Thromboresistance and Functional Healing in the COBRA Pzf Stent Versus Competitor DES: Implications for Dual Antiplatelet Therapy October 2018 October

CORONARY INTERVENTIONS

Euro PRECLINICAL RESEARCH Intervention

2019;15: e 342- e 353 published online353 Thromboresistance and functional healing in the COBRA PzF stent versus competitor DES: implications for dual antiplatelet therapy October 2018 October

Hiroyuki Jinnouchi1, MD; Hiroyoshi Mori1, MD; Qi Cheng1, MD; Matthew Kutyna1, MS; Sho Torii1, MD; Atsushi Sakamoto1, MD; Liang Guo1, PhD; Eduardo Acampado1, DVM; 2 1 1 1,2

published online Anuj Gupta , MD; Frank D. Kolodgie , PhD; Renu Virmani , MD; Aloke V. Finn *, MD

1. CVPath Institute, Gaithersburg, MD, USA; 2. University of Maryland, Baltimore, MD, USA

H. Jinnouchi and H. Mori contributed equally to this manuscript e

-edition July-edition 2019 This paper also includes supplementary data published online at: https://eurointervention.pcronline.com/doi/10.4244/EIJ-D-18-00740

KEYWORDS Abstract Aims: The aim of this study was to investigate the COBRA PzF stent (C-PzF) with respect to throm- • bare metal stent bogenicity and healing versus conventional drug-eluting stents (DES) in dedicated preclinical models to • coronary artery evaluate their suitability for short-term dual antiplatelet therapy (DAPT). disease Methods and results: • drug-eluting stent To examine acute thrombogenicity, the C-PzF durable polymer drug-eluting stent • preclinical (DP-DES), and a bioabsorable polymer DES (BP-DES) were compared in a porcine arteriovenous shunt research model and in a rabbit model to evaluate endothelial coverage at 14 days. Barrier function at 28 days in the rabbit was assessed in the former stents as well as in a polymer-free DES (PF-BES). The number of clots by scanning electron microscopy (SEM) was significantly less in the C-PzF and DP-EES versus the BP-EES. Endothelial coverage at 14 days was significantly greater in the C-PzF versus the DP-EES and BP-EES by CD31/PECAM-1 positive area in confocal microscopy (CM) (24.7% vs 11.9% vs 3.7%, respectively) and SEM (99.0% vs 29.6% vs 17.7%, respectively). Barrier protein expression by CM for p120/vascular- endothelial cadherin complex was significantly greater in the C-PzF versus the DP-EES, BP-EES, and D OI: OI: PF-BES (82.6% vs 12.8% vs 14.4% vs 18.1%, respectively). The percentage of Evan’s Blue positive area 10.4244/EIJ-D-18-00740 was least in the C-PzF versus all groups (22.0% vs 70.7% vs 66.4% vs 55.2%, respectively).

Conclusions: The C-PzF demonstrated a unique combination of thrombogenicity and healing advantages compared to contemporary DES and may be uniquely suitable for very short-term DAPT.

*Corresponding author: CVPath Institute Inc., 19 Firstfield Road, Gaithersburg, MD 20878, USA. E-mail: [email protected]

e e 343 e . - - - - - 6 . These 6 8 (Abbott Vascular, Vascular, (Abbott ® Rabbits 28 days C-PzF (n=4) PF-BES (n=4) BP-EES (n=4) DP-EES (n=4) . A recent preclinical study also A . 7 In vivo stent implantation model Preclinical assessment of COBRA of assessment Preclinical Editorial, see page 304 shunt model as compared to BMS ex vivo shunt model as compared 9 NA Rabbits 14 days C-PzF (n=6) BP-EES (n=6) DP-EES (n=6) Although recent trends favour the use of short-term DAPT (i.e., DAPT of short-term trends favour the use Although recent shows details of the methods; Sup- of the Supplementary Appendix 1 shows details supported the performance of the C-PzF showing favourable antirest supported the performance enotic and healing properties in the porcine model and resistance to in the porcine model and resistance enotic and healing properties thrombogenicity in an characteristics may be beneficial for use in high bleeding risk patients use in high bleeding may be beneficial for characteristics In a recent clinical be severely curtailed. duration must where DAPT goal for target a pre-specified performance the C-PzF achieved study, late myocardial infarction and no stentvessel failure with infrequent with BMS thrombosis as compared Methods of these used for each and devices models animal 1 lists the Table The test arm for all studies was the C-PzF. experiments. different Xpedition XIENCE available Commercially Results ACUTE THROMBOGENICITY IN A PORCINE ARTERIOVENOUS SHUNT MODEL There was no evidence of blood coagulation or platelet function abnor the measures of Additionally, malities in any of the animals studied. 2). coagulation were similar in all shunt runs (Supplementary Table including stents. The C-PzF equipped with this novel technology with this equipped The C-PzF stents. including on its surface, pre for binding serum albumin affinity exhibits a high and platelet adhesion cell, fibrinogen venting inflammatory relative the studies examining months), comparative one to three C-PzF versus current-genera the of healing and thrombogenicity tion DES (including durable, bioabsorbable, and polymer-free ver and polymer-free bioabsorbable, durable, tion DES (including Therefore, models have never been performed. sions) in preclinical characteristics specific these of this study was to evaluate the aim regarding decisions inform help to types stent different these in these devices. receiving in patients duration DAPT Santa Clara, CA, USA) durable polymer everolimus-eluting stents everolimus-eluting polymer CA, USA) durable Clara, Santa Marlborough, (DP-EES) and SYNERGY™ (Boston Scientific, stents everolimus-eluting polymer MA, USA) bioabsorbable the BioFreedom™ Additionally, (BP-EES) were compared. biolimus- (Biosensors, CA, Newport Beach, USA) polymer-free study. function stent (PF-BES) was added in a barrier eluting descriptions. 1 provides device plementary Table In vivo stent implantation model - - - 4 NA 1 hour Porcine . C-PzF (n=8) 3,4 BP-EES (n=8) DP-EES (n=8) AV shunt model Ex vivo AV Thrombogenicity study Endothelial coverage study Barrier function study . Fluorinated polymers have stent microscopy therapy 4 3 2 1 coronary intervention stent stent . It is estimated that 15% or more of patients 15% or more that . It is estimated ganic, high molecular weight polymer possess . The COBRA PzF™ coronary system (C-PzF; PzF™ The COBRA . 5 1,2 microscopy PzF -free biolimus-eluting stent -free biolimus-eluting Type of study Type drug-eluting everolimus-eluting percutaneous polymer-free polymer scanning electron vascular-endothelial bare metal COBRA cobalt-chromium confocal dual antiplatelet

Stent designs to allow shortening the duration of DAPT have of DAPT shortening the duration allow to designs Stent

Type of stent Type Animal Period Number of animals AV: arteriovenous; BP-EES: biodegradable polymer everolimus-eluting stent; C-PzF: COBRA PzF; DP-EES: durable polymer everolimus-eluting stent; arteriovenous; BP-EES: biodegradable polymer AV: biolimus-eluting stent NA: not applicable; PF-BES: polymer-free Model Table 1. Summary of animal model and stents tested. Table CeloNova Biosciences, San Antonio, TX, USA) is composed of a thincomposed USA) is TX, Antonio, CeloNova Biosciences, San (71 μm strut thickness) strut stent fluorinated Polyzene-F (PzF) polymer and coated with a unique for made of cobalt-chromium and mulation of poly(bis[trifluoroethoxy]phosphazene) without any added drugs. PzF is an inor rent-generation stents have thin-strut metallic backbones, polymericrent-generation stents have thin-strut metallic coatings distinguish one from another become a major area of focus. Ideal adaptations would confer bothbecome a major area of focus. Ideal adaptations While all cur antithrombotic and accelerated healing characteristics. shown particular promise because they have antithrombotic, anti- shown particular promise because they have and antirestenotic properties that may be conducive inflammatory, for curtailing DAPT SEM Introduction percuta- the field of Drug-eluting stents (DES) have revolutionised rates of Although DES reduce (PCI). neous coronary intervention stents (BMS), they also pro- metal with bare restenosis compared treatment repair process which requires extended long the arterial thieno- a and aspirin with (DAPT) therapy antiplatelet dual with use is associated with DAPT Overall, pyridine (e.g., clopidogrel). the studies have documented risk. Multiple bleeding increased outcomes, including association of bleeding after PCI and adverse mortality increased DAPT PF-BES VE ing a backbone of alternating nitrogen and phosphorus atoms and tri- fluoroethanol side groups that can be used to coat multiple substrates CM DES EES PCI PF Abbreviations BMS C-PzF CoCr undergoing PCI are at high risk for bleeding PCI undergoing BP-EES BP-EES DP-EES vs DP-EES vs BP-EES BP-EES C-PzF vs C-PzF vs Supplementary Table 3 Table Supplementary 0.0060.001 0.006 <0.001 0.438 0.041 0.415 <0.001 <0.001 0.003 0.1660.459 0.012 0.008 0.007 0.153 0.017 0.006 DP-EES DP-EES <0.001 0.003 0.098 <0.001 <0.001 0.01 <0.001 <0.001 0.001 C-PzF vs C-PzF vs

3.74 4.58 17.72 74.22 16.39 310.00 211.75 365.66 BP-EES (95% CI) (1.07-8.10) (2.69-12.57) (7.92-21.25) BP-EES Mean (70.00-80.78) (10.34-25.99) (255.00-371.00) (176.74-246.76) (187.45-713.28) shows representative SEM and CM images. C-PzF SEM and Figure 4 shows representative

summarise the results of the endothelial barrier function study barrier function study summarise the results of the endothelial - 3 sum Table cell coverage. for endothelial marker a non-specific and SEM CM by endothelial coverage results of the marises and - of quantifica in terms coverage strut greater showed significantly BP-EES struts by SEM versus coverage above tion of endothelial above struts of CD31 expression By CM, the percent and DP-EES. staining was significantly greateras evaluated by CD31/PECAM-1 regard to evaluation With and DP-EES. for C-PzF versus BP-EES positive area was significantly less in of macrophages, the RAM-11 3. to DP-EES and BP-EES, as shown in Table C-PzF when compared CM images with immunofluorescentFigure 5 shows representative in each of the groups. (RAM-11) staining against macrophages AND EXPRESSION IN IN VIVO RABBITBARRIER FUNCTION 28 DAYS MODEL AT Angiography in-life phase. All animals survived the study’s of the any in thrombosis or dissection of evidence no revealed 28 days by at was evaluated function barrier Endothelial stents. permeability endothelial (to evaluate dye staining Evans Blue (VE)- within the stents) and by CM for p120/vascular-endothelial borders in areas at cell which should co-localise complex cadherin stent during died One animal barriers. endothelial functioning with data for one were no Evans Blue uptake Therefore, there explant. 4 and PF-BES. Table one and BP-EES - 7.94 11.89 10.97 29.57 87.50 38.75 58.76 199.50 DP-EES (95% CI) (1.56-17.82) (1.47-20.85) (5.00-12.58) (3.74-73.76) DP-EES Mean (18.37-33.79) (83.64-89.27) (30.12-114.62) (143.50-313.75) 61.5 C-PzF 24.70 27.35 99.03 22.60 49.75 69.66 100.00 (95% CI) C-PzF Mean (19.00-150.5) (17.42-57.33) (16.11-49.57) (90.00-99.79) (14.26-35.83) (14.74-84.76) (95.73-100.00) (35.71-135.89) 1.67 (0.94-2.97) (0.36-2.13) 0.875 5.18 (3.05-8.79) show representative SEM and CM SEM and Figure 2 show representative Figure 1 and shows representative images from each group. There images from each group. Figure 3 shows representative summarises the results of platelet adhesion by CM and by platelet adhesion results of the 2 summarises Table staining against 2 lists the results of immunofluorescent Table BP-EES: biodegradable polymer everolimus-eluting stent; C-PzF: COBRA PzF; DP-EES: durable polymer everolimus-eluting stent; SEM: scanningBP-EES: biodegradable polymer everolimus-eluting stent; C-PzF: COBRA PzF; DP-EES: durable polymer electron microscopy CD31/PECAM-1 positive area above struts (%) CD31/PECAM-1 area between struts (%) RAM-11 positive cell density (number/mm²) Percent of coverage above strut by SEM (%) Percent of coverage between struts by SEM (%) BP-EES: biodegradable polymer everolimus-eluting stent; C-PzF: COBRA PzF; DP-EES: durable polymer everolimus-eluting stent; SEM: scanningBP-EES: biodegradable polymer everolimus-eluting stent; C-PzF: COBRA PzF; DP-EES: durable polymer electron microscopy Percent CD42b/CD61 positive area of total stent strut (%) Number of clots by SEM CD14 positive cell density (number/mm²) PM-1 positive cell density (number/mm²) Table 3. Endothelial coverage study in rabbit model at 14 days. Table Table 2. Thrombogenicity study in ex vivo arteriovenous shunt models. Table was significantly less area of PM-1 positive staining in C-PzF andwas significantly less whereas there was no significant differ DP-EES versus BP-EES, SEM. mark- staining against dual platelet immunofluorescent images with When total DP-EES, and BP-EES. in the C-PzF, ers (CD61/42b) area, the to stent surface area was normalised platelet fluorescence DP-EES,platelet adhesion versus significantly greater C-PzF had between the C-PzF and significant difference whereas there was no by SEM was signi- the number of clots counted BP-EES. However, - than BP-EES, whereas there was no signi ficantly less in the C-PzF and DP-EES. between the C-PzF ficant difference (PM-1) and a monocyte marker (CD14) bya neutrophil marker CM; ENDOTHELIAL COVERAGE IN AN IN VIVO RABBIT MODEL AT RABBIT MODEL AT ENDOTHELIAL COVERAGE IN AN IN VIVO 14 DAYS Angiography in-life phase. All animals survived the study’s of thein any or thrombosis dissection of no evidence revealed by CM using an anti- stents. Endothelial coverage was evaluated CD31 (PECAM-1),marker surface the endothelial against body ence between C-PzF and DP-EES. Similarly, CD14 immunostain- ence between C-PzF and DP-EES. Similarly, C-PzF and DP-EES asing area showed significantly less staining in comparable to DP-EES. compared to BP-EES, whereas C-PzF was

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e e 345 e PF-BES C-PzF vs BP-EES C-PzF vs 0.03 0.033 0.098 Preclinical assessment of COBRA of assessment Preclinical DP-EES <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.039 <0.001 <0.001 0.026 C-PzF vs S P-EE 73.85 65.29 55.19 18.92 18.12 (9.35-53.07) (62.21-87.50) (53.17-80.57) (50.38-61.73) (10.48-52.53) BP-EES

12 =0.0 58.22 49.91 66.43 15.49 14.40 p SB 66 (7.52-30.84) (7.77-29.62) (55.63-64.95) (46.65-56.52) (62.32-71.43) .1 =0 P-EE DP-EES p =0.003 p 66.06 57.12 70.68 15.70 12.81 (58.08-83.27) (49.18-77.31) (58.17-76.54) (12.86-20.72) (10.20-18.74) C-PzF

FD 0

50 40 30 20 10 per stent surface area (%) area surface stent per C-PzF DP-EES BP-EES PF-BES 99.72 99.58 22.02 78.30 82.61 Fluorescent positive area positive Fluorescent C-Pz (9.86-57.09) (99.65-99.93) (99.48-99.90) (32.40-95.80) (36.09-95.55)

B A by SEM (%) Percent of coverage between struts Percent of coverage above strut by SEM (%) Percentage of Evan’s Blue positive Percentage of Evan’s area Percentage of co-localised VE-cadherin and p120 between struts (%) BP-EES: biodegradable polymer everolimus-eluting stent; C-PzF: COBRA PzF; DP-EES: durable polymer everolimus-eluting stent; PF-BES: polymer freeBP-EES: biodegradable polymer everolimus-eluting stent; C-PzF: COBRA PzF; DP-EES: durable polymer biolimus-eluting stent; SEM: scanning electron microscopy; VE: vascular-endothelial Percentage of co-localised VE-cadherin and p120 above struts (%) Table 4. Barrier function study in rabbit model at 28 days. Table Representative images of CM using antibodies against CD62/42b in the swine arteriovenous shunt models. A) The left-hand images shunt models. images of CM using antibodies against CD62/42b in the swine arteriovenous Figure 1. Representative middle boxes at upper, to white images that correspond high-power the right-hand images are low-power images, whereas in each stent are there C-PzF showed thin platelet adhesion labelled by CD62/42b immunostaining. However, and lower positions in the low-power images. with C-PzF BP-EES exhibited clots of platelet as compared whereas of clumping consistent with platelet aggregation, areas no larger were per stent surface. positive area DP-EES. B) Graph showing fluorescent was much less platelet adhesion in there Additionally, and DP-EES. based on nested generalised linear mixed models to adjust are and p-values deviation for each group as mean±standard presented Data are PzF; DP-EES: durable stent; C-PzF: COBRA for variations between the shunt runs. BP-EES: biodegradable polymer everolimus-eluting stent polymer everolimus-eluting ) - 2 S P-EE BP-EES =0.006 p 17 SB =0.0 was significantly smaller in C-PzF and DP-EES versus BP-EES. smaller was significantly shunt same the in inflammation acute to regard with Similarly, cells fewer inflammatory significantly C-PzF demonstrated model, versus BP-EES and on the stent and monocytes) neutrophils (i.e., at 14 days, coverage to DP-EES. Endothelial degree a similar comparative studies examining the relative thrombogenicity and thrombogenicity the relative studies examining comparative for short-term suitability their and stents of different healing examined Here we specifically have not been conducted. DAPT PzF DES preclin- versus in dedicated current-generation COBRA and heal thrombogenicity models to compare their relative ical ing characteristics. Overall, DP-EES with fluoropolymer coating ing characteristics. staining with regard the least amount of platelet demonstrated similar platelet while C-PzF demonstrated to thromboresistance, the number of clots (>0.1 cm adhesion to BP-EES. However, DP-EES p P-EE 53 - .1 =0 p C-PzF

8 6 4 2 0

10 Number of clots per stent (n) stent per clots of Number FD C-Pz shows the higher power images Figure 7 shows the B A Discussion thought is months) three to one (i.e., DAPT short-term Although high risk for bleeding, at for patients solution best be the to Representative SEM images in a swine arteriovenous shunt model. A) On the left are representative low-power (15x magnification) representative A) On the left are SEM images in a swine arteriovenous shunt model. Figure 2. Representative to white high-power images (H-1: 50x; H-2: 200x; H-3: 600x; H-4: 4,000x) that correspond on the right are images of each stent, whereas clots in C-PzF on the strut surfaces with no larger showed minimal platelet aggregation boxes in the low-power images. C-PzF and DP-EES per stent by SEM was significantly less in C-PzF and BP-EES showed some clot formation. B) The number of clots and DP-EES, whereas based on nested and p-values are deviation for each group as mean±standard presented to BP-EES. Data are DP-EES as compared stent; biodegradable polymer everolimus-eluting generalised linear mixed models to adjust for variations between the shunt runs. BP-EES: microscopy stent; SEM: scanning electron PzF; DP-EES: durable polymer everolimus-eluting C-PzF: COBRA of CM with corresponding high-power SEM images. ing less endothelial permeability in C-PzF. Percent of co-locali- in C-PzF. permeability ing less endothelial by CM was and p120 as evaluated VE-cadherin between sation stents. in C-PzF compared to the other three greater significantly of percent greater C-PzF showed a significantly Additionally, groups. other above struts by SEM versus all coverage endothelial (Evans Blue, SEM and CM) images Figure 6 shows representative type. In addition, by stent by Evans Blue, CM and SEM. The area of Evans Blue uptake The area by Evans Blue, CM and SEM. to DP-EES and BP-EES, indicat was less in C-PzF as compared

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e e 347 e - . It has been shown that 8 S 01 Preclinical assessment of COBRA of assessment Preclinical P-EE <0.0 p 01 <0.0 p . PzF is also recognised as a fluorinated . PzF is also recognised 5 15 =0.4 . In the current study, although the percent area the percent although . In the current study, p 6 . In a previous porcine arteriovenous shunt study, shunt study, arteriovenous . In a previous porcine 9 C-PzF DP-EES BP-EES SB 0

250 200 150 100 CD14 (n) CD14 P-EE polymer, which is expected to show similar resistance to plate to resistance show similar to is expected which polymer, aggregation let of platelet staining was significantly smaller for DP-EES versus smaller was significantly staining of platelet both C-PzF and BP-EES, SEM showed a lower number of clots inflammatory in C-PzF and DP-EES versus BP-EES. Likewise, DP-EES coated with fluoropolymer (PVDF-HFP) with fluoropolymer are resistant DP-EES coated aggregation to platelet C-PzF had significantly less platelet aggregation versus COBRA- aggregation less platelet C-PzF had significantly BMS and V-BMS suggested that these polymers may have thromboresistant effects effects may have thromboresistant these polymers suggested that through preferen- adhesion and activation by reducing platelet of albumin adsorption and retention tial D - =0.007 p FD =0.008 p C-Pz =0.459 p C-PzF DP-EES BP-EES 0

800 700 600 500 400 300 200 100 PM-1 (n) PM-1 PM-1 PM-1 DAPI CD14 CD14 DAPI C B A ACUTE THROMBOGENICITY AND ACUTE INFLAMMATION e.g., polymers, studies using fluorinated Previous preclinical (PVDF-HFP), fluoride-co-hexafluoropropylene) poly(vinylidene botic and superior healing characteristics for C-PzF which may for C-PzF which characteristics healing and superior botic is necessary. DAPT for subjects in whom short-term be beneficial as assessed by CD31 expression and SEM analyses, was signi- greater in C-PzF as compared to DP-EES and BP-EES. ficantly barrier function, PzF exhibited with regard to endothelial Lastly, protein barrier function and endothelial barrier superior endothelial to DP-EES, and p120) as compared VE-cadherin expression (i.e., BP-EES and PF-BES. Overall, these data suggest both antithrom Representative CM images for PM-1 as a marker of neutrophils and CD14 as a marker of monocytes in a swine arteriovenous shunt and CD14 as a marker of monocytes in a swine arteriovenous CM images for PM-1 as a marker of neutrophils Figure 3. Representative PM-1 and CD14 were CD14, and demonstrate that the lower images (B) are whereas model. The upper images (A) show PM-1 in each group, (C) and number of CD14 positive cells on theless in C-PzF and DP-EES than in BP-EES. Number of PM-1 positive cells on the stent struts based on nested generalised linear mixed and p-values are deviation for each group as mean±standard presented stent struts (D). Data are stent; CM: confocal microscopy; everolimus-eluting models to adjust for variations between the shunt runs. BP-EES: biodegradable polymer stent PzF; DP-EES: durable polymer everolimus-eluting C-PzF: COBRA - . Minimal depo- . Minimal 11 . In order to prevent 12 8 .9 CD31 DAPI =0 p ES =0.003 p BP-E 01 <0.0 by confocal microscopy p Coverage above the struts C-PzF DP-EES BP-EES 0 80 60 40 20 % 100 C sition of platelet surrounding struts without apparent aggregation surrounding struts without apparent sition of platelet thin Therefore, healing. sign for early endothelial is an acceptable in C-PzF is likely adhesion without apparent aggregation platelet but that of heal to the process of thrombus formation not to lead our results suggest that, Overall, study. ing, as seen in the current do they coating, PzF surface the to adhere may platelets although of thromboresistance. suggesting some element not aggregate, ENDOTHELIAL COVERAGE is one of the after stent implantation coverage Delayed endothelial causes of stent thrombosis most important implantation of stents is platelet/fibrin deposition of stents is platelet/fibrin implantation CD31 DAPI - - ES DP-E 10 =0.0 p 01 <0.0 . Despite the increase in plate in increase the . Despite p 10 01 <0.0 CD31 DAPI p Coverage above the struts by scanning electron microscopy C-PzF DP-EES BP-EES F C-Pz 0 80 60 40 20 % 100 B A Representative SEM and CM images (CD31) in rabbit models at 14 days. A) Low-power (15x) images upper left, low-power (10x) CM SEM and CM images (CD31) in rabbit models at 14 days. Figure 4. Representative in C-PzF than DP-EES and is obviously greater (20x) CM images on the right. Note CD31 positive area images in the middle, and higher-power above strut surface by CM by stent type (C). area BP-EES. Graphs showing endothelial coverage in SEM at 14 days (B), and CD31 expression based on generalised estimating equation p-values between the groups and range for each group as median with interquartile presented Data are stent; CM: confocal microscopy; method to adjust for variations between the animals. BP-EES: biodegradable polymer everolimus-eluting microscopy stent; SEM: scanning electron PzF; DP-EES: durable polymer everolimus-eluting C-PzF: COBRA cell adherence was significantly less in both DP-EES and C-PzF adherence was significantly cell aggregates versus BP-EES, perhaps because of the lack of platelet cells inflammatory and activate can recruit which when activated monocytes and such as neutrophils let adhesion to C-PzF, fewer clots formed on the stent relative to fewer clots formed on the stent relative let adhesion to C-PzF, plate a thin High-power SEM demonstrated stents. comparator the let adhesive lining without apparent aggregation, causing a uni- this is due Whether form fluorescent signal throughout the stent. PVDF of PzF versus on platelet membrane effects to differential nm) C-PzF (50 of coating surface the that fact the or adhesion thinner than the PVDF-HPF on DP-EES is substantially coating process after first step in the healing A uncertain. (8 µm) remains

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e e 349 e . - 6 ES BP-E Preclinical assessment of COBRA of assessment Preclinical 41 =0.0 p 01 ES <0.0 is superior to the commercially available DES, which may allow available is superior to the commercially in DES. Differences to relative of DAPT duration shorter even an DES were probably C-PzF and competitor between strut coverage It is very likely that the due to the drug inhibiting endothelialisation. in since, coverage of speed the in role neutral plays a itself polymer models, C-PzF had compar studies in porcine previous preclinical endothe- showing mean V-BMS, to coverage endothelial able days by histopathology five at 98.33% even of coverage lial DP-E p 01 =0.0 p C-PzF DP-EES BP-EES . Whereas DES 13,14 0

400 300 200 100

) (n 11 M- RA F C-Pz 11 M- RAM-11 DAPI RAM-11 DAPI RA B A elute antiproliferative agents to prevent restenosis, C-PzF does not elute antiproliferative C-PzF in coverage endothelial show that data Our drug. a contain thrombotic events after stent implantation, early endothelial cov- early endothelial thrombotic events after stent implantation, is influenced coverage The speed of endothelial erage is crucial. of polymer, type thickness, as strut such factors contributing by The thickness of stent struts is type of drug, and amount of drug. cells through its the growth of endothelial well known to influence on shear stress and blood flow dynamics effect Representative images by CM for RAM-11 as a marker of macrophages in the rabbit model at 14 days. A) High-power images (20x) rabbit model at 14 days. in the as a marker of macrophages images by CM for RAM-11 Figure 5. Representative as presented on strut surfaces by stent type. Data are positive area on the strut surfaces. B) Graph showing RAM-11 positive areas of RAM-11 based on a generalised estimating are between the groups and p-values comparing differences deviation for each group mean±standard stent; CM: confocal everolimus-eluting equation method to adjust for variations between the animals. BP-EES: biodegradable polymer stent PzF; DP-EES: durable polymer everolimus-eluting C-PzF: COBRA microscopy; mediated 2+ ; Terumo Corp., Terumo ; ® Endothelial coverage by scanning electron microscopy C-PzF DP-EES BP-EES PF-BES . However, in that manuscript, endothelial . However, 17 0 80 60 40 20 (%) 100 ES ES -B p<0.05 vs C-PzF; p<0.05 vs DP-EES; p<0.05 vs BP-EES PF BP-E barrier function was never tested. Here we show for the first timefirst the for show we Here tested. never was function barrier by Evans Blue dye staining that barrier function is disrupted in certainly is almost This days. 28 at to C-PzF compared DES all exposurethat demonstrated previously we as effect, drug to due of rapamycin inhibitors (i.e., sirolimus or to a mammalian target everolimus) leads to impaired barrier function via Ca Tokyo, Japan), which is an abluminally coated biodegradable Tokyo, asVE-cadherin expression DES, showed better polymer-based compared to DP-EES in all groups. In a previous preclinical study, a biodegradable in all groups. In a previous preclinical study, (Ultimaster polymer sirolimus-eluting stent activation of protein kinase C alpha and downstream disruption ofdisruption C alpha and downstream kinase protein activation of . 15 expression above strut Endothelial cell barrier protein C-PzF DP-EES BP-EES PF-BES 0 80 60 40 20 . Among these, (%) 100 15 . In this study, VE-cadherin . In this study, 16 F ES C-Pz DP-E Evans Blue uptake C-PzF DP-EES BP-EES PF-BES 0 80 60 40 20 AB C (%) 100 expression was greater in C-PzF as compared to DP-EES, BP-EES, VE-cadherin and p120 and PF-BES. Consistent with results of co-localisation studies, Evans Blue dye staining was least in C-PzF Representative Evans Blue, SEM and CM for VE-cadherin and p120 images in a rabbit model at 28 days. A) Upper panels show model at 28 days. Evans Blue, SEM and CM for VE-cadherin and p120 images in a rabbit Figure 6. Representative VE-cadherin and SEM (low-power; 15x) and the lower panels are images (low-power) by stent type, the middle panels are Evans Blue gross coverage of the stent and and the greatest area, (low-power; 10x). Note that C-PzF showed the least Evans Blue positive p120 expression to the same positions lines correspond A. Red dotted High-power images of white boxes in panel co-localisation of VE-cadherin and p120. B) (middle), and (left), coverage above struts in SEM area images, SEM, and CM. C) Graphs showing Evans Blue positive in Evans Blue gross and p-values range for each group as median with interquartile presented p120 (right). Data are of VE-cadherin and co-localisation area estimating equation method to adjust for variations between the animals. BP-EES: biodegradable based on a generalised between the groups stent; PzF; DP-EES: durable polymer everolimus-eluting C-PzF: COBRA microscopy; stent; CM: confocal polymer everolimus-eluting VE: vascular-endothelial microscopy; biolimus-eluting stent; SEM: scanning electron PF-BES: polymer-free VE-cadherin is essential for normal endothelial barrier function barrier endothelial normal for essential is VE-cadherin BARRIER FUNCTION Endothelial permeability has been shown to play a role in types of atherosclerosis and neoatherosclerosis. Of the different junctional protein that exist on endothelial cells, adherens junctions play a vital role in endothelial barrier function Administration of an anti-VE-cadherin antibody to mice resultedmice to antibody anti-VE-cadherin an of Administration in increasing vascular permeability without modifying other types of interendothelial junction molecules

EuroIntervention 2019;15:e342-e353 350 e EuroIntervention 2019;15: 342- 353

e e 351 e - Preclinical assessment of COBRA of assessment Preclinical . The ongoing COBRA-REDUCE . 7 trial (NCT02594501) is examining whether, in patients undergoing in patients undergoing whether, trial (NCT02594501) is examining treat who also require oral anticoagulation, coronary intervention has higher PzF stent plus 14-day DAPT ment with the COBRA events com- safety and non-inferior outcomes for thromboembolic Administration-approved pared with a standard Food and Drug Our data sup- stent plus three- or six-month DAPT. drug-eluting if not better port such an approach, since C-PzF showed equivalent previous prospective clinical studies have demonstrated a very studies have demonstrated previous prospective clinical of stent thrombosis low rate . - 18 ES ES ES F -B PF BP-E DP-E B C-Pz A the VE-cadherin junctional formation in vascular endothelium the Endothelial barrier function in matched SEM and CM (VE-cadherin and p120) images. A) Representative high-power (40x) image A) Representative and CM (VE-cadherin and p120) images. Figure 7. Endothelial barrier function in matched SEM (white the right panel shows dissociation of VE-cadherin is on the left, whereas (yellow arrows) with co-localisation of VE-cadherin and p120 observed by CM (red between co-localised and dissociated VE-cadherin/p120 is clearly Note that the border arrows). and p120 (red arrows) clear border A by both SEM (50x) and CM (10x) using p120 and VE-cadherin antibodies. dotted line). B) The left images show matched areas to white images. The right images correspond is seen between incomplete and complete co-localisation of VE-cadherin and p120 on CM visualised on junctions which are of intact adherens SEM shows areas boxes in the left images (200x in SEM and 10x in CM). In C-PzF, of non-co- in DP-EES, BP-EES and PF-BES areas of p120 and VE-cadherin, whereas CM images showing co-localisation corresponding by SEM. BP-EES: biodegradable (yellow arrows) localised VE-cadherin and p120 on CM show platelet and white blood cells adherence stent; PzF; DP-EES: durable polymer everolimus-eluting C-PzF: COBRA microscopy; stent; CM: confocal polymer everolimus-eluting VE: vascular-endothelial microscopy; biolimus-eluting stent; SEM: scanning electron PF-BES: polymer-free tion, and early endothelial coverage and barrier function. In fact, In fact, function. barrier and coverage endothelial and early tion, CLINICAL IMPLICATIONS bene- has important data suggest that PzF coating Our preclinical reac inflammatory reduced in terms of antithrombogenicity, fits Whether other aspects of endothelial barrier function such as nitric oxide production might also be relevant towards reducing DAPT deserves further exploration. given its anti-aggregatory effects J Am 2015;66: Am Coll Cardiol. J Trollsas M, Hossainy S, Ratner BD. Trollsas , Greene S, Schuler G, Chevalier B. Why B. , Greene S, Schuler G, Chevalier , Giustino G, Witzenbichler B, Weisz G, Weisz B, Witzenbichler , Giustino G, T, Sakakura K, Pacheco E, Cheng Q, Zhao X, Q, Zhao Cheng E, K, Pacheco Sakakura T, , Cheng Q, Yahagi K, Acampado E, Sheehy A, Acampado E, Sheehy K, Yahagi , Cheng Q, Biointerphases. 2016;11:029806. , Abizaid A, Chevalier B, Greene S, Meredith I, S, Meredith B, Greene Chevalier A, Abizaid , DE, Garratt KN, Novack V, Barakat M, Meraj P, P, M, Meraj Barakat V, KN, Novack DE, Garratt Cutlip Szott LM, Irvin CA, Urban P Morice MC, Urban P Otsuka F Koppara Giustino G, Baber U, Sartori S, Mehran R, Mastoris I, Giustino G, Baber U, Généreux P AS, Sharma SK, Pocock SJ, Dangas GD. Duration of dual SJ, Dangas GD. Duration SK, Pocock Sharma AS,

7. 8. 3. 4. 5. 6. 1. 2. A, Jauhar R, Popma JJ, Stoler R, Silber S. L, Erglis Maillard Angiographic Outcomes of the COBRA and 9-Month Clinical Cardiovasc System. JACC Coronary Stent NanoCoated Polyzene-F 2017;10:160-7. Interv. used in drug eluting assessment of polymers Blood compatibility stent coatings. Kini LEADERS and design of the MC, Pocock S. Rationale Morice of the comparison double-blind randomized A trial: FREE in stent metal bare Gazelle vs the stent drug-coated BioFreedom course of dual risk using a short (1 month) at high bleeding patients Am Heart J. 2013;165:704-9. antiplatelet therapy. Am Coll Cardiol. stents? J are we still using coronary bare-metal 2013;61:1122-3. FD, Kolodgie K, Perkins LEL, SK, Sakakura K, Euller Yazdani of a Durable Polymer Thrombogenicity Acute R, Joner M. Virmani Drug-Eluting to Contemporary Stent Relative Everolimus-Eluting in Vivo Assessed Ex Coatings Polymer Biodegradable With Stents 2015;8:1248-60. Interv. a Swine Shunt Model. JACC Cardiovasc L, Ren J, Deshpande M, M, Maillard Barakat AV, E, Finn Acampado novel of a evaluation R. Preclinical Virmani FD, Joner M, Kolodgie 2016;222: Cardiol. J stent. Int modified surface polyphosphazene 217-25. References a system- therapy after drug-eluting stent implantation: antiplatelet trials. controlled of randomized review and meta-analysis atic 2015;65:1298-310. Coll Cardiol. TD, Rinaldi MJ, Neumann FJ, Metzger DC, Henry TD, Stuckey DP, M, Francese Yadav E, PL, Mazzaferri Cox DA, Duffy C, Mehran R, Stone GW. AJ, Litherland Kirtane T, Palmerini After Bleeding Predictors, and Impact of Post-Discharge Incidence, Percutaneous Coronary Intervention. 1036-45. Biotronik, Boston Scientific, CeloNova, Claret Medical, Cook Cook Medical, Claret CeloNova, Scientific, Boston Biotronik, Medtronic, MicroPort, Lifesciences, Cordis, Edwards Medical, Technology, SINO Medical ReCor, OrbusNeich, MicroVention, and Gore W.L. Corporation, Terumo Surmodics, Spectranetics, from 480 Biomedical, has received honoraria Virmani Xeltis. R. Lutonix, Cook Medical, Boston Scientific, Vascular, Abbott and is a consultant Gore, W.L. and Corporation Terumo Medtronic, Gore. W.L. Medtronic, and Vascular, Abbott for 480 Biomedical, Abbott, from Boston Scientific, honoraria A. Finn has received authors have no conflicts of interest to The other and CeloNova. declare. - . Additionally, in the process of staining, Additionally, . 20-22 . Finally, PECAM-1 is not a specific marker either forspecific a not is PECAM-1 . Finally, 7,19 Our data suggest that C-PzF has characteristics which make it might beThe usage of C-PzF beneficial for short-term DAPT. suitable especially for patients at high risk for bleeding. Impact on daily practice the stented artery halves were rinsed in phosphate buffered saline buffered the stented artery halves were rinsed in phosphate the pattern of stainingbefore staining with CD31/PECAM-1, and cells not platelets.resembles the cobblestone shape of endothelial antibody detects theTherefore, we believe that the anti-PECAM-1 on the surface of stents.presence of endothelial cells (not platelets) the coverage by SEM also supports the result In the current study, showing similar trends. of confocal microscopy using PECAM-1, thromboresistance to current-generation DES and BMS as well as as BMS as well DES and current-generation to thromboresistance tri- Clinical of functional healing. coverage and return earlier strut be cur could therapy DAPT how short to examine als are needed this novel stent system. with tailed Conflict of interest statement research support from institutional has received CVPath Institute Biosensors International, ART, Vascular, Abbott 480 Biomedical, This study was funded by CeloNova Biosciences, San Antonio, San This study was funded by CeloNova Biosciences, of data, and analysis collection The design of the study, TX, USA. by CVPath personnel. independently were performed Funding C-PzF showed a distinctive combination of advantages when of advantages combination C-PzF showed a distinctive its of endpoints designed to evaluate a combination examining be equi- might Thromboresistance DAPT. for short-term potential and DP-EES and BP-EES, valent or superior to current-generation healing was superior to the former stents as well as to PF-BES. very requiring patients to benefit of important be C-PzF might DAPT. short-term Conclusions Study limitations First, the results from in vivo andThis study has some limitations. directly to real-world patients whoex vivo models cannot be applied The atherosclerotic milieu arteries. have atherosclerotic coronary inflamma- thrombogenicity, that may influence has complex effects we should be cautious when extrapo- Therefore, tion, and healing. the clinic. Nonetheless, we believe that theselating these results to ofreliable to give some relative comparison models are generally between stents. Second, although our datathe endpoints examined functional endothelialisatoin for COBRA suggest quicker and more this does not mean that rates of restenosis would be equi- PzF, clinical studies havevalent to DES. Nonetheless, several previous lesion revascularisation as compared shown attractive rates of target to BMS endothelial cells or for platelet. However, CD31/PECAM-1 has CD31/PECAM-1 endothelial cells or for platelet. However, junctions and its util- been broadly used to evaluate endothelial cell ity has been proven

EuroIntervention 2019;15:e342-e353 352 e EuroIntervention 2019;15: 342- 353

e e 353 e - - Platelet-endothelial mean values from the values mean of all devices. AV, Quee SC, Coleman L, Coleman Quee SC, AV,

2013;33:2425-31. of barrier function study in study function barrier of Preclinical assessment of COBRA of assessment Preclinical 2001;226:686-91. Summary of the P-values The description

1. 2. 3.

A, Elkins M, Kozien D, Nakada MT. Regula MT. D, Nakada M, Kozien A, Elkins Table Table Table

amburino C, Capodanno D, Di Salvo ME, Sanfilippo A, A, Salvo ME, Sanfilippo Capodanno D, Di amburino C, T G, Finn Joner M, Nakazawa RayChaudhury Gratzinger D, Barreuther M, Madri JA.

20. 21. 22. 19. in vivo models. published online at: The supplementary data are https://eurointervention.pcronline.com/ doi/10.4244/EIJ-D-18-00740 Supplementary data Supplementary Appendix 1. Methods. Supplementary Supplementary quantification PPT), platelet (PT, animals of all blood coagulation and acti function (LTA), EST), platelet counts, platelet (platelet shunt model. (ACT) in a swine acute time clotting vated Supplementary P, G, Giacoppo D, Capranzano Longo V, Incardona Cascone I, - effective and Safety A. Manna La A, Monaco G, Ussia C, Sgroi clinical world coated stent in real Polyzene-F Catania the ness of 2 registry. ATLANTA results from the practice: 12-month 2012;7:1062-8. EuroIntervention. HK, X, Gold Q, Xu Cheng K, PS, Skorija Wilson E, Acampado com- between recovery cell R. Endothelial Virmani FD, Kolodgie Am Coll Cardiol. J drug-eluting stents. parator polymer-based 2008;52:333-42. endothelial endothelial barrier function through protein kinase C-α activation interac- cadherin endothelial p120-vascular the of disruption and Biol. Vasc Thromb tion. Arterioscler cell adhesion molecule-1 modulates endothelial migration through migration cell adhesion molecule-1 modulates endothelial tyrosine-based inhibitory motif. Biochem its immunoreceptor Biophys Res Commun. 2003;301:243-9. tion of PECAM-1 in endothelial cells during cell growth and migra- and growth cell during cells endothelial PECAM-1 in of tion tion. Exp Biol Med (Maywood).

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2006;113:2679-82. 2006;86:279-367. Physiol Rev. endothelial permeability. produced byAlterations in regional vascular geometry Pagel PS. of wall sheartheoretical stent implantation influence distributions using 3D computationalstress: analysis of a curved coronary artery 2006;5:40. fluid dynamics modeling. Biomed Eng Online. A, I, Stoppacciaro MG, Martin-Padura Brockhaus M, Lampugnani endothelial- Vascular E. Dejana PA, Ward DM, McDonald L, Ruco in integrity of microvascular determinant is an important cadherin A. 1999;96:9815-20. Acad Sci U S Natl vivo. Proc Supplementary data

Supplementary Appendix 1. Methods

The study protocol was approved by the Institutional Animal Care and Use Committee of the

MedStar Health Research Institute.

Animal models

Two different animal models were used in this study: 1) an ex vivo porcine arteriovenous shunt model to examine acute thrombogenicity and acute inflammation (n=4 animals), and 2) an in vivo rabbit iliofemoral stenting model to examine endothelialisation and endothelial barrier function at both 14 and 28 days (n=9 animals and 8 animals, respectively).

Test devices

Three different stents were compared in an ex vivo arteriovenous shunt model for assessment of acute thrombogenicity and inflammation and in an in vivo rabbit iliofemoral stenting model for assessment of healing and inflammation at 14 days. The test arm for both studies was a cobalt- chromium (CoCr) PzF stent, 3.0 x 18 mm (COBRA-PzF; CeloNova BioSciences, San Antonio,

TX, USA). Commercially available XIENCE Xpedition, durable polymer everolimus-eluting stents (DP-EES) 3.0 x 18 mm (Abbott Vascular, Santa Clara, CA, USA) and SYNERGY, bioabsorable polymer everolimus-eluting stents (BP-EES), 3.0 x 20 mm (Boston Scientific,

Marlborough, MA, USA) were compared. Additionally, the BioFreedom polymer-free biolimus- eluting stent (PF-BES), 3.0 x 18mm (Biosensors, Newport Beach, CA, USA) was added as a comparator to the DP-EES (3.0 x 18 mm) and BP-EES (3.0 x 16 mm) in the in vivo rabbit

iliofemoral model for assessment of endothelial coverage and function at 28 days (clinically the latter stent has been shown to require the shortest duration of dual antiplatelet therapy, i.e., one month, which potentially suggests an advantage with regard to healing and function).

Supplementary Table 1 shows a description of all devices used in this study.

Porcine ex vivo arteriovenous shunt model

A total of 4 healthy male Yorkshire cross domestic pigs were included in this study. A porcine ex vivo arteriovenous shunt model involving a test circuit of 3 different in-line test stents was performed for 60 minutes to evaluate platelet adherence, thrombus formation, and acute inflammation in the devices. Each shunt model had 3 different stents and each animal had 2 shunt experiments for a total of n=6 stents per animal.

Blood activated clotting time (ACT) was kept between 150 and 190 s using a bolus of heparin and maintenance low-dose intravenous heparin (100 IU/kg) to minimise the effect of platelet aggregation and thrombus formation on the surface of the stents. The current study was designed to examine inherent platelet-mediated thrombus formation induced by different types of stent.

At the end of each shunt run, stents were gravity perfused with Ringer’s lactate until cleared of blood and then fixed in 10% neutral buffered formalin, and longitudinally bisected where one half underwent dual immunofluorescent staining.

Rabbit model of iliac stent implantation

A total of 17 (9 for the 14-day endothelialisation study and 8 for the 28-day endothelial function study) healthy male New Zealand White rabbits (3.0 to 4.0 kg; Millbrook Laboratories, Amherst,

MA, USA) were included in the study. Animals were given aspirin (40 mg) orally 12 hours before stent implantation and once daily until euthanasia. Stents were deployed with each iliac of an animal randomly allocated to one of 3 groups (C-PzF, DP-EES, and BP-EES) in the 14-day coverage study and one of 4 groups (C-PzF, DP-EES, BP-EES and PF-BES) in the 28-day endothelial function study. Stents (all 3.0 mm in diameter) were deployed at nominal pressure immediately after vessel denudation and the iliac arteries after balloon denudation. Follow-up angiography was conducted to confirm stent patency before euthanasia at 14 days in the healing study and 28 days in the endothelial function study.

Imaging procedures: confocal microscopy (CM), scanning electron microscopy (SEM) and

Evans Blue test

In the shunt model, one half of the stent which was longitudinally bisected underwent dual immunofluorescent staining using antibodies against the antiplatelet marker CD61, a marker of platelet aggregation (Immunotec, Commerce, CA, USA) and CD42b, a marker of platelet adhesion (sc-7070; Santa Cruz, Dallas, TX, USA), to capture both originating and propagated platelet thrombus as well as an antibody against the monocyte marker CD14 (Novus Biologicals,

Littleton, CO, USA). The other half was immunostained using antibodies against the platelet markers CD61/CD42b and neutrophil marker PM-1 (BMA Biomedicals, Augst, Switzerland)

(dilution 1:800). Both halves were examined by SEM after CM (Zeiss LSM 800; Carl Zeiss

Microscopy, Jena, Germany). ZEN software (Zeiss ZEN 2012; Carl Zeiss Microscopy) quantitated the positive area of staining within predefined regions of interest. Areas staining positive for CD61/CD42b (mm2) within each stented segment were assessed. Platelet aggregate clots were counted by SEM in both stent halves.

In the rabbit model to assess healing at 14 days after implantation of stents, one half was immunostained using CD31/PECAM-1 (Dako, Carpinteria, CA, USA), while the other half was processed for RAM-11 (Dako) and examined by SEM after completion of CM image acquisition.

En face CM images were scanned both sides at 5x magnification with tiled Z-stack technology for whole surface view and at 20x magnification with single Z-stack for local regions of interest.

In the rabbit model to assess endothelial function at 28 days after implantation of stents, sterile- filtered Evans Blue dye administered i.v. was allowed to re-circulate for 1 hr to assess endothelial permeability. Evans Blue positive area was estimated visually after being bisected.

Evans Blue positive area was estimated per strut column and averaged for the entire stent. The whole stented segment was immunostained with dual staining using antibodies against VE- cadherin (R&D Systems Inc., Minneapolis, MN, USA) (dilution 1:200) and p120 (Santa Cruz)

(dilution 1:400). Subsequently, both immunostained stented halves were processed for SEM after imaging by CM. Both sides were scanned en face by CM at 5x magnification with tiled Z-stack technology for the entire stented segment surface view.

Statistical analysis

Normality of data distribution was checked by the Shapiro-Wilk test. Continuous variables were expressed as mean±standard deviation, median with interquartile range or estimated mean with

95% confidence interval (CI), as appropriate. In shunt models, a nested generalised linear mixed model was used to evaluate group differences considering multiple measurements per individual.

The experimental factor variables animal and shunt position were considered as nested random

effects and stent type was considered as a fixed effect. Vessel-level comparisons were tested by a generalised estimating equation method using a linear model or gamma with log link model between C-PzF and comparators, as appropriate. SPSS software, Version 22 (IBM Corp.,

Armonk, NY, USA) or JMP 9 (SAS Institute, Cary, NC, USA) was used for statistical analysis.

A p-value of p<0.05 was considered statistically significant.

Supplementary Table 1. The description of all devices.

Supplementary Table 2. Summary of the mean values from the blood coagulation of all animals (PT, PPT), platelet quantification (platelet counts, platelet EST), platelet function (LTA), and activated clotting time (ACT) in a swine acute shunt model.

Mean Fold change (%) (min - max) Test 1st shunt vs. 2nd shunt vs. 2nd shunt vs. Baseline After 1st shunt After 2nd shunt baseline baseline 1st shunt

10.50 Prothrombin time 9.10 10.37 (9.30 – 1.14 1.15 1.01 (seconds) (8.30 – 9.50) (9.50 – 11) 11.30)

PTT 12.27 54.47 68.73 4.44 5.60 1.26 (seconds) (10.50 – 13.40) (30 – 100) (23.70 – 100) Platelet count 277 269.33 234.33 0.97 0.85 0.87 (x1,000/μL) (165 – 479) (167 – 463) (136 – 430) Platelet EST Adequate Adequate Adequate N/A N/A N/A LTA (ADP = 20 um) 16.75 6.50 5.75 0.39 0.34 0.88 (% platelet aggregation) (0 – 51) (0 – 18) (0 – 17) LTA (ADP = 5 µm) 22.25 10.88 6.50 0.49 0.29 0.60 % platelet aggregation (11 – 30) (6.50 – 14.50) (3.50 – 10) ACT 97.00 152.55 158.77 157 1.64 1.04 (seconds) (94 – 99) (136 – 162) (150 – 168) Platelet EST was considered to be adequate when the platelet count was estimated to be within the reference interval. ADP: adenosine diphosphate; LTA: light transmission aggregometry; PTT: partial thromboplastin time

Supplementary Table 3. P-values of barrier function study in in vivo models.

BP-EES BP-EES DP-EES vs. PF-BES vs. DP-EES vs. PF-BES Percentage of co-localised VE-cadherin and p120 above struts (%) 0.373 0.522 0.089 Percentage of co-localised VE-cadherin and p120 between struts (%) 0.416 0.788 0.194 Percentage of Evans Blue positive area 0.002 0.721 0.007 Percent of coverage above struts by SEM (%) 0.013 0.114 0.579 Percent of coverage between struts by SEM (%) 0.010 0.106 0.519

BP-EES: biodegradable polymer everolimus-eluting stent; C-PzF: COBRA-PzF; DP-EES: durable polymer everolimus-eluting stent; PF-BES: polymer-free biolimus-eluting stent; SEM: scanning electron microscopy; VE: vascular-endothelial