Lütfiye Alpan

Impact of Silver Nanoparticles on Haemolysis, Platelet Function and Coagulation

Silver nanoparticles (Ag NPs) are increasingly used in biomedical applications because of their large antimicrobial spectrum. Data in the literature on the ability of Ag NPs to perform their desired function without eliciting undesirable effects on blood elements are very limited and contradictory. We studied the impact of Ag NPs on erythrocyte integrity, platelet function and blood coagulation. Erythrocyte integrity was assessed by spectrophotometric measurement of haemoglobin release. Platelet adhesion and aggregation was determined by light transmission aggregometry and scanning electron microscopy. The calibrated thrombin generation test was used to study the impact on coagulation cascade. We demonstrated that Ag NPs induced haemolysis. They also increase platelet adhesion without having any impact on platelet aggregation. Finally, they also had procoagulant potential. Bringing all data from these tests together, the no observed effect concentration is 5 μg/mL.

Effects of SiC nanoparticles orally administered in a rat model: Biodistribution, toxicity and elemental composition changes in feces and organs

BACKGROUND Silicon carbide (SiC) presents noteworthy properties as a material such as high hardness, thermal stability, and photoluminescent properties as a nanocrystal. However, there are very few studies in regard to the toxicological potential of SiC NPs. OBJECTIVES To study the toxicity and biodistribution of silicon carbide (SiC) nanoparticles in an in vivo rat model after acute (24h) and subacute (28days) oral administrations. The acute doses were 0.5, 5, 50, 300 and 600mg·kg(-1), while the subacute doses were 0.5 and 50mg·kg(-1). RESULTS SiC biodistribution and elemental composition of feces and organs (liver, kidneys, and spleen) have been studied by Particle-Induced X-ray Emission (PIXE). SiC and other elements in feces excretion increased by the end of the subacute assessment. SiC did not accumulate in organs but some elemental composition modifications were observed after the acute assessment. Histopathological sections from organs (stomach, intestines, liver, and kidneys) indicate the absence of damage at all applied doses, in both assessments. A decrease in the concentration of urea in blood was found in the 50mg·kg(-1) group from the subacute assessment. No alterations in the urine parameters (sodium, potassium, osmolarity) were found. CONCLUSION This is the first study that assesses the toxicity, biodistribution, and composition changes in feces and organs of SiC nanoparticles in an in vivo rat model. SiC was excreted mostly in feces and low traces were retrieved in urine, indicating that SiC can cross the intestinal barrier. No sign of toxicity was however found after oral administration.

A comparison of six major platelet functional tests to assess the impact of carbon nanomaterials on platelet function: A practical guide

Abstract The study of the haemocompatibility of nanomaterials that could be in contact with blood (e.g. nanoparticle (NP)-based drug-delivery system) is of major importance. The primary objective of this study was to compare the ability of six platelet functional tests to assess the impact of NPs on platelet function. The secondary objective was to determine an accurate and reliable screening test to measure the potential impact of NPs on primary haemostasis whatever their physicochemical properties. Four types of carbon NPs (carbon black, fullerenes, single-walled carbon nanotubes and multi-walled carbon nanotubes) were investigated on six platelet function tests: light transmission aggregometry, whole-blood impedance aggregometry, platelet function analyser-100 (PFA-100®) and Cone-and-Plate(let) analyser (Impact-R®), transmission- and field emission gun scanning electron microscopy (FEG-SEM). We considered that Impact-R® supported by FEG-SEM is the reference method to investigate the potential impact of NPs on platelet function.

Synthesis and pharmacological evaluation of 2-aryloxy/arylamino-5-cyanobenzenesulfonylureas as novel thromboxane A2 receptor antagonists

New series of original 2-aryloxy/arylamino-5-cyanobenzenesulfonylureas were synthesized and evaluated as thromboxane A2 receptor (TP receptor) antagonists. A functional pharmacological test was used, which consisted of measuring the inhibition of intracellular calcium mobilization in a model of mammalian cell line that specifically over-expressed the individual TPα or TPβ isoforms. 2-Arylamino-5-cyanobenzenesulfonylureas exhibited virtually identical affinity and/or functional activity than 2-aryloxy-5-cyanobenzenesulfonylureas for both TPα and TPβ, but some 2-aryloxy-substituted compounds showed increased selectivity for TPβ relative to TPα. Several compounds were found to be as potent as the 2-arylamino-5-nitrobenzenesulfonylurea reference compound BM-573, supporting the view that the bioisosteric replacement of the nitro group by a cyano group was tolerated. TP receptor antagonist activity of the most promising molecules was confirmed in a platelet aggregation assay using the TP receptor agonist U-46619 as a proaggregant. Three compounds (7e, 7h and 8h) were identified as leads for further non-clinical pharmacological and toxicological studies.

Can TiC nanoparticles produce toxicity in oral administration to rats

Background Titanium carbide (TiC) is used for ceramic metal composites in several industries and is regarded as a nanomaterial for catalyst and battery applications. However, there are very few studies in regard to the toxicological potential of TiC nanoparticles (NPs). Objective To study the toxicodynamics and toxicokinetics of TiC NPs in Sprague Dawley rats in acute (24 h) and subacute (28 days) oral administrations. The acute doses were 0.5, 5, 50, 300 and 1000 mg kg−1; the subacute doses were 0.5 and 50 mg kg−1. Results Organ histopathological examination (esophagus, stomach, intestines, spleen, liver, and kidneys) indicates the absence of damage at all applied doses, in both assessments. In the acute administration, alkaline phosphatases increased (5, 300 and 1000 mg kg−1), ASAT increased (1000 mg kg−1) and bile salts decreased (0.5 mg kg−1). No alterations in urine parameters (sodium, potassium, osmolarity) were found. Acute administration of TiC caused mineral changes in organs (liver, spleen, kidneys). TiC was mostly cleared by feces excretion 24 h after administration, in subacute administration causing variations in mineral absorption (Mg, Al, P, S, Ca, Zn). TiC could pass the intestinal barrier as TiC traces were detected in urine. Conclusion No sign of toxicity was found after oral administration. TiC was excreted mostly in feces producing mineral absorption alterations. Low traces were retrieved in urine, indicating that TiC can cross the intestinal barrier.

Application of a clot-based assay to measure the procoagulant activity of stored allogeneic red blood cell concentrates.

BACKGROUND Thrombotic effects are possible complications of red blood cell transfusion. The generation and accumulation of procoagulant red blood cell extracellular vesicles during storage may play an important role in these thrombotic effects. The objective of this study was to assess the value of a simple phospholipid-dependent clot-based assay (STA®-Procoag-PPL) to estimate the procoagulant activity of stored red blood cells and changes in this activity during storage of the blood component. MATERIALS AND METHODS Extracellular vesicles from 12 red blood cell concentrates were isolated at 13 storage time-points and characterised by quantitative and functional methods: the degree of haemolysis (direct spectrophotometry), the quantification and determination of cellular origin (flow cytometry) and the procoagulant activity (thrombin generation and STA®-Procoag-PPL assays) were assessed. RESULTS The mean clotting time of extracellular vesicles isolated from red blood cell concentrates decreased from 117.2±3.6 sec on the day of collection to 33.8±1.3 sec at the end of the storage period. This illustrates the phospholipid-dependent procoagulant activity of these extracellular vesicles, as confirmed by thrombin generation. Results of the peak of thrombin and the STA®-Procoag-PPL were well correlated (partial r=-0.41. p<0.001). In parallel, an exponential increase of the number of red blood cell-derived extracellular vesicles from 1,779/μL to 218,451/μL was observed. DISCUSSION The STA®-Procoag-PPL is a potentially useful technique for assessing the procoagulant activity of a red blood cell concentrate.

The euglobulin clot lysis time to assess the impact of nanoparticles on fibrinolysis

Abstract Nanoparticles (NPs) are developed for many applications in various fields, including nanomedicine. The NPs used in nanomedicine may disturb homeostasis in blood. Secondary hemostasis (blood coagulation) and fibrinolysis are complex physiological processes regulated by activators and inhibitors. An imbalance of this system can either lead to the development of hemorrhages or thrombosis. No data are currently available on the impact of NPs on fibrinolysis. The objectives of this study are (1) to select a screening test to study ex vivo the impact of NPs on fibrinolysis and (2) to test NPs with different physicochemical properties. Euglobulin clot lysis time test was selected to screen the impact of some NPs on fibrinolysis using normal pooled plasma. A dose-dependent decrease in the lysis time was observed with silicon dioxide and silver NPs without disturbing the fibrin network. Carbon black, silicon carbide, and copper oxide did not affect the lysis time at the tested concentrations.

Impact of functional inorganic nanotubes f-INTs-WS2 on hemolysis, platelet function and coagulation

Inorganic transition metal dichalcogenide nanostructures are interesting for several biomedical applications such as coating for medical devices (e.g. endodontic files, catheter stents) and reinforcement of scaffolds for tissue engineering. However, their impact on human blood is unknown. A unique nanomaterial surface-engineering chemical methodology was used to fabricate functional polyacidic polyCOOH inorganic nanotubes of tungsten disulfide towards covalent binding of any desired molecule/organic species via chemical activation/reactivity of this former polyCOOH shell. The impact of these nanotubes on hemolysis, platelet aggregation and blood coagulation has been assessed using spectrophotometric measurement, light transmission aggregometry and thrombin generation assays. The functionalized nanotubes do not induce hemolysis but decrease platelet aggregation and induce coagulation through intrinsic pathway activation. The functional nanotubes were found to be more thrombogenic than the non-functional ones, suggesting lower hemocompatibility and increased thrombotic risk with functionalized tungsten disulfide nanotubes. These functionalized nanotubes should be used with caution in blood-contacting devices.

Application of a clot based assay to measure the procoagulant activity of stored allogeneic red blood cell concentrates

Abstracts Supported bys Supported by ABSTRACTS OF THE 62ND ANNUAL MEETING OF THE SCIENTIFIC AND STANDARDIZATION COMMITTEE OF THE INTERNATIONAL SOCIETY ON THROMBOSIS AND HAEMOSTASIS MAY 25–28, 2016S OF THE 62ND ANNUAL MEETING OF THE SCIENTIFIC AND STANDARDIZATION COMMITTEE OF THE INTERNATIONAL SOCIETY ON THROMBOSIS AND HAEMOSTASIS MAY 25–28, 2016 Animal, Cellular and Molecular Models AMC01 Endothelial injury and hypoxia enhances neutrophil recruitment, extravasation, and apoptosis during thrombus formation Nishimura S, Seo K and Sakata A Jichi Med Univ, and the Univ of Tokyo, Tochigi, Japan; Jichi Med Univ, Tochigi, Japan Background: Primary trigger of cardiovascular events remains unclear due to the absence of direct analysis tools of in vivo thrombus formation. Aims: To elucidate blood cell behavior and vascular responses, we improved in vivo imaging technique based on multi-photon microscopy, and analyzed new three animal thrombus formation models. Methods:We combined multi-photon microscope technique, and lightmanipulations system, which enabled us to evaluate platelet aggregations by photochemical reactions. Results: First, we induced rapidly developing thrombi composed of discoid platelets, which was triggered by ROS production without endothelial damage. Discoid platelet activations and aggregations were induced without leukocyte recruitment in this system. Second, thrombus formation was also induced by direct endothelial cell disruption by femto-second laser irradiations. With the rapid recruitment of inflammatory neutrophils into damaged area, following fibrin net formation and tissue regenerative changes were observed, indicating tight association between endothelial damage and inflammatory reactions, Spontaneous apoptotic neutrophil death, and endothelial-neutrophilinteractions were induced after platelet aggregations. Last, platelet aggregations, and inflammatory neutrophil recruitment were also observed after transient ischemia and reperfusion. Combination of hypoxia and endothelial injury enhanced thrombus formation, and extravasation steps were remarkably enhanced. Leukocyte escaped from specific and limited “holes” in endothelial junctions. Conclusions: Intravital visualization of thrombus formation elucidated association of inflammation of endothelium, leukocyte recruitment, and platelet aggregations in vivo. Neutrophils were selectively recruited in early phase, which was enhanced by endothelial damages and hypoxic conditions. Our real-time imaging system can evaluate multicellular thrombotic processes and therapeutic strategies against them. AMC02 TMEM16F-mediated platelet pro-coagulant activity contributes to infarct progression after ischemic stroke Baig AA, Haining EJ, Geuss E, Beck S, Stegner D, Kleinschnitz C, Braun A and Nieswandt B University of W€ urzburg, Rudolf Virchow Center for Experimental Biomedicine, Wuerzburg, Germany; University Hospital of W€ urzburg, Department of Experimental Biomedicine, Wuerzburg, Germany; University Hospital of W€ urzburg, Department of Neurology, Wuerzburg, Germany Background: Transmembrane protein TMEM16F, mutated in patients with the bleeding disorder Scott syndrome, is important for the scrambling of phosphatidylserine (PS) to the platelet surface upon platelet activation. Thus, it plays an essential role in the platelet pro-coagulant response. However, the contribution of TMEM16F and the pro-coagulant activity of platelets to thrombo-inflammation after ischemic stroke is unknown. Aims: To investigate the pathophysiological role of TMEM16Fmediated platelet pro-coagulant activity in the setting of ischemic stroke. Methods: A platelet and megakaryocyte specific TMEM16F knockout (KO) mouse was generated by targeted deletion of exon 3 in the Anoctamin6 gene. The mice were assessed in the transient middle cerebral artery occlusion (tMCAO)model of ischemic stroke in addition tomodels of thrombosis and hemostasis, as well as in vitro platelet analyses. Results: TMEM16F KO platelets exposed significantly less PS and also failed to acquire a ballooned morphology after stimulation with ionomycin, indicating a reduced pro-coagulant potential. Likewise, thrombinoscope measurements showed that time to peak thrombin concentration was significantly delayed in KO platelet rich plasma (PRP), as compared to WT PRP. KO mice displayed significantly prolonged tail bleeding times, and were protected in a model of ferric chloride induced thrombosis in the carotid artery, confirming previous reports. These results highlighted the TMEM16F KO as a suitable model with which to investigate the pathophysiological significance of pro-coagulant platelets in cerebral infarct progression following tMCAO. Initial experiments indicate that female TMEM16F KO mice are protected from infarct growth after tMCAO. Further experiments are underway to test if this is also the case in male mice and to understand how pro-coagulant platelets contribute to thrombo-inflammation during ischemic-reperfusion injury. Figure 1