Muhammad Usman Ahmed

Valeria Kaneva

Stéphane Loyau

Dmitry Nechipurenko

Nicolas Receveur

Marion Le Bris

Emily Janus-Bell

Mélusine Didelot

Antoine Rauch

Sophie Susen

Nabil Chakfé

François Lanza

Elizabeth E Gardiner

Robert K Andrews

Mikhail Panteleev

Christian Gachet

Martine Jandrot- Perrus

Pierre H Mangin

Pharmacological Blockade of Glycoprotein VI Promotes Thrombus Disaggregation in the Absence of Thrombin

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

Introduction

Patelets play a central role in hemostasis and in arterial thrombosis. Upon vascular injury platelets adhere, become activated and aggregate to form a hemostatic plug in a healthy vessel and a mural thrombus that can become occlusive, in a diseased artery. [START_REF] Jackson | Arterial thrombosis-insidious, unpredictable and deadly[END_REF] It has been reported that the thrombus is heterogeneous in composition with (1) a core, rich in fibrin composed of highly activated platelets located close to the site of injury, in which important amounts of thrombin are generated; (2) a shell, in which platelets are less activated and no significant amounts of thrombin are generated, as evidenced by the absence of fibrin. [START_REF] Welsh | A systems approach to hemostasis: 1. The interdependence of thrombus architecture and agonist movements in the gaps between platelets[END_REF] From a therapeutic point of view, preventing the shell to grow or dissolving it appear as attractive strategies to prevent arterial occlusion and ischemic pathologies.

Platelets express a repertoire of adhesion receptors that engage with subendothelial adhesive matrix proteins after vessel wall injury. Under conditions of elevated blood flow found in arteries, the first step of platelet attachment is initiated by the GP (glycoprotein) Ib-IX-V complex interaction with VWF (von Willebrand factor) present in the subendothelium. Stable adhesion is supported by integrins of the β1 and β3 family, namely α2β1, α5β1, α6β1, αIIbβ3, and αvβ3 which bind to collagen, fibronectin, laminins, fibrinogen, and vitronectin, respectively. Concomitantly, these receptors together with the collagen receptor, GPVI (glycoprotein VI) initiate intracellular signals leading to platelet activation. [START_REF] Zahid | The future of glycoprotein VI as an antithrombotic target[END_REF] Activated platelets release soluble mediators, including thromboxane (Tx)A2 and ADP which reinforce platelet activation and increase the affinity of integrin αIIbβ3, for its main ligand, fibrinogen, allowing platelet aggregation. [START_REF] Versteeg | New fundamentals in hemostasis[END_REF] The thrombus composition is heterogeneous with a core containing highly activated platelets and a shell with weakly and reversibly activated platelets. [START_REF] Welsh | A systems approach to hemostasis: 1. The interdependence of thrombus architecture and agonist movements in the gaps between platelets[END_REF] The specific signaling mechanisms responsible for maintenance of platelets in this state is unclear, potential candidacies include GPIb (glycoprotein Ib)-dependent signalling, 5 soluble agonists, 2, [START_REF] Gachet | ADP receptors of platelets and their inhibition[END_REF] and we recently proposed GPVI. [START_REF] Mangin | Immobilized fibrinogen activates human platelets through glycoprotein VI[END_REF] GPVI is a member of immunoglobulin receptor family which is specifically expressed on megakaryocytes and platelets. 3 GPVI is considered as not being critical for hemostasis, based on the fact that GPVI-deficient subjects present a mild bleeding phenotype and GPVIdeficient mice show no spontaneous bleeding and no prolongation in tail-bleeding time. [START_REF] Dumont | Absence of collagen-induced platelet activation caused by compound heterozygous GPVI mutations[END_REF][START_REF] Hermans | A compound heterozygous mutation in glycoprotein VI in a patient with a bleeding disorder[END_REF][START_REF] Matus | An adenine insertion in exon 6 of human GP6 generates a truncated protein associated with a bleeding disorder in four Chilean families[END_REF] In contrast, it has been reported that absence of GPVI impairs experimental thrombosis in several models of localized vascular injury. 11, [START_REF] Mangin | Thrombin overcomes the thrombosis defect associated with platelet GPVI/FcRgamma deficiency[END_REF] Remarkably, GPVI-immunodepleted mice were also protected from thrombosis after ultrasound or mechanical injuries to atherosclerotic plaques in ApoE-deficient mice. 13, [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] Together, these observations suggest that GPVI is a particularly promising and potentially safe antithrombotic target. [START_REF] Zahid | The future of glycoprotein VI as an antithrombotic target[END_REF] GPVI physically associates with the FcR(Fc receptor) γ-chain and the Src family kinases Lyn and Fyn. [START_REF] Suzuki-Inoue | Association of Fyn and Lyn with the proline-rich domain of glycoprotein VI regulates intracellular signaling[END_REF] Ligand binding to GPVI promotes its clustering which results in phosphorylation of the immunoreceptor tyrosine-based motif of the FcR γ-chain by Src family kinases. [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] This leads to the recruitment of Syk and formation of signaling complexes which activate PLCγ2 (phospholipase Cγ2) resulting in mobilization of intracellular Ca 2+ stores, a key second messenger of platelets. GPVI engagement efficiently results in the release of soluble agonists such as ADP and TxA2 which enhance platelet activation. 17 GPVI is best recognized as a platelet activation receptor for fibrillar collagen and supports efficient platelet aggregation in vitro. [START_REF] Nieswandt | Platelet-collagen interaction: is GPVI the central receptor?[END_REF] We and others provided evidence that GPVI participates in platelet activation to laminins. 18, [START_REF] Schaff | Integrin α6β1 is the main receptor for vascular laminins and plays a role in platelet adhesion, activation, and arterial thrombosis[END_REF] We also reported that GPVI plays an important role in platelet adhesion and activation on fibrin. 20, [START_REF] Mammadova-Bach | Platelet glycoprotein VI binds to polymerized fibrin and promotes thrombin generation[END_REF] More recently, we proposed that human GPVI participates in platelet activation on immobilized fibrinogen, which could support a new role for GPVI in thrombus growth. [START_REF] Mangin | Immobilized fibrinogen activates human platelets through glycoprotein VI[END_REF] The primary aim of this study was to investigate the role of GPVI beyond the welldocumented initial stages, on the process of thrombus stability. The outcome of this study was to assess the potential of targeting this receptor in the setting of a treatment, when a thrombus had already formed.

We showed that growing thrombi experience elevated mechanical stress, which represents a major challenge to maintain their stability. We developed a numerical model which highlighted the importance of maintaining strong inter-platelet bonds to resist to the increase in mechanical stress when a thrombus grows. Using an experimental approach, we identified a key role of GPVI-mediated platelet activation in maintaining thrombus stability in the absence of thrombin, through its interplay with fibrinogen. These results indicate that targeting GPVI when a platelet-rich aggregate had already formed promotes disaggregation, opening new avenues for the use of anti-GPVI agents to prevent vessel occlusion. In addition, an anti-GPVI agent can also be combined to a thrombolytic agent to dissolve a fibrin-rich thrombus.

Materials and Methods

Patients

Two White patients suffering from congenital afibrinogenemia were collected for the study.

The patients were related and expressed the same homozygous mutation c.1001 G>A in exon 5, resulting in a nonsense p.W334X mutation of FGA (fibrinogen A) gene, previously reported to lead to premature termination of translation of fibrinogen α chain. [START_REF] Neerman-Arbez | Mutations in the fibrinogen aalpha gene account for the majority of cases of congenital afibrinogenemia[END_REF] For these 2 patients, plasma fibrinogen was undetectable by antigen and functional assays with unclottable prothrombin, activated partial thromboplastin, thrombin time, and reptilase time tests. Both patients suffered from spontaneous and post-traumatic bleeds but also had a past history of arterial or venous thromboembolism (ischemic stroke in patient 1, 2 episodes of superficial venous thrombosis, and 1 episode of deep venous thrombosis in patient 2; the 3 venous thrombosis events in patient 2 occurred under replacement therapy with fibrinogen concentrate) as reported previously. [START_REF] Simsek | Afibrinogenemia resulting from homozygous nonsense mutation in A alpha chain gene associated with multiple thrombotic episodes[END_REF] At the time of the study, the 2 patients were treated on demand with fibrinogen concentrates. No infusion of fibrinogen concentrate was performed in the previous 21 days before blood collection. The collection was approved by the French government under the number: DC-2008-642.

Computational Fluid Dynamics and Fluid-Structure Interaction Analysis

To assess the evolution of mechanical stress during thrombus growth, we used computational fluid dynamics combined with additional fluid-structure interaction analysis. Thrombi were represented as the hemi-spheres protruding into vessels of 600 µm diameter. Constant pressure boundary conditions were chosen for the blood flow corresponding to a wall shear rate (WSR) of 300 s -1 . Subendothelial matrix and thrombus were represented as isotropic elastic media with Young modulus value derived from thrombus nanoindentation analysis, [START_REF] Swieringa | Platelet control of fibrin distribution and microelasticity in thrombus formation under flow[END_REF] while blood was considered as a Newtonian fluid. A fluid-structure interaction module of Comsol Multiphysics was used for computation of flow and flow-induced mechanical stress throughout the system. See Figure I and Table I in the Data Supplement for more details.

Computational Model of Thrombus Formation

We developed a 2-dimensional model of thrombus formation with platelets represented by particles of different sizes. [START_REF] Paulus | Platelet size in man[END_REF] The model considers 2 types of inter-platelet interaction: (1) a primary GPIb-VWF-mediated interaction, modelled by stochastically associating and disassociating springs between platelets; the model was validated using experiments addressing platelet interactions with VWF-coated surfaces 26 ; (2) an αIIbβ3-fibrinogenmediated interaction modelled by Morse potential. Parameters of potential were acquired using experimental data on forces between single platelets. [START_REF] Nguyen | Rupture forces among human blood platelets at different degrees of activation[END_REF] The adhesive force increases with the time platelets spend in the thrombus. To study thrombus dynamics during the initial phase of thrombus growth, we decreased integrin-mediated force between platelets by 10% to 80%. Blood was considered as an incompressible Newtonian fluid described with Navier-Stokes and the continuity equations. The flow field was computed considering the partial vessel blockade by the thrombus. Equations describing both the flow profile and hydrodynamical forces that act on platelets were solved using the Open-FOAM (field operation and manipulation) simpleFoam solver. [START_REF] Trifanov | Developing quasi-steady model for studying hemostatic response using supercomputer technologies[END_REF] To infer how inter-platelet forces depend on both thrombus size and shear rate, the stationary configuration of tightly (hexagonally) packed equally sized disks under steady flow was analyzed. Disks, which represented platelets, were gathered in a semi-spherically shaped thrombus. To simplify the analysis, only Morse potential was used to describe platelet-platelet attraction. Injury was modelled as the array of adhesive interaction sites localized at the vessel wall.

In Vitro Flow-Based Adhesion Assay

Microfluidic flow chambers or glass microslides (for scanning electron microscopy experiments) were prepared as previously described. [START_REF] Maurer | Fibrillar cellular fibronectin supports efficient platelet aggregation and procoagulant activity[END_REF] For most experiments, human hirudinated blood (100 U/mL) was perfused through the microfluidic channels, with the exception of flows on plaque material for which we used nonrecalcified citrated blood. The human atherosclerotic plaque homogenates (1 mg/mL) prepared as previously described. [START_REF] Penz | Glycoprotein Ibalpha inhibition and ADP receptor antagonists, but not aspirin, reduce platelet thrombus formation in flowing blood exposed to atherosclerotic plaques[END_REF] To form fibrin-rich clots, citrated blood incubated with DIOC 6 (0.5 µmol/L) to label platelets and with DyLight 650-conjugated anti-fibrin (10 µg/mL) was recalcified with CaCl 2 (12.5 mmol/L) and MgCl 2 (3.5 mmol/L) immediately before perfusing it through collagen coated microfluidic chambers at 1500 s -1 . Disaggregation of a thrombus was defined as the detachment of at least 3 platelets or more in a field.

Light Transmission Aggregometry

Human washed platelets were prepared from ACD-anticoagulated blood by sequential centrifugation as previously described. [START_REF] Cazenave | Preparation of washed platelet suspensions from human and rodent blood[END_REF] The platelets were suspended at a concentration of 3×10 [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] /µL in Tyrode buffer containing 0.35% human serum albumin and 2 µL/mL apyrase.

Platelet aggregation was measured turbidimetrically after addition of different agonists, in the presence of human fibrinogen (0.8 mg/mL).

Scanning Electron Microscopy

Adherent platelets in glass microslides were fixed for 45 minutes with 25 mg/mL glutaraldehyde in 0.1 mol/L cacodylate buffer containing 20 mg/mL sucrose (305 mOsm, pH 7.3) and scanning electron microscopy was performed as previously described. 5

Statistical Analysis

Results are expressed as mean±SEM. All statistical analyses were performed with the GraphPad Prism program, version 5.0 (Prism, GraphPad, LaJolla, CA). Nonparametric Mann-Whitney U test was used when applicable, and χ 2 test with CI of 95% was used for categorical variables. One-way ANOVA or 2-way ANOVA followed with a post hoc Bonferroni was used for multiple comparisons. P<0.05 were considered statistically significant. Details are found in the figure legends.

Results

Blood Flow-Induced Mechanical Stress Challenges the Stability of a Growing Thrombus

A mural thrombus in an injured artery experiences forces applied by the flowing blood.

Computational fluid dynamics simulations indicated that surface shear stress was maximal at the thrombus apex and increases with thrombus height (Figure 1A through 1C). These external stress acting on the surface is redistributed throughout the thrombus and results in internal mechanical stress. Fluid-structure interaction analysis performed using the continuous 3-dimensional model indicated that internal mechanical stress (von Mises stress) was maximal at the thrombus base and was also increased with both thrombus height and blood flow velocities (Figure I in the Data Supplement). Similar results were obtained in a particle-based 2-dimensional model showing that inter-platelet forces within a thrombus were maximal at its base reaching elevated values of 350 pN for a thrombus of 26 µm in height and increased with thrombus height and WSR (Figure 1D through 1F). Together, these results indicate that both flow-dependent surface forces and internal platelet-platelet forces increase as a mural thrombus is growing in the vessel lumen. To assess the impact of flow-induced mechanical forces during thrombus growth, we developed a numerical particle-based model of thrombus formation dynamics which reproduces rheology found in arteries (750 s -1 ). This model recapitulates the main features of thrombosis with (1) platelet adhesion, (2) platelet activation depicted as they become darker (various levels of activation ranging from white (resting) to grey (activated), and (3) recruitment of circulating platelets which results in linear thrombus growth (Figure 1G and 1H; Movie I in the Data Supplement). The impact of elevated shear on thrombus stability was evidenced by the disruption of the thrombus occurring when a 3-fold increase in shear rate was applied at 30 seconds, (Figure 1I; Movie II in the Data Supplement). The importance of the inter-platelet bond strength in maintaining thrombus stability was shown by disaggregation taking place when the inter-platelet force was reduced by 40% at 30 seconds (Figure 1J; Movie III in the Data Supplement). This disaggregation highlighted the importance of a sustained platelet activation during thrombus growth to maintain its integrity. As GPVI is an activation receptor, we investigated whether it participates in maintaining thrombus ability to resist mechanical stress and whether its targeting could promote disaggregation.

Blockade of GPVI During Thrombus Growth Promotes Disaggregation in the Absence of Thrombin

Hirudinated human whole blood was flowed over collagen to preform aggregates followed by perfusion of 50 µg/mL of a blocking humanized anti-GPVI Fab fragment, ACT017, at an arterial WSR of 750 s -1 . Real-time video-microscopy showed that numerous individual platelets detached from the top of aggregates treated with ACT017 resulting in a clear disaggregation (Figure 2A; Movie IV in the Data Supplement). Quantification indicated a 4.9fold increase in the number of platelet thrombi disaggregating after treatment with ACT017 when compared with control buffer (Figure 2B). ACT017 also lowered the time to initiation of disaggregation by 45% (Figure 2C). The diaggregating effect of ACT017 was also evidenced when we maintained whole blood perfusion throughout the experiment (Movie V in the Data Supplement) and was not limited to hirudin as an anticoagulant, as a similar result was obtained with heparin and citrate (data not shown). Similar results were obtained on human atherosclerotic plaque material, where ACT017 promoted efficient disaggregation of platelet thrombi that were formed with citrated blood (Figure 2D). This disaggregation obtained by blocking GPVI was not restricted to ACT017, as another GPVI blocker, the Fab fragment of 1G5 mAb exhibited similar effects (Figure 2E). In contrast, thrombi did not disaggregate when equivalent concentrations of Fab fragments of the nonblocking anti-GPVI antibody 3J24 were perfused, suggesting that the inhibitory effect of ACT017 and 1G5 is related to their ability to disrupt ligand binding to GPVI (Figure 2F and2G). Finally, we observed that soluble GPVI-Fc, used at a concentration which inhibits collagen-induced platelet aggregation, did not disaggregate preformed aggregates (Figure 2F and2G).

Together, these results indicate that direct blockade of GPVI ligand binding, but not a competition approach, promotes disaggregation of preformed platelet thrombi, highlighting a novel role of GPVI in maintaining the stability of a thrombus during its build-up when thrombin generation was blocked. In the rest of our study, we have mainly used ACT017 as an agent that directly blocks GPVI. 3.2×10 -4 ±0.7×10 -4 disaggregating thrombi/µm 2 ; ACT017:

18.7×10 -4 ±1.9×10 -4 disaggregating thrombi/µm (50 µg/mL), 3J24.2 (50 µg/mL), GPVI-Fc (50 µg/mL), or control buffer diluted in PBS were perfused over the preformed aggregates at 750 s -1 for 12 min. Bar graphs represent the number of disaggregating thrombi (×10 -4 )/µm 2 ; control: 3.7×10 -4 ±0.9×10 -4 disaggregating thrombi/µm 2 ; 3J24.2: 3.5×10 -4 ±1.7×10 -4 disaggregating thrombi/µm 2 ; ns, P>0.5, n=4-8. G, Scatter plots represent time to initiation of disaggregation, expressed in minutes;

3.0×10 -4 ±1.4×10 -4 disaggregating thrombi/µm 2 ; non significant (ns); P>0.5, n=4-8. Bar is the mean±SEM; Bonferroni multiple comparison test; ***P<0.001.

Shear Forces Promote Disaggregation of Platelet Aggregates When GPVI Is Blocked

We next evaluated whether ACT017 promotes disaggregation in a Born-type aggregometer.

Washed human platelets were stimulated with collagen, U46619 or ADP, and ACT017 was added to the suspension once aggregation reached maximal values. We did not observe any disaggregation upon the addition of ACT017 (Figure 3A). Because shear is low in an aggregometer (<100 s -1 ), one hypothesis could be that ACT017-induced platelet disaggregation occurring in the blood perfusion assay depends on the presence of elevated shear forces. To examine that, we preformed aggregates in the flow system at 750 s -1 for 3 minutes and perfused ACT017 at WSR ranging from 150 s -1 up to 750 s -1 . In agreement with the result obtained in the light transmission aggregometer, ACT017 did not promote disaggregation of platelet thrombi under venous blood flow conditions (150 s -1 ), (Figure 3B).

In contrast, signs of disaggregation were observed at an arterial WSR of 300 s -1 with a maximal number of disaggregating thrombi being achieved at 750 s -1 (Figure 3B and3C).

Measuring the initial time of thrombus disaggregation further highlighted the importance of increasing blood flow conditions on ACT017-induced platelet thrombus disaggregation (Figure 3D). These results are in agreement with our observations in silico showing that elevated shear forces promote disaggregation when platelet activation is inhibited.

The Disaggregating Effect of GPVI Blockade Relies on the Inhibition of Platelet

Activation

As GPVI is an activation receptor, we hypothesized that ACT017-induced disaggregation in the absence of thrombin, is linked to an impairment of GPVI-mediated platelet activation in the growing thrombus. To test this hypothesis, we perfused elevated nonphysiological concentrations of soluble agonists (ADP or TRAP [thrombin receptor activator peptide]) over preformed aggregates to maximally activate platelets and overcome the GPVI activatory pathway, before injecting ACT017. We observed that under such conditions ACT017 was less efficient in promoting disaggregation and that the time of disaggregation was delayed, indicating that its effect is linked to the regulation of platelet activation (Figure 4A and4B).

In parallel, we examined thrombus contraction in real-time, as a readout of platelet activation.

While thrombi were contracting and appeared more compact over time in the control, a clear loosening and surface expansion was observed when ACT017 was perfused over contracted thrombi (Figure 4C; Movie VI in the Data Supplement). This was confirmed by quantification which showed that the surface occupied by the ACT017-treated thrombi was significantly increased in comparison with untreated control (Figure 4D). Scanning electron microscopy images confirmed the loosening of the aggregates treated with ACT017 and showed that individual platelets were also less contracted when compared with control, confirming that ACT017 impairs platelet activation (Figure 4E). As GPVI promotes platelet activation through a signaling pathway that has been well characterized, we evaluated the effect of inhibitors of several key immunoreceptor tyrosine-based motif signaling proteins such as srckinase (PP2), Syk (PRT060318), PI3Kβ (phosphoinositide 3-kinase; TGX-221), and PLCγ2 (U73122). The inhibitors increased the number of disaggregating thrombi by 3.6-fold, 2.6fold, 2.2-fold, and 4.4-fold, respectively, to a similar level as the once achieved with ACT017 (Figure 4F). Moreover, the initial time of thrombus disaggregation also showed a significant decrease for all 4 inhibitors when compared with control which was very close to the one of ACT017 (Figure 4G). The number of disaggregating thrombi and time of initiation of disaggregation were very similar between ACT017 and 1G5 (Figure 2) as compared with inhibition of the 4 immunoreceptor tyrosine-based motif signaling molecules, suggesting that disaggregation occurring when GPVI is blocked, relies on an impairment of GPVI-mediated platelet activation. s -1 for 12 min. F, Bar graphs represent the number of disaggregating thrombi (×10 -4 )/µm 2 ; control: 4.1×10 -4 ±0.8×10 -4 disaggregating thrombi/μm 2 ; ACT017:

18.7×10 -4 ±1.9×10 -4 disaggregating thrombi/µm 2 ; PP2: 15×10 -4 ±1.7×10 -4 disaggregating thrombi/µm 2 ; PRT060318: 18.2×10 -4 ±1.9×10 -4 disaggregating thrombi/µm 2 ; TGX-221: 16×10 -4 ±1.5×10 -4 disaggregating thrombi/µm 2 ; U73122: 17.5×10 -4 ±0.6×10 -4 disaggregating thrombi/µm

Disaggregation Induced When GPVI Is Blocked Relies on the Presence of Fibrinogen

We recently proposed that the role of GPVI in thrombus build-up relies on its ability to promote platelet activation on fibrinogen. [START_REF] Mangin | Immobilized fibrinogen activates human platelets through glycoprotein VI[END_REF] To evaluate whether thrombus disaggregation occurring when GPVI is blocked, is linked to this property, we performed experiments with blood of 2 patients with congenital afibrinogenemia. We observed that perfusion of hirudinated whole blood of both patients over immobilized collagen resulted in the formation of thrombi at 750 s -1 , even though they appeared smaller than the control (Figure 5A). This was expected since platelet aggregation in the absence of significant levels of fibrinogen has been previously reported and shown to rely on VWF. [START_REF] Marco | von Willebrand factor interaction with the glycoprotein IIb/IIa complex. Its role in platelet function as demonstrated in patients with congenital afibrinogenemia[END_REF] While ACT017-induced thrombus disaggregation in the control, little effect was observed on thrombi of either patient at 750 s -1 (Figure 5B). This result indicates that blockade of GPVI has no impact on thrombus disaggregation in the absence of normal levels of fibrinogen, even though these thrombi appeared looser than normal thrombi in agreement with a previous report. [START_REF] Remijn | Absence of fibrinogen in afibrinogenemia results in large but loosely packed thrombi under flow conditions[END_REF] Quantification confirmed the absence of ACT017-induced disaggregation with blood from the patients as compared with controls (Figure 5C). These results propose the existence of a link between fibrinogen and GPVI in allowing the stability of a growing thrombus. (×10 -4 )/µm 2 (n=2). C, Scatter plots represent the time at which disaggregation was initiated, expressed in minutes; ACT017: healthy human: 20.5×10 -4 ±1.5×10 -4 disaggregating thrombi/µm 2 ; afibrinogenemic patient-1 and 2: 0.0×10 -4 ±0.0×10 -4 disaggregating thrombi/µm 2 ; (n=2).

Combination of ACT017 and r-tPA Promotes Disaggregation of Fibrin-Rich Clots In Vitro

So far, we observed that GPVI contributes to the stabilization of a platelet-rich thrombus which contains no fibrin, suggesting that anti-GPVI agents could remove the part of the thrombus corresponding to the shell. [START_REF] Jamasbi | Differential inhibition of human atherosclerotic plaque-induced platelet activation by dimeric GPVI-Fc and Anti-GPVI antibodies: functional and imaging studies[END_REF] We next evaluated whether blockade of GPVI has also an impact on a thrombus core in which thrombin is found and which is stabilized by fibrin. Initially, we formed fibrin-rich thrombi by perfusing recalcified citrated human blood over collagen at 1500 s -1 . The high fibrin content of the thrombi was evidenced on 3-dimensional reconstructed images obtained with a fluorescently conjugated anti-fibrin antibody (Figure 6A). ACT017 had a minor effect on thrombus disaggregation with detaching platelets being mainly localized at the thrombus edge where less fibrin was present, which is explained by the ability of fibrin to entrap platelets and thereby stabilize the thrombus (Figure 6B; Movie VII in the Data Supplement). This result is in agreement with the view that fibrin stabilizes the clot. We next wondered whether ACT017 would cooperate with a thrombolytic approach based on r-tPA (recombinant tissue plasminogen activator) to enhance thrombus disaggregation. We showed that perfusion of r-tPA in platelet-poor plasma efficiently reduced the fibrin signal over time confirming its thrombolytic effect (Figure 6C and 6D). We observed that r-tPA alone promoted thrombus disaggregation which was probably linked to its ability to lyse fibrin and lower the stability of the thrombus (Figure 6E). Interestingly, combining r-tPA with ACT017 significantly increased thrombus disaggregation as compared with r-tPA alone, highlighting the potential of an anti-GPVI agent combined with a thrombolytic reagent to promote thrombus disaggregation (Figure 6E). Similar results were obtained when a combination of ACT017 and r-tPA was perfused over thrombi formed on a tissue factor-collagen coated surface with a clear loosening of platelet aggregates being evidenced by a marked increase in surface occupied by the thrombi (Figure 6F and6G). Together, these results suggest that combining a thrombolytic strategy with a blockade of GPVI could dissolve mural thrombi lodged in the vascular bed.

Discussion

In this study, based on the in silico model of thrombus dynamics, we propose that under conditions of arterial blood flow, thrombus dynamics is sensitive to platelet activation state which determines the critical platelet-platelet forces within the thrombus. A decrease in platelet activation level or an increase in the blood flow, result in platelet disaggregation in the numerical model. This in silico result is perfectly in line with the disaggregating effect of ACT017 observed in vitro, which is expected to lower platelet activation state within the thrombi and with the fact that this disaggregating effect increases with the higher values of blood flow (WSR). This study demonstrates that platelet activation is not a short-lived process which aims only to activate integrins to allow bond formation, but represents a long-lasting process that maintains strong inter-platelet bonds.

Most of the experiments performed in this study have been based on perfusions of anticoagulated blood in flow chambers which represents a limitation, as no thrombin is generated under such conditions and, therefore, differs from an in vivo situation. On the contrary, such a situation could mimic the part of the thrombus named the shell, in which it has been reported that no significant amount of thrombin is found based on the absence of fibrin.

We have recently reported that blockade of GPVI impairs the growth of a thrombus that had already formed most likely by preventing platelet activation and, therefore, aggregation. [START_REF] Mangin | Immobilized fibrinogen activates human platelets through glycoprotein VI[END_REF] We proposed that this effect was linked to an inhibition of GPVI/fibrinogen-mediated platelet activation, as platelets from GPVI-deficient patients were unable to spread on fibrinogen. The importance of GPVI/fibrinogen interaction in thrombus formation is further evidenced in the present article through the use of blood from afibrinogenemic patients. First, we confirmed that perfusing fibrinogen-poor blood over collagen formed thrombi, through a mechanism proposed to rely on VWF/αIIbβ3 interaction. [START_REF] Marco | von Willebrand factor interaction with the glycoprotein IIb/IIa complex. Its role in platelet function as demonstrated in patients with congenital afibrinogenemia[END_REF] We observed that while ACT017-induced disaggregation of control thrombi, it had little effect on thrombi formed with blood from 2 afibrinogenemic patients. Importantly, ACT017 exhibited almost no effect despite the fact that such thrombi were looser than control and, therefore, more prone to disaggregate. [START_REF] Remijn | Absence of fibrinogen in afibrinogenemia results in large but loosely packed thrombi under flow conditions[END_REF] This result suggests that the effect of GPVI blockade on thrombus disaggregation could be linked to its interplay with fibrinogen. Moreover, observation of thrombus disaggregation in realtime clearly shows that platelets detaching are the one on the top of the thrombus, that is, those interacting with fibrinogen, and never those attached to the surface through strong interaction with collagen.

It is well accepted that absence or blockade of GPVI prevents thrombus formation in vitro and in several in vivo models of experimental thrombosis. 11, [START_REF] Mangin | Thrombin overcomes the thrombosis defect associated with platelet GPVI/FcRgamma deficiency[END_REF] These results underscore a potential beneficial role of anti-GPVI agents in preventing arterial thrombosis, notably during percutaneous coronary intervention. Indeed, this procedure leads to exposure of highly reactive fibrillar collagen which initiates platelet activation and thrombus formation through GPVI. Therefore, blocking GPVI in such a setting now stands as a very promising antithrombotic strategy. Anti-GPVI agents could also be beneficial in patients with an ongoing thrombosis in addition to their use in prevention since we recently reported that an anti-GPVI blocking agent, ACT017, limits thrombus growth once a thrombus had already formed. [START_REF] Mangin | Immobilized fibrinogen activates human platelets through glycoprotein VI[END_REF] The results presented in this article extend these findings, showing that anti-GPVI agents promote disaggregation of thrombi, which was unexpected. This disaggregating effect was observed on fibrin-poor thrombi, which mimic the thrombus shell, representing the prominent part of the thrombus. This suggests that anti-GPVI agents could destabilize the shell, thereby preventing vessel occlusion. Additionally, we showed that thrombi rich in fibrin, a condition that mimics the core of the thrombus, are also destabilized by ACT017

when used in combination with r-tPA. The disaggregating effects of anti-GPVI agents used in combination with r-tPA on fibrin-rich thrombi opens new avenues for the use of such agents in clinical settings where a thrombus had already formed, such as during acute phases of myocardial infarction or stroke. This adds new arguments to previous observations that mice immunodepleted for GPVI were protected in a stroke model, in favor of a beneficial effect of anti-GPVI antibodies in stroke. [START_REF] Kleinschnitz | Targeting platelets in acute experimental stroke: impact of glycoprotein Ib, VI, and IIb/IIIa blockade on infarct size, functional outcome, and intracranial bleeding[END_REF] This study presented the results obtained exclusively from experiments in vitro with human blood. In this model, human whole blood is perfused over fibrillar collagen and forms a mural thrombus which is likely to mimic a pathological situation encountered during arterial thrombosis. We aimed to confirm these observations in vivo. As ACT017 blocks human but not mouse GPVI, we used a mouse strain in which murine GPVI was genetically replaced by its human counterpart. [START_REF] Mangin | A humanized glycoprotein VI (GPVI) mouse model to assess the antithrombotic efficacies of anti-GPVI agents[END_REF] To our surprise, ACT017 did not promote disaggregation of preformed mouse platelet thrombi in vitro, indicating that GPVI is not important to maintain the integrity of mouse thrombi (article in preparation). For these reasons, evaluating the effect of blocking human GPVI on thrombus stability during the initial phase of thrombus growth could not be achieved in a mouse. This is not the only example of major differences between platelets of the 2 species, other being related to the thrombin receptors and the presence of the immunoreceptor tyrosine-based motif receptor, FcγRIIa (Fc gamma receptor IIA).

Dimeric GPVI-Fc, unlike the 2 distinct blocking anti-GPVI Fab fragments ACT017 and 1G5, did not promote thrombus disaggregation. There is strong experimental evidence that dimeric GPVI-Fc inhibits collagen-induced platelet aggregation in vitro and ex vivo. GPVI-Fc also reduces experimental thrombosis probably through impairing GPVI interaction with collagen exposed at site of injury. 37, [START_REF] Massberg | Soluble glycoprotein VI dimer inhibits platelet adhesion and aggregation to the injured vessel wall in vivo[END_REF] The reason why GPVI-Fc has no effect on thrombus disaggregation could have 2 explanations. First, direct blockade of GPVI with an antibody has been proposed to be more efficient than using a competition approach based on GPVI-Fc in static and flow experiments. 34, [START_REF] Kleinschnitz | Targeting platelets in acute experimental stroke: impact of glycoprotein Ib, VI, and IIb/IIIa blockade on infarct size, functional outcome, and intracranial bleeding[END_REF] Second, we reported that dimeric GPVI-Fc does not bind fibrinogen, which is the ligand of GPVI in a growing thrombus, [START_REF] Mangin | Immobilized fibrinogen activates human platelets through glycoprotein VI[END_REF] and therefore, it is expected that such an agent would have no effect. This observation has important clinical implications as ACT017 and GPVI-Fc have both successfully completed phase I and are currently being evaluated in phase II clinical trials. 39, [START_REF] Ungerer | Novel antiplatelet drug revacept (Dimeric Glycoprotein VI-Fc) specifically and efficiently inhibited collagen-induced platelet aggregation without affecting general hemostasis in humans[END_REF] Here we describe that direct blockade of GPVI with Fab fragments, but not with GPVI-Fc, promotes disaggregation of both fibrin-poor and fibrinrich thrombi. Our data suggests that such agents should be evaluated in trials of patients with ischemic stroke treated with r-tPA. This is especially relevant since anti-GPVI agents do not appear to impact hemostasis and so carry a reduced bleeding risk when compared with other antiplatelet agents.

Figure 1 .

 1 Figure 1. In silico evidence that sustained platelet activation is crucial for the stability of a growing thrombus.A-C, Computational fluid dynamics simulations were performed to determine surface shear stress exerted on a semi-spherical thrombi of different sizes. A, Image representing the distribution of the shear stress on the surface of thrombi of 450 µm at flow conditions corresponding to wall shear rates of 300 s -1 (at sites distal to thrombus). B, Pointconnecting line graph represents the shear stress as a function of thrombus height at constant pressure drop boundary conditions. C, Images represent the distribution of the shear stress on the surface of thrombi of 300 µm, 450 µm, and 550 µm height in a channel of 600 µm at constant pressure drop boundary conditions. D, Distribution of inter-platelet forces in a 2dimensional (2D) thrombus subjected to the flow corresponding to 500 s -1 . Each inter-platelet force is shown as a line colored corresponding to the force value. Only forces corresponding to extended inter-platelet bonds are shown. Platelets contours are shown in gray color. E, The dependence of maximum inter-platelet bond extension force on thrombus height obtained in a 2D model (wall shear rate [WSR] of 500 s -1 ). F, The dependence of maximum inter-platelet bond extension force on a wall shear rate obtained for 2D thrombus of 26 µm height. G, Representative images of the dynamics of thrombus formation in silico. The injury region is shown in black; platelets are represented by circles of different colors (darkening color reflects the extent of platelet activation). WSR is 750 s -1 ; arrow indicates flow direction. Scale bar represents 5 µm. H-J, The graphs plot thrombus height over time at 750 s -1 . I, The arrow represents the time at which a 3-fold increase in WSR was applied (H and I; n=6). J, The arrow represents the time at which the force between platelets was reduced by 40% (n=10). Bar graph represents area under curve (AUC). Nonparametric Mann-Whitney U Test; ***P<0.001.

Figure 2 .

 2 Figure 2. GPVI (glycoprotein VI) blockade promotes disaggregation of preformed thrombi in vitro.A-C and E-G, Hirudinated (100 U/mL) human whole blood was perfused for 3 min at 750 s -1 through microfluidic flow chambers coated with a solution of type I fibrillar collagen (200 µg/mL) to preform aggregates. A-C, ACT017 (50 µg/mL) or control buffer was perfused over the preformed aggregates at 750 s -1 for 12 min. A, Representative differential interference contrast microscopy images of thrombi subjected to ACT017 or control buffer diluted in PBS. The black arrow indicates the flow direction. Scale bars represent 20 µm. B, Bar graphs represent the number of disaggregating thrombi (×10 -4 )/µm 2 ; control:3.2×10 -4 ±0.7×10 -4 disaggregating thrombi/µm 2 ; ACT017:

Figure 3 .

 3 Figure 3. Shear regulates ACT017-induced disaggregation of preformed thrombi.A, Human washed platelets (3.0×10 5 /µL) were stimulated with collagen (2.5 µg/mL), U46619 (2 µmol/L) or ADP (20 µmol/L) in the presence of fibrinogen (64 µg/mL). ACT017 (50 µg/mL) or control buffer was added at maximal aggregation (indicated by an arrow). Aggregation profiles are representative of 3 separate experiments (n=3). B-D, Hirudinated (100 U/mL) human whole blood was perfused over immobilized collagen (200 µg/mL) for 3 min at 750 s -1 to preform aggregates. Then, ACT017 (50 µg/mL) diluted in PBS was perfused over preformed aggregates at different wall shear rate (WSR; 150 s -1 , 300 s -1 , and 750 s -1 ) for 12 min. B, Representative differential interference contrast microscopy images of thrombi subjected to ACT017 or control buffer diluted in PBS at indicated WSR. The black arrow indicates the flow direction. Scale bars represent 20 µm. C, Bar graph represents the percentage of stable thrombi (n=3). χ 2 test with CI of 95%; *P<0.05 and ***P<0.001. D, Interleaved bars represent time to initiation of disaggregation after perfusion of ACT017 or

Figure 4 .

 4 Figure 4. The disaggregating effect of ACT017 relies on impairment of GPVI (glycoprotein VI)-mediated platelet activation.A-B, Hirudinated (100 U/mL) human whole blood was perfused for 3 min at 750 s -1 through microfluidic flow chambers coated with a solution of type I fibrillar collagen (200 µg/mL) to preform aggregates, before perfusing buffer containing or not ADP (50 µmol/L) or TRAP (thrombin receptor activator peptide; 50µmol/L) at 750 s -1 for 2 min and then at 150 s -1 for 3 min, followed by ACT017 (50 µg/mL) or control buffer diluted in PBS at 750 s -1 for 12 min. A, Bar graphs represent the number of

Figure 5 .

 5 Figure 5. ACT017-induced thrombus disaggregation depends on fibrinogen. Hirudinated (100 U/mL) human whole blood from healthy volunteers or patients suffering from afibrinogenemia was perfused for 3 min at 750 s -1 through microfluidic flow chambers coated with a solution of type I fibrillar collagen (200 µg/mL) to preform aggregates, before perfusing blood containing ACT017 (50 µg/mL) or control buffer for 7 min. A, Representative differential interference contrast microscopy images of thrombi subjected to ACT017 or control buffer. The black arrow indicates the flow direction. Scale bars represent 20 µm. B, Bar graphs represent the number of disaggregating thrombi

Figure 6 .

 6 Figure 6. Effect of ACT017 and r-tPA (recombinant tissue plasminogen activator) on disaggregation of fibrin-rich clots. Citrated (3.2%) human whole blood was labelled with a DyLight 650-coupled anti-fibrin antibody (10 µg/mL) and was recalcified by adding CaCl 2 (12.5 mmol/L) and MgCl 2 (3.5 mmol/L) just before perfusing it through collagen coated 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 platelet-poor plasma (PPP) obtained from the same donor (hirudinated blood) in the presence of ACT017 (50 µg/mL) or r-tPA (50 µg/mL) alone or in combination, at 3000 s -1 for 35 min. A, Representative 3-dimensional (3D) reconstructed confocal microscopy image of thrombi. Fibrin is stained in red and platelets in gray. B, Bar graphs represent the number of disaggregating thrombi (×10 -4 )/µm 2 (n=3). Mean±SEM; nonparametric Mann-Whitney U test; ns denotes not significant. C, Representative 3D reconstructed confocal microscopy image of thrombi subjected to r-tPA or control buffer diluted in PPP. D, Connecting line graphs represent the surface area coverage of fibrin (expressed as percentage of control) over time (n=3); mean±SEM. E, Bar graphs represent the number of disaggregating thrombi (×10 -4 )/µm 2 (n=3). Mean±SEM; Bonferroni multiple comparison test; ***P<0.001. F and G, Citrated (3.2%) human whole blood was labelled with a DyLight 650coupled anti-fibrin antibody (10 µg/mL) and was recalcified by adding CaCl 2 (12.5 mmol/L) and MgCl 2 (3.5 mmol/L) just before perfusing it through collagen and TF-coated microfluidic flow chambers at 1500 s -1 . F, Representative bright field/epifluorescence microscopy images of r-tPA pretreated thrombi subjected to ACT017 or control buffer diluted in PPP. Fibrin appears in red. G, Bar graphs represent increase in surface occupied (expressed in percentage) by the disaggregating thrombi (n=3). Mean±SEM; χ 2 test with CI of 95%; control: 10.5±2.9% increase in surface area; ACT017: 32.5±2.0% increase in surface area ***P<0.01; n=3.

  mg/mL) to preform aggregates, before perfusing ACT017 (50 µg/mL) or control buffer in the same blood at 300 s -1 for 12 min. Bar graphs represent the number of

	disaggregating	thrombi	(×10 -4 )/µm 2 (n=4).	mean±SEM;	nonparametric	Mann-
	Whitney U Test; ***P<0.001. E, 1G5 (50 µg/mL) or Fab IgG diluted in PBS was perfused
	over preform aggregates at 750 s -1 for 12 min. Bar graphs represent the number of
	disaggregating thrombi (×10 -4 )/µm 2 (n=3). Two-tailed paired t test; **P<0.01. F, ACT017

2 ; ***P<0.001, n=8. C, Scatter plots represent the time at which disaggregation was initiated, expressed in minutes; mean±SEM; nonparametric Mann-Whitney U Test; control: 9.6±0.3 min; ACT017: 4.4±0.1 min; ***P<0.001, n=8 (D) Citrated (3.2%) human whole blood was perfused for 4 min at 300 s -1 through glass microslides coated with a solution of human atherosclerotic plaque homogenates (1

Acknowledgments

We thank Wolfgang Siess for providing atherosclerotic plaque material as a control.

Sources of Funding

This work was supported by ARMESA (Association Recherche et Developpement Médical en santé publique) and by a grant from Concours mondial de l'innovation: TherAVC 2.0 (BPI France). M.U. Ahmed was supported by PhD fellowship from HEC (Higher Education Commission) Pakistan. Disclosures M. Jandrot-Perrus is the founder of Acticor Biotech. C. Gachet is the cofounder of Acticor Correspondence to: Pierre H Mangin, Université de Strasbourg, INSERM, EFS Grand-Est, BPPS UMR-S1255, FMTS, F-67065 Strasbourg, France. Email pierre.mangin@efs.sante.fr