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

Catheter-induced thrombosis is a major contributor to infectious and mechanical complications of biomaterials that lead to device failure. Herein, a dualfunction submicron textured nitric oxide (NO)-releasing catheter was developed. The hemocompatibility and antithrombotic activity of vascular catheters were evaluated in both 20 h in vitro blood loop and 7 d in vivo rabbit model. Surface characterization assessments via atomic force microscopy show the durability of the submicron pattern after incorporation of NO donor S-nitroso-N-acetylpenicillamine (SNAP). The SNAP-doped catheters exhibited prolonged and controlled NO release mimicking the levels released by endothelium. Fabricated catheters showed cytocompatibility when evaluated against BJ human fibroblast cell lines. After 20h in vitro evaluation of catheters in a blood loop, textured-NO catheters exhibited a 13-times reduction in surface thrombus formation compared to the control catheters, which had 83% of the total area covered by clots. After the 7 d in vivo rabbit model, analysis on the catheter surface was examined via scanning electron microscopy, where significant reduction of platelet adhesion, fibrin mesh, and thrombi can be observed on the NO-releasing textured surfaces. Moreover, compared to relative controls, a 63% reduction in the degree of thrombus formation within the jugular vein was observed. Decreased levels of fibrotic tissue decomposition on the jugular vein and reduced platelet adhesion and thrombus formation on the texture of the NO-releasing catheter surface are indications of mitigated foreign body response. This study demonstrated a biocompatible and robust dual-functioning textured NO PU catheter in limiting fouling-induced complications for longer-term blood-contacting device applications. STATEMENT OF SIGNIFICANCE: Catheter-induced thrombosis is a major contributor to infectious and mechanical complications of biomaterials that lead to device failure. This study demonstrated a robust, biocompatible, dual-functioning textured nitric oxide (NO) polyurethane catheter in limiting fouling-induced complications for longer-term blood-contacting device applications. The fabricated catheters exhibited prolonged and controlled NO release that mimics endothelium levels. After the 7 d in vivo model, a significant reduction in platelet adhesion, fibrin mesh, and thrombi was observed on the NO-releasing textured catheters, along with decreased levels of fibrotic tissue decomposition on the jugular vein. Results illustrate that NO-textured catheter surface mitigates foreign body response.

In vitro assessment and phase I randomized clinical trial of anfibatide a snake venom derived anti-thrombotic agent targeting human platelet GPIbα.

The interaction of platelet GPIbα with von Willebrand factor (VWF) is essential to initiate platelet adhesion and thrombosis, particularly under high shear stress conditions. However, no drug targeting GPIbα has been developed for clinical practice. Here we characterized anfibatide, a GPIbα antagonist purified from snake (Deinagkistrodon acutus) venom, and evaluated its interaction with GPIbα by surface plasmon resonance and in silico modeling. We demonstrated that anfibatide interferds with both VWF and thrombin binding, inhibited ristocetin/botrocetin- and low-dose thrombin-induced human platelet aggregation, and decreased thrombus volume and stability in blood flowing over collagen. In a single-center, randomized, and open-label phase I clinical trial, anfibatide was administered intravenously to 94 healthy volunteers either as a single dose bolus, or a bolus followed by a constant rate infusion of anfibatide for 24 h. Anfibatide inhibited VWF-mediated platelet aggregation without significantly altering bleeding time or coagulation. The inhibitory effects disappeared within 8 h after drug withdrawal. No thrombocytopenia or anti-anfibatide antibodies were detected, and no serious adverse events or allergic reactions were observed during the studies. Therefore, anfibatide was well-tolerated among healthy subjects. Interestingly, anfibatide exhibited pharmacologic effects in vivo at concentrations thousand-fold lower than in vitro, a phenomenon which deserves further investigation.Trial registration: Clinicaltrials.gov NCT01588132.

Assessment of the effects of Syk and BTK inhibitors on GPVI-mediated platelet signaling and function.

Spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (BTK) play critical roles in platelet physiology, facilitating intracellular immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling downstream of platelet glycoprotein VI (GPVI) and GPIIb/IIIa receptors. Small molecule tyrosine kinase inhibitors (TKIs) targeting Syk and BTK have been developed as antineoplastic and anti-inflammatory therapeutics and have also gained interest as antiplatelet agents. Here, we investigate the effects of 12 different Syk and BTK inhibitors on GPVI-mediated platelet signaling and function. These inhibitors include four Syk inhibitors, Bay 61-3606, R406 (fostamatinib), entospletinib, TAK-659; four irreversible BTK inhibitors, ibrutinib, acalabrutinib, ONO-4059 (tirabrutinib), AVL-292 (spebrutinib); and four reversible BTK inhibitors, CG-806, BMS-935177, BMS-986195, and fenebrutinib. In vitro, TKIs targeting Syk or BTK reduced platelet adhesion to collagen, dense granule secretion, and alpha granule secretion in response to the GPVI agonist cross-linked collagen-related peptide (CRP-XL). Similarly, these TKIs reduced the percentage of activated integrin αIIbß3 on the platelet surface in response to CRP-XL, as determined by PAC-1 binding. Although all TKIs tested inhibited phospholipase C γ2 (PLCγ2) phosphorylation following GPVI-mediated activation, other downstream signaling events proximal to phosphoinositide 3-kinase (PI3K) and PKC were differentially affected. In addition, reversible BTK inhibitors had less pronounced effects on GPIIb/IIIa-mediated platelet spreading on fibrinogen and differentially altered the organization of PI3K around microtubules during platelets spreading on fibrinogen. Select TKIs also inhibited platelet aggregate formation on collagen under physiological flow conditions. Together, our results suggest that TKIs targeting Syk or BTK inhibit central platelet functional responses but may differentially affect protein activities and organization in critical systems downstream of Syk and BTK in platelets.

Biocompatibility assessment of cyclic olefin copolymers: Impact of two additives on cytotoxicity, oxidative stress, inflammatory reactions, and hemocompatibility.

This work reports the biocompatibility evaluation of cyclic olefin copolymers (COC) as candidates for implantable medical devices. The focus was to establish the influence of two major additives (antioxidant and lubricant) on the overall biocompatibility. The cytotoxicity was evaluated according to ISO 10993-5 guidelines using L929 fibroblasts, HUVEC, and THP-1-derived macrophages. Oxidative stress (ROS, GSH/GSSG, and SOD analysis) and pro-inflammatory cytokines (Il-6 and TNF-α secretion) were quantified using THP-1 cells in direct contact with films. Hemocompatibility was assessed through haemolysis testing, dynamic blood coagulation, platelet adhesion, and activation (membranous P-selectin expression). Results show that the different types of COC have successfully passed the in vitro biocompatibility tests. The presence of antioxidant induces however a slight decrease in ROS production in correlation with a high SOD activity and a modification in blood coagulation profile probably linked to antioxidant recrystallization phenomenon on the surface of COC. The lubricant presence reduced haemolysis, fibrinogen adhesion, and platelet activation. Surface nanotopography of COC highlights different types of needles and globules according to the present additive. Those primary results indicate that COC are promising biomaterial. However, additives influenced some biological parameters pointing out the necessity of a global approach of risk analysis for biocompatibility evaluation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3333-3349, 2017.

Biodegradability and platelets adhesion assessment of magnesium-based alloys using a microfluidic system.

Magnesium (Mg)-based stents are extensively explored to alleviate atherosclerosis due to their biodegradability and relative hemocompatibility. To ensure the quality, safety and cost-efficacy of bioresorbable scaffolds and full utilization of the material tunability afforded by alloying, it is critical to access degradability and thrombosis potential of Mg-based alloys using improved in vitro models that mimic as closely as possible the in vivo microenvironment. In this study, we investigated biodegradation and initial thrombogenic behavior of Mg-based alloys at the interface between Mg alloys' surface and simulated physiological environment using a microfluidic system. The degradation properties of Mg-based alloys WE43, AZ31, ZWEK-L, and ZWEK-C were evaluated in complete culture medium and their thrombosis potentials in platelet rich plasma, respectively. The results show that 1) physiological shear stress increased the corrosion rate and decreased platelets adhesion rate as compared to static immersion; 2) secondary phases and impurities in material composition induced galvanic corrosion, resulting in higher corrosion resistance and platelet adhesion rate; 3) Mg-based alloys with higher corrosion rate showed higher platelets adhesion rate. We conclude that a microfluidic-based in vitro system allows evaluation of biodegradation behaviors and platelets responses of Mg-based alloys under specific shear stress, and degradability is related to platelets adhesion.

Lack of effect of supplementation with EPA or DHA on platelet-monocyte aggregates and vascular function in healthy men.

BACKGROUND AND AIMS: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) found in fish oil are postulated to have favourable effects on platelet, endothelial and vascular function. We investigated whether EPA has differential effects on in vivo platelet aggregation and other markers of cardiovascular risk compared to DHA. METHODS AND RESULTS: Following a 2 wk run-in taking encapsulated refined olive oil, 48 healthy young men were randomly allocated using a parallel design to receive EPA-rich (3.1 g EPA/d) or DHA-rich (2.9 g DHA/d) triglyceride concentrates or refined olive oil (placebo), for a total supplementary lipid intake of 5 g/d. The specified primary outcome was change in platelet monocyte aggregates (PMA); secondary outcomes were capillary density, augmentation index, digital pulse volume measurements, 24 h ambulatory BP, plasma 8-isoprostanes-F2α. Changes in the proportions of DHA and EPA in erythrocytes and non-esterified fatty acid composition indicated compliance to the intervention. There was no significant treatment effect on PMA (P = 0.382); mean changes (%) (95% CI) were placebo -0.5 (-2.0, 1.04), EPA 0.4 (-0.8, 1.6), DHA 0.3 (-1.5, 2.0). R-QUICKI, an index of insulin sensitivity, was greater following EPA compared to placebo (P < 0.05). No other significant differences were noted. CONCLUSION: Neither EPA- nor DHA-rich fish oil supplementation influence platelet-monocyte aggregation or several markers of vascular function after 6 wk in healthy young males. This trial was registered at clinicaltrials.gov as NCT01735357.

Sonorheometry assessment of platelet function in cardiopulmonary bypass patients: Correlation of blood clot stiffness with platelet integrin αIIbß3 activity, aspirin usage, and transfusion risk.

BACKGROUND: Impaired platelet function may underlie bleeding associated with cardiopulmonary bypass (CPB) and at present is incompletely evaluated with existing diagnostic technologies. Sonorheometry (SR) is a recently developed ultrasound-based technology that quantifies hemostasis and platelet activity from a blood sample by measuring ex vivo clot stiffness (S). We hypothesized that impaired platelet-fibrin interactions as assessed by SR would correlate with transfusion during CPB and history of prior aspirin therapy. METHODS: Thirty-nine patients undergoing elective cardiopulmonary bypass (CPB) were enrolled following informed consent (University of Virginia IRB#14050) in a prospective observational pilot study to assess pre-operative platelet function and transfusion frequency. To assess platelet activity, abciximab was added to blood prior to SR and native S versus abciximab treated S created a differential test for platelet activity. Patient blood samples were activated with kaolin and SR was then used to measure clot stiffness. Patients were transfused with blood products as directed by clinical practice, with the surgical team blinded to SR results. RESULTS: Blood clot stiffness with and without abciximab, was compared in a ratio test (S/Sabciximab) named the Platelet Function Index (PFI). PFI was hypothesized to be positively correlated with platelet contributions through integrin αIIbß3 to clot stiffness. PFI for CPB subjects was lower for those receiving transfusions than those not receiving transfusions (p<0.006). A receiver-operator characteristics (ROC) analysis correlating the PFI with the blinded surgical team's decision on transfusions that included platelet concentrates generated an area under the curve (AUC) of 0.79 (p<0.001). Additionally, the mean value of PFI for subjects on aspirin therapy was lower than for those not on aspirin therapy (p<0.02) and correlated with a 1.73-fold enhanced risk of receiving a peri-operative transfusion. CONCLUSION: Evaluation of platelet function with SR may help in the specification of blood transfusion needs in cardiac surgery and in the assessment of aspirin effects on risk of surgical bleeding.

Next generation covered stents made from nanocomposite materials: A complete assessment of uniformity, integrity and biomechanical properties.

Covered stents are stents wrapped with a thin polymeric membrane, and are typically used to treat vessel aneurysms and seal perforated arteries. Current covered stents suffer from restenosis due to limitations in material and fabrication methods which leaves metallic struts directly exposed to blood. We have developed a biocompatible and haemocompatible nanocomposite polymer, polyhedral oligomeric silsesquioxane poly(carbonate-urea) urethane (POSS-PCU). We devised a novel combination of ultrasonic spray atomisation system and dip-coating process to produce small calibre covered stents with metal struts fully embedded within the membrane, which also yields greater coating uniformity. Stent-polymer bonding was enhanced via silanisation and coating of reactive pre-polymer. Platelet studies supported the non-thrombogenicity of POSS-PCU. Biomechanical performances including diametrical compliance, bending strength, radial strength and recoil were evaluated and optimised. This proof-of-principle manufacturing technique could lead to the development of next-generation small calibre adult and paediatric covered stents. These stents are currently undergoing preclinical trial. From the Clinical Editor: The use of stents to treat vascular diseases is now the standard of care in the clinical setting. Nonetheless, a major problem of the current stents is the risk of restenosis and thrombosis. The authors developed a nanocomposite material using polyhedral oligomeric silsesquioxane and poly(carbonate-urea) urethane (POSS-PCU) and incorporated into metallic stents. Preliminary data have already shown promising results. It is envisaged that this would further lead to better stent technology in the future.

In stent restenosis and thrombosis assessment after EP224283 injection in a rat model.

OBJECTIVE: After stent implantation, platelet aggregation and thrombus formation are thought to play a key role in the early phase of in-stent restenosis (ISR). Drug-eluting stents have reduced ISR, but are associated with healing-related issues or hypersensitivity reactions, leading to an increased risk of late acute stent thrombosis. EP224283 is a new dual-action antithrombotic molecule combining a GPIIbIIIa antagonist and a factor Xa inhibitor. We investigated its efficacy on restenosis in a rat model of ISR and on platelet adhesion. METHODS AND RESULTS: Rat aortas were stented and the animals received either EP224283 or vehicle subcutaneously every 48 h. At day 7 and day 28 after surgery, the stented aortas were removed and processed for morphometric analysis or protein analysis. At day 28, EP224283 significantly reduced neointima growth (in the range of 20%). Protein analysis revealed that EP224283 reduced cell proliferation pathways: ERK1/2 and Akt were down-regulated and p38 up-regulated. Expression of Ki67 was also reduced. In vitro assessment depicted a reduction of platelet activation and platelet adhesion among treated rats. CONCLUSION: These results show a beneficial effect of EP224283 on in-stent restenosis and on stent thrombogenicity that may improve results after stent implantation. Further investigations are required to assess the efficacy of a local delivery of EP224283 on both acute thrombosis and ISR.

Superior efficacy of clopidogrel plus acetylsalicylic acid compared with extended-release dipyridamole plus acetylsalicylic acid in preventing arterial thrombogenesis in healthy volunteers.

INTRODUCTION: Recent ex vivo platelet aggregometry data indicate that clopidogrel 75 mg/day plus acetylsalicylic acid (ASA) 75 mg/day is a more potent antiplatelet regimen than the marketed combination of dipyridamole+ASA. The present study was designed to assess the antithrombotic effect of both dual antiplatelet regimens using a human ex vivo model of arterial thrombosis. MATERIALS AND METHODS: This was a randomized, double-blind, placebo-controlled, crossover study. During two 10-day treatment periods separated by a 14-day washout period, 23 healthy male volunteers received once-daily clopidogrel 75 mg plus acetylsalicylic acid 75 mg, or twice-daily extended-release dipyridamole 200 mg plus acetylsalicylic acid 25 mg. Assessments were made at baseline and on Day 10 of each period. Arterial thrombus formation was induced ex vivo by exposing a collagen-coated surface in a parallel-plate perfusion chamber to native blood for 3 min (arterial wall shear rate 2600 s(-1)). Total platelet and fibrin deposition was determined by immunoenzymatic methods. RESULTS: Compared with baseline values, the mean inhibition of total platelet deposition was 63.9+/-5.9% with clopidogrel plus acetylsalicylic acid, compared with 18.4+/-5.6% for extended-release dipyridamole plus acetylsalicylic acid (67% reduction; 95% CI, 49-79%; p<0.0001). Corresponding figures for fibrin deposition were 64.9+/-4.8% and 18.3+/-9.7%, respectively (58% reduction; 95% CI, 45-67%; p<0.0001). Both treatments were well tolerated. CONCLUSIONS: Clopidogrel plus acetylsalicylic acid showed significantly superior antithrombotic efficacy compared with extended-release dipyridamole plus acetylsalicylic acid in preventing arterial thrombogenesis in humans.

An assessment of athrombogenic properties of electret polyethylene film.

This paper shows the results of an investigation into the effect of an electric charge on blood platelet adhesion. All of the experiments were made on a polyethylene film. The electrets were formed using the electron beam method. The assessment of the electret effect on blood platelet adhesion was performed microscopically. It was found out that an electric charge plays a major role in the process of adhesion of blood morphological elements.

In vitro assessment of the hemocompatible properties of polymers.

Based on the available data in the literature and personal results, a system of in vitro tests, consisting of two levels of selection, was proposed for assessment of hemocompatible properties of polymers. The first level consists of rapid methods for preliminary assessment (clotting time, hemolytic activity of the sample, and number of adhesive platelets). The second level involves methods for final selection and consists of measurement of the kinetics of the initial stages of adsorption of albumin, the degree of activation of the complement system, the intensity of radical oxidation of the lipid fraction of the blood, and the degree of platelet spreading. Comparative experimental assessment of these methods was conducted with a number of polymers and revealed the confidence limits for the application of each test and suggested quantitative criteria for selection.

An assessment of platelet aggregation induced by 5-hydroxytryptamine.

A method of expressing platelet aggregation response after incubation relative to the response before incubation has been used in preference to using direct measurements, and some reasons are given for this choice. The effect of pre-incubating human platelets with reserpine has been compared with the effect of pre-incubation with 5-hydroxytryptamine (5HT). Reserpine inhibited 5HT-induced aggregation more slowly than 5HT and on this basis their actions could be distinguished. It was found that the aggregation response of human platelets to 5HT without pre-incubation (R) is inversely proportional to the natural 5HT content of whole blood, and an explanation has been suggested.A decrease in the aggregation response of human platelets to 5HT without pre-incubation (R) with increasing age has been noted, and is commented upon with reference to the release of amines from blood platelets. Platelets in plasma obtained from control subjects and incubated with methysergide and from migrainous patients taking methysergide both have reduced aggregation responses to 5HT. Platelets incubated with clonidine (St155) do not have reduced aggregation responses. A potentiating effect was noted after 20 minutes' incubation, but platelets from migraine patients taking St155 behaved normally. Tyramine affected the response of platelets to 5HT, giving results similar to those reported for reserpine. The differences between the aggregation responses to 5HT of platelets from migraine patients and normal control subjects are discussed.