A Recombinant Fusion Construct between Human Serum Albumin and NTPDase CD39 Allows Anti-Inflammatory and Anti-Thrombotic Coating of Medical Devices

Medical devices directly exposed to blood are commonly used to treat cardiovascular diseases. However, these devices are associated with inflammatory reactions leading to delayed healing, rejection of foreign material or device-associated thrombus formation. We developed a novel recombinant fusion protein as a new biocompatible coating strategy for medical devices with direct blood contact. We genetically fused human serum albumin (HSA) with ectonucleoside triphosphate diphosphohydrolase-1 (CD39), a promising anti-thrombotic and anti-inflammatory drug candidate. The HSA-CD39 fusion protein is highly functional in degrading ATP and ADP, major pro-inflammatory reagents and platelet agonists. Their enzymatic properties result in the generation of AMP, which is further degraded by CD73 to adenosine, an anti-inflammatory and anti-platelet reagent. HSA-CD39 is functional after lyophilisation, coating and storage of coated materials for up to 8 weeks. HSA-CD39 coating shows promising and stable functionality even after sterilisation and does not hinder endothelialisation of primary human endothelial cells. It shows a high level of haemocompatibility and diminished blood cell adhesion when coated on nitinol stents or polyvinylchloride tubes. In conclusion, we developed a new recombinant fusion protein combining HSA and CD39, and demonstrated that it has potential to reduce thrombotic and inflammatory complications often associated with medical devices directly exposed to blood.


Introduction
Cardiovascular diseases such as ischemic heart disease and stroke are the world's leading causes of death. The World Health Organization states that 16% of total deaths can be traced back to these diseases [1]. Treatment of patients with cardiovascular problems often includes the invasive application of medical devices. Often these medical devices will be directly exposed to blood, e.g., vascular grafts, stents, permanently implantable biosensors The ectonucleoside triphosphate diphosphohydrolase-1, an NTPDase (CD39), is a promising anti-thrombotic and anti-inflammatory agent [36][37][38][39][40]. Normally expressed on the surface of endothelial cells (ECs), CD39 prevents platelet activation and attachment through hydrolysis of the phosphate residue of ATP and ADP [38,[41][42][43]. ATP triggers pro-inflammatory pathways, so the degradation of ATP to ADP by CD39 reduces the proinflammatory effect of ATP. ADP is a major player in the platelet-activation cascade [39,44]. Through further hydrolysis of ADP to AMP by CD73, CD39 is responsible for a shift from a pro-inflammatory to an anti-inflammatory environment [39,40,45]. Several studies have confirmed that CD39 activity is substantively reduced in injured or rejected grafts, and that administration of soluble CD39 may be a useful substitute post implantation [41,46].
Here, we have designed, generated and analysed a novel anti-thrombotic and antiinflammatory recombinant fusion protein consisting of HSA and CD39 as a highly promising bioactive coating for medical devices and PVC tubes to guarantee an active, safe and natural interface between blood and medical devices.

Materials and Methods
A more detailed description of the methods is provided in the Supplementary Material.

Generation of Recombinant Fusion Construct, Production, Expression of Protein and Purification
Details of HSA-CD39 origin, polymerase chain reaction (PCR)-based fusion, mammalian production (HEK293 cells) and purification are provided as Supplementary Methods. The quantity of the purified protein was measured using a Pierce Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA). The samples from purification steps were loaded onto a 12% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis under denaturing conditions and visualized via Coomassie staining. The same samples were also stained on a Western blot (BioRad, Hercules, CA, USA) using an anti-Penta-His antibody (Roche, Basel, Switzerland) coupled with horseradish peroxidase.

Blood Sampling from Healthy Human Volunteers
All blood sampling procedures were approved by the Research and Ethics Unit of the University of Tübingen, Germany (project number 270/2010BO1) and the Ethics Committee of the Alfred Hospital, Melbourne, Australia. Unless otherwise specified, blood was collected by venepuncture from healthy volunteers who provided informed consent and was anticoagulated with citrate. All subjects were free of platelet-affecting drugs for ≥14 days.

Preparation of Platelet-Rich Plasma
Citrated blood from volunteers was centrifuged at 180× g for 10 min. Platelet-rich plasma (PRP) was collected and stored at 37 • C. Before usage it was diluted 1:10 with phosphate-buffered saline plus (PBS+; 100 mg/L calcium chloride, 100 mg/L magnesium chloride; ThermoFisher Scientific, Waltham, MA, USA). Blood and PRP were used within the first 6 h after venepuncture.

Flow Cytometry
The efficiency and functionality of the HSA-CD39 protein were determined using flow cytometry. The protein was incubated with 20 µM ADP (MoeLab, Langenfeld, Germany) or 5 µL PBS for 20 min. The active protein will hydrolyse ADP to AMP. Diluted PRP was added and incubated for 5 min. Platelet activation status was measured by a fluorescein isothiocyanate (FITC)-labelled monoclonal antibody PAC-1 (BD Bioscience, Franklin Lakes, NJ, USA), a R-phycoerythrin (PE)-labelled monoclonal antibody directed against CD62P (P-Selectin) (BD Bioscience, Franklin Lakes, NJ, USA) or their respective isotype antibody controls (ThermoFisher Scientific, Waltham, MA, USA). Samples were fixed using Cellfix (BD Bioscience, Franklin Lakes, NJ, USA) and analysed via fluorescence-activated cell sorting (FACS) Calibur (BD Bioscience, Franklin Lakes, NJ, USA). A total of 10,000 events were acquired in each sample.

ADP Bioluminescence Assay
HSA-CD39 s function to directly hydrolyse ADP was measured using an ATP bioluminescence assay according to the manufacturer's description (Kit CLS II, Roche, Basel, Switzerland) [39,41]. HSA-CD39 was incubated with 20 µM ADP for 20 min. The remaining ADP was converted to ATP by the pyruvate kinase reaction, and measured using the ATP bioluminescence assay via a microplate luminometer (Mithras LB 940, Berthold Technologies, Bad Wildbad, Germany). Different concentrations of ADP, PBS and HSA (Alburex Human albumin 5%, CSL Behring, Hattersheim am Main, Germany) were also used as controls.

Lyophilisation of Protein
To analyse the possibility of lyophilising the HSA-CD39 protein, different concentrations were lyophilised using the CoolSafe ScanVac (LaboGene ApS, Lynge, Denmark) according to the manufacturer's description. The lyophilised samples were stored for 14 days at room temperature (RT) before rehydration and analysis of platelet activation using flow cytometry.

Coating of Stent Material for In Vitro Analysis
HSA-CD39 and HSA (CSL Behring, Hattersheim am Main, Germany) proteins were passively adsorbed by the different materials. The samples were diluted in PBS, added, incubated and dried on the materials with HSA-CD39 and HSA (CSL Behring, Hattersheim am Main, Germany). Materials were then stored for 24 h before flow cytometric analysis of CD39 activity. Polystyrene (BD Bioscience, Franklin Lakes, NJ, USA), 316L stainless-steel plates, Ti plates (Acandis, Pforzheim, Germany), polyurethane-coated stents (Acandis, Pforzheim, Germany) and nitinol BlueOxide stents (Acandis, Pforzheim, Germany) were coated with different protein concentrations (0.05 µg, 0.1 µg, 0.25 µg and 0.5 µg) and PBS as the control. Coated 316L stainless-steel plates were washed 3× with PBS and dried again prior to functional testing. Coated Ti plates were sterilized with ethylene oxide (EO) according to the sterilization protocol for medical devices of the University Hospital of Tübingen, Germany. Long-term-coated material was stored at RT. DERIVO nitinol BlueOxide stents (3.3 × 15 mm, Acandis, Pforzheim, Germany) were coated by dip-coating of stents with 100 µg/mL HSA-CD39 in PBS 10× and dried with argon gas between dipping steps. Coated stents were also sterilized by EO according to the sterilization protocol.

In Vitro Haemocompatibility Testing Using Roller Pump and Modified Chandler Loop Model
To investigate the influence of HSA-CD39-coated nitinol BlueOxide stents (nitinol BlueOxide DERIVO embolisation device, Acandis, Pforzheim, Germany) in vitro, coated plates were loaded into heparin-coated Tygon tubes (Saint Gobain Performance Plastics, Wertheim, Germany). PVC tubes (inner diameter 3.2 mm, length 75 cm) were coated with heparin by Ension (Pittsburgh, PA, USA). Through this model, the haemocompatibility of the coated stents, i.e., activation of the coagulation cascade, the complement system and inflammation, were analysed after perfusion of blood, as described in detail by Krajewski et al. [48]. Human whole blood was anticoagulated with heparin (1.5 IE/mL, Ratiopharm GmbH, Ulm, Germany). Then, each tube was filled with 6 mL freshly heparinised human blood, connected by a silicon connection tubing and circulated by a roller pump (BVP Ismatec, Wertheim, Germany) in a water bath at 37 • C for 60 min at 150 mL/min. For each of the 5 donors, 6 mL heparinised blood was used for baseline measurements before circulation. Before and after circulation, blood was taken, measured with a haematolyser (ABX Micros 60, Axon Lab AG, Baden, Switzerland) for blood cell count and further used for enzyme-linked immunosorbent assays (ELISA) (Echelon Biosciences, Salt Lake City, UT, USA) [45,46]. For measuring thrombin-antithrombin III complex (TAT complex; Enzygnost TAT Micro, Siemens Healthcare, Erlangen, Germany) via ELISA, blood was directly filled in citrate S-Monovettes ® (Sarstedt AG & Co, Nümbrecht, Germany) and centrifuged at 1800× g at 22 • C for 18 min. Resulting plasma was deep-frozen in liquid nitrogen and stored at −20 • C until performance of ELISA, according to the manufacturer's description. Stents were prepared for scanning electron microscopy (SEM).
In a second experimental setup, the previously established modified chandler loop was used to test the haemocompatibility of coated ECC tubes [10,48]. PCV tubes (lengths of 50 cm; Raumedic ® ECC BloodLine 1/4 × 1/16, Raumedic AG, Helmbrechts, Germany) were coated via rotating incubation with 12 mL of 20 µg/mL (240 µg) HSA or 20 µg/mL (240 µg) HSA-CD39 at RT for 3 h followed by storage at 4 • C overnight. The pH of the protein solutions were adjusted to 4.6 prior to incubation. After incubation, tubes were rinsed with PBS prior to being filled with blood. An untreated tube without blood contact, an untreated tube and a commercially available heparin-coated tube (Carmeda BioActive Surface ® , Medtronic, Dublin, Ireland) were used as controls. Coated tubes were filled with fresh, pooled and heparinised blood (1 IE/mL) and closed into a ring. Blood was circulated in a water bath at 37 • C for 90 min (30 rotations/min). Afterwards, tubes were washed with PBS and fixed with 2% glutaraldehyde (GA), then PVC pieces were prepared for SEM.

Statistical Analysis
Unless otherwise specified, data are represented as mean ± standard deviation (SD). All analyses containing more than two groups were analysed with one-way analysis of variance (ANOVA), comparing all groups with one another, corrected by post hoc Bonferroni analysis or Dunnett's/Sidak's test, and the corrected p-values are given. Multiple comparisons were analysed with two-way ANOVA and Dunnett's multiple comparison. All analyses for two groups were performed using Student's t-tests. The statistical analyses were performed with the statistical software package GraphPad Prism (version 6.0, GraphPad Software, San Diego, CA, USA). Statistical significance was defined as p < 0.05.

Generation, Production and Enzymatic Activity of Recombinant Fusion Protein HSA-CD39
For the generation of our recombinant fusion protein consisting of HSA and CD39, the DNA sequence of HSA was inserted into a previously described pSectag2A vector containing the CD39 sequence [39]. The resulting HSA-CD39 was further digested, purified and inserted into a gWiz vector to yield a higher production rate ( Figure 1A). Following double digestion of both constructs, the HSA-CD39 insert was visualised via agarose gel at 3235 bp ( Figure 1A). Confirmation of successful molecular biology was made by colony screening of clones via PCR sequencing, where positive clones resulted in a 2149 bp  Figure 1C). After DNA sequencing confirmation, the DNA was produced by HEK293F cells and purified afterwards. The protein purity of the HSA-CD39 fusion protein was analysed on SDS-PAGE and a band was observed between the 100 kDa and 150 kDa marks ( Figure 1D). Specificity of the HSA-CD39 construct was shown by Western blotting via the use of an anti-Penta-His antibody, which was coupled with horseradish peroxidase (141 kDa, Figure 1E). 150 kDa marks ( Figure 1D). Specificity of the HSA-CD39 construct was shown by Western blotting via the use of an anti-Penta-His antibody, which was coupled with horseradish peroxidase (141 kDa, Figure 1E).
Direct analysis of ADP dephosphorylation by HSA-CD39 was conducted using an ATP bioluminescence assay. Increasing concentrations of HSA-CD39 resulted in linear and significant reductions in ADP concentration in comparison to the HSA control ( Figure  4A). Similar effects were observed using lyophilised HSA-CD39, which demonstrates its long-term stability ( Figure 4B). The functionality of HSA-CD39 was investigated every week for two months (dried in polystyrene tubes and stored at RT) via flow cytometry ( Figures 3C and S2). HSA-CD39 hydrolysed ADP and stopped platelet activation at ≥0.25 µg protein after rehydration, compared to samples without HAS-CD39 after week 1 (0.40 ± 0.17 vs. 59.33 ± 30.28; p < 0.001) and week 8 (3.80 ± 2.33 vs. 43.23 ± 6.73; % activated platelets ± SD, p < 0.001). The enzymatic properties of HAS-CD39 were similarly active through the 8 weeks of storage.
Direct analysis of ADP dephosphorylation by HSA-CD39 was conducted using an ATP bioluminescence assay. Increasing concentrations of HSA-CD39 resulted in linear and significant reductions in ADP concentration in comparison to the HSA control ( Figure 4A). Similar effects were observed using lyophilised HSA-CD39, which demonstrates its longterm stability ( Figure 4B).

HSA-CD39 Fusion Protein Coating Allows for Endothelialisation
Fluorescence microscopy images of the HSA-CD39-coated nitinol BlueOxide plates displayed good endothelialisation performance. Sterilised plates were coated with HSA-CD39 or just PBS. hECs were seeded onto the coated plates, followed by incubation for 48 h. No differences between the DAPI-stained hECs regarding cell morphology, cell growth and cell count could be detected compared to the non-coated bare nitinol BlueOxide plates ( Figure 6A,B). Additional quantitative analysis of the microscope pictures using ImageJ confirmed this result ( Figure 6C).

Haemocompatibility and In Vitro Proof of Function of HSA-CD39-Coated Nitinol Blue Oxide Stents and PVC Tubes
HSA-CD39-coated nitinol BlueOxide stents and uncoated stents were loaded into PCV tubes and incubated with fresh human blood to determine their haemocompatibility and thrombogenicity [22,38]. PVC tubes without stents were used as an additional control. A baseline reading was analysed before the blood was placed into circulation through the stents or tubes. At the endpoint, the blood was collected for comparison analysis. No significant changes were measured in white blood cells, red blood cells, haemoglobin or haematocrit compared to the baseline, the control tube without stent and the uncoated stent ( Figure 7A-D). Significant reductions in the number of platelets were found when blood was circulated in the PVC control tube and the uncoated groups, compared to the baseline (221,000.6 ± 23,000.84 and 133,000.2 ± 24,000.3 vs. 259,000 ± 36,000, respectively; number of platelets/µL ± SD, p < 0.05). However, no difference was observed in the HSA-CD39-coated nitinol BlueOxide stents as compared to the baseline reading (229,000.0 ± 27,000.74 vs. 259,000 ± 36,000; ns) ( Figure 7E). These results indicate that the platelets in the PVC tube control and the uncoated groups aggregated, whereas no aggregation occurred in the HSA-CD39-coated nitinol BlueOxide stents. Activation of the coagulation cascade was determined by measuring the formation of the TAT complex before and after perfusion ( Figure 7F). An increased readout of the TAT complex for the uncoated stent group was shown compared to the baseline, the control and also the HSA-CD39-coated stent group (446.4 ± 225.5 vs. 2.37 ± 0.45; 24.02 ± 11.45; 42.72 ± 11.26, respectively; µg/L TAT complex formation ± SD, p < 0.01).

HSA-CD39 Fusion Protein Coating Allows for Endothelialisation
Fluorescence microscopy images of the HSA-CD39-coated nitinol BlueOxide plates displayed good endothelialisation performance. Sterilised plates were coated with HSA-CD39 or just PBS. hECs were seeded onto the coated plates, followed by incubation for 48 h. No differences between the DAPI-stained hECs regarding cell morphology, cell growth and cell count could be detected compared to the non-coated bare nitinol BlueOxide plates ( Figure 6A,B). Additional quantitative analysis of the microscope pictures using ImageJ confirmed this result ( Figure 6C).

Haemocompatibility and In Vitro Proof of Function of HSA-CD39-Coated Nitinol Blue Oxide Stents and PVC Tubes
HSA-CD39-coated nitinol BlueOxide stents and uncoated stents were loaded into PCV tubes and incubated with fresh human blood to determine their haemocompatibility and thrombogenicity [22,38]. PVC tubes without stents were used as an additional control. A baseline reading was analysed before the blood was placed into circulation through the stents or tubes. At the endpoint, the blood was collected for comparison analysis. No significant changes were measured in white blood cells, red blood cells, haemoglobin or haematocrit compared to the baseline, the control tube without stent and the uncoated stent ( Figure 7A-D). Significant reductions in the number of platelets were found when blood was circulated in the PVC control tube and the uncoated groups, compared to the baseline (221,000.6 ± 23,000.84 and 133,000.2 ± 24,000.3 vs. 259,000 ± 36,000, respectively; number of platelets/µL ± SD, p < 0.05). However, no difference was observed in the HSA-CD39-coated nitinol BlueOxide stents as compared to the baseline reading (229,000.0 ± 27,000.74 vs. 259,000 ± 36,000; ns) ( Figure 7E). These results indicate that the platelets in the PVC tube control and the uncoated groups aggregated, whereas no aggregation occurred in the HSA-CD39-coated nitinol BlueOxide stents. Activation of the coagulation cascade was determined by measuring the formation of the TAT complex before and after perfusion ( Figure 7F). An increased readout of the TAT complex for the uncoated stent group was shown compared to the baseline, the control and also the HSA-CD39-coated stent group (446.4 ± 225.5 vs. 2.37 ± 0.45; 24.02 ± 11.45; 42.72 ± 11.26, respectively; µg/L TAT complex formation ± SD, p < 0.01). After circulation, uncoated and HSA-CD39-coated stents were also analysed via SEM. Representative SEM images of each stent from the same blood donor, displayed at different magnifications, showed distinct differences in the blood cell adhesion (Figure 8). The uncoated stent showed homogenous adhesion of several blood cells, especially platelets, and an increased fibrin network for all blood donors, such that only a few platelets could be detected on the surface of the HSA-CD39-coated stent (Figure 8). SEM imaging of HSA-CD39 coating on the PVC tubes showed a reduction in cell adhesion on the inner surface of the HSA-CD39-coated tube after circulation as compared to the other control groups (Figure 9). In particular, the non-treated and HSA-coated tubes showed more cell Figure 7. HSA-CD39 coated onto nitinol BlueOxide stents shows no effect on blood haematology or haemocompatibility using a dynamic in vitro thrombogenicity model. Haematology analysis of coated stents before and after circulation for 60 min at 150 mL/min (thrombogenicity model). After circulation, uncoated and HSA-CD39-coated stents were also analysed via SEM. Representative SEM images of each stent from the same blood donor, displayed at different magnifications, showed distinct differences in the blood cell adhesion (Figure 8). The uncoated stent showed homogenous adhesion of several blood cells, especially platelets, and an increased fibrin network for all blood donors, such that only a few platelets could be detected on the surface of the HSA-CD39-coated stent (Figure 8). SEM imaging of HSA-CD39 coating on the PVC tubes showed a reduction in cell adhesion on the inner surface of the HSA-CD39-coated tube after circulation as compared to the other control groups (Figure 9). In particular, the non-treated and HSA-coated tubes showed more cell adhesion compared to the HSA-CD39-coated tube.
After circulation, uncoated and HSA-CD39-coated stents were also analysed via SEM. Representative SEM images of each stent from the same blood donor, displayed at different magnifications, showed distinct differences in the blood cell adhesion (Figure 8). The uncoated stent showed homogenous adhesion of several blood cells, especially platelets, and an increased fibrin network for all blood donors, such that only a few platelets could be detected on the surface of the HSA-CD39-coated stent (Figure 8). SEM imaging of HSA-CD39 coating on the PVC tubes showed a reduction in cell adhesion on the inner surface of the HSA-CD39-coated tube after circulation as compared to the other control groups (Figure 9). In particular, the non-treated and HSA-coated tubes showed more cell adhesion compared to the HSA-CD39-coated tube.

Discussion
Medical devices that are directly exposed to blood are often associated with inflammation and thrombus formation [2,3]. The lack of biocompatibility of foreign materials triggers inflammatory processes and activation of the coagulation cascade, as well as activation and aggregation of platelets in the blood [49][50][51]. The use of drug-eluting materials and extensive anti-platelet therapy after surgery have shown improvements in safety

Discussion
Medical devices that are directly exposed to blood are often associated with inflammation and thrombus formation [2,3]. The lack of biocompatibility of foreign materials triggers inflammatory processes and activation of the coagulation cascade, as well as activation and aggregation of platelets in the blood [49][50][51]. The use of drug-eluting materials and extensive anti-platelet therapy after surgery have shown improvements in safety and efficiency. However, adverse drug interactions, pro-thrombotic events, poor endothelialisation, hypersensitivity and bleeding complications still occur frequently [24]. Therefore, research on a natural, non-allergic, bio-and haemo-compatible medical coating is required. In this study, we genetically designed the fusion of HSA to CD39 in order to engineer a recombinant multifunctional fusion protein which provides an ideal coating strategy for blood-contacting material. The HSA component allows adherence of our fusion protein to be passively adsorbed onto the materials, whilst the attached CD39 component prevents thrombotic actions from occurring. The data indicate that our HSA-CD39 fusion protein is stable in storage and is still highly functional in reducing platelet activation and adhesion for up to 8 weeks (Figures 3 and 4B). HSA-CD39 also protects platelet activation and inflammation processes, which are commonly evoked by foreign materials such as stainless steel, Ti, nitinol (an alloy of nickel and Ti), polyurethane stents and PVC ( Figure 5).
CD39 is a membrane-bound enzyme constitutively expressed on intact ECs. This NTPDase hydrolyses the nucleotides ATP and ADP [39,42,45]. CD39 has attracted major attention as a pharmacological agent [39,42,52,53]. Several studies have shown that the administration of CD39 decreases the risk of thrombosis and protects against myocardial infarction and stroke [54,55]. A hallmark study conducted in transgenic mice expressing CD39 demonstrated increased resistance to thrombosis when challenged by an acute ferricchloride-induced injury to their carotid artery [56]. Furthermore, overexpression of CD39 in rat aortas diminishes the proliferation of smooth vascular cells and prevents neointimal formation after angioplasty [46]. However, direct injection of CD39 is associated with concentration-dependent bleeding complications [39,54]. To overcome this obstacle, our laboratory genetically fused CD39 to a single-chain antibody that was specific to activated platelets, resulting in a successful and bleeding-free targeted therapy in vivo [38][39][40]. We further investigated this construct in a murine model of myocardial ischemia/reperfusion injury, where we demonstrated that the activated-platelet-targeted CD39 provides significant myocardial protection and preserves heart function [40]. Furthermore, using CD39 mRNA, we showed the CD39 protein has active enzymatic properties and can hydrolyse ADP to AMP, thereby preventing platelet activation and proving the therapeutic potential of CD39 [42]. In this current study, we harness the enzymatic properties of CD39 and further utilise HSA for coating on several medically used materials. To demonstrate the anti-thrombotic effects of this fusion protein, we used two markers of platelet activation, the monoclonal antibody PAC-1 (specific for activated GPIIb/IIIa) and an antibody against P-selectin (anti-CD62P) ( Figure S1). Upon platelet activation, GPIIb/IIIa changes from a low-affinity state to a high-binding-affinity state for fibrinogen/fibrin, thereby mediating platelet aggregation [57][58][59]. Being the most abundant platelet receptor, with the high density of 60,000 to 80,000 receptors per platelet, the activation of GPIIb/IIIa and its resulting aggregation is a main contributor to thrombosis [58,60]. P-selectin's role in platelet aggregation is not as dominant, but it is seen as a sensitive marker of platelet activation [41]. Since most blood-contact medical device failures are due to thrombosis, our study has chosen to focus on platelet activation as a readout. Overall, our studies indicated HSA-CD39 fusion protein is highly functional in hydrolysing ADP, a major player in the platelet activation cascade, and at preventing platelet activation, adhesion and aggregation.
HSA has been widely used for the coating of medical products, possibly owing to its inferred safety and stability given its abundance in human serum [61][62][63][64]. Serum hypoalbuminemia has been observed during inflammatory processes and in cardiovascular events [29]. HSA is physiochemically stable and has been studied extensively in relation to clinical use for the maintenance of blood homeostasis in medical conditions [61,63].
Blood contact with foreign materials leads to the adherence of pro-thrombotic plasma proteins (e.g., fibrinogen) on the materials' surfaces, but studies have shown that adsorbed albumin is able to passivate various materials, thereby providing an anti-thrombotic effect by minimising platelet adhesion [26,64,65]. Furthermore, HSA is known to provide an antioxidant effect, reducing complement cascade activation [62,65,66]. Clinically, HSA is used in combination therapy with various drugs and bioactive proteins, or as an encapsulation agent [64].
HSA coated on an arterial polyester prosthesis (Dacron ® ) displayed reduction in platelet adhesion, less activation of the coagulation cascade and decreased formation of fibrinopeptide A, as an index for decreased thrombin action, highlighting the importance of structural design and surface chemistry [67,68]. Additionally, a HSA/polyethylenimine multilayer coating on plasma-treated PVC was shown to resist platelet adhesion effectively [69].
Ti is a material frequently used for orthopaedic implants and cardiovascular devices. Adsorption of HSA into Ti has been shown to prevent adhesion of other blood proteins and reduce bacterial adherence [70,71]. We harnessed these advantages of HSA, especially its passive binding capacity, as part of our fusion protein to improve the biocompatibility of medical devices. Our HSA-CD39 fusion protein provides protection against deviceinduced platelet activation and inflammation processes, and thus minimises bleeding risk and promotes adaptation of the surrounding tissue to the foreign material in situ. We demonstrate the maintained functionality of our generated fusion protein HSA-CD39 on Ti even after sterilisation with EO ( Figure 5E). After radiation, sterilisation via EO is the most commonly used process in the medical device industry and is performed after standard protocols [72,73]. Therefore, we demonstrate a highly stable device coating already suited to clinical translation.
The application of therapeutic recombinant proteins for a safe, biocompatible interface on medical devices has attracted major interest in the biopharmaceutical industry. This includes the pursuit of a perfectly haemocompatible, biopassive surface and the progress in the application of active therapeutic compounds [74,75]. In the development of stents, nitinol combines the properties of elasticity, biocompatibility and the shape-memory effect, which makes it suitable for self-expanding stents. The native oxide layer formed on the surface prevents nickel ions from binding to Ti, resulting in a nickel-free environment and substantially reducing allergic reactions and toxicity [74,75]. To analyse our HSA-CD39 fusion protein in vitro, we used a flow diverter nitinol BlueOxide DERIVO embolisation device, which has been evaluated for the treatment of intracranial aneurysms in clinical trials (Figures 7 and 8) [76].
In the area of coating strategies, antibodies against CD34 and CD133 coated onto stents have been evaluated in a rabbit model, showing reduced intimal proliferation and re-stenosis as compared to bare metal stents and gelatine-coated stents [62,63,75,76]. Using these antibodies to attract vascular-circulating endothelial progenitor cells (EPCs) leads to adhesion of a functional endothelial layer of EPCs on the stent surface after vascular injury. Murine monoclonal CD34-coated stents (GenousTM, OrbusNeich) were proven to be safe and enhance endothelialisation in various clinical trials [77,78]. Our study demonstrates good endothelialisation rates and a reduction in platelet activation ( Figure 6).
Haemocompatibility analysis of our HSA-CD39 protein showed no influence on whole human blood. Using uncoated bare metal nitinol BlueOxide stents, we noted a significant reduction in platelet count, which also implies increased platelet aggregation. Our HSA-CD39-coated stents, on the other hand, showed no significant decrease in platelet count and additionally showed a reduced TAT complex, indicating minimal platelet aggregation and minimal coagulation cascade activation, respectively (Figure 7) [28].
Addressing the issue of the handling and storage of sensitive medical products, our data demonstrate preserved enzymatic activity of HSA-CD39 after drying, lyophilisation, coating, washing, sterilisation and 8 weeks of storage. We have shown that HSA-CD39 can be used as a new coating strategy across various devices and blood-contacting materials.
Our HSA-CD39 protein approach reduces platelet adhesion, activation and further inflammatory processes, therefore providing a great clinical advancement in the realm of bioprostheses by minimising the need for anti-thrombotic therapy, which is inherently linked to potential bleeding complications. There are some limitations to our study. We have shown that HSA-CD39 coating on our materials was present after washing steps were conducted and remained highly functional in its activity to hydrolyse ADP. However, we have not directly measured the amount of protein lost. Different materials may require other coating methods, which may expose our fusion protein to heat or other storage conditions. Although we have demonstrated that our fusion protein is more effective in reducing platelet activation, adhesion and aggregation, we have not systematically defined which of the individual components, HSA or CD39, are the cause of the described benefits. Further characterisations of HSA-CD39 should include the contributions of the individual fusion protein components. We have conducted ex vivo blood circulation and demonstrated that HSA-CD39 coating resulted in less platelet aggregation; however, future in vivo experiments will be conducted to determine the anti-thrombotic and antiinflammatory properties of the materials post implantation. Additionally, the contribution of reduced ADP levels, in comparison to the generation of adenosine via the use of P2Y receptor inhibitors or A2A adenosine receptor blockers in vivo, will allow us to define the effects of HSA-CD39 more thoroughly. In addition, future investigations into the coating strategies, temperature changes and storage conditions, as well as a diverse range of biomaterials, will be explored.

Conclusions
In this study, we have generated a recombinant fusion protein combining the antithrombotic and anti-inflammatory properties of CD39 with HSA as a suitable coating for medical devices in order to reduce foreign-material-associated complications. Our newly designed HSA-CD39 fusion protein is highly functional in preventing platelet activation, adhesion and aggregation. It is also stable after EO sterilisation and can be coated onto several materials typically used in medical devices. HSA-CD39 coating can mitigate the healing process, improve the incorporation of foreign material into the surrounding tissue and reduce interactions with blood components such as coagulation proteins, platelets and leukocytes. Overall, our HSA-CD39 fusion protein is a natural bioactive interface which is highly potent in the prevention of platelet activation and inflammation; therefore, its use for medical device coating provides potential benefits for patients.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/pharmaceutics13091504/s1, Figure S1: Representative images of fluorescence histograms via flow cytometry demonstrating HSA-CD39 prevents platelet activation using two markers of platelet activation; Figure S2: Flow cytometry demonstrating HSA-CD39 can be dried in polystyrene tubes and stored at RT for up to 7 weeks.