In Vitro Susceptibility of Wistar Rat Platelets to Hydrogen Peroxide and AAPH-Induced Oxidative Stress

Hydroxyl and peroxyl radicals are biologically active species because of their likelihood to damage cellular constituents. An in vitro study on Wistar rats was conducted to investigate the influence of hydrogen peroxide (H₂O₂) and 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) on platelets and compare the vulnerability of platelets to oxidative stress (OS) induced by these two free radical initiators. Isolated platelets were divided into controls (without free radical initiators; n = 5) and experimentals (with free radical initiators; n = 5). Different concentrations (0.5, 1.0 and 2.0) of free radical initiators H₂O₂ and AAPH were used to treat the platelets and incubated for 5, 15 and 30 min. Biomarkers such as platelet aggregation, superoxide generation, lipid peroxidation (thiobarbituric acid reactive substances, conjugate dienes), protein oxidation (protein carbonyls, sulfhydryls) and antioxidant enzymes were assessed. In H₂O₂ and AAPH treated platelets, though OS was observed at concentrations of 0.5 and 1.0 mM, platelets could tolerate the oxidative insult. Treatment of platelets with 2.0 mM H₂O₂ demonstrated the onset of irreversible changes in platelets as observed in the results of increased superoxide generation and lipid peroxidation products. In 2.0 mM AAPH platelets, the oxidative damage was evident as indicated through increased aggregation, superoxide generation and conjugate dienes and lower protein sulfhydryls. Platelets were more susceptible to AAPH than H₂O₂, as AAPH acted on both lipids and proteins whereas H₂O₂ acted only on lipids. This study gives insight on platelet survival under different OS situations.     

Role of N-13 ammonia PET/CT in diagnosing pancreatic necrosis in patients with acute pancreatitis as compared to contrast enhanced CT – Results of a pilot study

BackgroundContrast enhanced computerized tomography (CECT) is used to determine severity of acute pancreatitis based upon the presence and extent of necrosis. However limitations do exist precluding its applicability in renal failure. Positron emission tomography (PET) imaging for cardiac perfusion shows good uptake of N-13 ammonia (¹³NH₃) metabolites in pancreas owing to high perfusion.AimTo evaluate the role of ¹³NH₃ PET/CT in acute pancreatitis and compare it with CECT in diagnosing and quantifying pancreatic necrosis.Material and methodsPatients presenting within 1 week of acute pancreatitis were studied. Static PET images were acquired after intravenous injection of 370–740 MBq of ¹³NH₃. ¹³NH₃ PET/CT was followed by CECT in the absence of renal impairment. Maximum standard-uptake-value (SUVmax) of pancreas (P) and liver (L) were taken and their ratio (P/L) was estimated to determine perfusion. Areas within pancreas with no tracer uptake were considered necrotic. These patients were managed as per institutional protocol. Patients undergoing ¹³NH₃ PET/CT for coronary artery disease were used as controls.Results29 patients (72% males) were studied of whom 6 had elevated serum creatinine. ¹³NH₃ PET/CT was done in all patients along with 9 controls while CECT was carried out after PET/CT in 23 patients. Median levels of SUVmax (P/L) in the controls, uninvolved pancreas and necrotic areas were 1.0 (0.86–1.03), 0.66 (0.50–0.92) and 0.12 (0.07–0.21) respectively (p < 0.001). Necrosis estimation was similar in 22/23 patients without renal failure while in one patient only ¹³NH₃ PET/CT picked up necrosis (<30%). 5/6 patients with renal failure had necrosis on ¹³NH³ PET/CT which was confirmed on surgery or subsequent CECT after improvement of renal failure.ConclusionThis pilot study is the first in literature to diagnose necrosis in patients with acute pancreatitis using ¹³NH₃ PET/CT. With minimal additional radiation burden, it is possible to estimate the absolute tissue perfusion as well. With no adverse renal side effects, this can be an alternative to CECT in patients with renal failure giving similar information. It has good agreement with CECT with a good interobserver acceptability.     

Measurement of the Expiratory Ammonia Concentration and its Clinical Significance

Alghough gaseous ammonia (NH₃) can freely enter cells through the plasma membrane where NH₃ is cyto(neuro)toxic, NH₃ and ionic ammonia (NH₄ ⁺) contents have not been studied in biological materials. We developed a new method for measurement of expiratory NH₃ concentration, which may reflect blood NH₃ concentrations. The method is a sensor tube type-gas assay system. Expiratory NH₃ concentrations in patients with chronic liver diseases increased when their blood ammonia (NH₄ ⁺+NH₃) concentrations increased above 90 μg/dl (normal range; 12–66 μg/dl). However, cirrhotic patients, who had relatively higher expiratory NH₃ concentration compared to blood NH₃ concentrations (calculated from Henderson-Hasselbalch formula), were found to have subclinical encephalopathy. Measurement of experatory NH₃ concentration may be of clinical significance for the diagnosis of encephalopathy associated with hyperammonemia.     

Increased platelet responsiveness following coronary stenting: Heparin as a possible aetiological factor in stent thrombosis

Aims Platelet activation may be a determinant of thromboticand restenotic complications following intracoronary stenting.In order to measure the effect of stenting on platelet activationantigen expression we used whole blood flow cytometry in 18patients undergoing Palmaz–Schatz stenting (treated withfull anticoagulation) and compared these with a group of 18patients undergoing elective angioplasty. The effects of lowmolecular weight heparin and unfractionated heparin on plateletbehaviour were also studied, both in vitro and in vivo to determinethe contribution of prolonged heparin therapy to platelet activationfollowing stenting. Methods and results Fibrinogen binding to activated GPIIb-IIIa,and surface expression of P-selectin, GPIb and GPIIb-IIIa antigenswere measured in unstimulated peripheral blood samples (rest)and on stimulation with adenosine diphosphate (0·1–10µmol.l^–1)and thrombin (0·02–0·16U.ml^–1). Nochanges were seen in resting samples following angioplasty orstenting. Agonist responsiveness was unaltered after angioplasty,but in stented patients antigen expression in response to thrombinwas significantly reduced (P0·04), whilst the adenosinediphosphate response was significantly increased (P=0·01).Similar effects were observed in patients with unstable anginatreated with either low molecular weight heparin or unfractionatedheparin in vivo. In vitro, both unfractionated and low molecularweight heparin inhibited thrombin-induced platelet activation,but stimulation of adenosine diphosphate responses was moremarked with unfractionated than low molecular weight heparin. Conclusions There was a significant increase in platelet responsivenessto adenosine diphosphate following intra-coronary stenting inpatients treated with conventional anticoagulants. This wasprobably a consequence of treatment with heparin. Activationof platelets by heparin may explain the increased rate of stentthrombosis in patients treated with anticoagulant therapy. Lowmolecular weight heparins stimulate platelets less than unfractionatedheparin.     

Evaluation of blood collection methods and anticoagulants for platelet function analyses on C57BL/6J laboratory mice

Abstract

The exploration of thrombotic mechanisms relies on the application of blood collection methods from laboratory mice with a minimal pre-activation of platelets and the clotting system. So far, very little is known on how the blood collection method and the anticoagulant used influence pre-activation of mouse platelets and coagulation. To determine the most suitable blood collection method, we systematically compared blood collection by heart puncture, Vena cava puncture, and puncture of the retro-orbital vein plexus and the use of citrate, heparin, and EDTA as frequently used anticoagulants with regard to platelet activation and whole blood clotting parameters. The activation of platelet-rich plasma diluted in Tyrode’s buffer was analyzed by flow cytometry, analyzing the exposure of P-selectin and activated integrin α_IIbβ₃. Clotting of whole blood was profiled by thrombelastometry. Puncture of the retro-orbital vein plexus by plastic capillaries is not superior in terms of blood volume and platelet pre-activation, whereas heart puncture and Vena cava puncture resulted in similarly high blood volumes. Cardiac puncture and Vena cava puncture did not result in pre-activated platelets with citrate as an anticoagulant, but the use of EDTA resulted in increased levels of integrin α_IIbβ₃ activation. Puncture of the retro-orbital vein plexus by plastic capillaries resulted in increased platelet integrin α_IIbβ₃ activation, which could be prevented by soaking with citrate or coating with heparin. Further, activation of coagulation in citrated whole blood by puncture of the retro-orbital vein plexus using a blunt plastic capillary was observed by thromboelastometry. The use of citrate is the optimal anticoagulant in mouse platelet assays. Blood collections from the heart or Vena cava represent reliable alternatives to retro-orbital puncture of the vein plexus to avoid pre-activation of platelets and coagulation.     

Effect of heparin on platelet count and platelet aggregation

The in vitro effect of heparin on platelet aggregation was studied in three groups: in 26 subjects recently treated with heparin, in 18 subjects on maintenance hemodialysis, and in 20 normal controls. With the aid of Technicon H6000, platelet counts and platelet aggregations were compared in whole blood samples collected in ethylenediaminetetraacetic acid (EDTA) and in heparinized tubes. Although there was no significant difference between platelet count of heparinized and EDTA blood in the control group, the dialysis group and the group recently treated with heparin showed significantly lower platelet counts and more platelet aggregation in heparinized tubes than in EDTA tubes. We speculate that the majority of subjects exposed to heparin develop an antibody or a proaggregator which can aggregate or agglutinate platelets in the presence of heparin and causes destruction of platelets; but only in a small percentage of subjects receiving heparin is this reaction severe enough to cause thrombocytopenia.     

Propensity for young reticulated platelet recruitment into arterial thrombi

Atherosclerosis reduces platelet survival and thereby increases the percentage of younger platelets in the circulation assuming steady-state thrombocytopoiesis. We hypothesized that younger platelets have an increased propensity for arterial thrombus participation compared to older counterparts. Platelet-rich thrombi were generated by perfusing human heparinized whole blood from normal donors over arterial cross-sections under shear conditions (3,350 s^?1) corresponding to significant coronary artery stenosis using a perfusion chamber. Harvested thrombi were disaggregated, stained with thiazole orange, anti-integrin β₃, glycoprotein (GP) Ibα, GP IX and P-selectin, and compared to paired whole blood samples from the same donor by flow cytometry. Thiazole orange staining intensity provides a measure of platelet m-RNA content and age. Thiazole orange staining intensity (MN ± SEM) of platelets harvested from thrombi (62 ± 13) was twofold greater compared to paired intra-individual whole blood samples (31 ± 1). Integrin β₃ receptor density was also greater for thrombus platelets (12.0 ± 1.0) compared to whole blood platelets (7.0 ± 0.6; p < 0.0001). GPs Ibα and IX were reduced from thrombus platelets possibly reflecting shedding. Younger “reticulated” platelets appear to have a greater propensity for thrombus participation under shear conditions of coronary artery stenosis compared to older counterparts. This predisposition may be explained by an increased receptor density of integrin β₃ in younger platelets. By this mechanism, the atherosclerotic process may enhance the individual propensity for arterial thrombosis.     

Homocysteine and its thiolactone may promote apoptotic events in blood platelets in vitro

The actions of homocysteine and its major metabolite, cyclic thioester, homocysteine thiolactone on endothelial cells, blood platelets, plasmatic fibrinogen and plasminogen – the important major components of haemostasis, regulating the flowing properties of blood – are complex and sometimes controversial. Homocysteine (Hcys) can promote apoptosis in endothelial cells, but the role of Hcys and its thiolactone in the apoptotic process in blood platelets is unknown. In order to study the appearance of apoptosis in platelets after treatment with the reduced form of Hcys or its thiolactone different markers were chosen: annexin V binding (phosphatidylserine exposure), platelet microparticle formation, mitochondrial membrane depolarization and α_IIbβ₃ expression in vitro. Apoptotic events and platelet activation were measured by a flow cytometer. In gel-filtered platelets treated with different concentrations of the reduced form of Hcys (25, 50 and 100 µM, 10 min) a significant increase of phosphatidylserine exposure (about 37% at the highest concentration, p < 0.001) and platelet microparticle formation were observed. Homocysteine caused also a dose-dependent depolarization of mitochondrial potential. The same apoptotic markers appeared in HTL-treated platelets (0.2 and 1 µM). Moreover, resveratrol (25 µM), a well known antioxidant, distinctly reduced the level of apoptotic markers. The obtained results indicate that Hcys and its thiolactone may promote in vitro apoptotic events in human gel-filtered platelets.     

Nucleic acid scavengers inhibit thrombosis without increasing bleeding

Development of effective, yet safe, antithrombotic agents has been challenging because such agents increase the propensity of patients to bleed. Recently, naturally occurring polyphosphates such as extracellular DNA, RNA, and inorganic polyphosphates have been shown to activate blood coagulation. In this report, we evaluate the anticoagulant and antithrombotic activity of nucleic acid-binding polymers in vitro and in vivo. Such polymers bind to DNA, RNA, and inorganic polyphosphate molecules with high affinity and inhibit RNA- and polyphosphate-induced clotting and the activation of the intrinsic pathway of coagulation in vitro. Moreover, [NH₂(CH₂)₂NH₂]∶(G = 3);dendri PAMAM(NH₂)₃₂ (PAMAM G-3) prevents thrombosis following carotid artery injury and pulmonary thromboembolism in mice without significantly increasing blood loss from surgically challenged animals. These studies indicate that nucleic acid-binding polymers are able to scavenge effectively prothrombotic nucleic acids and other polyphosphates in vivo and represent a new and potentially safer class of antithrombotic agents.     

Dual effects of heparin on VEGF binding to VEGF receptor-1 and transduction of biological responses

Vascular endothelial growth factor (VEGF) regulates blood vessel formation by binding to the receptor tyrosine kinases VEGF receptor-1 (Flt-1) or VEGF receptor-2 (KDR) and to the structurally unrelated neuropilins. As exon 7-containing isoforms of VEGF bind to heparin, angiogenesis may be modulated by heparin/heparan sulfate. We analyzed the effect of heparin on VEGF₁₆₅-binding and activation of VEGF receptor-1 in porcine aortic endothelial cells, which lack expression of VEGF receptor-2 and neuropilins. Heparin decreased binding of ¹²⁵I-VEGF to 50% at 5 μg/ml and cross-linking of ¹²⁵I-VEGF to VEGF receptor-1 on intact cells was similarly decreased. Schatchard analyses showed that the affinity for binding of ¹²⁵I-VEGF to VEGF receptor-1 was decreased in the presence of heparin. In contrast, VEGF receptor-1 kinase activity was elevated when cells were treated simultaneously with VEGF and heparin. In accordance, VEGF-induced tyrosine phosphorylation of phospholipase Cγ (PLCγ) and DNA synthesis were augmented by heparin. However, basal PLCγ tyrosine phosphorylation and DNA synthesis levels were to some extent increased by incubation of cells with heparin alone. We conclude that although heparin decreases binding of VEGF to VEGF receptor-1, the remaining binding results in more efficient kinase activation. Taken together, there is no loss of VEGF/VEGF receptor-1 function in the presence of heparin. This revised version was published online in June 2006 with corrections to the Cover Date.     

Backflux of ammonia from brain to blood in human subjects with and without hepatic encephalopathy

In patients with hepatic encephalopathy (HE) the blood concentration of ammonia is usually highly elevated. Ammonia readily enters brain cells from the blood, and toxic effects of ammonia on brain metabolism and neurotransmission are believed to play a key role in the pathogenesis of HE. It has, however, been a matter of great controversy whether backflux of unmetabolized ammonia (NH₃ + NH₄ ⁺) from brain cells to the blood occurs in man. In the present analysis of data from a dynamic PET study of brain ¹³N–ammonia metabolism in healthy subjects and cirrhotic patients with and without HE, we provide the first unambiguous evidence for backflux of ammonia from brain cells to the blood in man. The high temporal and spatial resolution of modern PET technology was employed to distinguish between unidirectional blood-brain transport of ammonia and subsequent metabolism of the ammonia in the brain. In all 16 subjects, clearance of the unidirectional transport of ¹³N–ammonia from the blood to brain cells (K₁) was higher than the metabolic clearance of ¹³N–ammonia from the blood (K_met=K₁ k₃/(k₂+k₃). This can only be explained by backflux (k₂) of ammonia from brain cells to the blood. In conclusion, backflux of ammonia from the brain to the blood does indeed occur in both healthy subjects and cirrhotic patients with and without hepatic encephalopathy.     

Can the surface electrocardiogram be used to predict myocardial viability?

OBJECTIVE—To investigate whether QRS morphology on the surface ECG can be used to predict myocardial viability.
DESIGN—ECGs of 58 patients with left ventricular impairment undergoing positron emission tomography (PET) were studied. ¹³N-Ammonia (NH₃) and ¹⁸F-fluorodeoxyglucose (FDG) were the perfusion and the metabolic markers, respectively. The myocardium is scarred when the uptake of both markers is reduced (matched defect). Reduced NH₃ uptake with persistent FDG uptake (mismatched defect) represents hibernating myocardium. First, the relation between pathological Q waves and myocardial scarring was investigated. Second, the significance of QR and QS complexes in predicting hibernating myocardium was determined.
RESULTS—As a marker of matched PET defects, Q waves were specific (79%) but not sensitive (41%), with a 77% positive predictive accuracy and a poor (43%) negative predictive accuracy. The mean size of the matched PET defect associated with Q waves was 20% of the left ventricle. This was not significantly different from the size of the matched PET defects associated with no Q waves (18%). Among the regions associated with Q waves on the ECG, there were 16 regions with QR pattern (group A) and 23 regions with QS pattern (group B). The incidence of mismatched PET defects was 19% of group A and 30% of group B (NS).
CONCLUSIONS—Q waves are specific but not sensitive markers of matched defects representing scarred myocardium. Q waves followed by R waves are not more likely to be associated with hibernating myocardium than QS complexes.


Keywords: electrocardiography; myocardial viability; positron emission tomography; myocardial scarring     

Studies on the biological effects of ozone: 9. Effects of ozone on human platelets

During the course of ozonated autohaemotherapy (O₃-AHT) using heparin as an anticoagulant, it was occasionally observed that a few clots were retained in the filter during blood reinfusion. This observation prompted an investigation on the effect of ozone (O₃) on human platelets. We have now shown, both by biochemical and morphological criteria, that heparin in the presence of O₃ can promote platelet aggregation. In contrast, after Ca²⁺ chelation with citrate, platelet aggregation is much reduced. The potential role of the transient formation of hydrogen peroxide (H₂O₂) in the presence of Ca²⁺ with the possible expression of adhesion molecules is briefly discussed.     

Direct cytotoxicity of hypoxia-reoxygenation towards sinusoidal endothelial cells in the rat

Abstract: Aims/Background: Sinusoidal endothelial cells are the primary target of ischemia-reperfusion injury following liver preservation. The present study was undertaken to examine the susceptibility of sinusoidal endothelial cells to hypoxia-reoxygenation and the potential role of oxygen free radicals in the induction of cell injury. Methods: Sinusoidal endothelial cells were isolated from rat liver. After 2–3 days of primary culture, the cells were exposed to hypoxia (N₂/CO₂ 95/5) for 120 min and reoxygenation (O₂/CO₂ 95/5) for 90 min. Control cells were exposed to hypoxia alone, to 95% O₂ alone or were maintained under normoxic conditions. Human umbilical vein endothelial cells were used as a model of vascular endothelial cells and submitted to the same protocol. Cell viability and lipid peroxidation were assessed by LDH leakage and malondialdehyde production, respectively. In order to test the potential role of xanthine oxidase and mitochondria dysfunction in cell injury, the cells were treated with allopurinol and potassium cyanide (KCN) respectively. Results: The different gaseous treatments did not affect LDH leakage in human umbilical vein endothelial cells. In sinusoidal endothelial cells, the sequential hypoxia-reoxygenation caused a significant increase in LDH release, malondialdehyde production and xanthine oxidase activity while hypoxia alone had no effect except on xanthine oxidase activity. Allopurinol inhibited xanthine oxidase without preventing cell injury or lipid peroxidation in this latter cell type. Conclusions: The results suggest that sinusoidal endothelial cells, as opposed to vascular endothelial cells, are susceptible to a direct cytotoxic effect of hypoxia-reoxygenation. This effect occurs in combination with an increase in xanthine oxidase activity and lipid peroxidation, although cell injury is mediated at least in part by mechanisms independent of xanthine oxidase such as mitochondrial dysfunction.     

Defining the domains of type I collagen involved in heparin- binding and endothelial tube formation

Cell surface heparan sulfate proteoglycan (HSPG) interactions with type I collagen may be a ubiquitous cell adhesion mechanism. However, the HSPG binding sites on type I collagen are unknown. Previously we mapped heparin binding to the vicinity of the type I collagen N terminus by electron microscopy. The present study has identified type I collagen sequences used for heparin binding and endothelial cell–collagen interactions. Using affinity coelectrophoresis, we found heparin to bind as follows: to type I collagen with high affinity (K_d ≈ 150 nM); triple-helical peptides (THPs) including the basic N-terminal sequence α1(I)87–92, KGHRGF, with intermediate affinities (K_d ≈ 2 μM); and THPs including other collagenous sequences, or single-stranded sequences, negligibly (K_d 10 μM). Thus, heparin–type I collagen binding likely relies on an N-terminal basic triple-helical domain represented once within each monomer, and at multiple sites within fibrils. We next defined the features of type I collagen necessary for angiogenesis in a system in which type I collagen and heparin rapidly induce endothelial tube formation in vitro. When peptides, denatured or monomeric type I collagen, or type V collagen was substituted for type I collagen, no tubes formed. However, when peptides and type I collagen were tested together, only the most heparin-avid THPs inhibited tube formation, likely by influencing cell interactions with collagen–heparin complexes. Thus, induction of endothelial tube morphogenesis by type I collagen may depend upon its triple-helical and fibrillar conformations and on the N-terminal heparin-binding site identified here.     

Low-Pressure H2, NH3 Microwave Plasma Treatment of Polytetrafluoroethylene (PTFE) Powders: Chemical,Thermal and Wettability Analysis

Functionalization of Polytetrafluoroethylene (PTFE) powders of ~6 μm particle size is carried out using low-pressure 2.45 GHz H₂, NH₃ microwave plasmas for various durations (2.5, 10 h) to chemically modify their surface and alter their surface energy. The X-ray Photoelectron Spectroscopy (XPS) analyses reveal that plasma treatment leads to significant defluorination (F/C atomic ratio of 1.13 and 1.30 for 10 h NH₃ and H₂ plasma treatments, respectively vs. 1.86 for pristine PTFE), along with the incorporation of functional polar moieties on the surface, resulting in enhanced wettability. Analysis of temperature dependent XPS revealed a loss of surface moieties above 200 °C, however, the functional groups are not completely removable even at higher temperatures (>300 °C), thus enabling the use of plasma treated PTFE powders as potential tribological fillers in high temperature engineering polymers. Ageing studies carried over a period of 12 months revealed that while the surface changes degenerate over time, again, they are not completely reversible. These functionalised PTFE powders can be further used for applications into smart, high performance materials such as tribological fillers for engineering polymers and bio-medical, bio-material applications.     

Role of anti-Naka antibody,monocytes and platelets in the development of transfusion-related acute lung injury

Background and Objectives Transfusion-related acute lung injury (TRALI) is one of the most serious side-effects of transfusion. We report here the first two cases of TRALI caused by anti-Nak^a (anti-CD36) antibody from a single blood donor. The aim of this study was to clarify the role of the anti-Nak^a antibody in TRALI development. Materials and Methods Human lung microvascular endothelial cells were co-cultured with Nak^a-positive monocytes and Nak^a-positive platelets together with serum prepared from blood products of a TRALI-caused anti-Nak^a antibody-carrying donor. Expressions of leukotriene B₄ (LTB₄) and tumour necrosis factor α (TNF-α) in the co-culture supernatants were determined. Results The expressions of LTB₄ and TNF-α were found to be markedly increased, particularly in the presence of both Nak^a-positive monocytes and platelets. The expressions of these mediators were almost completed within 4 h after the initiation of co-culture. Monocyte contribution seemed to be stronger than that of platelets. In the absence of human lung microvascular endothelial, no significant LTB₄ or TNF-α release was observed. Conclusion Anti-Nak^a antibody may be strongly implicated in lung microvascular endothelial dysfunctions that lead to TRALI in a monocyte- and platelet-dependent manner.     

Unexplained recurrent venous thrombosis in a patient with MYH9-related disease

Bioactive substances found in numerous foods can be successfully and safely used to modify various cellular functions and affect the oxidative stress. Aronia melanocarpa fruits (Rosaceae) are one of the richest plant sources of phenolic substances shown to have anti-inflammatory, antitumor, antioxidative and antiplatelet activities. We investigated antioxidant properties of the extract from berries of A. melanocarpa by the estimation of the selected and other biomarkers of oxidative stress, i.e. the level of 8-epi-prostaglandin F₂ (8-EPI) (by immunoassay kit) and the amount of glutathione (by HPLC method) in control platelets and platelets treated with H₂O₂. The expression of α_IIbβ₃ (a marker of platelet activation) was measured by flow cytometer. The antioxidative and antiplatelet properties of the tested extract were also compared with the action of a well characterized antioxidative and antiplatelet commercial monomeric polyphenol–resveratrol. The extract from berries of A. melanocarpa (at the highest tested concentration ?100 µg/ml) decreased the production of 8-EPI (a marker of lipid peroxidation) in control blood platelets and platelets treated with H₂O₂ (2 mM). A combined action of the tested plant extract and H₂O₂ evoked a significant increase of reduced form of glutathione in platelets compared with cells treated with H₂O₂ only. Moreover, the tested plant extract (at the highest used concentration ?100 µg/ml) reduced the expression of α_IIbβ₃ on blood platelets. Comparative studies indicate that the tested plant extract was found to be more reactive in blood platelets than the solution of pure resveratrol.     

A Scanning Electron Microscopic Investigation of Effects of Prostacyclin on Platelet Retention in Glass Bead Columns

As whole blood passes through a column of glass beads, platelets are activated and all 3 basic platelet properties (adhesion, release reaction and aggregation) displayed, an aspect similar to platelets being involved in a hemostatic process. For this reason, we chose the platelet retention test as the tool to study the effect of synthetic prostacyclin (PGI₂) on platelet function and behavior. PGI₂ inhibited platelet retention with its major impact on platelet aggregation. The extent of inhibition was not proportional to the concentration of PGI₂. It appears that 2 levels of platelet aggregation take place inside the column. One, triggered by a weak stimulus, is readily inhibitable by low concentrations of PGI². The other, brought about by a strong stimulus, requires higher concentrations of PGI₂ to achieve complete inhibition. Even with as much as 100 ng PGI₂/ml blood, a 15% retention remained which probably represented platelet adhesion to glass beads. Scanning electron microscopic examination of glass beads showed massive platelet aggregation and adhesion in control blood samples. No platelet aggregate was observed on beads filtered with blood containing 20 ng/ml or more PGI₂. Individual platelets were found on glass beads filtered with samples containing any concentration of PGI₂; these platelets exhibited extensive morphologic changes.     

Impact of non-inhibited platelet supplementation on platelet reactivity in patients treated with prasugrel or ticagrelor for an acute coronary syndrome: An ex vivo study

Abstract

Managing bleeding in patients receiving P2Y₁₂ inhibitors is challenging. Few data are available regarding the efficacy of platelet transfusion in patients treated with prasugrel or ticagrelor. The aim of this study was to evaluate the minimal amount of platelet supplementation (in terms of ratio of non-inhibited platelets to inhibited platelets) necessary to restore platelet reactivity in platelet-rich plasma (PRP) of patients treated with aspirin and a prasugrel or ticagrelor loading dose for an acute coronary syndrome. PRP samples from patients were mixed ex vivo with increasing proportions of pooled PRP from healthy volunteers. Platelet reactivity was challenged with adenosine diphosphate (ADP), arachidonic acid, collagen or thrombin receptor activating peptide using light transmission aggregometry. The primary endpoint was the proportion of patient samples recovering an ADP-induced maximal aggregation (ADP-Agg_max) value above 40%. In patients treated with prasugrel (n?=?32), ADP-Agg_max increased progressively with supplements of pooled PRP, with an average increase of 7.9% (95% CI [7.1; 8.8], p?<?0.001) per each 20% increase in the ratio of non-inhibited platelets to inhibited platelets. A ratio of 60% was associated with 90% of patients reaching the primary endpoint. In patients treated with ticagrelor (n?=?15), ADP-Agg_max did not significantly increase with any level of supplements. In conclusions, ex vivo addition of non-inhibited platelets significantly improved ADP-Agg_max in patients treated with prasugrel with a dose-dependent effect. There was no evidence of such a reversal in patients treated with ticagrelor. These results suggest that platelet transfusion may be more effective in blunting bleeding in patients treated with prasugrel, than those treated with ticagrelor.