beta1 is the main receptor for vascular laminins and plays a role in platelet adhesion, activation, and arterial thrombosis[END_REF].

We should nevertheless remain cautious as the injuries applied in the different experimental models might not expose sufficient amounts of fibronectin and therefore not favor the observation of a defect in PF4Cre-5-deficient mice. Moreover, in patients, arterial thrombosis generally occurs in a diseased vessel after rupture of an atherosclerotic plaque which is rich in fibronectin [START_REF] Rohwedder | Plasma fibronectin deficiency impedes atherosclerosis progression and fibrous cap formation[END_REF]. Therefore, an analysis of thrombus formation in a plaque rupture model in mice deficient for α5β1 integrin would have been definitely interesting and important to fully estimate the role of this integrin in vivo. To reproduce atherosclerosis, α5β1 deficient mice could be crossed with mice deficient in apolipoprotein E (ApoE) and the plaque could be ruptured by a needle or ultrasound to induce thrombosis (Hechler and Gachet, 2011a).

Finally, our observations result from mouse studies, and it remains possible that the role of α5β1 might be more important in humans.

Platelet plug formation is a dynamic process which could be divided into three stages:

the transient build-up of a sub-occlusive thrombus, which is followed by disaggregation and ends up in the stabilization of a residual patch. Such a profile has been observed in numerous injury model of the mesenteric venule (Kamocka et al., 2010). However, there is no other study of stabilized (after embolization) thrombus composition under venous conditions. Therefore, an analysis of fibrin distribution in a remaining thrombus after vein injury would have been definitely interesting and important to fully estimate the role of fibrin in the arrest of thrombus growth.

Our results indicate that the fibrin layer covering the thrombus switches off the process of thrombus growth. However, we did not fully investigate the mechanism of this process. An obvious explanation of the arrest of thrombus growth is that the fibrin layer is poorly adhesive and reactive so that when platelets adhere to it, they do not become fully activated and do therefore not represent a nucleation site for further growth. Another explanation could be that fibrin physically or chemically traps thrombin within the thrombus limiting its diffusion and avoiding it to sustain platelet activation and enhance coagulation. Therefore, in vivo analysis of thrombin diffusion within the thrombus during its growth could be interesting for further investigation of the mechanism of the arrest of the hemostatic response.

Under hemostatic conditions, the platelet plug should be formed as quickly as possible to limit blood loss after injury. Depending on the injury severity the plug geometry has to be different. Indeed, an occlusive aggregate is required after the rupture of small vessels while a non-occlusive plug has to be formed in case of smaller injuries, when the circulation still persists, but blood is pouring out of the vessel. This is essential to avoid an excessive response which could result in unnecessary occlusion in vessels presenting modest injuries, thereby avoiding reduction of blood supply to downstream tissues. Our study hypothesized that this process of arrest of hemostasis is finely tuned by fibrin. If fibrin indeed stops the hemostatic process one could wonder how a plug can become occlusive in the case of a vessel transection.

Interestingly, we observed that in such a case, the thrombus forming is very rich in platelets but does not contain significant amounts of fibrin, which explains its ability to fully grow in the lumen. It remains to be determined why after vessel transection, no significant levels of fibrin are formed, which will require further investigations.

To conclude, my work is part of a global perspective of characterizing the role of fibrin and platelet receptors in hemostasis and thrombosis, which could not only help to better appreciate and discriminate hemostasis from thrombosis, but also open new avenues for specific antithrombotic strategies. My work indicates that targeting high shear rates is not a specific antithrombotic strategy since high shear also occurs in injured healthy vessels. In my second study focused on integrin α5β1, I could show that this integrin plays an important role in platelet accumulation on fibronectin, but not in experimental thrombosis in mice, which suggests that this receptor might not be a relevant antiplatelet target. Finally, I identified of a role for fibrin in turning off the physiological response of plug formation and preventing further platelet incorporation.

Bibliography

L'adhérence, l'activation et l'agrégation des plaquettes assurent l'hémostase, mais sont également à l'origine de la thrombose artérielle responsable de pathologies ischémiques graves. Toutes

  Christian Gachet who allowed me to become a part of this laboratory in the beginning of my way. My thanks to Christophe Dubois, Hind Hamzeh-Cognasse and Florence Toti for your interest in this subject by accepting to judge this work. I would like to thank Campus France, especially Sophie Bussinger, Pierre Blouin-Hulin and Pauline Kracher, and Embassy of France, especially Olivier Dubert, Abdo Malac and Anaëlle Durand, for granting me a scholarship. I am grateful to François Lanza for reading my thesis, providing important corrections and discussions not only on my thesis, but also on my scientific projects. Many many thanks to Catherine Bourdon who has been teaching me, working with me and supporting me during all these 6 years in France. You are the person I really could count on! Thank you for family diners, weekend hiking, tasting food from your garden, chatting on many subjects and all this care and attention to me! I am really grateful to our "laboratory guru" Nicolas Receveur. Thank you for your criticism, music, chatting at any time, practicing English with me and teaching it during my first year in France! And of course thank you for your incredible cuisines! You have shown me the World of French cheeses and wine I have shed a lot of tears because of your comments, but thank you for that, it made me better and stronger! And, of course, I would like to thank all other members of Pierre's team: Emily, Usman, Clarisse, Axelle, Luc, Kevin, Marianne, Marion and Margot, --for our meetings, your help in realization of projects, the jokes during working time and a lot of conversations. Thanks a lot to Valentin, Quentin, Lea and Cyril for your help in my integrating into life outside the laboratory. Thank you for diners, weekends together, saving my life and the life of my cat, and huge amount of conversations! My many thanks to Monique, Catherine Z, Ketty, Nolwenn and Lauriane for teaching me, helping in my projects and, especially, to Monique who forgave all my stunts  I would like to thank Anita, Jean-Yves and Fabienne who taught me and helped me with doing electron microscopy. Thank you for your time and patience! I am thankful to Veronique, Patricia and Sylvie for their working advices and also for conversations, support and jokes during this time. Many thanks to Valerie who helped me with document preparation, tickets and laboratory's purchases. Thank you for your emotional involvement and concern about me. I would like to thank Nathalie Brouard, Blandine Maitre, Catherine Strassel, Beatrice Hechler, Catherine Leon, Catherine Angenieux, Stephanie Benoit, Josiane Weber, Henri de la Salle, Catherine Ravanat, Yotis Senis and Alexandra Mazharian for their help in improving my work, for all comments and advices. Thanks a lot to Elmina, Adele, Julie, Alicia, Thao, Marie-Belle, Camille, Yannick, Inès, Anaïs, Desline, Floriane, David, Gabriel, Sébastien and Morgane for this time in the laboratory! Your help, jokes and advices made this time more colorful! I am thankful to my first scientific tutor Nikita Gudimchuk for teaching me how to read scientific papers, analyze data and write my first computational model (sorry that I failed in the latter!).

Figure 1 :

 1 Figure 1: Schematic representation of platelet ultrastructure ...................................................

Figure 2 :

 2 Figure 2: The molecular mechanisms of primary hemostasis ..................................................

Figure 3 :

 3 Figure 3: The signaling pathway triggered by the GPIb-IX-V complex ................................

Figure 4 :

 4 Figure 4: The signaling pathway triggered by the GPVI/FcRγ complex .................................

Figure 5 :

 5 Figure 5: The signaling pathway triggered by the CLEC-2 receptor .......................................

Figure 6 :

 6 Figure 6: Molecular mechanisms of platelet integrin activation (inside-out signalling) .........

Figure 7 :

 7 Figure 7: The outside-in signaling of integrin αIIbβ3 ..............................................................

Figure 8 :

 8 Figure 8: Signaling pathways triggered by soluble agonist receptors P2Y1, P2Y12, P2X1 and

Figure 9 :

 9 Figure 9: The signaling pathway triggered by PAR receptors .................................................

Figure 10 :

 10 Figure 10: Schematic coagulation cascade ...............................................................................

Figure 11 :

 11 Figure 11: Spatial-temporal regulation of extrinsic and intrinsic pathways ............................

Figure 12 :

 12 Figure 12: Fibrinogen, fibrin, fibrinolysis ................................................................................

Figure 13 :

 13 Figure 13: Schematic fibrinolytic cascade ...............................................................................

Figure 14 :

 14 Figure 14: The molecular mechanisms of atherothrombosis ...................................................

Figure 15 :

 15 Figure 15: The mechanism of atherothrombosis ......................................................................

Figure 16 :

 16 Figure 16: Pharmacological treatment of arterial thrombosis ..................................................

Figure 17 :

 17 Figure 17: FeCl3-induced model of arterial thrombosis ...........................................................

Figure 18 :

 18 Figure 18: Laser-induced model of arterial thrombosis ...........................................................

Figure 19 :

 19 Figure 19: Electrolytic model of arterial thrombosis ...............................................................

Figure 20 :

 20 Figure 20: Mechanical model of arterial thrombosis ...............................................................

Figure 21 :

 21 Figure 21: Blood flow rheology ...............................................................................................

Figure 22 :

 22 Figure 22: Pathological blood flow condition .......................................................................... 92

Figure 1 :

 1 Figure 1: Schematic representation of platelet ultrastructure (Adapted from(Brisson et al.,

Figure 4 :

 4 Figure 4: The signaling pathway triggered by the GPVI/FcRγ complex. DAG,

Figure 5 :

 5 Figure 5: The signaling pathway triggered by the CLEC-2 receptor. Btk, Bruton tyrosine

  Most of the signaling pathways stimulated by these agonists lead to phospholipase C (PLC) β or PLCγ activation. PLC generates diacylglycerol (DAG) and inositol-1,4,5triphosphate (IP3), activating protein kinase C (PKC) and mobilizing intracellular Ca 2+ ,

Figure 6 :

 6 Figure 6: Molecular mechanisms of platelet integrin activation (inside-out signalling).

Figure 7 :

 7 Figure 7: The outside-in signaling of integrin αIIbβ3. Akt, protein kinase B alpha; DAG,

Figure 8 :

 8 Figure 8: Signaling pathways triggered by soluble agonist receptors P2Y1, P2Y12, P2X1

Figure 9 :

 9 Figure 9: The signaling pathway triggered by PAR receptors. AC, adenylate cyclase;

Figure 10 :

 10 Figure 10: Cascade de coagulation schématique. TF, facteur tissulaire (Adapté de (Palta et

4 .

 4 Régulation spatio-temporelle des voies extrinsèques et intrinsèques La coagulation est un processus complexe, strictement régulé dans l'espace et dans le temps, qui conduit à la formation d'un caillot de fibrine à un endroit donné. Ce processus est subdivisé en trois phases : initiation, amplification et propagation. L'initiation de la cascade de coagulation et son amplification commencent au niveau de la paroi vasculaire endommagée. A

Figure 11 :

 11 Figure 11: Régulation spatio-temporelle des voies extrinsèques et intrinsèques. APC,

Figure 12 :

 12 Figure 12: Fibrinogène, fibrine, fibrinolyse. FpA, fibrinopeptide A; FpB ; fibrinopeptide B

Figure 13 :

 13 Figure 13: Cascade fibrinolytique schématique. Les flèches indiquent la stimulation et

Figure 14 :

 14 Figure 14: The molecular mechanisms of atherothrombosis. ADP, adenosine diphosphate;

Figure 15 :

 15 Figure 15: The mechanism of atherothrombosis. LDL, low density lipoprotein; SMC,

Figure 16 :

 16 Figure 16: Pharmacological treatment of arterial thrombosis. AA, arachidonic acid; COX-

Figure 17 :

 17 Figure 17: FeCl3-induced model of arterial thrombosis. (Figure from (Shim et al., 2021)).

Figure 18 :

 18 Figure 18: Laser-induced model of arterial thrombosis. (Figure from(Hechler et al., 2010;

Figure 19 :

 19 Figure 19: Electrolytic model of arterial thrombosis. (Figure from[START_REF] Huang | Prevention of arterial thrombosis by intravenously administered platelet P2T receptor antagonist AR-C69931MX in a canine model[END_REF]).

Figure 20 :

 20 Figure 20: Mechanical model of arterial thrombosis. The injury was induced by using the

Figure 21 :

 21 Figure 21: Blood flow rheology. The blood vessel section illustrates a typical flow profile

Figure 22 :

 22 Figure 22: Pathological blood flow condition. The blood vessel section illustrates a typical

  Figure/Table count: 5 Reference count: 32 Scientific Categories: Thrombosis and Hemostasis; Vascular Biology

Figure 1 .

 1 Figure 1. The dynamics of the thrombus formation after vessel wall injury. A-B,

Figure 2 .

 2 Figure 2. Fibrin covers the hemostatic plug. A-C, Thrombus formation was induced

Figure 3 .

 3 Figure 3. Comparison of thrombus growth between fibrin-poor and fibrin-rich

Figure 4 .

 4 Figure 4. The role of fibrin in thrombus formation after vessel wall injury in vivo.

Figure 5 .

 5 Figure 5. The thrombus formation after vessel wall injury in FibAEK mice. A-B,

  les étapes des deux processus sont contrôlées par le flux sanguin dont les paramètres diffèrent selon le contexte (patho)physiologique. L'objectif de ce travail a été de mieux comprendre les mécanismes impliquant les plaquettes et la fibrine en hémostase et en thrombose artérielle et de caractériser les conditions rhéologiques de l'hémostase. L'identification de taux de cisaillement élevés au niveau d'un site de lésion a élargi la gamme de paramètres rhéologiques physiologiques trouvés dans un contexte hémostatique. L'utilisation de souris déficientes en intégrine α5β1 montre le caractère modeste de cette intégrine dans l'hémostase et la thrombose artérielle. Enfin, l'identification du rôle de la fibrine dans l'arrêt de la croissance du clou hémostatique a montré sa capacité à désactiver la réponse physiologique et à limiter le recrutement de plaquettes.Mots clés : taux de cisaillement, hémostase, intégrines, plaquettes, fibrine, thrombose artérielleThe role of platelets, fibrin and hemodynamics in hemostasis and arterial thrombosisPlatelet adhesion, activation and aggregation ensure hemostasis but can also lead to arterial thrombosis inducing serious ischemic pathologies. All steps of both processes are controlled by blood flow whose parameters differ depending on the (patho)physiological context. The aim of this work was to improve our understanding of the mechanisms implicating platelets and fibrin in hemostasis and arterial thrombosis and to characterize the rheological conditions occurring during hemostasis.Identification of elevated shear rates after vessel lesion expanded the range of physiological rheological parameters relevant to hemostasis. The use of α5β1 integrin deficient mice shows the modest role of this integrin in hemostasis and arterial thrombosis. Identification of a role for fibrin in the arrest of platelet plug formation confirmed its ability to turn off the physiological response by preventing further platelet incorporation.

Table 1 :

 1 Platelet granules component…………………………………………………………26

Table 2 :

 2

Table 3 :

 3 Time-average values of shear rate within the human vasculature vessel…………….85

		Abbreviations
	AA	arachidonic acid
	AC	adenylate cyclase
	ADAMTS13	A Desintegrin and Metalloproteinase with ThromboSpondin type
		1 repeat 13
	ADP	adenosine diphosphate
	Akt	protein kinase B alpha
	APC	activated protein C
	AT	antithrombin
	ATIII	antithrombin
	ATP	adenosine triphosphate
	Btk	Bruton tyrosine kinase
	CalDAG-GEFI	calcium and DAG-regulated guanine-nucleotide-exchange factor
	cAMP	cyclic adenosine monophosphate
	CDM	cell-derived extracellular matrix
	CLEC-2	C-type lectin-like type II transmembrane receptor
	COX-1	cyclooxygenase-1
	CTLD	carbohydrate-recognizing domain
	DAG	diacylglycerol
	Erk	extracellular signal-regulated kinase
	F	factor
	FAK	focal adhesion kinase

Table 1 . Platelet granules components Type of granules Type of granule components Components

 1

		Adhesive proteins	Fibrinogen, fibronectin, vWF, vitronectin
		Membrane glycoproteins	GPIb, αIIbβ3, GPVI
		Coagulation factors	FV, FXI, FXIII, TF, kininogens
		Coagulation inhibitors	TFPI, protein S, protease nexin-2
	α-granules	Fibrinolysis components	PAI-1, TAFI, α2-antiplasmin, plasminogen, uPA
		Inflammatory, pro-atherogenic, wound healing and antimicrobial proteins	P-selectin, PSLGP-1, thrombospondin, chemokines and cytokines, TLT-1, osteonectin, complement components, VEGF
			ATP, ADP, serotonin (5-HT),
	Dense granules	Small molecules	calcium, polyphosphates,
			pyrophosphate
	Lysosomes	Enzymes	Hydrolases, metalloproteases

Table 2 . ADP-P2Y12 inhibitors (Patti et al., 2020; Schneider et al., 2015; Secco et al., 2013)

 2

	Title	Structure	Mechanism of action Administration	Side effects
	Ticlopidine thienopyridine prodrug, irreversible	the	use	is	neutropenia,	aplastic	anemia,
				discouraged		thrombotic	thrombocytopenic
							purpura and gastrointestinal distress
	Clopidogrel thienopyridine prodrug, irreversible	oral, once daily dark purple bruise, itching, pain,
							redness, or swelling
	Prasugrel	thienopyridine prodrug, irreversible	oral, once daily increased risk of major and life-
							threatening bleedings
	Ticagrelor	cyclo-	noncompetitive,	oral, twice daily high rate of major bleedings,
		pentyltriazolop	reversible				bleeding gums, blurred vision, chest
		yrimidine					pain, tightness, or discomfort,
							confusion etc
	Cangrelor	adenosine	competitive, reversible IV, continuous	abdominal or stomach pain, back
		triphosphate		infusion		pain, blood in the eyes, blood in the
		analogue					urine
		2.1.3. Dual-antiplatelet therapy

Table 3 . Time-average values of shear rate within the human vasculature vessel (Hathcock, 2006)

 3

	Vessel	Diameter, mm Shear rate, s -1
	Ascending aorta	23-45	50-300
	Femoral artery	5	300
	Common carotid artery	5.9	250
	Left main coronary artery 4	460
	Small arteries	0.3	1,500
	Arterioles	0.03	1,900
	Large veins	5-10	200
	Inferior vena cava	20	40-60

Blood flow is creating a tangential force between fluid layers called shear stress (τ)

  1,2 , Muhammad Usman Ahmed 1 , Catherine Bourdon1 , et al.,

	1 Université de Strasbourg, INSERM, EFS Grand-Est, BPPS UMR-S1255, FMTS, F-
	67065 Strasbourg, France; 2 Center for Theoretical Problems of Physicochemical
	Pharmacology, Moscow 119991, Russia;
	Corresponding authors: Pierre H. Mangin, Université de Strasbourg, INSERM, EFS
	Grand-Est, BPPS UMR-S1255, FMTS, 10 rue Spielmann, F-67065 Strasbourg,

Acknowledgements

First and foremost, I am really really grateful to my teacher, supervisor, boss and my French "uncle" Pierre Mangin for advising me, showing me how "a big scientific machine" ACKNOWLEDGEMENTS We thank J.N. Mulvihill for reviewing the English of the manuscript and J.Y. Rinckel and F. Proamer for help in preparing the samples for SEM. This work was supported by the Ministry of Education and Science of Russia (075- 15-2022-242).

General introduction

The central role of fibrin in arrest of the hemostatic response Publication 3: « Fibrin on the thrombus surface turns off the hemostatic response» Alexandra Yakusheva 1,2 , Muhammad Usman Ahmed 1 , Catherine Bourdon 1 et al. 1 Université de Strasbourg, INSERM, EFS Grand-Est, BPPS UMR-S1255, FMTS, F-67065 Strasbourg, France; 2 Center for Theoretical Problems of Physicochemical Pharmacology, Moscow 119991, Russia

In preparation

ABSTRACT

Following an injury of a healthy vessel, the subendothelium proteins are exposed to the flowing blood and initiate the hemostatic response. While the mechanism allowing platelet aggregation is very well characterized, the one switching off the hemostatic response is still unclear. The aim of this work was to evaluate the role of fibrin in the dynamics of the formation of a hemostatic plug, beyond its stabilizing role. Using a flow-based assay, we observed that the presence of a fibrin layer on top of an aggregate prevents its growth. Mechanical removal of the thrombus after vessel flicking resulted in continuous regrowth which stopped only when fibrin reached its maximal level within the thrombus surface and covered it. In vivo experiments with pharmacological agents removing fibrin during thrombus growth lead to thrombus embolization, which allowed the aggregate to grow again, and this regrowth process could be repeated many times. Finally, the dynamic of thrombus formation in FibAEK mice was profoundly altered with as expected a lack of thrombus stabilization, but additionally we observed that the hemostatic process was prolonged compared to a control with successive growth and embolization phases. In conclusion, these findings identify a central role of fibrin in the arrest of the hemostatic response, by limiting platelet adhesion to the plug. In addition to its very well-established role in stabilizing the clot, fibrin is also instrumental in regulating the hemostatic response by turning off the physiological response of plug formation and preventing further platelet incorporation.

AUTHORSHIP

AY acquired and analyzed the data; MUA performed in vitro experiments; AY and CB performed in vivo experiments; PM conceived and designed the research and handled funding and supervision.

DISCLOSURES

The authors declare no conflicts of interest.

Supplementary Figure 1. The role of fibrin in thrombus formation after vessel wall injury in vivo. A, Thrombus formation was induced by FeCl3 (7.5%)-injury of the left common carotid artery of wild type mice and was followed by injection of 20 mg/kg hirudin 20 min after thrombus initiation. The time represents the time of the injection.

The curve shows the area of the platelets aggregate and fibrin as a function of the time after injection.

General Discussion and Perspectives

in vivo studies in large and small vessels under different types of injuries (Denis and Wagner, 2007;Nonne et al., 2005;Smith et al., 2012;Tang et al., 2016;Wang et al., 2017). While the process of platelet adhesion, activation and aggregation at the site of injury is very well understood, the mechanism limiting extensive platelet aggregation to switch off the process remains fully unknown, which became the aim of my third project.

Using intravital microscopy of laser-induced cremaster muscle arteriole thrombosis, the group of Lawrence Brass has reported that a thrombus is heterogeneous in composition with a core of highly activated platelets close to the site of injury, surrounded by a shell of less activated platelets [START_REF] Stalker | Hierarchical organization in the hemostatic response and its relationship to the platelet-signaling network[END_REF]. The core region is composed of tightly packed platelets and is the primary site of thrombin activity and fibrin deposition. A limitation of this view is that it is a snapshot at one given time point and does not take into account the dynamic aspect of thrombus formation which is very important. Our findings took into account the dynamics of thrombus formation and focused on the end stage when the plug formation ends. Our results indicate that the arrest of thrombus growth appeared after embolization of the shell, when only a fibrin-rich core was exposed to the blood. We propose that this core is covered with a fibrin layer, which exhibits a protective function, plays an important role in the passivation of the thrombus and promotes thereby the arrest of the hemostatic response.

In our study, we provided evidence for the formation of a fibrin layer on the thrombus surface in needle-and FeCl3-injury models in the carotid artery formed under arterial blood flow conditions. The limitation of our study is the lack of evidence of fibrin layer formation under venous blood flow conditions. The group of Rosen E. demonstrated that fibrin was a main component of the stabilized thrombus area by using a fibrin(ogen) antibody in a laser-induced