Micropatterned surfaces as biointerface to reduce platelet adhesion in cardiovascular devices

Surface micropatterning is being explored as a strategy to mitigate thrombus formation and reduce long-term anticoagulation requirements in left ventricular assist devices (LVADs). This study investigated whether specific micro-topographies can modulate platelet deposition under LVAD supraphysiological wall shear stress (WSS) conditions. A custom microfluidic platform was developed to generate a linear WSS gradient from 16 to 130Pa. Microchannels were patterned with reverse cones and hemispheres in small (1-3µm) and large (3-9µm) sizes using two-photon polymerization and nanoimprinting. Human blood was perfused through the microchannels, and platelet deposition was quantified over time as the area coverage ratio (Aₚₗₜ/Aₜₒₜ) and area under the curve (AUC). Platelet detachment events were counted as an indicator of thrombus stability, and computational simulations supported the interpretation of local shear conditions. Consistent trends emerged,although no statistical differences were observed relative to flat controls. Aₚₗₜ/Aₜₒₜ increased with WSS for all surfaces. At 16Pa, small and large cones reduced platelet adhesion by approximately 84 and 98%, respectively, compared to flat controls. At 49Pa, the reduction was about 95% for small cones and 80% for large cones. Conical geometries also promoted platelet washout at higher WSS. Small hemispherical features showed more than 50% lower platelet adhesion than flat surfaces for WSS > 16Pa, with limited thrombus growth. Conical micropatterns may be most effective at limiting platelet adhesion at lower WSS, while small hemispheres may perform better at higher WSS. These trends suggest a surface-shear interaction that warrants further investigation for LVAD surface optimization.

Platelet aggregation, in fundamental terms, is considered a biological end point that contributes to the occurrence of clinical events among patients with advanced atherosclerotic coronary artery disease. Acute coronary syndrome, including non–ST elevation myocardial infarction (NSTEMI) and ST elevation myocardial infarction (STEMI), accounts for upward of 733 000 hospital admissions yearly in the United States.1 The primary pathophysiological mechanism responsible for the majority of acute coronary syndromes is endothelial plaque disruption with and subsequent platelet adhesion, activation, and thrombus formation.2 The end result is formation of thrombus within a coronary artery, leading to subtotal vessel occlusion with NSTEMI and complete occlusion of the artery with STEMI. In this review, we provide a contemporary view of platelet adhesion as a highly coordinated and teleologically conserved process achieved by surface receptors, protein ligands, and matrix proteins operating at the platelet–subendothelium interface. We also discuss drugs in development, including monoclonal antibodies, inhibitory peptides, and oligonucleotides; preclinical data; and, where available, clinical trial results, highlighting the potential translation of fundamental constructs in platelet biology to patient care. Platelets are derived from a hematopoietic bone marrow stem cell precursor.3 Through a highly conserved program of cellular differentiation, this stem cell precursor becomes a megakaryocyte at a rate of approximately 100 000 000 megakaryocytes produced per day.3 Subsequently, each individual megakaryocyte will give rise to approximately 500 platelets via additional developmental steps within the bone marrow that involve a proplatelet intermediate stage.3 Once released into circulation, a mature platelet has an expected life span of 7 to 10 days.3 Platelet maturation and development involve the expression of receptors on the platelet cell surface. These receptors facilitate platelet adhesion and activation, and they promote thrombus development through receptor–ligand interactions, with several ligands expressed on the surface of endothelial cells, within the …

Introduction: We have developed a novel photoreactive peptide that can be universally modified onto the lumen surfaces of native vessels and cardiovascular devices through brief UV exposure. This peptide enhances endothelial cell recruitment while preventing platelet adhesion. We hypothesize that this photoreactive peptide can improve the blood-contact surfaces of cardiovascular devices, including autologous grafts, synthetical conduits, patches, and tissue-engineered vascular grafts, to promote endothelialization and reduce the risk of restenosis. Material and Methods: The hemocompatible peptide (His–Gly–Gly–Val–Arg–Leu–Tyr; HGGVRLY) was conjugated with a phenylazide group, enabling its modification onto various materials within 2 minutes of UV exposure. Tests were conducted on native and decellularized rat abdominal aorta (AA) as well as expanded polytetrafluoroethylene (ePTFE). The binding of endothelial cells and platelets to the modified surfaces was assessed. Additionally, in vivo performance of the modified decellularized vascular grafts was evaluated through implantation. Results: The photoreactive peptide demonstrated efficient and stable modification onto native and decellularized vessels as well as ePTFE, highlighting its universal photo-induced properties (Fig. 1). The modification process, involving immersion in the peptide solution followed by a 2-minute UV exposure, was completed within 5 minutes. Endothelial cell binding on vessels and ePTFE was significantly enhanced after modification, accompanied by reduced platelet adhesion (Fig. 2). In vivo studies of modified decellularized vascular grafts implanted into rat AA showed a significantly reduced incidence of occlusion during medium-term follow-up. Moreover, the photoreactive peptide remained stable for up to two months post-implantation, confirming the durability of the photoreactive conjugation (Fig. 3). Conclusions: This photoreactive peptide can be efficiently and stably modified onto a variety of material surfaces, including native tissue and synthetic materials. By promoting endothelial recruitment and reducing platelet adhesion, this peptide has the potential to enhance the performance of autologous native grafts and cardiovascular devices in surgical applications.

The endothelium is a critical mediator of homeostasis on blood-contacting surfaces in the body, serving as a selective barrier to regulate processes such as clotting, immune cell adhesion, and cellular response to fluid shear stress. Implantable cardiovascular devices, including stents, vascular grafts, heart valves, and left ventricular assist devices, are in direct contact with circulating blood and carry a high risk for platelet activation and thrombosis without a stable endothelial cell (EC) monolayer. Development of a healthy endothelium on the blood-contacting surface of these devices would help ameliorate risks associated with thrombus formation and eliminate the need for long-term antiplatelet or anticoagulation therapy. Although ECs have been seeded onto or recruited to these blood-contacting surfaces, most ECs are lost upon exposure to shear stress due to circulating blood. Many investigators have attempted to generate a stable EC monolayer by improving EC adhesion using surface modifications, material coatings, nanofiber topology, and modifications to the cells. Despite some success with enhanced EC retention in vitro and in animal models, no studies to date have proven efficacious for routinely creating a stable endothelium in the clinical setting. This review summarizes past and present techniques directed at improving the adhesion of ECs to blood-contacting devices. Impact statement Clinical success of blood-contacting devices such as vascular grafts, stents, and heart valves has remained limited by postimplantation problems, including thrombosis and loss of patency. Without a stable endothelial cell (EC) monolayer, blood-contacting devices are at risk for platelet activation and thrombosis. Methods to improve EC adhesion on these devices have not translated to long-term in vivo success, as many ECs are lost after exposure to circulating blood. In this study, we summarize methods to improve EC adhesion and retention. Successful endothelialization of blood-contacting devices may improve patient outcomes after device implantation and limit the need for long-term antiplatelet or anticoagulation therapy.

Although coronary stents have improved the early and long-term consequences of arterial lesions, the prevention of restenosis and late stent thrombosis is key to prevent a new obstruction of the vessel. Here we aimed at improving the tissue response to stents through surface modification. For that purpose, we used two different approaches, the use of nanostructuration by electrochemical anodization and the addition of a quercitrin (QR) coating to the Ti surface. Four surfaces (Ti, NN, TiQR and NNQR) were characterized by atomic force microscopy, scanning electronic microscopy and contact angle analysis and QR content was evaluated by fluorescent staining. Cell adhesion, cytotoxicity, metabolic activity and nitric oxide (NO) production was evaluated on primary human umbilical cord endothelial cells (HUVECs). Platelet adhesion, hemolysis rate and Staphylococcus epidermidis CECT 4184 adhesion at 30 min were analyzed. Nanostructuration induced an increase on surface roughness, and QR coating decreased the contact angle. All surfaces were biocompatible, with no hemolysis rate and lower platelet adhesion was found in NN surfaces. Finally, S. epidermidis adhesion was lower on TiQR surfaces compared to Ti. In conclusion, our results suggest that NN structuration could improve biocompatibility of bare metal stents on endothelial cells and reduce platelet adhesion. Moreover, QR coating could reduce bacterial adhesion.

Reduced platelet adhesion and blood coagulation on cross-linked albumin films

In vivo assessment of dual-function submicron textured nitric oxide releasing catheters in a 7-day rabbit model

Polyaniline is a promising conducting polymer with still increasing application potential in biomedicine. Its surface modification can be an efficient way how to introduce desired functional groups and to control its properties while keeping the bulk characteristics of the material unchanged. The purpose of the study was to synthetize thin films of pristine conducting polyaniline hydrochloride, non-conducting polyaniline base and polyaniline modified with poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPSA) and investigate chosen parameters of their hemocompatibility. The modification was performed either by introduction of PAMPSA during the synthesis or by reprotonation of polyaniline base. The polyaniline hydrochloride and polyaniline base had no impact on blood coagulation and platelet adhesion. By contrast, the polyaniline reprotonated with PAMPSA completely hindered coagulation thanks to its interaction with coagulation factors Xa, Va and IIa. The significantly lower platelets adhesion was also found on this surface. Moreover, this film maintains its conductivity at pH of 6, which is an improvement in comparison with standard polyaniline hydrochloride losing most of its conductivity at pH of 4. Polyaniline film with PAMPSA introduced during synthesis had an impact on platelet adhesion but not on coagulation. The combined conductivity, anticoagulation activity, low platelet adhesion and improved conductivity at pH closer to physiological, open up new possibilities for application of polyaniline reprotonated by PAMPSA in blood-contacting devices, such as catheters or blood vessel grafts.

Recent Advances in the Understanding of Thrombosis.

The adhesion of human blood platelets onto vascular catheters was studied using a specially designed perfusion chamber. Polyurethane catheters were exposed to citrated human blood for different periods (up to 20 min) and at different wall shear rates (190, 260, 330 sec-1). The rate of platelet adhesion was determined using 111In-labeled platelets, while the morphology of adhering platelets was investigated using scanning electron microscopy. A linear increase in platelet adhesion was found within the first 10 min of perfusion, after which a plateau value was reached. The number of adhering platelets did not vary significantly with the shear rates applied, which may indicate that within the range of shear rates studied, the adhesion of platelets onto the catheter surface is mainly determined by the rate of the reaction between the platelets and the material surface. Catheters coated with a conjugate of heparin and albumin showed a four- to five-fold reduction in platelet adhesion as compared to uncoated catheters. This reduction in platelet adhesion was not only due to the presence of albumin moieties at the surface but also to the presence of heparin residues in the adsorbed albumin-heparin conjugate.

Poly(etherurethane)s are widely used as blood-contacting biomaterials due to their good biocompatibility and mechanical properties. Nevertheless, their blood compatibility is still not adequate for the more demanding applications. Surface modification is an effective way to improve the blood compatibility and retain the bulk properties of biomaterials. The purpose of present study was to design and synthesize a novel nonthrombogenic biomaterial by modifying the surface of poly(etherurethane) with zwitterionic monomer. Films of polyurethane were grafted with sulfobetaine by a three-step procedure. In the first step, the film surfaces were treated with hexamethylene diisocyanate (HDI) in toluene at 50 degrees C in the presence of di-n-butyl tin dilaurate (DBTDL) as a catalyst. The extent of the reaction was measured by ATR-IR spectra; a maximum number of free NCO group was obtained after a reaction time of 90 min. In the second step, the hydroxyl group of 4-dimethylamino-1-butanol (DMAB) was allowed to react in toluene with isocyanate groups bound on the surface. In the third step, sulfobetaine was formed on the surface through the ring-opening reaction between tertiary amine of DMAB and 1,3- propane-sultone (PS). It was characterized by ATR-IR, XPS. The data showed that the grafted surfaces were composed of sulfobetaine. The results of the contact angle measurements showed that they were strongly hydrophilic. The state of platelet adhesion and shape variation for the attached platelets was described. The modified surface shows excellent blood compatibility feature by the low platelet adhesion.

Platelet adhesion is a key event in thrombus development on blood-contacting medical devices. It has been demonstrated that changes to the chemistry of a material surface can reduce platelet adhesion. In this work, it is hypothesized that sub-micron surface textures may also reduce adhesion via a decrease in the surface area of material with which platelets can make contact, and hence a decreased probability of interaction with adhesive ligands. A polyether(urethane urea) was textured with two different sizes of sub-micron pillars using a replication molding technique that did not alter the material surface chemistry. Adhesion of platelets was assessed in a physiologically relevant shear stress range of 0-67 dyn/cm2 using a rotating disk system. Platelets were immunofluorescently labeled and adhesion was compared on smooth and textured samples. Platelet adhesion was greatest at low shear stress ranging from 0 to 5 dyn/cm2, and sub-micron textures were observed to reduce platelet adhesion in this range. Additionally, non-adherent platelets did not demonstrate large-scale activation after exposure to textured samples. We conclude that surface textures with sub-platelet dimensions may reduce platelet adhesion from plasma to polyether(urethane urea) at low shear stress.

Shear-induced platelet activation (SIPA) has been identified to induce platelet adhesion and thrombus formation in continuous flow left ventricular assist devices (LVAD). Platelet glycoprotein (GP) IIb/IIIa receptor inhibitors are effective to prevent SIPA. However, systemic GP IIb/IIIa receptor inhibitor application is associated with severe bleeding complications. The aim of the study was to evaluate (i) the feasibility of absorption and elution of the GP IIb/IIIa receptor blocker TAK-029 from the Ti6Al4V surface of the pump; and (ii) the effect of local GP IIb/IIIa receptor blocker delivery regarding platelet adhesion on the surface of a continuous flow VAD model. Saturating concentrations of TAK-029 were adsorbed on the surface of a centrifugal pump. Whole human blood was perfused in circulatory mock loops using untreated (control), albumin-coated, or TAK-029-coated pumps. Peripheral resistance of the circulatory systems were adjusted accordingly to generate 5 L flow per min with impeller rotational speeds of 3500 (high-shear group) and 1500 rpm (low-shear group), respectively. Platelet adhesions on the respective impellers were quantified by ELISA and scanning electron microscopy (SEM). TAK-029 elution and half-life time were determined by ELISA. Compared with control, albumin-coated pumps showed 64 and 20% less platelet adhesions in the high- and low-shear group, respectively. TAK-029 coated pumps reduced platelet adhesion by additional 33 and 65%, respectively, compared with the albumin group. Elution of TAK 029 was initially very rapid and continued slowly. The results show that it is possible to adsorb and elute a small molecular weight GP IIb/IIIa receptor blocker from the pump surface. This drug elution reduced platelet adhesion on the pump significantly. Further studies are necessary to find a suitable drug bonding that will prolong the antiplatelet effect and preclude any bleeding complication caused by this procedure.

Ten specially synthesized polyurethanes (PUs) were used to investigate the effects of surface properties on platelet adhesion. Surface composition and hydrophilicity, fibrinogen (Fg) and von Willebrand's factor (vWf) adsorption, monoclonal anti-Fg binding, and platelet adhesion were measured. PUs preadsorbed with afibrinogenemic plasma or serum exhibited very low platelet adhesion, while adhesion after preadsorption with vWf deficient plasma was not reduced, showing that Fg is the key plasma protein mediating platelet adhesion under static conditions. Platelet adhesion to the ten PUs after plasma preadsorption varied greatly, but was only partially consistent with Fg adsorption. Thus, while very hydrophilic PU copolymers containing PEG that had ultralow Fg adsorption also had very low platelet adhesion, some of the more hydrophobic PUs had relatively high Fg adsorption but still exhibited lower platelet adhesion. To examine why some PUs with high Fg adsorption had lower platelet adhesion, three monoclonal antibodies (mAbs) that bind to sites in Fg thought to mediate platelet adhesion were used. The antibodies were: M1, specific to gamma-chain C-terminal; and R1 and R2, specific to RGD containing regions in the alpha-chain N- and C-terminal, respectively. Platelet adhesion was well correlated with M1 binding, but not with R1 or R2 binding. When these mAbs were incubated with plasma preadsorbed surfaces, they blocked adhesion to variable degrees. The ability of the R1 and R2 mAbs to partially block adhesion to adsorbed Fg suggests that RGD sites in the alpha chain may also be involved in mediating platelet adhesion and act synergistically with the C-terminal of the gamma-chain.

BACKGROUND: Titanium-alloys (Ti) are widely used as blood biocompatible material for blood pumps and coronary stents. Thrombus formation on the Ti surface often results from high shear stress induced-platelet activation, and it is contributory to pump failure and coronary reocclusion, respectively. Although heparin and aspirin are routinely used to prevent thrombus formation in clinical settings, the efficacy is not satisfactory. The aim of this study was to elucidate the mechanisms of thrombus formation on Ti surface induced by high shear stress and to evaluate the efficacy of antithrombotic agents. METHODS: A cone-and-plate viscometer and a parallel flow chamber were used for shear-induced platelet aggregation and adhesion studies, respectively. Platelet microaggregation of citrate anticoagulated platelet rich plasma samples were measured by flow cytometry. Anti-GPIIIa antibody was used to identify the number of platelets adhering to the Ti surface after infusion of heparinized whole blood. RESULTS: Heparin (10 unit/ml) enhanced shear (108 dynes/cm2) induced human platelet aggregation by 22%. Absorptions of vWF and fibrinogen onto Ti surface were increased within 60 min after incubation with plasma and both plasma proteins enhanced platelet adhesion induced by high shear stress (111 dynes/cm2). Indomethacin (30 mM) had no effect on shear-induced platelet adhesion. Maximal concentration of TAK-029, a GPIIb/IIIa antagonist, prevented the platelet adhesions to non-, vWF- and fibrinogen-coated Ti surface by 89%, 75% and 98%, respectively. CONCLUSION: Treatment with a GPIIb/IIIa antagonist is an appropriate option to prevent shear-induced thrombus formation on Ti surface.