Comparison of acute thrombogenicity and albumin adsorption in three different durable polymer coronary drug-eluting stents

Introduction

Stent thrombosis (ST) continues to be one of the most feared complications after percutaneous coronary interventions1. Thus, thromboresistance is an ideal property for coronary stents. Previous data have suggested that polymeric coatings influence blood-materials interactions depending upon their adsorbing capacity with regard to specific plasma proteins which drive the stent’s interaction with platelets and leukocytes, potentially leading to cell adhesion, activation and thrombosis2^,3^,4. For biomaterials, it has been proposed that surfaces with high levels of albumin adsorption are desirable as albumin could passivate the polymer surface by preventing more reactive components, such as platelets and fibrinogen, from binding5. It is well known that fluorinated polymers such as poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) used in both the CoCr everolimus-eluting stents (Abbott Vascular, Santa Clara, CA, USA) and the platinum-chromium everolimus-eluting stent (Boston Scientific, Marlborough, MA, USA) possess this reaction4. While it is generally believed that polymeric coatings, regardless of type, have some antithrombotic effect compared to bare metal surfaces, the relative effects of different durable polymers used in coronary DES have never been examined with respect to thrombogenicity and albumin adsorption and retention.

We compared the relative thromboresistance of three currently available durable polymer DES with distinct coatings using an ex vivo porcine shunt model and their albumin binding capacity to understand the association between albumin adsorption and thromboresistance for each polymeric DES (i.e., fluoropolymer everolimus-eluting stent [FP-EES; Abbott Vascular, BioLinx™ polymer-coated zotarolimus-eluting stent [BL-ZES; Medtronic, Minneapolis, MN, USA] and CarboSil® elastomer polymer-coated ridaforolimus-eluting stent [EP-RES; Cordis, Milpitas, CA, USA]).

Methods

Detailed methods are described in Supplementary Appendix 1.

TEST DEVICES

The control arm for three different studies was the XIENCE Alpine™ (Abbott Vascular) (FP-EES). Comparators included two contemporary durable polymer DES: 1) BL-ZES which is coated with a mixture of hydrophilic C10 polymer, hydrophilic C19 polymer, and polyvinylpyrrolidone (PVP) (BioLinx polymer), and 2) EP-RES which is coated with a blend of CarboSil and PBMA. The MULTI-LINK 8™ BMS (Abbott Vascular) served as the third comparator. Table 1 lists experimental models and devices, and Supplementary Table 1 shows the description of all devices.

SWINE EX VIVO ARTERIOVENOUS SHUNT MODEL

For assessment of acute thrombogenicity and inflammation, platelets (CD61/CD42b), monocytes (CD14) and neutrophils (PM-1) were evaluated by confocal microscopy (CM)3. For quantification of platelet aggregation, the areas of positive staining within each stented segment were measured by ZEN software (Zeiss ZEN 2012; Carl Zeiss Microscopy, Jena, Germany). The number of inflammatory cells was counted manually and expressed as cell density (cells/mm²) relative to strut surface area.

ALBUMIN

PREPARATION OF STENTS

Stents were cut longitudinally to make uniform lengths (6-7 mm) and inserted into a bottomless channel slide used for perfusion application (sticky-Slide I Luer 0.8 mm channel height, Cat. No. 80198; ibidi GmbH, Gräfelfing, Germany) (Supplementary Figure 1A, Supplementary Figure 1B).

ALBUMIN IN VITRO FLOW LOOP

Labelled human albumin was circulated through silicone tubing in line with the ibidi slide using a perfusion pump. The first 30 minutes under flow was for albumin binding (Supplementary Figure 1C), and the second 30 minutes under flow was for washing with FluoroBrite™ DMEM (Thermo Fisher Scientific, Waltham, MA, USA) (Supplementary Figure 1D). Albumin binding was imaged only during latter phases because of background noise generated by dissolved fluorescent proteins during the first 30 minutes.

Randomly selected regions of interest from one different area of each stent were scanned. In addition, the entire area of each stent was scanned by CM and quantified by ZEN software. High and low signals of fluorescence were defined in each experiment to assess all stents in the same condition. Each image was recorded at one-minute intervals.

ASSESSMENT OF ALBUMIN ADHERENCE TO VARYING STENT SURFACES

The positive area of albumin labelling (green channel) was quantitated within defined regions of interest. Intensity scale was defined as the average of total light intensity of the green signal from every pixel by the strut area. The analysis was performed every one minute, and the covered strut area was quantified and expressed as percent albumin coverage (%) and intensity scale of albumin.

A REAL-TIME MANNER USING HUMAN PLATELET IN VITRO FLOW LOOP

In addition, human platelet adherence to stent surfaces was evaluated in a real-time manner using an in vitro flow loop live-cell assay to determine the temporal distribution of platelet adherence.

STATISTICAL ANALYSIS

In an ex vivo shunt model and an albumin flow loop model, nested generalised linear mixed models with Dunnett's correction for multiple testing were employed in order to investigate group differences in consideration of multiple measurements per individual. Within these models, stent type was considered as fixed effect, while the experimental factor variables animal or experiment, shunt number and linear position were considered as nested random effects. Values are expressed as estimated mean with 95% confidence interval. The analyses were performed with SPSS Advanced Statistics, Version 25 (IBM Corp., Armonk, NY, USA). The statistical tests were two-tailed and a value of p<0.05 was considered to indicate statistical significance.

Results

ACUTE THROMBOGENICITY IN A PORCINE ARTERIOVENOUS SHUNT MODEL

There was no evidence of blood coagulation or platelet function abnormalities in any of the animals studied. Additionally, the measures of coagulation were similar in all shunt runs (Supplementary Table 2).

Table 2 summarises the results of platelet adhesion by CM. Figure 1 shows representative CM images with immunofluorescent staining against dual platelet markers (CD61/42b) in FP-EES, BL-ZES, EP-RES and BMS. When total platelet fluorescence area was normalised to stent surface area, platelet adhesion was lower in the order FP-EES, BL-ZES, EP-RES and BMS. Table 2 lists the results of immunofluorescent staining against a neutrophil marker (PM-1) and a monocyte marker (CD14) by CM; Figure 2 shows representative images from each group. The cell density of PM-1 was least for FP-EES

Figure 1. Representative confocal microscopic images using immunofluorescent staining against dual platelet markers (CD61/CD42b) in an ex vivo shunt model. A) Low (upper) and high (lower) power confocal microscopic images showed minimal platelet aggregation in FP-EES, whereas obvious platelet aggregation was observed in BL-ZES, EP-RES and BMS. B) Graph shows fluorescent positive area per stent surface. Data are presented as mean±standard deviation for each. * p<0.05 vs FP-EES; ? p<0.05 vs BL-ZES; ?? p<0.05 vs EP-RES.

Figure 2. Representative confocal microscopic images using immunofluorescent staining against a neutrophil marker (PM-1) and a monocyte marker (CD14) in an ex vivo shunt model. A) The upper panels (A1 and A2) show confocal microscopic images with PM-1 staining and the lower panels show confocal microscopic images with CD-14 staining. FP-EES showed minimal neutrophil and monocyte attachment. However, BL-ZES, EP-RES and BMS showed many neutrophils and monocytes on the stent surfaces. B) Graphs show the number of PM-1 (B1) and CD-14 (B2) positive cells on the stent struts. The number of PM-1 and CD-14 was significantly the least in FP-EES relative to BL-ZES, EP-RES and BMS. Data are presented as mean±standard deviation for each group. * p<0.05 vs FP-EES.

ALBUMIN ADSORPTION AND RETENTION IN A FLOW LOOP MODEL USING HSA

The amount of fluorescent albumin on the BMS surface increased over time; there were significant differences between times 0 and 30 minutes and between 30 minutes and 48 hours (Supplementary Figure 2). Also, BMS with 48-hour exposure to albumin showed a significantly lesser amount of platelet attachment as compared to BMS with no exposure to albumin (Supplementary Figure 3, Supplementary Table 3). Table 3 summarises the results of albumin coverage by CM, and Figure 3 shows representative CM images with immunofluorescent albumin in FP-EES, BL-ZES, EP-RES and BMS. Figure 4 shows representative quantitated images in all stents as analysed by ZEN software. When total albumin fluorescence area was normalised to stent surface area, an area of total albumin coverage was significantly greater in FP-EES, BL-ZES and EP-RES than in BMS, whereas there were no significant differences in area between FP-EES, BL-ZES and EP-RES. However, the area of albumin coverage as determined by high signal intensity was greater in FP-EES versus all other stents. Although FP-EES, BL-ZES and EP-RES showed nearly complete albumin coverage on the stent struts, the majority of sampled areas showed high albumin signal intensity in FP-EES, whereas it was a low signal intensity in BL-ZES and EP-RES (Figure 4).

Figure 3. Representative confocal microscopic images using fluorescent human serum albumin in the flow loop model. The low (upper) and high (lower) power images show the stent surface fully covered by an obvious strong signal of fluorescent albumin in FP-EES. On the other hand, although BL-ZES and EP-RES also showed near complete coverage by albumin, the albumin signals (green) on the surface in BL-ZES and EP-RES were less intense relative to FP-EES. Minimal albumin signal was observed in BMS.

Figure 4. Quantitated images of confocal microscopy with fluorescent human albumin. A) High signal of fluorescent albumin was coloured yellow, whereas low signal of albumin was coloured grey. FP-EES was fully covered by a yellow colour, whereas BL-ZES and EP-RES were covered mostly by grey. BMS showed minimal grey colour. B), C) & D) Graphs show high signal of albumin, low signal and total signal in each stent. Data are presented as mean±standard deviation for each group. * p<0.05 vs FP-EES; ? p<0.05 vs BL-ZES; ?? p<0.05 vs EP-RES.

A real-time analysis over the 30-minute washing phase also showed that coverage of albumin was significantly greater in FP-EES, BL-ZES and EP-RES relative to BMS (Supplementary Table 4, Figure 5, Moving image 1). The intensity of albumin, indicative of a higher amount of adsorption, was greater in the order FP-EES, BL-ZES, EP-RES and BMS. All stents did not show a significant decrease in albumin retention over the 30-minute washing phase (Supplementary Table 5). Albumin intensity correlated negatively with platelet attachment in the shunt model (Supplementary Figure 4).

Figure 5. Percent coverage and intensity of albumin in each group over 30 minutes during washing phases. A) These images show quantitated coverage and intensity of albumin at the end of washing phases. All durable polymer DES showed nearly full albumin coverage. However, albumin intensity was higher in the order FP-EES, BL-ZES and EP-RES. Coverage and intensity of albumin were minimal in BMS. B) & C) Graphs show estimated mean percent coverage and intensity over 30 minutes during washing phases. There were significant differences in FP-EES, BL-ZES, and EP-RES versus BMS, although significant differences were not observed between FP-EES, BL-ZES and EP-RES. FP-EES had the highest intensity, followed by BL-ZES and EP-RES, with the least being in BMS. Significant differences were seen in all comparisons. In percent coverage and intensity, minimal changes were observed over 30 minutes.

PLATELET ADHESION IN A REAL-TIME FLOW LOOP MODEL USING HUMAN PLATELETS

A novel in vitro flow loop model using freshly isolated fluorescently labelled human platelets from healthy volunteers was constructed. Stents were exposed to circulating platelets, imaged and analysed by CM over the course of 60 minutes. Consistent with the shunt study, the amount of platelet aggregation was numerically least in FP-EES, followed by BL-ZES and EP-RES and then BMS (n=3 per group), although there were non-significant differences between each (Supplementary Table 6, Supplementary Figure 5, Moving image 2). Platelet adhesion in the flow model correlated nicely with that in the shunt model (Supplementary Figure 6).

Discussion

The current study evaluated acute thrombogenicity and albumin retention in currently available durable polymer DES. The main findings of this preclinical study were as follows: 1) antithrombotic and anti-inflammatory reactions were greater in the order FP-EES, BL-ZES, EP-RES and BMS in an ex vivo porcine shunt model; 2) albumin coverage was greater in all durable polymer DES relative to BMS, and 3) albumin intensity indicating concentration of albumin was significantly the greatest in FP-EES, followed by BL-ZES and EP-RES, with the least being in BMS. Taken together, these data suggest that durable polymer coatings provide some level of thromboresistance compared to BMS, which may be the mechanism by which polymeric coatings to some extent have thromboresistant properties. From this standpoint, durable polymers used on DES are not equivalent.

DIFFERENT DURABLE POLYMER COATING DES IN AN EX VIVO SHUNT MODEL

This shunt study suggests that polymeric coating provides a protective barrier against acute thrombus formation. Clinical data also suggest a protective effect of polymer-coated stents in patients with coronary artery diseases6. Among durable polymer DES, fluoropolymer EES showed the strongest thromboresistance. BL-ZES showed some degree of thromboresistance when compared with BMS as the control. This suggests that the BioLinx polymer confers some ability to avoid platelet adhesion. The outer surface of the polymer consists of hydrophilic polyvinyl-pyrrolidinone which inhibited monocyte adhesion in one study7 and, according to our study, allows some degree of albumin adsorption. On the other hand, PBMA/CarboSil coating showed comparable thrombogenicity to BMS. Previous in vitro testing showed initially good adsorption of albumin to PBMA polymer with very little retention after detergent washing and a relatively low albumin/fibrinogen binding ratio compared to the PVDF-HPF fluoropolymer4. These data support the observation that, even if durable polymers are applied, these polymers show differing individual potential with respect to thrombogenicity even in the current era.

Platelet aggregation on the surface of stent struts exists along with circulating leukocytes consisting mainly of neutrophils and monocytes8. Moreover, leukocyte tissue factors are transferred to platelets, promoting propagation of thrombus9^,10. In the current study, the density of inflammatory cells on the surface of struts was consistent with the amount of platelet adherence to the stent surfaces.

ALBUMIN BINDING AND RETENTION IN DIFFERENT POLYMER COATINGS IN FLOW LOOP MODELS

Albumin has traditionally been used as a passivating protein. High albumin retention on the surface of the strut might be a beneficial mechanism to prevent a thrombogenic profile. The albumin-rich layer tempers host-material interactions and competitively inhibits the adhesion of fibrinogen and von Willebrand factor4^,11. However, in an in vivo setting, surface adsorption is a reversible process; the composition of adsorbed proteins may change over time, a phenomenon known as the Vroman effect12. Albumin can be replaced with other proteins leading to thrombosis such as von Willebrand factor and fibrinogen, etc. However, absorptivity between albumin and stent surfaces may vary to a great extent based on materials. In fact, previous preclinical studies showed various capability of keeping albumin on stent surfaces by different materials after washing by sodium dodecyl sulphate4. Previously, Szott et al reported that fluoropolymer can retain non-significantly more albumin on the stent surface relative to PBMA and polystyrene-b-polyisobutylene-b-polystyrene (Kaneka, Japan; SIBS-1 and SIBS-2) even after using sodium dodecyl sulphate, which is supposed to detach albumin from the surface of struts. In addition to differences in albumin retention, the superior thromboresistance of FP-EES was shown. Each stent offers a different amount of albumin adsorption; the level of affinity of albumin on the stent surface appears to affect thrombogenicity.

APPLICATION TO SHORT DUAL ANTIPLATELET THERAPY (DAPT)

Differences in intrinsic thrombogenicity between stents is important when considering shortening DAPT therapy as stent struts may not be fully covered by endothelium at the time DAPT is discontinued. Bare struts, especially those which do not possess intrinsic thromboresistance, may become a nidus for thrombus formation. Superior thromboresistant features of polymers may contribute to a short duration of DAPT (i.e., <3 months).

In multicentre prospective trials using a standard strategy for DAPT duration, the rate of ST was very low (0.1-0.6%) and comparable in different types of current DES6^,13^,14^,15. However, information about the rate of ST after a short DAPT strategy (i.e., 1 to 3 months) is limited. To date, a few studies are available where a one-month duration of DAPT was evaluated16. The LEADERS FREE trial was a landmark trial of one-month DAPT in which polymer-free DES were compared with BMS with the same one-month DAPT protocol. However, this study showed a high rate of ST in both groups due to greater thickness and bare surface although it also suggested that it is feasible to perform a short duration of DAPT after DES implantation. More recently, the STOP DAPT II trial also showed the feasibility of one-month DAPT after implantation of FP-EES when compared to 12-month DAPT17. The current preclinical study further supports these results. The potential differences in thromboresistance seen within this study for different DES suggest that a one-month DAPT duration for different types of DES may not be standardised (regardless of DES type) and that each needs to be tested in dedicated clinical trials.

Limitations

There are some limitations in this study. First, this study was performed in a swine ex vivo shunt model without antiplatelet agents and with low-dose heparin to evaluate the potential of the material itself. Therefore, this result cannot simply be applied to the clinical setting where patients are treated with mono or dual antiplatelet therapy. Second, the drug can affect inflammatory cell adhesion and platelet attachment of the adjacent stent in this model18. However, the effect of the drug on platelet attachment is minimal (Supplementary Figure 7). Third, an in vitro flow loop model was used in this study to assess human serum albumin adsorption and retention to different DES. Although this cannot completely mimic in vivo conditions, we believe the results of these studies simulate in vivo conditions since the concentration of albumin used was similar to that in human blood. Fourth, this study focused on albumin since albumin is considered to be a protein protecting against platelet attachment. Other types of protein may also play an important role in protecting against thrombogenicity. Previous data regarding the fact that fluoropolymer binds greater amounts of albumin as well as fibrinogen sounds counterintuitive given that fibrinogen4 is a known promoter of clot formation through its conversion to fibrin by thrombin. Some emerging data suggest that albumin has an inhibitory property against fibrin polymerisation and that this may be one of the mechanisms by which fluoropolymers promote thromboresistance19. Further basic research is needed for a greater understanding of this relationship. Finally, studying platelet adherence using the in vitro flow loop model is different from that seen in the setting of vascular injury.

Conclusions

Overall, antithrombogenicity was better in the order FP-EES, BL-ZES, EP-RES and BMS. Although three durable polymer DES showed greater albumin coverage as compared to BMS, albumin intensity indicating concentration of albumin was not equivalent in three durable polymer DES. The results of albumin adsorption might be the cause of differences in platelet adhesion seen in the three different types of durable polymer DES and BMS, although other factors such as the adhesion of leukocytes also probably play a role. Because we also showed that the antiproliferative agents loaded onto DES have some thromboresistant properties, it is important to acknowledge the potential contribution of the antiproliferative agents as contributing to the thromboresistant effects seen in the different DES studied. These experiments may provide insight into the suitability of different durable polymer DES for shortening DAPT duration.

Funding

This study was funded by Abbott Vascular, Santa Clara, CA, USA. CVPath Institute, Inc., Gaithersburg, MD, USA, provided full support for this work. CVPath Institute has received institutional research support from 480 Biomedical, Abbott Vascular, ART, Biosensors International, Biotronik, Boston Scientific, CeloNova, Claret Medical, Cook Medical, Cordis, Edwards Lifesciences, Medtronic, MicroPort, MicroVention, OrbusNeich, ReCor, Sino Medical Technology, Spectranetics, SurModics, Terumo Corporation, W.L. Gore and Xeltis.

Conflict of interest statement

R. Virmani has received honoraria from 480 Biomedical, Abbott Vascular, Boston Scientific, Cook Medical, Lutonix, Medtronic, Terumo Corporation and W.L. Gore, and is a consultant for 480 Biomedical, Abbott Vascular, Medtronic, and W.L. Gore. A.V. Finn has received honoraria from Boston Scientific, Abbott Vascular, and CeloNova. L. Perkins is a full-time employee of Abbott Vascular. S. Hossainy is a full-time employee of Abbott Vascular. S. Pacetti is a full-time employee of Abbott Vascular. The other authors have no conflicts of interest to declare.

Supplementary data

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