Shannen J Deconinck

Christoph Nix

Svenja Barth

Eveline Bennek-Schöpping

Antoine Rauch

An-Sofie Schelpe

Elien Roose

Hendrik B Feys

Inge Pareyn

Aline Vandenbulcke

Joshua Muia

Christophe Vandenbriele

Sophie Susen

Bart Meyns

Claudia Tersteeg

Steven Jacobs

Simon F De Meyer

Prof. Dr Karen Vanhoorelbeke

ADAMTS13 inhibition to treat acquired von Willebrand syndrome during mechanical circulatory support device implantation

Keywords: Acquired von Willebrand syndrome, ADAMTS13, Bleeding, Mechanical circulatory support, von Willebrand factor

Background-Acquired von Willebrand syndrome (aVWS) is common in patients with mechanical circulatory support (MCS) devices. In these patients, the high shear stress in the device leads to increased shear-induced proteolysis of von Willebrand factor (VWF) by ADAMTS13 (A Disintegrin And Metalloprotease with Thrombospondin type 1 repeats, number 13). As a result, the high molecular weight (HMW) VWF multimers are lost, leading to a decreased VWF function and impaired haemostasis which could explain the bleeding complications that are frequently observed in these patients. To counteract this abnormal VWF degradation by ADAMTS13, we

Introduction

It is estimated that more than 30 million people suffer from heart failure worldwide. [START_REF] Miller | Evolution of left ventricular assist device therapy for advanced heart failure: A review[END_REF] The advanced stage of heart failure occurs in 6 to 25% of the heart failure patients and is associated with an unacceptably high mortality when treated with medical therapy alone. [START_REF] Chaudhry | Advanced Heart Failure: Prevalence, Natural History, and Prognosis[END_REF] A small number of the patients with advanced heart failure can be referred for cardiac transplantation. Unfortunately, only around 5000 transplantations are annually performed worldwide, limited by the availability of donor organs. [START_REF] Miller | Use of Ventricular Assist Devices and Heart Transplantation for Advanced Heart Failure[END_REF] Hence, this has led to an advancement in the field of mechanical circulatory support (MCS) devices. MCS devices to treat advanced stage heart failure can either be long-term or short-term depending on the clinical needs and potential of cardiac recovery. [START_REF] Stulak | Ventricular assist device selection: Which one and when?[END_REF] Left ventricular assist devices (LVADs) are long-term support devices (e.g. Heartmate II™ or III™) which pump blood from the left ventricle to the aorta. [START_REF] Rose | Long-Term Use of a Left Ventricular Assist Device[END_REF][START_REF] Slaughter | HeartWare ventricular assist system for bridge to transplant: Combined results of the bridge to transplant and continued access protocol trial[END_REF] Devices providing urgent/emergent MCS to treat patients with cardiogenic shock (e.g. Impella® or extracorporeal membrane oxygenation (ECMO)) are classified as short-term support devices, whose primary goal is to restore normal haemodynamics. [START_REF] Sayago | Impella CP® Circulatory Support Device as a Bridge to Heart Transplantation: First Experience in Spain[END_REF][START_REF] Lauten | Percutaneous left-ventricular support with the impella-2.5-assist device in acute cardiogenic shock results of the impella-EUROSHOCK-Registry[END_REF] Although MCS devices tremendously improve the survival of advanced stage heart failure patients, thrombotic and bleeding complications are serious side effects. [START_REF] Susen | Circulatory support devices: Fundamental aspects and clinical management of bleeding and thrombosis[END_REF] Importantly, gastrointestinal bleeding is one of the most common severe adverse events of LVAD therapy with an incidence of 20-30%. [START_REF] Susen | Circulatory support devices: Fundamental aspects and clinical management of bleeding and thrombosis[END_REF][START_REF] Guha | Gastrointestinal bleeding after continuousflow left ventricular device implantation: review of pathophysiology and management[END_REF][START_REF] Cushing | Gastrointestinal Bleeding Following LVAD Placement from Top to Bottom[END_REF] Gastrointestinal bleeding in LVAD patients was initially linked to the use of anticoagulation following device implantation. Bleeding rates are, however, higher in LVAD patients compared with patients treated with anticoagulation for other indications. [START_REF] Crow | Gastrointestinal bleeding rates in recipients of nonpulsatile and pulsatile left ventricular assist devices[END_REF] Hence, the use of anticoagulation only partially explains the haemorrhagic complications in these patients. Recently, it was suggested that bleeding in MCS device patients might be caused by a loss of function of the haemostatic protein von Willebrand factor (VWF). [START_REF] Geisen | Non-surgical bleeding in patients with ventricular assist devices could be explained by acquired von Willebrand disease[END_REF] VWF is a multimeric protein and its high molecular weight multimers (HMW) are crucial for its haemostatic activity. However, HMW VWF multimers disappear in LVAD patients within minutes after device implantation, [START_REF] Geisen | Non-surgical bleeding in patients with ventricular assist devices could be explained by acquired von Willebrand disease[END_REF][START_REF] Uriel | Acquired von Willebrand syndrome after continuous-flow mechanical device support contributes to a high prevalence of bleeding during long-term support and at the time of transplantation[END_REF][START_REF] Heilmann | Acquired Von Willebrand syndrome is an early-onset problem in ventricular assist device patients[END_REF][START_REF] Van Belle | Von Willebrand factor as a biological sensor of blood flow to monitor percutaneous aortic valve interventions[END_REF] leading to decreased VWF function and increased risk for bleeding, known as acquired von Willebrand syndrome (aVWS). aVWS is therefore hypothesized to be a major contributing factor for the bleeding episodes observed in these patients. Likewise, aVWS is also observed in patients implanted with an Impella® or with an ECMO device and also in those patients the loss of HMW VWF multimers might contribute to the frequently observed bleedings in these patients. [START_REF] Lansink-Hartgring | Hemorrhagic complications during extracorporeal membrane oxygenation -The role of anticoagulation and platelets[END_REF][START_REF] Manzo-Silberman | Percutaneous left ventricular assistance in post cardiac arrest shock: Comparison of intra aortic blood pump and IMPELLA Recover LP2.5[END_REF][START_REF] Flierl | Acquired von Willebrand syndrome in cardiogenic shock patients on mechanical circulatory microaxial pump support[END_REF][START_REF] Kalbhenn | Early diagnosis of acquired von Willebrand Syndrome (AVWS) is elementary for clinical practice in patients treated with ECMO therapy[END_REF] Remarkably, removal of the device and subsequent heart transplantation results in a restoration of the HMW VWF multimers and VWF function and corrects the bleeding. [START_REF] Uriel | Acquired von Willebrand syndrome after continuous-flow mechanical device support contributes to a high prevalence of bleeding during long-term support and at the time of transplantation[END_REF] Current treatment of LVAD-induced gastrointestinal bleeding consists of supportive care (iron supplementation, blood transfusion), endoscopic therapy (argon plasma coagulation, placement of endoscopic haemoclips or contact thermal therapy) or a reduction in antithrombotic treatment [START_REF] Guha | Gastrointestinal bleeding after continuousflow left ventricular device implantation: review of pathophysiology and management[END_REF]. However, identifying and accessing the lesions in the gastrointestinal tract is not straightforward and recurrent bleeding occurs in 50% of the patients despite endoscopic procedures. [START_REF] Cushing | Gastrointestinal Bleeding Following LVAD Placement from Top to Bottom[END_REF] Moreover, a downshift in antithrombotic therapy poses the patients at high thrombotic risk, again increasing morbidity and mortality. [START_REF] Maltais | PREVENtion of HeartMate II Pump Thrombosis Through Clinical Management: The PREVENT multi-center study[END_REF][START_REF] Starling | Unexpected abrupt increase in left ventricular assist device thrombosis[END_REF] Hence, there is a need for a targeted therapy to treat the underlying cause of MCS deviceinduced bleeding, which is thought to be the acquired VWF defect.

During the past few years, efforts have been made to determine the mechanism by which HMW VWF multimers are lost in MCS device patients. First it was hypothesized that VWF degradation was mainly caused by mechanical destruction of VWF but novel insights into the role of ADAMTS13 in the shear-induced VWF degradation were provided by in vitro LVAD studies. [START_REF] Jilma-Stohlawetz | Acquired von Willebrand factor deficiency caused by LVAD is ADAMTS-13 and platelet dependent[END_REF][START_REF] Rauch | Antibody-based prevention of von Willebrand factor degradation mediated by circulatory assist devices[END_REF] Due to the high shear stress, caused by MCS devices, HMW VWF unfolds, exposing the cleavage site for the VWF-cleaving protease ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats, number 13) (Figures S1 andS2). [START_REF] Susen | Circulatory support devices: Fundamental aspects and clinical management of bleeding and thrombosis[END_REF][START_REF] Geisen | Non-surgical bleeding in patients with ventricular assist devices could be explained by acquired von Willebrand disease[END_REF] As a consequence, these excessively cleaved VWF multimers have a reduced potential for binding to collagen and platelets and thus a diminished haemostatic activity, thereby rendering MCS device patients more prone to bleeding.

We therefore aimed to consolidate the role of ADAMTS13 in the shear-induced proteolysis of VWF in vitro by using a specific ADAMTS13 inhibitor and to determine whether blocking ADAMTS13 using this specific inhibitor can rescue the loss of HMW VWF multimers in a preclinical MCS device animal model.

Methods

Monoclonal anti-human ADAMTS13 antibodies

Murine anti-human ADAMTS13 monoclonal antibodies (mAbs) 3H9 and 5C11 are in house generated mAbs. [START_REF] Feys | Thrombotic thrombocytopenic purpura directly linked with ADAMTS13 inhibition in the baboon (Papio ursinus)[END_REF] mAb 3H9 inhibits ADAMTS13 activity and is directed against the metalloprotease (M) domain of ADAMTS13, mAb 5C11 does not inhibit ADAMTS13 and has an epitope in the second thrombospondin type 1 repeat (T2) of ADAMTS13. [START_REF] Feys | Thrombotic thrombocytopenic purpura directly linked with ADAMTS13 inhibition in the baboon (Papio ursinus)[END_REF] Both mAbs have been extensively characterized both in vitro and in vivo. [START_REF] Feys | Thrombotic thrombocytopenic purpura directly linked with ADAMTS13 inhibition in the baboon (Papio ursinus)[END_REF] Murine anti-human ADAMTS13 mAb 17C7 has been recently generated and is also directed against the M domain of ADAMTS13. [START_REF] Schelpe | Antibodies that conformationally activate ADAMTS13 allosterically enhance metalloprotease domain function[END_REF] mAb 17C7 is known to inhibit ADAMTS13 activity but was not further characterized. Further characterization of mAb 17C7 was done in the current study. Methods used for characterization of the murine mAb 17C7 are provided in Supplemental Methods.

Human and bovine blood

Human citrated blood was collected from the 'Etablissement Français du Sang' blood bank (Lille, FR) and from the Belgian Red Cross-Flanders Blood Establishment (Mechelen, BE). Citrate (CPDA 12.5%, Cantaert Medical, Melle, BE) and heparin (5000 IU/L, VWR, Pennsylvania, US) anti-coagulated bovine blood was provided by the animal facility of Gasthuisberg (Leuven, BE). Blood collections were approved by the institutional ethics committee; human blood donors consented to the use of their blood donation in scientific research and the study was in accordance with the Declaration of Helsinki.

In vitro Impella CP® and 5.5® system

The Impella® (Abiomed, Aachen, DE) pump, a micro-axial continuous-flow (CF) device commonly used for short-term LVAD support (Figure S2), was localized inside a closed tubing system containing a valve, resistance clamp and sampling device (Figure 1A). The system was filled with 750 mL citrate anti-coagulated human blood (Red Cross) or bovine blood (animal facility Gasthuisberg). Where needed, blood was supplemented with 20 μg/mL of the inhibitory anti-ADAMTS13 mAb 3H9 or mAb 17C7 or the control anti-ADAMTS13 mAb 5C11 or with an equal volume of phosphate buffered saline (PBS). The pump rotor was set at 44,000 (Impella CP®) or 30,000 (Impella 5.5®) rotations per minute (rpm); speeds applied in patients with cardiogenic shock. [START_REF] Sayago | Impella CP® Circulatory Support Device as a Bridge to Heart Transplantation: First Experience in Spain[END_REF] Blood was sampled 5 minutes before the onset of perfusion (BL) and 5 (T5), 30 (T30), 60 (60) and 180 (T180) (and 300 (T300) minutes for bovine blood) minutes after the onset of perfusion (Figure 1B). Plasma was obtained through centrifugation (2000g, 15 min) of the whole blood samples. Human quality CP® and 5.5® pumps were manufactured by Abiomed Europe GmbH and underwent manufacturing verification. Technical parameters such as pump performance level (pump flow) and motor current were carefully monitored and displayed in real time using the clinically approved Automated Impella Controller™ (interface for the Impella platform).

In vitro Heartmate II™ system using human blood

To investigate the degradation of VWF in a device used for long-term LVAD support, an axial CF Heartmate II™ (manufactured and provided by Thoratec Corp., Pleasanton, CA) device was used [START_REF] Heilmann | Acquired Von Willebrand syndrome in patients on long-term support with HeartMate II[END_REF] (Figure S1) and in vitro perfusion of human blood through the device was performed as described [START_REF] Rauch | Antibody-based prevention of von Willebrand factor degradation mediated by circulatory assist devices[END_REF]. The inlet and outlet ducts of the HeartMate II® device were connected to 2 tubings to make a closed system. In the closed system, a sampling device was included (Figure S3A). The system was filled with 250 mL citrate anti-coagulated whole human blood (blood bank 'Etablissement Français du Sang'). Where needed, human blood was supplemented with 20 μg/mL of the inhibitory anti-ADAMTS13 mAb 3H9 or control anti-ADAMTS13 mAb 5C11. The pump rotor was set at 9000 rpm; a speed applied in patients with heart failure [START_REF] Frazier | First clinical use of the redesigned Heartmate II left ventricular assist system in the United States: a case report[END_REF]. Blood was sampled 5 minutes before the onset of perfusion (T0) and 5 (T5), 30 (T30) and 180 (T180) minutes after the onset of perfusion (Figure S3B). Plasma was obtained through centrifugation (2000g, 15 min) of the whole blood samples. Clinical pump parameters (e.g. pump flow, speed and power) were displayed in real time by the Heartmate II system monitor.

VWF multimer analysis

VWF multimer analysis was performed as described before [START_REF] Meyer | Restoration of plasma von willebrand factor deficiency is sufficient to correct thrombus formation after gene therapy for severe von willebrand disease[END_REF][START_REF] Deconinck | Differences in von Willebrand factor function in type 2A von Willebrand disease and left ventricular assist device-induced acquired von Willebrand syndrome[END_REF]. Briefly, VWF multimers present in the plasma samples were separated on a sodium dodecyl sulphate (SDS) isoelectric focusing (IEF) 1.2% agarose gel. After electrophoresis, the gel, bound to a Gelbond (Cambrex Bio Science Rockland Inc., Rockland, ME, USA), was dried and the VWF multimer pattern was visualized with anti-human VWF immunoglobulins labelled with alkaline phosphatase (AP) and the AP-conjugate-substrate kit (BioRad, Hercules, CA, USA). Densitometric analysis was performed to determine the percentage of HMW VWF multimers using ImageJ software (version 1.47, NIH, MD). The low molecular weight (LMW, band 1-5), the medium molecular weight (MMW, band 6-10), and high molecular weight (HMW, band >10) multimers were selected and the density of the HMW multimers relative to the total multimer density was calculated as a percentage.

von Willebrand factor (VWF) antigen VWF antigen (VWF:Ag) levels in the plasma samples obtained from the in vitro LVAD systems, were quantified by ELISA as described [START_REF] Deconinck | Differences in von Willebrand factor function in type 2A von Willebrand disease and left ventricular assist device-induced acquired von Willebrand syndrome[END_REF][START_REF] Vanhoorelbeke | A reliable and reproducible ELISA method to measure ristocetin cofactor actiivity of von Willebrand factor[END_REF]. Briefly, a 96-well microtiter plate was coated with 4.1 μg/mL of polyclonal anti-human VWF antibodies (Dako, Glostrup, DK), blocked with a 3% milk powder solution and a 1/80 dilution of human or bovine plasma was added and the samples were further diluted ½ in PBS containing 0.3% milk. Next, bound VWF was detected using polyclonal anti-human VWF antibodies labelled with horse radish peroxidase (HRP) (Dako). Colouring reaction was performed with orthophenylene diamine (OPD) and H 2 O 2 . The reaction was stopped with 4M sulfuric acid. Absorbance was measured at 490nm. A normal human plasma pool from 20 individuals (NHP) or a normal bovine plasma pool from 6 calves (NBP) was used to set up a calibration curve and undiluted plasma was set at 100%. The anti-VWF antibodies are known to cross react with bovine VWF.

VWF collagen binding activity

VWF collagen binding activity (VWF:CB) was determined using ELISA as described [START_REF] Favaloro | Development of a simple collagen based ELISA assay aids in the diagnosis of, and permits sensitive discrimination between Type I and Type II, von Willebrands disease[END_REF][START_REF] Vanhoorelbeke | A reliable and reproducible ELISA method to measure ristocetin cofactor activity of von Willebrand factor[END_REF]. Briefly, 25 μg/mL of human collagen type III (Sigma-Aldrich, Saint-Louis, MO, USA) for binding of human VWF or 25 μg/mL of human collagen type I (Sigma-Aldrich) for binding of bovine VWF was coated onto a 96-well microtiter plate and the plate was blocked with a 3% milk powder solution. Next, a 1/80 dilution of plasma was added and the samples were further diluted ½ in PBS containing 0.3% milk. Bound VWF was detected with HRP-labelled rabbit anti-human VWF antibodies (Dako) and visualized as described above. The VWF:CB was calculated using NHP as a reference of which the VWF:CB was set at 100%.

Surgical procedure

New Jersey calves (Leurs GbR, Kerken, GE weighing 85-150 kg (1 male, 3 females), 211-236 days old with baseline ADAMTS13 activity between 50-150% and baseline platelet counts between 100-800×10 9 /L [START_REF] James | Laboratory Animal Medicine[END_REF]) were premedicated with Ketamin (Nimatek 15 mg/kg intramuscular, Dechra, Northwich, UK) and Xylazine (XYL-M 2%, Inovet (V.M.D. nv, Arendonk, BE) and general anesthesia was induced with Isoflurane (5% in O 2 100%, Dechra). The animals were intubated and general anesthesia was maintained using Isoflurane (2%, Dechra). An arterial line was placed in the right ear and a central venous line in the left jugular vein. The skin was disinfected with Povidone-iodine and antibiotic prophylaxis was given (Penicillin (40,000 IU/kg, Dechra) and Gentamycin (6.6 mg/kg intravenous, Inovet (V.M.D. nv)). Next, a left-sided thoracotomy was made through the 4 th intercostal space. The descending aorta was exposed and a side-biting vascular clamp was applied on the aorta after administration of Heparin (200 IU/kg, Leo Pharma nv, Lier, BE). An 8 mm Dacron graft was put end-to-side on the aorta with Prolène 5-0. Through this graft, the Impella 5.5® pump (manufactured by Abiomed Europe GmbH) was inserted using the Seldinger technique and under fluoroscopic guidance placed in the left ventricle [START_REF] Seldinger | Catheter Replacement of the Needle in Percutaneous Arteriography: A new technique[END_REF]. Once a proper placement was achieved, the Impella® pump was fixed in its position by fixation of the driveline in the 8 mm Dacron graft. The driveline was tunneled to the back of the animal. A thorax drain was left in the left pleural space and the thoracic wall was closed in layers. Metacam (1.5 mL, Boehringer Ingelheim, Ingelheim am Rein, DE) was administered post-operatively and the chest wound was inspected daily. Impella 5.5® pumps ran at a speed that is applied in patients with cardiogenic shock (30,000 rpm). All animal experiments were performed by the rules as set by the animal ethical committee of the KU Leuven (P218-2015) and according to the "Principles of Laboratory Animal Care," formulated by the National Society for Medical Research, and the Guide for the Care and Use of Laboratory Animals, prepared by the Institute of Laboratory Animal Resources (National Institutes of Health). Injections and blood sampling were performed by venipuncture of the jugular vein.

Study design

Calves were implanted with an Impella®, which was expected to reduce the size of the VWF multimers, as has been observed in patients [START_REF] Flierl | Acquired von Willebrand syndrome in cardiogenic shock patients on mechanical circulatory microaxial pump support[END_REF][START_REF] Davis | Development of Acquired von Willebrand Syndrome During Short-Term Micro Axial Pump Support[END_REF]. Next, after 168 hours (7 days) of Impella® implantation, one bolus of PBS (volume based on the volume of the administered mAb 17C7) was injected and blood was sampled 2 (T2), 24 (T24), 48 (T48), 72 (T72) and 168 (T168) hours after PBS injection. As PBS injection was not expected to have an effect on VWF multimers, the same calf was treated with the inhibitory anti-ADAMTS13 mAb 17C7 since this allowed us to reduce the number of animals needed for this study. Subsequently, 168 hours after PBS injection, one bolus of 600 μg/kg of the inhibitory mAb 17C7 was given intravenously and blood was sampled, 2 (T2), 24 (T24), 48 (T48), 72 (T72) and 168 (T168) hours after mAb 17C7 injection (Figure 4A). Blood was collected on citrate, heparin and EDTA collection tubes (BD, New Jersey, US) right before Impella® implantation (BL) and at 2 (T2), 24 (T24), 48 (T48), 72 (T72) and 168 (T168) hours after Impella® implantation and PBS/mAb 17C7 injection (Figure 3A).

Blood analysis

Plasma was obtained through centrifugation (2000g, 15 min) of the whole blood samples. Total blood cell counts, lactate dehydrogenase (LDH), hemoglobin and creatinine were determined blinded using automated and standardized methods (XE-5000 analyzer, Sysmex, Kobe, JP for full blood count and hemoglobin and Cobas 8000 modular analyzer series, Roche Diagnostics, Mannheim, DE for LDH and creatinine) in the Medicine laboratory at Gasthuisberg (Leuven, BE). Analyses of ADAMTS13 and VWF parameters and mAb load were performed as described in Supplemental Methods.

Statistical analysis

Statistical significance between T0 and all other time points (T5, T30, T60, T180, T300) within one group (within control (PBS or mAb 5C11) or within treated (mAb 3H9 and 17C7) groups) was assessed with the Friedmann test with corrections for multiple comparisons (Dunn's multiple comparisons test) for the in vitro MCS device experiments. This test was also used to determine statistical significance between baseline (BL) and all time points (T2, T24, T48, T72 and T168) of the different conditions (Impella implantation, PBS and mAb 17C7 injections) for the preclinical Impella®-induced aVWS animal study. All statistical analysis was performed using Graphpad Prism version 8.0.1 (Graphpad Software, Inc., La Jolla, CA, USA). All values are expressed as mean ± standard deviation and p-values were calculated using p<0.05 as a cut-off for significance.

Results

Inhibition of human ADAMTS13 prevents the loss of HMW VWF multimers in in vitro MCS device systems

To test whether inhibition of ADAMTS13 can prevent cleavage of HMW VWF multimers during MCS device implantation, we first performed an in vitro study using an Impella CP® device (Figure 1A). This micro-axial device was selected for our in vitro studies as it can be easily implanted in large animals, a prerequisite for our subsequent in vivo experiments (see below). [START_REF] Meyns | Left ventricular support by cathetermounted axial flow pump reduces infarct size[END_REF] Our previously developed and well characterized mAb 3H9 was used to block ADAMTS13 activity. [START_REF] Feys | Thrombotic thrombocytopenic purpura directly linked with ADAMTS13 inhibition in the baboon (Papio ursinus)[END_REF] Human blood was preincubated with either placebo (PBS) or mAb 3H9 and was next perfused through an Impella CP® pump for 180 min at 37°C at a speed that is used in the clinical setting (44,000 rotations per minute (rpm)). Blood was sampled at 5, 30, 60 and 180 min (Figure 1B) and VWF multimers and VWF function were determined as described in the Methods section.

As expected, a time-dependent decrease in HMW VWF multimers was observed when human blood, supplemented with PBS, was perfused through the system (38.13 ± 2.83% HMW VWF multimers before versus 11.56 ± 1.92% at 180 min after perfusion, p=0.0003) (Figure 1C andD). This loss in HMW VWF multimers was reflected by a loss in VWF function as the VWF multimers had a significantly lower affinity for collagen. The VWF collagen binding activity over VWF antigen (VWF:CB/VWF:Ag) ratio decreased from 1.07 ± 0.08 to 0.52 ± 0.10 at 180 min after perfusion (p=0.001) (Figure 1E). Interestingly, blocking ADAMTS13 activity using mAb 3H9 at 20 μg/mL prevented the loss of HMW VWF multimers (39.51 ± 2.03% HMW VWF multimers before versus 35.49 ± 2.78% at 180 min after perfusion) (Figure 1C andD). When using mAb 3H9, VWF function was mainly preserved except for a slight decrease in VWF:CB/VWF:Ag after 180 min of perfusion (Figure 1E).

To assess whether blocking ADAMTS13 can also prevent aVWS using the Heartmate II™ device, a device that is FDA approved for both destination therapy and bridge-totransplantation [START_REF] Kirklin | Eighth annual INTERMACS report: Special focus on framing the impact of adverse events[END_REF], we next studied the efficacy of the mAb 3H9 in preventing the loss of HMW VWF multimers and VWF function in vitro using our previously described in vitro Heartmate II™ system (Figure S3A). [START_REF] Rauch | Antibody-based prevention of von Willebrand factor degradation mediated by circulatory assist devices[END_REF] Similar as with the Impella CP® device, blocking ADAMTS13 activity with mAb 3H9 prevented the loss of HMW VWF multimers (36.27 ± 1.79% HMW VWF multimers before versus 30.99 ± 6.50% at 180 min after perfusion) (Figure S3C andD) and of VWF function (VWF:CB/VWF:Ag ratio was 0.93 ± 0.09 before perfusion versus 0.92 ± 0.16 at 180 min) (Figure S3E). Taken together, these in vitro experiments show that pharmacological inhibition of ADAMTS13 prevents the loss of HMW VWF multimers and safeguards VWF function.

mAb 17C7 prevents the loss of HMW VWF multimers in an in vitro Impella 5.5® system perfused with bovine blood

Based on our promising in vitro findings, we next wanted to expand our study by testing the efficacy of ADAMTS13 inhibition to treat aVWS after MCS device implantation in a clinically-relevant large animal model. Since mAb 3H9 could not inhibit ADAMTS13 from large animal species that are frequently used for LVAD testing (e.g. sheep, pigs and calves) [START_REF] Chan | Shear Stress-Induced Total Blood Trauma in Multiple Species[END_REF], we developed a novel inhibitory anti-ADAMTS13 mAb, mAb 17C7. mAb 17C7 is one of a novel panel of in-house generated mAbs against ADAMTS13 [START_REF] Schelpe | Generation of anti-idiotypic antibodies to detect anti-spacer antibody idiotopes in acute thrombotic thrombocytopenic purpura patients[END_REF] and was further characterized in depth in this study. The inhibitory mAb 17C7 has an apparent dissociation constant (Kd) for binding to human ADAMTS13 of 0.51 ± 0.06 μg/mL (Figure S4A) and a half maximal inhibitory concentration (IC50) of 0.48 ± 0.06 μg/mL (Figure S4B). As shown in Figure S5, mAb 17C7 not only inhibits human ADAMTS13, but also efficiently blocks the activity of bovine, ovine, cynomolgus monkey and baboon ADAMTS13. Hence, mAb 17C7 could be used to test the efficacy of ADAMTS13 inhibition to treat aVWS after MCS device implantation in a calf model. We selected the calf as a preclinical animal model as these animals are suitable for long-term fixation in the stable, necessary to prevent damage or tangling of the pump cable that is connected to the non-wearable automated Impella controller (AIC).

Before testing the efficacy of our novel therapy in the calves, we first evaluated in vitro whether mAb 17C7 could prevent the loss of HMW VWF multimers in bovine blood using the Impella 5.5® device. We here used the recently developed Impella 5.5® device instead of the Impella CP®, as the Impella 5.5® was specifically designed for longer duration of support (i.e. 30 days compared to 6 days with the Impella CP®), which is necessary for the later in vivo study in the calves. For the in vitro experiments, the same in vitro set-up was used as with the Impella CP® device (Figure 1A) and bovine blood was perfused for 300 min in the presence of PBS or mAb 17C7 (Figure 2A). Similar as with the experiments with human blood, perfusion of bovine blood supplemented with PBS resulted in a time-dependent loss of HMW VWF multimers (54.19 ± 3.44% HMW VWF multimers before versus 29.83 ± 4.01% at 300 min after perfusion, p=0.005) (Figure 2B and C). This loss of HMW VWF multimers resulted in a loss of VWF function as indicated by a decrease of the VWF:CB/VWF:Ag ratio (from 0.98 ± 0.13 before perfusion to 0.42 ± 0.18 at 300 min after perfusion, p=0.01) (Figure 2D). Addition of 20 μg/mL mAb 17C7 prevented VWF proteolysis by ADAMTS13 (HMW VWF multimers were 54.98 ± 4.00% before versus 50.47 ± 8.69% at 300 min after perfusion) (Figure 2B andC) and prevented the loss of VWF function (VWF:CB/VWF:Ag ratio was 0.89 ± 0.09 before perfusion and remained 0.87 ± 0.15 at 300 min after perfusion) (Figure 2D). A similar protective effect of mAb 17C7 was observed in human blood using the in vitro Impella CP® system (Figure S6).

Blocking ADAMTS13 in a preclinical Impella 5.5® calf model rescues the loss of HMW VWF multimers

To further validate our approach in vivo, Impella 5.5® pumps were implanted in calves (n=4). To assess the specific effect of mAb 17C7, calves were injected at 168 hours (7 days) after pump implantation with PBS, and subsequently (168 hours after PBS injection) with mAb 17C7 (Figure 3A). In line with patient observations and as shown in Figure 3B and C, Impella 5.5® implantation in the calves resulted in a significant decrease of HMW VWF multimers at 72 hours (51.42 ± 4.37% before versus 26.04 ± 7.61% at 72 hours after Impella® implantation, p= 0.003), which persisted for the full observation period of 168 hours after Impella® implantation (with 31.00 ± 3.66% HMW VWF multimers at 168 hours after Impella® implantation, p=0.007). Subsequent injection of PBS did not alter the VWF multimeric profiles as decreased percentages were maintained 2, 24 and 72 hours after PBS injection (respectively 28.57 ± 4.66% (p=0.002), 32.54 ± 5.17% (p=0.03) and 31.89 ± 2.94% (p=0.03) HMW VWF multimers) (Figure 3B andC). The decrease in HMW VWF multimers after Impella® implantation and after PBS injection resulted in a decreased VWF function indicated by a reduced VWF:CB/VWF:Ag ratio at 72 hours after Impella® implantation (0.80 ± 0.09 compared to 1.03 ± 0.03 before implantation, p=0.04) and at 48 hours and 72 hours after PBS injection (0.75 ± 0.12 (p=0.01) and 0.83 ± 0.06 (p=0.04), respectively) (Figure 3D). Interestingly however, injection of one bolus of 600 μg/kg (dose predetermined with an in vitro ADAMTS13 activity assay [START_REF] Muia | An optimized fluorogenic ADAMTS13 assay with increased sensitivity for the investigation of patients with thrombotic thrombocytopenic purpura[END_REF]) of mAb 17C7 resulted in a rescue of HMW VWF multimers as the decrease in HMW VWF multimers was restored after 2 hours, 24 hours, 48 hours, 72 hours and 168 hours (42.13 ± 4.96%; 43.69 ± 1.31%; 44.10 ± 2.78%; 34.55 ± 13.62% and 42.62 ± 4.16% HMW VWF multimers at 2 hours, 24 hours, 48 hours, 72 hours and 168 hours after mAb injection respectively compared to before Impella® implantation (51.42 ± 4.37%)) (Figure 3B andC), hence demonstrating the therapeutic potential to rescue aVWS after device implantation. Accordingly, VWF function was restored after blocking ADAMTS13 activity with mAb 17C7 (VWF:CB/VWF:Ag ratio was 0.93 ± 0.09 at 2 hours, 0.97 ± 0.07 at 24 hours, 0.93 ± 0.16 at 48 hours, 0.86 ± 0.22 at 72 hours and 0.94 ± 0.14 at 168 hours (7 days) after mAb injection) (Figure 3D).

Besides the analysis of VWF, we also assessed other critical parameters. First, we followed ADAMTS13 activity throughout the experiment and we confirmed full inhibition of ADAMTS13 activity until the end of the experiment (168 hours) after mAb 17C7 injection (Figure S7A). However, it is noteworthy to mention that despite full ADAMS13 inhibition, HMW VWF multimers did not restore to baseline values after mAb injection (51.42 ± 4.37% HMW VWF multimers), indicating that possibly an additional mechanism (besides ADAMTS13 proteolysis) might play a minor role in the residual loss of HMW VWF multimers. Inhibition of ADAMTS13 activity was in line with the detection of mAb 17C7 in plasma, which was still 3.70 ± 0.87 μg/mL at 168 hours after mAb injection (Figure S7B). ADAMTS13 antigen levels were not affected after the injection of mAb 17C7, indicating that the antibody did not accelerate clearance of ADAMTS13 from circulation (Figure S7C).

Finally, an important concern when blocking ADAMTS13 is the risk of inducing thrombotic thrombocytopenic purpura (TTP) symptoms which include severe thrombocytopenia, hemolytic anemia and organ failure (e.g. increased creatinine and LDH). No severe thrombocytopenia (Figure 4A), hemolytic anemia (Figure 4B andC) nor organ damage (Figure 4D andE) was observed in the 7-day time frame post-injection of mAb 17C7 during which the calves were monitored. Of note, 2 hours after device implantation, creatinine levels were slightly elevated (Figure 4E), whereas LDH levels (Figure 4D) and WBC count (Figure 4F) were increased 24 hours post-implantation, reflecting temporary changes that were most likely surgery related.

Patients with MCS device-associated gastrointestinal bleeding often present with melena or rectoral bleeding and laboratory analysis may reveal anemia. [START_REF] Ahsan | Clinical Approach to Manage Gastrointestinal Bleeding with a Left Ventricular Assist Device (LVAD)[END_REF][START_REF] Gurvits | Bleeding with the artificial heart: Gastrointestinal hemorrhage in CF-LVAD patients[END_REF] However, no signs of bleeding were observed in the calves as confirmed by normal RBC counts and hemoglobin levels (Figure 4B andC) and the absence of melena. The fact that no active gastrointestinal bleeding was observed in our calf model, might be explained by the absence of an underlying mechanism (e.g. angiodysplasia) since gastrointestinal bleeding occurs most often from arteriovenous malformations in the gastrointestinal tract. [START_REF] Guha | Gastrointestinal bleeding after continuousflow left ventricular device implantation: review of pathophysiology and management[END_REF][START_REF] Muthiah | Increased incidence of angiodysplasia of the gastrointestinal tract and bleeding in patients with continuous flow left ventricular assist devices (LVADs)[END_REF][START_REF] Cochrane | Gastrointestinal angiodysplasia is associated with significant gastrointestinal bleeding in patients with continuous left ventricular assist devices[END_REF]

Discussion

Nowadays, treatment of gastrointestinal bleedings in LVAD patients relies on endoscopic intervention or downshifting of antithrombotic therapy or a reduction in pump speed. [START_REF] Guha | Gastrointestinal bleeding after continuousflow left ventricular device implantation: review of pathophysiology and management[END_REF][START_REF] Cushing | Gastrointestinal Bleeding Following LVAD Placement from Top to Bottom[END_REF] However, none of these conventional treatment strategies target the molecular mechanism, i.e. excessive degradation of VWF by ADAMTS13, that could be responsible for the bleeding. [START_REF] Bartoli | Pathologic von Willebrand factor degradation is a major contributor to left ventricular assist device-associated bleeding: pathophysiology and evolving clinical management[END_REF] Our data show that targeting ADAMTS13 using an inhibitory anti-ADAMTS13 mAb, is a successful strategy to prevent aVWS in in vitro MCS devices and reverse aVWS in a preclinical Impella®-induced aVWS calf model. These data further underline the idea that the main cause of VWF degradation in LVADs is increased VWF proteolysis of VWF by ADAMTS13 rather than shear-induced mechanical destruction of VWF or clearance of VWF-platelet complexes, as previously suggested. [START_REF] Dassanayaka | Mechanistic Pathway(s) of Acquired Von Willebrand Syndrome with a Continuous-Flow Ventricular Assist Device[END_REF][START_REF] Nascimbene | von Willebrand factor proteolysis by ADAMTS-13 in patients on left ventricular assist device support[END_REF] Nevertheless, mechanical destruction of VWF or platelet binding to VWF and subsequent clearance cannot be excluded and could possibly account for the observation that there was no 100% reversal of HMW VWF multimers loss upon ADAMTS13 inhibition (Figure 3B andC). In addition, our study corroborates previous in vitro work showing that an anti-VWF mAb inhibiting the binding of VWF to ADAMTS13 was also able to prevent shear-induced degradation of VWF using an in vitro Heartmate II™ system. [START_REF] Rauch | Antibody-based prevention of von Willebrand factor degradation mediated by circulatory assist devices[END_REF] Similarly, no loss of HMW VWF multimers was observed when perfusing blood of patients with a congenital deficiency in ADAMTS13 through an in vitro Heartware HVAD® system. [START_REF] Jilma-Stohlawetz | Acquired von Willebrand factor deficiency caused by LVAD is ADAMTS-13 and platelet dependent[END_REF] The main concern when inhibiting ADAMTS13 is the induction of TTP-like symptoms. However, when blocking ADAMTS13 in vivo, we did not observe signs and symptoms of TTP during a 7-day follow-up. These results fit with the requirement of a secondary trigger that is needed to induce full-blown TTP. [START_REF] Hovinga | Thrombotic thrombocytopenic purpura[END_REF] Indeed, ADAMTS13-deficient mice or rats in which ADAMTS13 is inhibited only develop TTP when a secondary trigger (injection of shigatoxin or rVWF) is used. [START_REF] Schiviz | A new mouse model mimicking thrombotic thrombocytopenic purpura : correction of symptoms by recombinant human ADAMTS13[END_REF][START_REF] Motto | Shigatoxin triggers thrombotic thrombocytopenic purpura in genetically susceptible ADAMTS13-deficient mice[END_REF][START_REF] Tersteeg | Potential for Recombinant ADAMTS13 as an Effective Therapy for Acquired Thrombotic Thrombocytopenic Purpura[END_REF] Similarly, patients with deficient ADAMTS13 can live for years without the development of TTP signs and symptoms. [START_REF] Mariotte | Epidemiology and pathophysiology of adulthood-onset thrombotic microangiopathy with severe ADAMTS13 deficiency (thrombotic thrombocytopenic purpura): a cross-sectional analysis of the French national registry for thrombotic microangiopathy[END_REF] However, since infections and multiple adverse events leading to the activation of inflammatory pathways are common complications in MCS device patients, it should be considered that these conditions may potentially evoke an acute TTP episode. [START_REF] Grosman-Rimon | New therapy, new challenges: The effects of long-term continuous flow left ventricular assist device on inflammation[END_REF] On the other hand, inhibition of ADAMTS13 function in baboons led to an early and mild stage of TTP including thrombocytopenia and haemolytic anemia but no life-threatening complications. [START_REF] Feys | Thrombotic thrombocytopenic purpura directly linked with ADAMTS13 inhibition in the baboon (Papio ursinus)[END_REF] The reasons for these differences between species remain unclear. Of note, mild TTP signs and symptoms in baboons only appeared after 48 hours. [START_REF] Feys | Thrombotic thrombocytopenic purpura directly linked with ADAMTS13 inhibition in the baboon (Papio ursinus)[END_REF] As the anti-ADAMTS13 therapy would be used to treat acute bleedings, a short-term treatment is warranted (24 to 48 hours), thereby minimizing the potential risk for full-blown TTP development. Although our preclinical study in the calves demonstrated no TTP-like signs and symptoms, the safety profile of the drug warrants further investigation and will be mapped during future studies. However, it is important to acknowledge that a small risk for early stage TTP remains a concern when inhibiting ADAMTS13. Caplacizumab, which is currently approved in the EU and US for the treatment of acquired TTP could be used as a counteracting agent, in case that TTP might set in in the initial clinical studies in humans. [START_REF] Sargentini-Maier | Clinical pharmacology of caplacizumab for the treatment of patients with acquired thrombotic thrombocytopenic purpura[END_REF] Although the aim of our current study was to demonstrate proof of concept to reverse aVWS, the observation that calves on Impella® support do not show signs of active bleeding as confirmed by the absence of melena and anemia (no drop in RBC count and hemoglobin), is a limitation of this study. Hence, the current model could not be used to evaluate if aVWS reversal was sufficient to treat bleedings and thus plays a major contributing factor in the development of gastrointestinal bleedings. To our knowledge, no clinically significant LVAD-induced bleeding model exists so future clinical studies will need to point out if targeted inhibition of ADAMTS13 can treat the bleedings in the acute setting.

In conclusion, inhibiting ADAMTS13 function could become a promising therapeutic strategy to rescue aVWS-induced bleeding, not only in LVAD patients, but in any disorder where increased VWF proteolysis leads to the loss of HMW VWF multimers like in aortic stenosis, ECMO patients and VWD type 2A patients. Especially, since there currently exist no targeted pharmacotherapeutics to treat bleedings in these patients, this novel drug answers the unmet medical need. Although our initial results demonstrate no major safety issues, the potential risks that are linked to ADAMTS13 inhibition should be carefully evaluated in future clinical trials.

Essentials:

•

Patients with a heart pump suffer from the acquired von Willebrand syndrome (aVWS)

•

A targeted therapy to treat heart pump-induced aVWS is currently unavailable (A) Schematic representation of the in vitro Impella CP® system. (B) A blood sample was taken before the onset of perfusion (BL) after which the human blood was supplemented with either PBS or 20 μg/mL of the inhibitory anti-ADAMTS13 mAb 3H9. After a 5-minute incubation period, the blood was added to the in vitro Impella CP® system and perfusion of the blood through the pump was started. Next, blood was sampled 5 (T5), 30 (T30), 60 (T60) and 180 (T180) minutes after the start of perfusion (A) A blood sample was taken before the onset of perfusion (BL) after which the bovine blood was supplemented with either PBS or 20 μg/mL of the inhibitory anti-ADAMTS13 mAb 17C7. After a 5-minute incubation period, the blood was added to the in vitro Impella 5.5® system and perfusion of the blood through the pump was started. Next, blood was

Figure 1 .

 1 Figure 1. Direct blocking of ADAMTS13 activity in an in vitro Impella CP® system with human blood prevents the loss of HMW VWF multimers and preserves VWF collagen binding activity.

Figure 2 .

 2 Figure 2. Direct blocking of ADAMTS13 activity in an in vitro Impella 5.5® system with bovine blood prevents the loss of HMW VWF multimers and preserves VWF collagen binding activity.

sampled 5 (

 5 T5), 30 (T30), 60 (T60), 180 (T180) and 300 (T300) minutes after the start of perfusion. (B) Representative VWF multimeric pattern was obtained by separating VWF on SDS-agarose gels resulting in the separation of the low molecular weight (LMW), medium molecular weight (MMW) and high molecular weight (HMW) VWF multimers. (C) The percentage of HMW VWF multimers was determined via densitometry. (D) VWF collagen binding activity (VWF:CB) and VWF antigen (VWF:Ag) was determined using ELISAand the ratio of VWF:CB/VWF:Ag is depicted. Data are represented as mean ± standard deviation (n=3 independent biological replicates). NBP= normal bovine pooled plasma. p values; *p < 0.05, **p < 0.01. Friedmann test with corrections for multiple comparisons (Dunn's multiple comparisons test). Images of the other gels that support the findings of this study are available from the corresponding author upon request.

Figure 3 .

 3 Figure 3. Blocking ADAMTS13 in the LVAD-induced aVWS calf model rescues the loss of HMW VWF multimers.(A) Impella 5.5® pumps were implanted in calves (black arrow) and 168 hours (T168) after pump implantation, the calves were injected with one bolus of PBS intravenously (black arrow). Next, 168 hours (T168) after injection with PBS, one bolus (600 μg/kg) of inhibitory mAb 17C7 was injected (black arrow). Plasma samples were obtained from calves before Impella 5.5® implantation (BL) and at 2 (T2), 24 (T24), 48 (T48), 72 (T72) and 168 (T168) hours after Impella 5.5® implantation and subsequent PBS and inhibitory mAb 17C7 injection. (B) Representative VWF multimeric pattern was obtained by separating VWF on SDS-agarose gels resulting in the separation of the low molecular weight (LMW), medium molecular weight (MMW) and high molecular weight (HMW) VWF multimers.

Figure 4 .

 4 Figure 4. Inhibiting ADAMTS13 does not cause thrombocytopenia, hemolytic anemia nor organ damage in the LVAD-induced aVWS calf model.(A) Platelet counts, (B) hemoglobin levels, (C) red blood cell (RBC) counts, (D) Lactate dehydrogenase (LDH) levels, (E) creatinine levels and (F) white blood cell (WBC) counts were measured in blood or plasma samples of calves before Impella 5.5® implantation (BL) and at 2 (T2), 24 (T24), 48 (T48), 72 (T72) and 168 (T168) hours after Impella 5.5® implantation and subsequent PBS and inhibitory mAb 17C7 (600 μg/kg) injection, using automated or standardized methods. Data are represented as mean ± standard deviation (n=4 independent biological replicates). p value; *p < 0.05. Friedmann test with corrections for multiple comparisons (Dunn's multiple comparisons test).

  of the low molecular weight (LMW), medium molecular weight (MMW) and high molecular weight (HMW) VWF multimers. (D) The percentage of HMW VWF multimers was determined via densitometry. (E) VWF collagen binding activity (VWF:CB) and VWF antigen (VWF:Ag) was determined using ELISA and the ratio of VWF:CB/ VWF:Ag is depicted. Data are represented as mean ± standard deviation (n=5 independent biological replicates). NHP= normal human pooled plasma. p values; *p < 0.05, **p < 0.01, ***p < 0.001. Friedmann test with corrections for multiple comparisons (Dunn's multiple comparisons test). Images of the other gels that support the findings of this study are available from the corresponding author upon request.

. (C) Representative VWF multimeric pattern was obtained by separating VWF on SDS-agarose gels resulting in the separation

J Thromb Haemost. Author manuscript; available in PMC 2022 December 01.

Acknowledgements

The authors thank Ulrich Budde for the expertise and helpful discussions; Senna Staessens for the technical assistance with the in vitro MCS device systems; and Mieke Ginckels and David Celis for their excellent technical assistance during the animal trials. This work was supported by the 'Fund for scientific research Flanders (FWO Vlaanderen)' [grant number 1S60917N] awarded to Shannen J Deconinck and the KU Leuven [grant numbers PF/10/014, C32/17/012 and C3/20/116] awarded to Karen Vanhoorelbeke. Joshua Muia was supported by the National Institutes of Health, National Institute of General Medical Sciences grant R35 GM142926.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.


Mykola Isaiev
email: mykola.isaiev@knu.ua

Sergii Burian

Leonid Bulavin

William Chaze

Michel Gradeck

Guillaume Castanet

Samy Merabia

Pawel Keblinski

Konstantinos Termentzidis
email: konstantinos.termentzidis@univ-lorraine.fr

Alain Mcgaughey

Thierry Biben

Gibbs Adsorption Impact on a Nanodroplet Shape: Modification of Young-Laplace Equation

Keywords: nanoscale droplet, external field, molecular dynamics, adsorption, Young-Laplace equation

come   L'archive ouverte pluridisciplinaire

Introduction

The interfaces between two or more different phases predominate a variety of physical and chemical properties, especially at the nanometer scale compared to bulk materials, as the surfaceto-volume fraction become very high. In particular, interactions between different phases are crucial for the wettability, solubility, chemical reactions etc. The prediction of the interaction of different phases is a key challenge for a wide range of applications in chemical industry, catalysis, material science, microfluidics, pharmacology, medicine, etc.

As an example, we can note, the wetting properties of a liquid droplet on a solid substrate. For the case of nanoscale droplet additionally to the surface tension, the line tension influence on the wetting angle should be taken into account. These excesses of free energy per unit length separating three distinct phases respectively should be taken into account to achieve stability of deformable surfaces. The general Young equation can be presented as follows:

cos(𝜃 𝑤 ) = 𝛾 𝑣𝑠 -𝛾 𝑙𝑠 𝛾 𝑣𝑙 - 𝜏 𝛾 𝑣𝑙 𝑟 , (1)
where 𝜃 𝑤 is the wetting angle, 𝛾 𝑣𝑠 , 𝛾 𝑙𝑠 , and 𝛾 𝑣𝑙 are the vapor/solid, liquid/solid, and vapor/liquid surface tensions respectively, 𝜏 is the line tension, and 𝑟 is the contact radius of a three-phase line.

Barisic and Beskok [START_REF] Barisik | Wetting characterisation of silicon (1,0,0) surface[END_REF] have simulated spherical droplets with different volumes located on the silicon substrate with molecular dynamics. They measured a wetting angle as a function of the curvature of three phase contact line (1/r). With the use of a linear dependence (Equation ( 1)), they estimated the line tension and they defined several parameters of the interaction between water molecules and silicon atoms with the use of the experimental wetting angles of macrosize droplet as input. Later on, it was shown in [START_REF] Isaiev | Efficient tuning of potential parameters for liquid-solid interactions[END_REF] that the simulation of a cylindrical droplet is more efficient than this of spherical droplets for the evaluation of the interaction parameters between a droplet and a substrate, since the curvature of the three-phase contact line is equal to zero [START_REF] Peng | The impact of line tension on the contact angle of nanodroplets[END_REF] in http://arxiv.org/abs/1707.08844

the case of cylindrical droplets. Leroy et al [START_REF] Leroy | Parametrizing Nonbonded Interactions from Wetting Experiments via the Work of Adhesion: Example of Water on Graphene Surfaces[END_REF] have shown recently that the evaluation of the forcefields parameterization using experimental wetting angles of droplet has limitations. They have stated that the dependence of water surface tension on the chosen water model should be taken into account. Thus, additional markers for reliable description of water-solid interfacial properties should be found, such as "work of adhension" [START_REF] Leroy | Parametrizing Nonbonded Interactions from Wetting Experiments via the Work of Adhesion: Example of Water on Graphene Surfaces[END_REF]. Numerous MD studies [START_REF] De Ruijter | Dynamic wetting studied by molecular modeling simulations of droplet spreading[END_REF][START_REF] Barisik | Temperature dependence of thermal resistance at the water/silicon interface[END_REF][START_REF] Thomas | Density, distribution, and orientation of water molecules inside and outside carbon nanotubes[END_REF][START_REF] Grigera | Wall-Water Interface. A Molecular Dynamics Study[END_REF] of solid-liquid interfaces refer to the density fluctuations of fluids near the solid free surfaces. These fluctuations appear as a result of molecules or atoms absorbed in or adsorbed on the solid substrate. The density of adsorbents strongly depends on how these atoms or molecules interact with the substrate. The density variation can even lead to experimentally perceptible contraction of the volume of the liquid containing nanoparticles [START_REF] Pastoriza-Gallego | A study on stability and thermophysical properties (density and viscosity) of Al2O3 in water nanofluid[END_REF][START_REF] Korolovych | Influence of single-walled carbon nanotubes on thermal expansion of water[END_REF]. Moreover, the altered structure of confined water near the wall can lead to a drastic change of its physical properties [START_REF] Schlaich | Water Dielectric Effects in Planar Confinement[END_REF][START_REF] Cerveny | Confined Water as Model of Supercooled Water[END_REF][START_REF] Han | Thermal Transport at Solid-Liquid Interfaces: High Pressure Facilitates Heat Flow through Nonlocal Liquid Structuring[END_REF].

An important question that one should address in all cases, is if one can treat nanoscale droplets with macroscopic approaches? Which are the limitations of such approaches, or what should be the corrections to achieve convergence of dimensions? In this paper we focus on the influence of the absorbed layer on the droplet shape and droplet volume. We study the contraction of a nanoscale cylindrical water droplet on a silicon substrate with means of MD. We applied a homogeneous external field with different intensities to tune the contact area. We correlate the shape of a nanoscale droplet modeled and simulated by MD with the one obtained by a macroscopic method of hydrostatic similarity of droplet shape in gravity field. The deviations between the results of the MD simulations and the analytical approach will be explained.

Furthermore, we propose a new model accounting the contraction of the droplet volume due to adsorption phenomena. The proposed model can be also relevant of experimentally observed volume contraction of nanocolloidal solutions [START_REF] Pastoriza-Gallego | A study on stability and thermophysical properties (density and viscosity) of Al2O3 in water nanofluid[END_REF][START_REF] Korolovych | Influence of single-walled carbon nanotubes on thermal expansion of water[END_REF] or nanoporous materials filled with liquids http://arxiv.org/abs/1707.08844 [START_REF] Isaiev | Thermal Elasticity Stresses Study in Composite System "Porous Silicon -Liquid[END_REF][START_REF] Andrusenko | Photoacoustic effects in nanocomposite structure "porous silicon-liquid[END_REF]. These systems are accessible to experimental measurements and they have high ratio of surface/volume.

Molecular dynamics simulations of water droplet on silicon substrate

MD Methodology

All MD simulations presented here are performed using LAMMPS code (Large-scale Atomic/Molecular Massively Parallel Simulator) [START_REF] Plimpton | Fast Parallel Algorithms for Short-Range Molecular Dynamics[END_REF]. We have considered cylindrical droplet on a smooth silicon substrate with and without an external homogeneous field. Additionally, cylindrical droplet without substrate was simulated as reference. We use cylindrical droplets to avoid the influence of line tension on the contact angle. In both cases the simulation box dimensions are 108.6 × 217.2 × 200 Å in x, y and z-direction respectively. http://arxiv.org/abs/1707.08844

The initial configurations of the atoms of the droplet without/with substrate are presented in Figure 1a and 1b respectively. Initially, a rectangular crystalline water droplet with size 108.6 × 43.4 × 43.4 Å was built. The total number of water molecules in the droplet is equal to 6860.

The distance between oxygen atoms of water molecules was set to 3. Interactions between water molecules are described by the extended simple point charge (SPC/E)

water model [START_REF] Berendsen | The missing term in effective pair potentials[END_REF], with parameters taken by Orsi [START_REF] Orsi | Comparative assessment of the ELBA coarse-grained model for water[END_REF]. The 12-6 Lenard-Jones interatomic potential was used for the interactions between oxygen and silicon atoms. The parameters of the interaction were chosen as εSi-O = 15.75 meV and σSi-O= 2.6305 Å [START_REF] Isaiev | Efficient tuning of potential parameters for liquid-solid interactions[END_REF]. In our study we have neglected the interactions between hydrogen and silicon atoms. The interactions between silicon atoms the Stilling-Weber potential [START_REF] Stillinger | Computer simulation of local order in condensed phases of silicon[END_REF] is used. Additionally, the harmonic force was applied to atoms of the lower four atomic layers (z <2a0) to tether them to their initial positions. This is done to prevent moving the silicon slab in Z direction during the simulations.

The initial configurations were thermostated during 2 ns with the Nosé-Hoover thermostat in the canonical ensemble to achieve equilibration at 300 K. The timestep was set equal to 1 fs. The resulting droplets are presented in Figure 1c and 1d for the free droplet and the droplet on the substrate.

Droplet without external field

The water density profiles were collected every 0.1 ps and then averaged during 0.5 ns. To obtain the density profile, the three-dimensional space was divided by square cross-section rectangular (1×1 Å2, in YZ plane). The resulting density profile of the free droplet and the dependence of the http://arxiv.org/abs/1707.08844 The density of bulk water inside the droplet was found to be equal to 997.6 kg/m 3 and the density of the equilibrium vapor is 0.01474 kg/m 3 and pressure equal to 0.05 atm. These densities are in a good agreement with the results published previously for the same water model [START_REF] Ghoufi | Mesoscale modeling of the water liquid-vapor interface: A surface tension calculation[END_REF] and http://arxiv.org/abs/1707.08844

with experimental data. The liquid-vapor interface of the droplet is relative sharp (Figure 2a and 2b), and it has thickness approximately equal to 10 Å. We define the liquid/vapor interface as a constant density surface equal to the half of the bulk water density. In Figure 2a Additionally, in Figure 2c the density map of the droplet on the silicon substrate in the case of absent of external forces is depicted. We have evaluated the wetting angle which has been found to be equal to 86.6 o . This value is in a good agreement with previous results [START_REF] Isaiev | Efficient tuning of potential parameters for liquid-solid interactions[END_REF]. In Figure 2d the dependence of the averaged water density in the plane perpendicular to Y-axis and crossing the droplet's centre of mass as a function of z is presented. As it was mentioned above, a layering effect appears close to the substrate. The density of this layer can be higher than the density of bulk water under normal conditions for more than 30 %. The thickness of this layer is calculated to be approximately 4±1 Å. The presence of this layer leads to the droplet volume contraction, as the volume per unit length in this case becomes equal to 1803.1Å 2 (without the presence of the substrate it was found to be equal to 1977.6 Å 2 )

The impact of the external homogeneous field

In order to study the interaction between a liquid and a solid in an external homogeneous field, we added gravity-like field, with which all water atoms are subject to an additional force in direction towards the negative values of the z-axis. This additional field was added during the thermalization process described above. In Figure 3a and 3b MD snapshots of the droplet in the external field with intensities 10.1×10 12 and 51.3×10 12 m/s 2 are depicted. These values correspond to radius of curvature 2 and 64 times greater than for the case of absence of gravity. We are aware that these gravity fields are enormous, but we can justify them by two reasons. First of all, as it was explained http://arxiv.org/abs/1707.08844 in the introduction our aim is to reproduce macroscopic behavior of droplets with means of molecular dynamics.

𝑣 ̃= 𝑣 -𝜀𝑎 , (2)
where 𝑣 ̃ is a volume of contracted droplet, a is the contact area per unit length, ε is the slope of the dependence of volume on the contact area 𝑣 ̃(𝑎)(Figure 4b). We fitted the results of the molecular dynamic simulations with the equation ( 2), and found that the best fitting value is ε = 0.875 Å. http://arxiv.org/abs/1707.08844

From the estimated ε, the surface excess or adsorption per unit area Γ [START_REF] Tadros | Gibbs Adsorption Isotherm[END_REF] can be calculated (in mol/m 2 ) as follows

Γ = 𝜀𝜌 𝑏 𝑀 𝐻2𝑂 , (3)
where ε should be taken in m. Then, we estimate that Γ equals to 4.8 μmol/m 2 .

At this point, it is important to check possible effects due to capillary forces. If capillary forces were the origin of the phenomena observed in our case, one could have observed a gradient of water pressure in the z direction. This is not the case as it can been seen if Figure 5, where one can observe that indeed the pressure is higher close to the water/silicon interface (this is related to the adsorption layer), but in the main volume of the water droplet we observe that there is no systematic way that the pressure varies along the z-direction.

Analytical model of a nanodroplet in an external field

In the previous section the simulation of the nanoscale droplet in an external field with MD is presented. In this section an analytical approach of the droplet volume contraction due to the enhancement of the contact area will be presented. As it was mentioned previously, we assume that the volume of a droplet decreases due to the influence of the potential field of the atoms of the substrate. Direct approach requires calculation of the field created by the atoms of the substrate.

Since this field is strongly heterogeneous, the calculation of the droplet shape can be very cumbersome from practical point of view. Therefore, we consider the influence of this field phenomenologically due to considered adsorption of water molecules on silicon surface.

http://arxiv.org/abs/1707.08844

For the quantitative description of the droplet shape in an external field at the macroscale the approach based on the Young-Laplace equation is usually used [START_REF] Río | Axisymmetric Drop Shape Analysis: Computational Methods for the Measurement of Interfacial Properties from the Shape and Dimensions of Pendant and Sessile Drops[END_REF][START_REF] Dimitrov | Using Interfacial Tension Measurements To Study the Kinetics of Ferritin and Sugar Adsoprtion onto Mercury[END_REF]. The Young-Laplace equation describes the mechanical equilibrium condition for two homogeneous fluids separated by an interface that is assumed to be a surface of zero thickness. In the case of the absence of external field, the pressures difference Δ𝑃 across a curved interface caused by the surface tension can be presented as follows

𝛾 ( 1 𝑅 1 + 1 𝑅 2 ) = Δ𝑃, (4)
where, γ is the surface tension, 𝑅 1 and 𝑅 2 are the principal radii of the surface curvature. For the considered case of the cylindrical droplet one of the principal curvature radius is trend to be equal infinity (𝑅 2 → ∞). In the frame of the assumption of the incompressible isotropic fluid in an external homogeneous gravity-like field, the equation of the force equilibrium can be presented as follows [START_REF] Landau | Fluid Mechanics[END_REF] Δ𝑃 = Δ𝑃 0 + Δ𝜌𝑔(𝑧 -𝑧 0 ),

http://arxiv.org/abs/1707.08844

where Δ𝑃 0 is the pressure difference in reference point 𝑧 = 𝑧 0 , Δ𝜌 is the difference in densities between the two phases, and g is the external field intensity. In the Equation 5the direction of the z-axis is chosen the same as it is presented in Figure 6 (invers to the direction of z -axis in MD simulation).

In the case when the origin of the coordinate system and the reference point are located at the droplet apex, the equation ( 5) can be presented as follows

1 𝑅 1 = 1 𝑟 + 𝑧 𝜅 2 , ( 6
)
where r is the radius of curvature at the droplet apex, 𝜅 = √𝛾/(Δ𝜌𝑔) is the capillary length. In this case we neglected the influence of the size dependence of the surface tension [START_REF] Burian | Size dependence of the surface tension of a free surface of an isotropic fluid[END_REF].

Reflectional symmetric drop shape analysis

For the description of a droplet shape in a gravity field, Axisymmetric Drop Shape Analysis (ADSA) [START_REF] Rotenberg | Determination of surface tension and contact angle from the shapes of axisymmetric fluid interfaces[END_REF] proposed by Rotenberg et al is most commonly used. Regarding cylindrical droplet, the modification of ADSA can be used taking into account reflectional symmetry of the droplet shape for the analysis (RDSA). In this case the curvature can be related to the arc length s and the angle of inclination of the interface to the horizontal angle 𝜑 by:

1 𝑅 1 = 𝑑𝜑 𝑑𝑠 . (7)
Therefore, the equation ( 4) can be presented as follows

𝑑𝜑 𝑑𝑠 = 1 𝑟 + 𝑧 𝜅 2 . ( 8
)
The equation ( 6) is supplemented with the geometrical relations

𝑑𝑦 𝑑𝑠 = cos(𝜑) ; 𝑑𝑧 𝑑𝑠 = sin(𝜑) ; 𝑑𝑣 𝑑𝑠 = 2ysin(𝜑), (9)
http://arxiv.org/abs/1707.08844

where 𝑣 is the droplet volume per unit length. The system formed by Equation 7, 8 and 9 is solved numerically using the Runge-Kutta method taken as initial conditions 𝑦(0) = 𝑧(0) = 𝜑(0) = 𝑣(0) = 0. Integration is stopped when φ reaches the value of the contact angle 𝜑 𝑐 = 86.6 𝑜 that is a priori known parameter.

The dependence of the droplet height and its contact radius as a function of the gravity intensities evaluated numerically (filled squares) and with MD simulation (filled circles) are presented in Figure 7a and 7b respectively. As one can see, there is a deviation between results of the numerical approach and results of MD simulations. This discrepancy might arise because of the presence of the adsorbed water layer.

Since the physical basis of the Equation 4is the assumption of the incompressibility of the liquid, the volume of a droplet under different gravities is the same. Although the volume of the macrosize droplet conserved in the presence of gravity field, the volume of the nanoscale droplet is change due to the presence of the adsorbed layer. To take this into account we decomposed the droplet into two parts: adsorption layer and the droplet with capillary shape.

In this case the mass of the droplet 𝑚 𝑑 per unit of length can be determined by:

𝑚 𝑑 = 𝜌 𝑏 (𝑣 -𝑣 𝑎 ) + 𝜌 𝑎 𝑣 𝑎 , (10)
where 𝜌 𝑏 and 𝜌 𝑎 are the densities of the bulk water and adsorbed layer respectively; 𝑣 𝑎 is the volume per unit length of the adsorbed layer. To allow fair comparisons with the MD simulations, the mass of the droplet was kept the same. For that, the parameter 𝑟 in Equation 8 was varied iteratively. The value of 𝑟 is increased if 𝑚 𝑑 is bigger than the requested liquid mass, or decreased in the contrary.

The result of the numerical simulations of the droplet contact radius and radius at the droplet top considering the adsorbed layer are presented in Figure 7a and 7b with green squares.

http://arxiv.org/abs/1707.08844

The values of the adsorbed layer thickness (Ha = 4 Å) and its density (1220 kg/m 3 ) was taken from the results of MD simulations. As one can see in figure-7, there is an excellent agreement of the results of numerical integration of Eq [START_REF] Thomas | Density, distribution, and orientation of water molecules inside and outside carbon nanotubes[END_REF] and ( 8) with the results of molecular dynamics simulations. Therefore, a significant impact of the adsorbed layer presence on the droplet shape contraction can be stated.

The Gibbs adsorption in this case can be estimated as follows

Γ = 𝐻 𝑎 (𝜌 𝑎 -𝜌 𝑏 ) 𝑀 𝐻2𝑂 = 4.9 μmol m 2 , ( 11
)
where MH2O is the molar mass of water. The Γ values predicted with Equation 3 and 11 are in excellent agreement.

Bashforth-Adams equation with adsorption layer

Another approach for the droplet shape treatment is based on the Bashforth-Adams equation (BAE) solution. This equation can be obtained from the Equation 6, after replacement of the curvature with expression in the following form:

http://arxiv.org/abs/1707.08844

1 𝑅 1 = 𝑑 2 𝑧 𝑑𝑦 2 (1 + ( 𝑑𝑧 𝑑𝑦 ) 2 ) 3 2 ⁄ . (12)
The dimensionless BAE in this case can be presented as follows

𝜕 2 𝑧∂ 𝑦 2 (1 + ( 𝜕𝑧∂ 𝑦 ) 2 ) 3 2 = 1 + 𝐵𝑜 • 𝑧, (13)
where 𝑧̃= 𝑧/𝑟 is the dimensionless z coordinate, 𝐵𝑜 = Δ𝜌𝑔𝑟 2 𝛾 ⁄ = 𝑟 2 𝜅 2 ⁄ is dimensional invariant, which defines the droplet shape in gravity field, called Bond number.

In our case the parameters, which can be correlated with the results of MD simulation are the volume, the height and the contact radius of a droplet. Therefore, it is better to rewrite the Equation 12in the coordinate system where the origin is located on the contact radius (point (0, H)) and axis z directed in opposite site than in previous case (Figure 6). In this case, it can be presented as follows:

𝑑 2 𝑧 𝑑𝑦 2 (1 + ( 𝑑𝑧 𝑑𝑦 ) 2 ) 3 2 = ∆𝜌𝑔 𝛾 ( 𝑣 2 -𝑧(𝑦)𝑅 2 ) + sin𝜃, (14)
Additionally, the condition of the volume conserving gives the following dependence

sin𝜃 - 𝑅 𝑟 = ∆𝜌𝑔 𝛾 (𝐻 • 𝑅 - 𝑣 2 ), (15)
The Figure 7 presents the comparison of the droplet contact radius and radius at the droplet top evaluated with the MD simulations (points) and with Equation 14and 15. As one can see in Figure 6, there are some differences between the results of the simulations and the analytical approach.

Especially the contact area is systematically overestimated with the BAE model and the http://arxiv.org/abs/1707.08844 discrepancy increases in increasing the intensity of the external field. These differences can be overcome with the modification of volume used in the Equation 14 and 15 and Equation 2.

The dependences of the droplet height and contact radius as a function of external force intensities without (dashed-line) and with (solid-line) considered adsorption are presented in Figure 7a and 7b respectively. As one can see, the solid line fits better to the MD simulations. Therefore, the adsorption layer is an important factor that should be taken into account when dealing with nanoscale water droplets.

Discussion and conclusions

In our study, we have considered a nanoscale droplet in an external homogeneous field. We prove that the macroscale approach based on Young-Laplace equation has limitations in the description of the droplet shape. In particularly, in addition to the external field, one should take into account the interatomic interaction of the atoms of the liquid with the atoms of the substrate at the interface between the two phases. A thin adsorbed layer with higher or lower density, depending the interaction between the liquid and the solid, compared to the liquid bulk value under the same conditions appears. The presence of this layer leads to the droplet contraction/expansion, which can be more or less important depending on the volume to surface ratio. In figure 8 the dependence of the ratio of the number of adsorbed particles to the total one as a function of the droplet radius is presented. In particular, for droplets with radius bigger than 100 nm, this ratio is equal to 0.1 %, and the adsorption does not significantly influence the size of the droplet (Figure 8).

We propose a new phenomenological model that takes into account the adsorption of liquid molecules on a solid substrate. We show also that the application of a homogenous external field allows to tune the contact area between liquid and solid for droplets with constant number of http://arxiv.org/abs/1707.08844

particles. As a result a reliable evaluation of the specific adsorption of the liquid atoms/molecules on a solid substrate can be evaluated. Thus, one can predict the volume contraction/expansion of the solution of a liquid with embedded nanoparticles with atomistic simulations. From the other hand, the presented approach can be easily adopted for the inverse problem of the tuning of the potential interaction between solids and liquids. For example, the parameterization of interatomic potential can be obtained by fitting of macroscale experiment of the contraction/expansion of the mentioned above solution. In this case, the Equation 11 can be considered as a dependence of the mixture volume on the specific surface of the nanoparticles.

Figure 1 .

 1 Figure 1. Initial (a,b) and after thermostating (c,d) configurations of a cylindrical nanoscale water droplet for the cases of a droplet (a,c) without substrate and a droplet on a silicon substrate (b and d).

  1 Å, and this represents approximately the density of the bulk water under normal conditions. The surface of the crystalline silicon substrate has the orientation of the (1, 0, 0) facet, with lattice constant equals to a0 = 5.43 Å. The silicon slab has dimensions 108.6 × 217.2 × 27.15 Å (40 × 80 × 10 atoms).

  density as a function of the distance from the droplet centre are presented in Figure2a and 2brespectively.

Figure 2 .

 2 Figure 2. a) Averaged density map of a free droplet; b) Droplet density profile as a function of the distance from the droplet centre; c) Averaged density map of a droplet on a silicon substrate; d) Averaged density profile of a droplet in the plane perpendicular to y-axis and crossing the droplet's centre of mass as a function of z.

  and 2b the liquid/vapor interfaces are presented as bold black solid lines. The radius of the droplet is approximately equal to 25.1 Å and its volume per unit length is v = 1977.6 Å 2 (Figure 2a and 2b).

Figure 3 .

 3 Figure 3. Molecular dynamics snapshots and the density profiles (upper insets) of the droplet density map in an external field with intensities 10.1×10 12 (a) and 51.3×10 12 N/kg (b), corresponding density profiles in the middle of water droplet for the same intensities of external field (c) (d)

Figure 4 .

 4 Figure 4. a) The dependence of the contact length on the external field intensity; b) The dependence of the droplet volume per unit length of cylinder as a function of the contact area per unit length

Figure 5 .

 5 Figure 5. Pressure maps of the droplet under absence (a) and presence of external force field 20 (b) and 50 (c) 10 12 N/kg. No gradient of pressure is observed in any case.

Figure 6 .

 6 Figure 6. A schematic representation of the geometry of the studied droplet

Figure 7 .

 7 Figure 7. Dependences of the droplet height (a) and contact radius (b) as a function of the intensity of gravity field

Figure 8 .

 8 Figure 8. Dependence of the number of the adsorbed particles to the total one as a function of the droplet radius.

Acknowledgements

This work was supported by the Institute CARNOT ICEEL for the project "CAMTRASTE" (Controle et AMelioration des TRAnsferts par Structuration des surfaces d'Echange). Calculations


Hervé Mayer, Université This edited collection on Disney's Star Wars is a much-needed assessment of the impact of Disney's Lucasfilm purchase on production, promotion and reception of the Star Wars franchise across media. The collection brings together contributors from the film industry and academics specializing in media and visual studies as well as communication and fan studies. The twenty-one chapters are organized in two parts: production and promotion, and reception and participation. The book proposes a well-researched overview of the impact of the buyout in various areas and is noteworthy for its contributions in corporate production studies and fan studies..

In their introduction, Proctor and McCulloch discuss the purchase of Lucasfilm in 2012 in light of George Lucas's prior relationship to Disney and contradictory declarations about a sequel trilogy, arguing that Disney's main strategy was "not to rebrand Star Wars by constructing a binary between Lucasfilm and Disney but, rather, by unifying them in order to instill trust and belief" (11). The editors then announce the aim of the collection, which is to distinguish "key shifts that have occurred at the level of industry, narrative, and reception" since the Disney acquisition and "examine the ways in which ownership, conglomeration, and branding operate within the postmillenial mediascape" (12).

The first part of the volume, "Production and Promotion," opens with three chapters focusing on brand synergy. Matthew Freeman's chapter on "Rebuilding Transmedia Star Wars" analyzes the ways in which Disney shifted the status and content of the Star Wars Expanded Universe to create a discourse of legitimacy and canon. Disney rebranded the Expanded Universe Star Wars Legends and presented its texts as alternatives, using imagination as a specific world-building approach connecting Disney's Star Wars productions to Lucasfilm's. Similarly, Matt Hills' chapter on "Transmedia Spectacle and Transownership Storytelling as Seen on TV" examines the position of televisual productions within the Star Wars universe, pre-and post-acquisition. It compares the Holiday Special (1978) to Rebels (2014Rebels ( -2018) ) and shows that the latter used aesthetics and narrative as another bridge between past and present owners. The brand synergy is further exemplified in Lincoln Geraghty's "Rebuilding the Force, Brick by Brick" stressing the role of LEGO and LEGO-book publisher Dorling Kindersley in the process. Dan Hassler-Forest work on "Fan Labor and Brand Management" discusses how the promotion of The Force Awakens relied on a form of immaterial fan labor and collaborative, yet asymmetric, brand management. The film's promotion rested on nostalgia for the original trilogy, but also incorporated fan participation: "fan culture's transformative creative work is itself transformed into something that can be used to rebrand not only the Star Wars franchise but also the conglomerate of which it is a part" (80). In "Rebellions Are Built on Realism," Joshua Wucher argues that the use of special effects in the film Rogue One is heir to the Lucasfilm tradition. Both Rogue One and promotional paratexts reprised the original trilogy's analog and digital special effects traditions in ways that ensured fidelity. Colin B. Harvey's "Binding the Galaxy Together" stresses further the link with tradition and investigates the role of memory in the construction of the Star Wars franchise's transmedia network. He compares memory practices pre-and post-acquisition in relation to the increasingly connective digital practices of fans' engagement with the franchise.

The first part closes with three chapters that study the effects of Disney's purchase at the narrative and aesthetic level across different media. Ross Garner's "The Mandalorian Variation" focuses more specifically on the limiting impact of Disney's acquisition on the franchise's gender identity and dynamics, and more particularly on the construction of the lead action female character in the TV series Star Wars Rebels. Douglas Brown analyses the impact of the acquisition on Star Wars videogames in "To Disney Infinity and Beyond." Comparing classic Star Wars games with three post-acquisition releases, Brown finds that preacquisition games were bolder in experimenting articulations to the franchise. Finally, Rebecca Williams's "From Star Tours to Galaxy's Edge" examines the ways in which the history of Lucasfilm and Disney intertwined before the Disney buyout through the Star Tours immersive rides featured in Disney's theme parks since the mid-1980s. She argues the rides completed the transmedia world of Star Wars by extending fan experiences "from the textual into the bodily and the spatial" (136), making them more immersive and personal.

Part II of the collection on "Reception and Participation" starts with two chapters addressing the critical responses to Disney productions and the role played by nostalgia in both promotion and reception. Jonathan Gray's chapter responds to the critical appraisal of The Force Awakens as an unimaginative remake of A New Hope. The author addresses the question of franchises' relationship to originality, nostalgia, and repetition, arguing that The Force Awakens's mode of relation to A New Hope is one of reflexivity. Another way of redefining nostalgia was to appeal to fans disappointed by the Disney acquisition through subcultural branding as argued by Emma Pett in "Real Life is Rubbish", her examination of the promotion and reception of Secret Cinema's 2015 immersive production of The Empire Strikes Back (1980).

The next two chapters explore gendered politics in Disney Star Wars. Paul Booth's "Disney's Princess Leia" focuses on controversy regarding the character and Disney's handling of the conversation. One way to avoid criticisms of gender normativity was to shift toy manufacturer for the Princess toys from Mattel to Hasbro-a company mostly making toys for boys. Another was to promote Leia to the role of general in The Force Awakens. But Disney also emphasized the more princess-like attributes of Princess Leia in the Disney-Marvel's five-issue comics Princess Leia (2015) which bridged the gap in the character arc from princess to general-when the original trilogy had constructed a figure of female empowerment by denying those characteristics. Lorna Jowett pursues the exploration of Disney Star Wars gendered politics through the analysis of merchandising strategies for The Force Awakens in "Rey, Mary Sue, and Phasma Too." The film's merchandising was met with negative responses from female fans as product lines initially obliterated completely the character of Rey. These responses fueled the discussion on gender politics and inclusivity in popular culture.

The chapters by Jones and Kent discuss the creative appropriation of content by fans. Bethan Jones's "Jafar Wars" examines the role played by fan productions in framing and anticipating the narrative Fan reactions and their incorporation by the studio are further analyzed in the next four chapters. William Proctor's "Fear of a #BlackStormtrooper" argues that the fan controversy surrounding the appearance of John Boyega as a Stormtrooper in the original teaser for The Force Awakens was less about race than it was about canonical authenticity. Proctor shows that the meanings of the controversy were partly shaped by media responses to it and advocates a more complex reading of online fan activity than the stereotyped binary between reactionary and progressive fans. Tom Phillips analyzes fans' reaction videos to trailers for The Force Awakens and compares them to actors Daisy Ridley and John Boyega's reaction videos. He then argues that the film's promotion used fannish discourses as an act of reassurance for audiences and a perpetuation of the film's culture of anticipation. Bridget Kies discusses the economy of knowledge surrounding film releases and how fans actively manage and engineer their expectations and access to information prior to release in "Spoiler Culture, Marathon Screenings, and Affective Responses to The Force Awakens." Using her own fan experience, Kies argues that such fan activities as accessing or avoiding spoilers and behind-the-scene information, practicing screening marathons, and sharing energy through group events meaningfully shape the screening experience, while such experience retains its inherent power to surprise and affect fans in unexpected ways. Taking the opposite perspective, Lucy Bennett discusses her own experience as a non-Star Wars viewer to explore the processes and meanings that can occur when one remains unexposed to a cultural text. Being a non-viewer means being confronted with the cultural shame of not being familiar with a canonical work. It also means having mediated knowledge of, and expectations regarding, the unseen text charged through popular culture. Eventual viewing comes to confirm or contradict expectations in complex ways, leading to an experience the author terms "out-of-sync canonical viewing" (278).

The closing chapters in part II adopt a broader look on the franchise from the perspectives of narrative development and reception. Mark J. P. Wolf's "Beyond Vader: The Franchise Reawakens" examines the four post-Lucas Star Wars feature filmsfrom The Force Awakens to Solofrom a worldbuilding perspective as the franchise under Disney moves beyond the central character in the first six episodes, Darth Vader. Wolf discusses the production choices regarding the new trilogy's direction and the ensuing mixed contribution of The Force Awakens to the Star Wars storyworld. Finally, William Proctor's "A New Hate? The War for Disney's Star Wars" addresses the online debate among fans and critics about franchise fatigue and toxic fandom in the wake of the releases of The Last Jedi and Solo, and how the franchise has lately been ensnared in the new culture wars. Proctor argues that the media's preeminent focus on the most virulent elements of Star Wars fandom constructed a biased narrative that dramatized toxic narratives at the expense of a fan majority.

The first edited collection focusing on Disney's purchase of Lucasfilm, Disney's Star Wars is a rich, incisive, and valuable contribution to the study of transmedia franchises. The variety of perspectives, approaches, and methodologies featured in the collection illustrates the multilayered consequences of the purchase. One may regret the timing of the publication, with contributions that focus primarily on events prior to and surrounding the release of The Force Awakens (2015), but this leaves room for future publications that can offer an overarching perspective on the transmedia developments surrounding the Disney Star Wars trilogy.

  surrounding Disney's acquisition of the Star Wars franchise. Jones analyses fan fiction, art, and videos that mix Disney and Star Wars texts and fan responses to these paratexts to show how they can play a role in fan reception of the original texts. In "Fans Negotiating Disney's (De)Stabilized Star Wars Canon," Michelle Kent discusses how fans negotiated Disney's rebranding of the Expanded Universe and announcements of narrative departures in the up-coming trilogy. Analyzing responses from the two largest international Star Wars costuming fan clubs, Kent shows that Disney's changes impacted real-world fan practice. Many fans use Star Wars as a cultural object to enrich their lives through acts of civic engagement (such as supporting children's charities), and the decanonization of the Expanded Universe means disrupting such uses, thus making fandom less meaningful.