The Carmat Bioprosthetic Total Artificial Heart Is Associated With Early Hemostatic Recovery and no Acquired von Willebrand Syndrome in Calves

Author’s Accepted Manuscript Carmat Bioprosthetic Total Artificial Heart is Associated With An Early Hemostatic Recovery and No Acquired Willebrand Syndrome in Calves David M. Smadja, Sophie Susen, Antoine Rauch, Bernard Cholley, Christian Latrémouille, Daniel Duveau, Luca Zilberstein, Denis Méléard, MarieFazia Boughenou, Eric Van Belle, Pascale Gaussem, Antoine Capel, Piet Jansen, Alain Carpentier

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S1053-0770(17)30246-X http://dx.doi.org/10.1053/j.jvca.2017.02.184 YJCAN4034

To appear in: Journal of Cardiothoracic and Vascular Anesthesia Cite this article as: David M. Smadja, Sophie Susen, Antoine Rauch, Bernard Cholley, Christian Latrémouille, Daniel Duveau, Luca Zilberstein, Denis Méléard, Marie-Fazia Boughenou, Eric Van Belle, Pascale Gaussem, Antoine Capel, Piet Jansen and Alain Carpentier, Carmat Bioprosthetic Total Artificial Heart is Associated With An Early Hemostatic Recovery and No Acquired Willebrand Syndrome in Calves, Journal of Cardiothoracic and Vascular Anesthesia, http://dx.doi.org/10.1053/j.jvca.2017.02.184 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Carmat bioprosthetic total artificial heart is associated with an early hemostatic recovery and no acquired Willebrand syndrome in calves

David M. Smadja1,2,3, Sophie Susen4, Antoine Rauch4, Bernard Cholley2,5, Christian Latrémouille2,6, Daniel Duveau7, Luca Zilberstein8, Denis Méléard5, Marie-Fazia Boughenou5, Eric Van Belle9, Pascale Gaussem1,2,3, Antoine Capel10, Piet Jansen10, Alain Carpentier6

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AP-HP, European Georges Pompidou Hospital, Hematology Department, Paris, France 2 Université Paris Descartes, Sorbonne Paris Cité, Paris, France 3 Inserm UMR-S1140, Paris, France 4 Hematology Department, University Hospital, Inserm UMR-S 1176, Lille-IIUniversity, Lille, France 5 AP-HP, European Georges Pompidou Hospital, Anesthesia and intensive care Department, Paris, France 6 AP-HP, European Georges Pompidou Hospital, Cardiovascular surgery Department and Biosurgical Research Laboratory, Paris, France 7 Cardiovascular surgery Department, University Hospital Guillaume and René Laennec, Nantes, France 8 Alfort National Veterinary School, Maisons Alfort, France 9 Cardiology Department, University Hospital, Inserm UMR-S 1176, Lille-IIUniversity, Lille, France 10 Carmat SA, Vélizy-Villacoublay, France Address for correspondence: Prof. David Smadja European Hospital Georges Pompidou, Hematology Department 20 rue Leblanc, 75015 Paris, France, Tel: +3156093933, Fax: +3156093393 E-mail: [email protected] 1

Declaration of interests A Carpentier is cofounder and shareholder of Carmat SA. DMS, CL, BC and DD received consulting fees from Carmat. P Jansen and A Capel are employed by Carmat SA.

Acknowledgments

Funding for the study was provided by Carmat SA and the Alain Carpentier Foundation. David M Smadja research lab is funded by Conny-Maeva Charitable Foundation. We thank the nursing staff of the cardiovascular intensive care unit and surgery departments. We also thank the technicians of hematology department of Georges Pompidou hospital, in particular Nadège Ochat, Yann Burnel and Florence Desvard. We thank Christiane Veneziani for expert assistance with manuscript preparation.

Abstract: Objectives: To determine haemostasis perturbations, including von Willebrand factor multimers, after implantation of a new bioprosthetic and pulsatile total artificial heart (TAH) Design: Preclinical study Setting: Single-center Biosurgical Research Laboratory Participants: Female Charolais calves, 2-6 months of age and weighing 102-122 kg Interventions: Surgical implantation of TAH through a mid-sternotomy Measurements and Main Results: 4/12 calves had a support duration of several days (4, 4, 8 and 10 days) allowing us exploring early steps of haemostasis parameters, including Prothrombin Time (PT), coagulation factor levels (II, V, VII+X and fibrinogen) and platelet count. Multimeric analysis of von Willebrand factor (VWF) was performed to detect a potential loss of high molecular weight (HMW)-multimers, as previously described for continuous flow rotary blood pumps. Despite the absence of anticoagulant treatment given in the postoperative phase, no signs of coagulation activation were detected. Indeed, after an immediate post-surgery decrease of PT, platelet count and

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coagulation factor levels, most parameters returned to baseline values. HMW-multimers of VWF remained stable either after initiation or during days of support. Conclusions: Coagulation parameters and platelet count recovery in post-operative phase of Carmat TAH implantation in calves, in the absence of anticoagulant treatment, and associated with the absence of decrease in HMW-multimers of VWF is in line with an early haemocompatibility that is currently being validated in human clinical studies.

Key words: total artificial heart, transplantation, haemocompatibility, haemostasis, von Willebrand factor, cardiac surgery Introduction

The Carpentier-Matra (Carmat) Total Artificial Heart (C-TAH) has been developed as a heart replacement device for patients at risk of imminent death from biventricular failure, and who are not eligible for heart transplantation. It is designed for orthotopic placement, and therefore uniquely fits in the human thoracic cavity. The C-TAH has also been designed to minimize infections; thromboembolism and bleeding that are common complications of left ventricular assist devices (LVAD). One of the main bleeding complications of LVAD is acquired von Willebrand syndrome (AVWS), which is characterized by a loss of high molecular weight (HMW) multimers when the entire blood volume is exposed to high shear stress (1-3). High shear stress induces unfolding and cleavage of the VWF A2 domain, in vivo, and can occur within 200 seconds, in response to acute changes in shear conditions (4) or turbulence. This is considered to be induced as a result of oxidative stress (5, 6), an abnormal sensitivity to ADAMTS-13 proteolysis (7), and involves interactions of VWF with platelets (8). Blood-contacting surfaces of C-TAH consist of expanded polytetrafluorethylene (ePTFE) and bovine pericardial tissue processed in glutaraldehyde (9, 10). These materials have demonstrated a high

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level of biocompatibility in applications such as bioprosthetic cardiac valve replacement (10, 11). In vitro haemocompatibility tests of these surfaces showed a limited consumption of fibrin and minor platelet adhesion/activation on the surfaces (9). In a series of implanted calves we previously demonstrated that the C-TAH provided adequate blood flow, low levels of hemolysis and no visible evidence of thromboembolic depositions in major organs and device cavities, for up to 10 days (12). The current study was conducted on a subset of calf experiments (4 animals) where the prime objective was surgical training. The purpose was to monitor and characterize the early biocompatibility of the C-TAH. We studied the coagulation profiles and the consumption of coagulation factors, platelets and high molecular weight (HMW) multimers of von Willebrand Factor (VWF) after C-TAH implantation. Materiel and Methods

Carmat Total Artificial Heart (C-TAH) The C-TAH is a biventricular pulsatile, electrically powered, hydraulically actuated blood pump with all components embodied in a single device, mimicking the natural heart, and implanted in the pericardial sac (Figure 1). It’s most original feature is the use of bioprosthetic materials, similar to those used for bioprosthetic heart valves that are well known for having good haemocompatibility properties. This device comprises a left and right ventricle, each with a blood compartment and an hydraulic fluid compartment, separated by a hybrid membrane made of two layers glued together. One layer, in contact with the hydraulic fluid, is made of polyurethane, the other layer in contact with the blood, is made from bovine pericardial tissue, chemically treated with glutaraldehyde to achieve long-term tolerance and haemocompatibility. The blood compartments of each ventricle have their static surfaces lined with expanded polytetrafluoroethylene, a blood compatible material currently used in vascular surgery. Membrane pumping action is actuated by two electrohydraulic rotary pumps creating a systolic and diastolic phase by rapidly reversing the direction of hydraulic fluid 4

flow that in turn pushes and pulls the membranes. The pumps are regulated so as to mimic the viscoelastic contractility of the natural heart, thus providing physiological pressure and flow curves. This preserves valve durability and optimizes blood circulation within the ventricles. Electronics and microprocessors that drive the system are incorporated into the C-TAH. Sensors in the ventricles allow instantaneous monitoring of pressures providing continuous assessment of right and left ventricular preload and afterload. The positions of the membranes are monitored by ultrasound transducers. A control algorithm responds to changes in preload and afterload by adjusting heart rate (from 35 to 150 beats per min) and stroke volume (30–65 mL), while assuring complete ejection, to prevent stasis. A polyurethane sac containing hydraulic fluid that serves as a compliance chamber when stroke volume varies surrounds the entire prosthesis. Energy is provided by lithium ion batteries connected to the C-TAH by a single transcutaneous highly flexible driveline, 8 mm in diameter. A detailed description of the C-TAH components has been previously provided (13). The infection risk was assessed in the C-TAH development by three corrective actions: First, the driveline Dacron® coating was reduced to 10 cm close to the skin crossing and the implant procedure mentions that the Dacron® has to be only subcutaneously. Secondly, the implant procedure also recommends that the driveline subcutaneous route, in U-form, has to be the longest as possible from the pericardial bag to reduce the pericarditis risk. Thirdly, the traction forces on the wound are diminished; even remove, by the use of a belt. Animal model and surgical procedure A sub-set of four Charolais calves weighing 102-120 kg and 3-6 months of age, were used for the study. This animal model had been selected because it is the closest to the human thoracic anatomy (30). The study was approved by the local animal care ethics committee and took place at the Biosurgical Research Laboratory of the Georges Pompidou European Hospital in Paris, France (Protocol registration: CEEA34.AC.108.12). The detailed surgical procedure had been developed in previous work with calves (12) and in humans (13). Extubation was done as soon as the animal was

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completely awake and with satisfactory spontaneous breathing and deglutition. Awake animals were transferred to the animal care facility, typically on the first postoperative day, where careful monitoring was managed by a team of clinicians, veterinarians and technical staff. Blood volume replacement was by matched calf blood only. Transfusion was necessary only during surgical procedure. Monitoring comprised arterial and central venous pressures; central venous oxygen saturation; urine output and C-TAH blood flow. Animals judged to be compromised by respiratory dysfunction, severe blood loss, cerebral dysfunction or other complications, were euthanized. Plasma samples and coagulation assays Peripheral venous blood was collected in tubes containing 0.105 M sodium citrate (1:9 v/v). Poor platelet plasma was obtained by centrifugation at 2,300g for 15 min. PT, fibrinogen and coagulation factors have been quantified with classic methods of mechanical clot detection on a coagulometer STAR®evolution from Stago, with human reagents. Platelet count was realized by impedance with the LH750® from Beckman Coulter. Multimeric structure of plasma von Willebrand factor The multimeric structure of plasma von Willebrand factor was analyzed by electrophoresis and the percentage of multimers of the highest molecular weight (more than 15 mers) was determined after densitometric scanning, as previously described (2, 14). A pool of normal platelet-poor plasma was used as a reference in each gel electrophoresis (NPP; standard human plasma, Siemens healthcare diagnostics, Marburg, Germany; coefficient of variation=11%). The same amount of VWF antigen (VWF:Ag) was loaded for each sample. The result of a given sample is expressed as the ratio of the value of HMW multimers in the sample versus the value in the “normal pool plasma” (HMWmultimers-ratio).

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Statistical analysis Because of the small number of calves studied, we performed non-parametric analysis, which did not reveal any significant modification in HMW- multimers of Willebrand factor. Significant differences between time points were identified by the Kruskal-Wallis test. For other biological studies, we demonstrated individual analysis without any statistical analysis. Statistical analysis was performed using the Stat View software package (SAS, Cary, NC). Differences with P<0.05 were considered significant.

Results Hemostatic recovery after C-TAH implantation Twelve female Charolais calves were implanted with the C-TAH between January and June 2013 as part of a surgical training program. Subsets of 4 animals were maintained for 4 to10 days and were included in this biocompatibility study (Table 1). The two animals kept alive for 8 and 10 days, had recovered their enteral function. In the two 4 day survivors (animals 2 and 10), euthanasia was prompted by respiratory failure (animal 2) and rear limb damage, secondary to a fall and subsequent rhabdomyolysis (animal10). A further calf (animal 12) survived 8 days, and died of respiratory failure. Device explant analysis and histological examination of kidneys and brain failed to reveal any thromboembolic complications in any animal and the devices were all clean (table 1). Figure 2 shows the chronology of prothrombin time (PT) and coagulation factor II, V and VII+X levels; Figure 3 shows the chronology of platelet count and fibrinogen. At basal level, mean (±SD) of PT was at 54.5±7 %, platelet count at 303±69 G/l, and fibrinogen level at 2.6±0.1 g/l. Compared to humans, calves showed high baseline levels of factor V (596 ±79%), while factor II was at 34±5%

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and factors VII+X at 66±4% (reference values for humans ranging 70-140%). A decrease in PT, platelet count and fibrinogen level was observed, as expected, during the immediate post-operative period A subsequent rise in PT, fibrinogen and platelet count was observed starting at Day 1 in all animals, except for animal 10 (Figure 4B). This calf was the heaviest of the 4 animals enrolled (122 Kg). No modification of HMW-multimers in calves undergoing C-TAH implantation No time dependent loss of HMW-multimers was observed after initiating the device (Figure 5A and 5B) and no significant quantitative decrease was evidenced (Figure 5C) at 5, 30 or 60 min after CTAH start up (p=0.25). Neither was there any decrease in HMW-multimers at day 1, 4 or 10, (data not shown). The C-TAH was operated at a mean flow of 9.3 L/min in this group of 4 animals, which is in the top flow range of the device. These results could therefore be regarded as testing in “worst case” conditions with regard to blood handling.

Discussion.

Mechanical circulatory support (MCS) is a therapeutic option for patients with end-stage heart failure. However, thromboembolism and bleeding events are still the most common complications after implantation of a LVAD or the Syncardia temporary TAH (15-20). These complications are due to continuous activation of coagulation factors and platelets on artificial surfaces despite therapeutic anticoagulation. A bioprosthetic artificial heart has been developed, based on the bioprosthetic valve experience (10). Indeed, bioprosthetic heart valves are commonly used for replacement of diseased heart valves. These valves are fabricated from bovine pericardium and preserved and rendered nonimmunogenic using a chemical pretreatment with glutaraldehyde (10). These valves provide a prosthesis which is non-thrombogenic. This acquired haemocompatibility is probably attributed to depositions of fibrin strands and blood cells (10, 21). Thus, the C-TAH has been developed with

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blood-contacting surfaces comprising expanded polytetrafluorethylene (ePTFE) and processed bovine pericardial tissue (10, 21). These materials have demonstrated a high level of haemocompatibility in applications such as cardiac valve replacement and vascular grafts (11, 22). Moreover, we previously described that blood-contacting surfaces of the C-TAH ventricles demonstrated good haemocompatibility after exposure to circulating human blood in vitro (9). Indeed, activation of coagulation and platelets was lower for ePTFE and pericardial tissue compared to a silicone surface, and at the same level as heparin-coated medical grade PVC, which is one of the most haemocompatible materials routinely used in extracorporeal circuits (23). The results of this present study demonstrate that a transient activation of coagulation is observed (decreased PT, coagulation factors and platelet count), immediately after C-TAH implantation but this is followed by a return to preoperative levels after 3 days. This normalization is in line with previous data demonstrating that coagulation activation typically happens during the first hours of blood–tissue contact (24). This early normalization of coagulation parameters and the absence of hemolysis, previously described (12), provides a considerable advantage compared to other TAH systems that only recover after several months (25). Moreover, the calves did not receive any anticoagulant treatment after implantation. The regime employed with the Syncardia TAH, comprises aspirin at post-op day 2 (26), and heparin as soon as possible, because of the high level of thrombotic complications (19, 20). In this study there was no evidence of abnormal bleeding (gastric bleeding is common in other assist devices) and there were no thrombotic complications or pathological evidence of thrombus, despite the absence of anticoagulation medications once weaned from bypass. This finding has allowed us to consider a minimal anticoagulation regime with the ongoing human clinical trial with the C-TAH. While uncontrolled bleeding events are a common complication of MCS, not all bleeding can be explained by the need for anticoagulant therapy. Several studies have shown that bleeding events can be partly explained by impaired platelet function in patients on LVAD support (27, 28). However, these data remain controversial (29) and platelet dysfunction may

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be partly related to the overall impact of end-stage heart failure in this patient population. Moreover, cardiopulmonary bypass (CPB) is also frequently responsible for transient thrombocytopenia, induced by hemodilution and/or platelet activation resulting from the contact of blood with the extracorporeal circuit (30-32). However, most CPB studies show a return to normal platelet count values at day 3 or 4 (33). Thus we believe that our normalization of platelet count in animals presented here, after 4 to10 days, is a good marker for postoperative hemostatic recovery. VAD associated bleeding complications can also be partly due to AVWS, which is characterized by loss of HMW-multimers of VWF. Loss of multimers can occur as VWF is subjected to increased shear stress, which has been shown to occur with continuous-flow VADs (1, 3, 29, 34). HMW multimers loss is associated with all types of continuous flow devices and occurs immediately after activation (18). However it has not been observed in the Syncardia TAH (34), which is a pulsatile flow system, and this study also showed no such distortion of HMV-multimers. All devices inducing AVWS have in common an increased shear stress and turbulence that induce potential oxidative stress (5, 6), an abnormal sensitivity to ADAMTS-13 proteolysis (7) and involve interactions of VWF with platelets (8). Absence of AVWS observed in the C-TAH in calves is in line with the rheology achieved with this device in reproducing cardiac pulsatility without inducing pathological shear stress or turbulence. In the C-TAH, optimized pump flow dynamics were developed using a computer model with the object of reproducing physiological blood flow and pressure dynamics and the reduction of stasis by targeting full ejection at each beat. The resulting electrohydraulic actuation of the C-TAH produces a visco-elastic deployment of the hybrid membranes, thereby minimizing shear stress on blood components and valves. This combination of features in the C-TAH would be expected to yield the result we found, of no AVWS. Since hemorrhage is a classic complication of cardiac surgery and cardiac assistance (not only because of anticoagulation), we believe that the avoidance of AVWS should form a significant feature of future patient management regimes and device deigns. 10

One of the biological limitations is the absence of confirmation of absence of AVWS with other biological exploration used in human and not realizable here because we have calves. Quantification of HMW multimers should include bedside whole blood assessment (with PFA-100 analyzer) and VWF propeptide quantification in plasma. It is well known that increased blood flow can induce an acute release of VWF by the vascular endothelium (35, 36), and this release of VWF can be detected by VW propeptide quantification (37). This propeptide quantification has never been demonstrated in calves and should help to confirm our results. Moreover, several animal models reproducing acquired Willebrand disease exist in rabbit (2) or pig (38). We cannot speculate that surgical and/or shear stress between species in human and calve is different. Another limitation of our study is about the animal model used. Indeed, since the C-TAH has been designed for human use, applications to the calf model have several limitations, including the difficulty of supporting chronic studies. So, we do not have any data about potential chronic coagulopathy that often appear several days postoperatively. The high mortality rate can be is explained by several issues related to the model. The maximum output of the C-TAH is 9L/min while these calves have a normal resting cardiac output of some 12 – 15 L/min. Since the surgical approach for the C-TAH requires a sternotomy incision (that have an increased delayed recovery and a risk of sternal infection, sepsis and pain), the animal is placed in an unnatural dorsal position which compromises several key organs, by compression, for an extended time. Left thoracotomy used in other types of assist devices is not possible because of geometric configuration of the C-TAH. The heaviest calf (animal 10) was also the oldest (6 months) and required the C-TAH to be continuously operated at maximum flow of 11 L/min. This resulted in an increased device temperature of up to 41+/- 1.8 °C. This could provide an explanation for the absence of platelet recovery in this calf (Figure 3B) despite the body temperature stabilization at 37.5 +/-0.7°C (39). Prolonged duration of sub-optimal flow did not seem to have a negative impact on organ perfusion in this series (12), but should we have been able to progress to a more chronic study, the rapid growth of the animals would certainly have limited their exercise

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capacity and subsequently induced heart failure. Finally, our sample size can appear small. French authorities required 3 animals with a sub-acute implantation to start the feasibility study in human. Thus, we realized these 4 animals to be able to start human clinical trials. However, we believe our results are reliable because they are in line with what we observed in early human implantation. We recently published two first cases (13) and found a very good hemocompatibility. Indeed, for example, the first patient implanted had a 50-day anticoagulant-free period because postoperative bleeding disorders. Autopsy did not detect any relevant thrombus formation within the bioprosthesis nor the different organs in accordance with hemocompatibility described in this report. In conclusion, this small biological study after implantation of bioprosthetic total artificial heart in calves has demonstrated encouraging results, with respect to early normalization of coagulation factors and formed elements, despite the lack of anticoagulation. There was no AVWS, which is a common finding in other assist devices, and thought to be partly responsible for the observed coagulopathies and the need for significant anticoagulation therapies. Validation of these findings will follow in the upcoming human clinical studies of the C-TAH.

Figure 1: The C-TAH and its blood contacting surfaces A: Schematic view of the TAH connected to the atria and great vessels. Two rotary pumps shuttle the hydraulic fluid) from right to left (yellow arrows), creating filling and ejection of the blood chambers. B: the hybrid membrane with polyurethane on the actuating fluid side and bovine pericardium on the blood-contacting side. C: the hybrid membrane is sealed onto the blood cavity, which is covered with ePTFE

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Figure 2: Prothrombin time (PT), factor II, V, and VII+X activity measurement after C- TAH implantation

Figure 3: Platelet count and Fibrinogen level after C-TAH implantation

Figure 4: Absence of loss of HMW-multimers after C-TAH implantation in calves A- Separation of the multimers contained in plasma is done according to their size by electrophoresis and blotting. A second step of colorimetric immunostaining of the electrophoresis using alkaline phosphatase labelled polyclonal antibodies allows immunoenzymatic visualization of VWF multimers. A representative time course of HMW multimers after initiating the C-TAH. Black arrows indicate the front migration. NP indicates normal human pooled plasma. B- Densitometric analysis of a representative time course of HMW-multimers after C-TAH start-up. C- Quantitative analysis of HMW multimers (relative to baseline) in 3 consecutives calves after densitometric integration of the gel. The HMW-multimers ratio at baseline is 1 (by definition). No significant loss of HMW multimers occurred over time. T5, T30 and T60 correspond respectively to 5, 30 and 60 minutes after beginning of C-TAH.

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Table 1: Calves' Clinical data

Case Age Animal Thoracic Support number (Months) weight (kg) perimeter (cm) duration (d)

Avg Pump output (L/min)

Extubated

Postop anticoag.

Cause of death/euthanasia

2

4

107

106

4

8,5+/- 0,2

Yes

No

Respiratory failure

7

3

115

112

10

8,9+/- 0,1

Yes

No

Anemia

10

6

120

115

4

11+/- 2

Yes

No

Lower limb paralysis, rhabdomyolysis

12

3

102

105

8

8,8+/- 0,3

Yes

No

Sepsis, respiratory failure

18

19

20