Addition Of Adenosine Diphosphate Research Articles

Metabolomics analysis of the lactobacillus plantarum ATCC 14917 response to antibiotic stress

BackgroundLactobacillus plantarum has been found to play a significant role in maintaining the balance of intestinal flora in the human gut. However, it is sensitive to commonly used antibiotics and is often incidentally killed during treatment. We attempted to identify a means to protect L. plantarum ATCC14917 from the metabolic changes caused by two commonly used antibiotics, ampicillin, and doxycycline. We examined the metabolic changes under ampicillin and doxycycline treatment and assessed the protective effects of adding key exogenous metabolites.ResultsUsing metabolomics, we found that under the stress of ampicillin or doxycycline, L. plantarum ATCC14917 exhibited reduced metabolic activity, with purine metabolism a key metabolic pathway involved in this change. We then screened the key biomarkers in this metabolic pathway, guanine and adenosine diphosphate (ADP). The exogenous addition of each of these two metabolites significantly reduced the lethality of ampicillin and doxycycline on L. plantarum ATCC14917. Because purine metabolism is closely related to the production of reactive oxygen species (ROS), the results showed that the addition of guanine or ADP reduced intracellular ROS levels in L. plantarum ATCC14917. Moreover, the killing effects of ampicillin and doxycycline on L. plantarum ATCC14917 were restored by the addition of a ROS accelerator in the presence of guanine or ADP.ConclusionsThe metabolic changes of L. plantarum ATCC14917 under antibiotic treatments were determined. Moreover, the metabolome information that was elucidated can be used to help L. plantarum cope with adverse stress, which will help probiotics become less vulnerable to antibiotics during clinical treatment.

Ratiometric fluorescence probe constructed using metal–organic frameworks and nitrogen-doped carbon dots for specific detection of adenosine monophosphate

The selective detection of adenosine monophosphate (AMP) is of great significance for the study of cellular energy metabolism. In this study, a ratiometric fluorescent probe was designed based on europium-based metal–organic frameworks (Eu-MOF) and nitrogen-doped carbon dots (CDs) that could specifically recognize AMP. Europium nitrate and Disodium Terephthalate (Na2BDC) were used to synthesize Eu-BDC with red fluorescence. The fluorescence of the Eu-BDC was effectively suppressed due to water molecules coordinating with the Eu3+. However, the addition of AMP resulted in a significant restoration of the fluorescence of Eu-BDC, while the fluorescence remained suppressed upon addition of adenosine diphosphate (ADP) and adenosine triphosphate (ATP). We developed a ratiometric fluorescence sensor that could accurately detect AMP by utilizing the blue fluorescence emitted from CDs as a reliable reference signal. Under optimal conditions, the Eu-BDC/CDs system for AMP detection exhibited a linear detection range of 1.0–80.0 μmol/L and a low limit of detection (LOD) of 0.25 μmol/L. The method displayed simple preparation, high selectivity and low cost for AMP detection. To our knowledge, this is the first highly-specify double-response biosensor for AMP.

Excited state dynamics of Zn–salophen complexes

Zn–salophen complexes are a promising class of fluorescent chemosensors for nucleotides and nucleic acids. We have investigated, by means of steady state UV–Vis, ultrafast transient absorption, fluorescence emission and time dependent density functional theory (TD-DFT) the behavior of the excited states of a salicylidene tetradentate Schiff base (Sal), its Zn(II) coordination compound (Zn–Sal) and the effect of the interaction between Zn–Sal and adenosine diphosphate (ADP). TD-DFT shows that the deactivation of the excited state of Sal occurs through torsional motion, due to its rotatable bonds and twistable angles. Complexation with Zn(II) causes rigidity so that the geometry changes in the excited states with respect to the ground state structure are minimal. By addition of ADP to a freshly prepared Zn–Sal ethanol solution, a longer relaxation constant, in comparison to Zn–Sal, was measured, indicative of the interaction between Zn–Sal and ADP. After a few days, the Zn–Sal–ADP solution displayed the same static and dynamic behavior of a solution containing only the Sal ligand, demonstrating that the coordination of the ADP anion to Zn(II)leads to the demetallation of the Sal ligand. Fluorescence measurements also revealed an enhanced fluorescence at 375 nm following the addition of ADP to the solution, caused by the presence of 2,3-diamino naphthalene that is formed by demetallation and partial decomposition of the Sal ligand. The efficient fluorescence of this species at 375 nm could be selectively detected and used as a probe for the detection of ADP in solution.Graphical abstract

Effects of electronic cigarette flavorants on human platelet aggregation ex vivo

Because little is known about the effects of individual flavorants in electronic cigarette (e-cig) fluids on human platelet aggregation, we tested for the direct effects of 15 common e-cig flavorants on adenosine diphosphate (ADP)-induced human platelet aggregation ex vivo. To better understand a potential mechanism of action of flavorants, we quantified 2 phases of aggregation. Human platelet-rich plasma (PRP) was obtained from whole blood of healthy volunteers and used in a platelet aggregometry assay. PRP was incubated with 1 of 15 different flavorant compounds (e.g., benzyl alcohol, eugenol, citronellol, menthol, menthone, diacetyl, maltol, limonene, methylbutyric acid, isoamyl acetate, acetylpyridine, eucalyptol, 2,5-dimethylpyrazine, cinnamaldehyde, and vanillin) at 100 µM for 5 min at 37 °C prior to addition of ADP (10 µM). Subsequent ADP-induced platelet aggregation was tracked for 5 min using an aggregometer. Aggregation curves were analyzed for flavorant-induced effects on total (%) aggregation, Phase 1 and Phase 2 components, and compared with their ADP-only control via One-Way ANOVA. Notably, eugenol significantly inhibited total aggregation; an effect due solely to inhibition of Phase 2. No other flavor tested had any effect on total or phase-specific ADP-induced platelet aggregation. These results indicate that parent flavorant compounds commonly found in e-cig liquids neither activate nor inhibit ADP-induced human platelet aggregation. However, as flavorants are chemically altered during heating of e-cig, thermally-derived products of flavorants (e.g., flavor acetals) also will need to be tested for effects on platelet activation.

The Antifungal Activity of Loquat (Eriobotrya japonica Lindl.) Leaves Extract Against Penicillium digitatum.

This study was performed to determine the antifungal activity of loquat (Eriobotrya japonica Lindl) leaf extract (LLE) against the citrus postharvest pathogen Penicillium digitatum (P. digitatum). The LLE exhibited an antifungal activity against P. digitatum, with a minimum inhibitory concentration (MIC) of 0.625 mg/ml and a minimum fungicidal concentration (MFC) of 1.25 mg/ml. Significant inhibitory effects of LLE on mycelial growth and spore germination of P. digitatum were seen in a dose-dependent manner. Simultaneously, to investigate possible antifungal mechanisms by LLE, we analyzed their influence on morphological changes, cell membrane permeability, cell wall and cell membrane integrity, and adenosine phosphates (ATP, ADP, and AMP) levels. Alterations, such as sunken surface and malformation, occurred in the LLE-treated P. digitatum spores. Furthermore, intracellular inclusion content decreased after LLE treatment, indicating an increase in cell membrane permeability. Besides, the LLE treatment induced a significant decline in the level of adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) with a noticeable addition of extracellular ATP, ADP, and AMP during the entire treatment period. Overall, the results manifested that the antifungal activity of LLE against P. digitatum can be attributed to the derangement of cell membrane permeability and disordered energy metabolism. This is the first report on the mechanism of antifungal activity of LLE and could be useful in the development of targeted fungicides from natural origin.

Platelet and neutrophil responses to Porphyromonas gingivalis in human whole blood.

Porphyromonas gingivalis is a causative agent for periodontal disease. Binding of platelets to this gram-negative anaerobe can regulate host hemostatic (thrombus forming) and immune (neutrophil interacting) responses during bacterial infection. Additionally, in response to bacterial pathogens neutrophils can release their DNA, forming highly prothrombotic neutrophil extracellular traps (NETs), which then further enhance platelet responses. This study evaluates the role of P. gingivalis on platelet expression of CD62P, platelet-neutrophil interactions, and labeled neutrophil-associated DNA. Human whole blood was preincubated with varying P. gingivalis concentrations, with or without subsequent addition of adenosine diphosphate (ADP). Flow cytometry was employed to measure platelet expression of CD62P using PerCP-anti-CD61 and PE-anti-CD62P, platelet-neutrophil interactions using PerCP-anti-CD61 and FITC-anti-CD16, and the release of neutrophil DNA using FITC-anti-CD16 and Sytox Blue labeling. Preincubation with a high (6.25×106 CFU/mL) level of P. gingivalis significantly increased platelet expression of CD62P in ADP treated and untreated whole blood. In addition, platelet-neutrophil interactions were significantly increased after ADP stimulation, following 5-22min preincubation of blood with high P. gingivalis CFU. However, in the absence of added ADP, platelet-neutrophil interactions increased in a manner dependent on the preincubation time with P. gingivalis. Moreover, after ADP addition, 16min preincubation of whole blood with P. gingivalis led to increased labeling of neutrophil-associated DNA. Taken together, the results suggest that the presence of P. gingivalis alters platelet and neutrophil responses to increase platelet activation, platelet interactions with neutrophils, and the level of neutrophil antimicrobial NETs.

Water Hyacinth (Eichhorniacrassipes) Ethanolic Extract Anti Platelet Activity

Cardiovascular diseases (CVDs) are a group of disorders of the heart and blood vessels. CVDs are responsible for 37% of deaths in Indonesia. Thrombosis is the main factor causing blood vessel clost in CVDs. Blood vessel clots then affect narrowed blood vessel. This causes strokes. The aim of this study is to determine the antithrombotic effect of water hyacinth ethanolic extract, the effective dose, and the potential to become anticoagulant agent. The water hyacinth was extracted by ethanol 96%. The extract was tested on five groups of mice, each group containing five mice. Each group was tested for fourteen days. Group I and II are fed by extract suspension with varied dose 1 mg/BW and 2 mg/BW. Group III is fed by warfarin as standard. Group IV and V are the positive and negative group, the mice were fed by NaCMC. On the 7th and 14th days, prothrombin time (PT) and activated partial thromboplastin time (aPTT) were conducted. The plasma of group 1-IV mixed with adenosine diphosphate (ADP) as inducer, except plasma of group V used for negative control, did not mixed with ADP. Data analysed by ANOVA and LSD to obtain effective dose. The results showed the difference between PTT and aPTT among the tested groups. Group I and II showed prolonged PT which is 22 and 32 seconds respectively. The control positive showed PT 9.48 seconds on day 7th and 8.22 seconds on day 14th. LSD analysis showed that there is the significant difference between positive control and the other groups. It showed platelet aggregation performed due to the addition of ADP as inducer. There was a significant difference between dose 2 and the negative control group, and there is no significant difference of dose 1. In conclusion, water hyacinth has the antithrombotic activity, the effective dose is 1 mg/BW, and it potential to further developed as anticoagulant agent.
 
 Keywords: antiplatelet, cardiovascular diseases, water hyacinth.

Platelet activation and hypercoagulability in patients with early primary biliary cholangitis compared with healthy controls.

BackgroundPatients with primary biliary cholangitis (PBC) who have advanced disease are hypercoagulable, with no thrombophilic factors compared to non-cholestatic cirrhotics. We investigated whether hypercoagulability is present in early-stage PBC.MethodsPBC patients with biopsy-documented early disease and healthy controls matched by sex and age were asked to participate in the study. All were evaluated using rotational thromboelastometry (ROTEM), platelet aggregation, and flow cytometry. Four ROTEM parameters were evaluated (clotting time, clotting formation time, α-angle, and maximum clot firmness [MCF]). Platelet aggregation was determined as the maximal change in light transmission after the addition of adenosine diphosphate, collagen and epinephrine. Flow cytometry was used to evaluate the expression of glycoprotein (GP) IIb, GPIIa, and P-selectin on the platelet surface.ResultsWe enrolled 50 individuals in the study (25 PBC patients, 25 controls). Prothrombin time and activated partial thromboplastin time did not differ significantly between PBC patients and controls (P-value not significant). In ROTEM, aaaaaaaa-angle and MCF parameters were abnormally elevated in 9 (36%) PBC patients compared to 3 (12%) healthy controls and the difference was statistically significant (P=0.026). Platelet aggregation in PBC patients was not significantly different from controls. In flow cytometry, GPIIb and P-selectin expression was greater in PBC patients than in the control group and the difference was statistically significant (P=0.005 and P=0.006 respectively).ConclusionIn this study, we used a combination of sophisticated methods to detect evidence of platelet activation and hypercoagulability in patients with early PBC. Our findings may have important clinical implications and merit further investigation.

CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module

Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45−/− mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.

Suppression of platelet reactivity during dialysis by addition of a nitric oxide donor to the dialysis fluid

BackgroundDialysis membranes that release nitric oxide (NO) from their surface, mimicking one of the functions of endothelial cells, may suppress platelet reactivity during hemodialysis treatment. The aim of the present study was to examine whether the addition of a NO donor to the dialysis fluid can suppress platelet reactivity during dialysis.MethodsPorcine whole blood was circulated for 4 h through a polysulfone (PS) dialyzer or polymethylmethacrylate (PMMA) dialyzer. After the blood was circulated through the blood circuit and dialyzer, sodium nitroprusside was added to the dialysis fluid as a NO donor. The changes in the platelet reactivity, measured by the platelet aggregation activity by the addition of adenosine diphosphate or collagen in the blood sample, were evaluated during ex vivo dialysis experiments in the presence of a dialysis fluid containing or not containing a NO donor.ResultsThe platelet aggregation activity was significantly decreased at 30 min after the start of the experiment in the case where nitroprusside was added to the dialysis fluid (the NO (+) condition) as compared to the case where no nitroprusside was added to the dialysis fluid (the NO (−) condition), for both the PS and PMMA membranes. The suppression of the platelet reactivity in the NO (+) condition was sustained until the end of the experimental period (240 min). The platelet cyclic guanosine monophosphate level was also significantly increased in the NO (+) condition as compared to the NO (−) condition.ConclusionsNO in the dialysis fluid appears to be capable of suppressing the increase of the platelet reactivity observed during dialysis.

Montelukast inhibits RANKL‑induced osteoclast formation and bone loss via CysLTR1 and P2Y12.

Osteoclasts (OCs) are resorptive cells responsible for bone erosion in diseases, including osteoporosis, periodontitis and rheumatoid arthritis. Montelukast is a cysteinyl leukotriene receptor 1 (CysLTR1) antagonist clinically used for the treatment of asthma. In the present study, the role of CysLTR1 on OC formation and bone loss was investigated using montelukast. Montelukast inhibited receptor activator of nuclear factor‑κB ligand (RANKL)‑induced OC formation in cultures of mouse bone marrow macrophages. Additionally, montelukast suppressed actin ring formation and bone resorption activity of differentiated OCs. The inhibitory effect of montelukast was associated with impaired activation of extracellular signal‑regulated kinase, AKT serine/threonine kinase, and/or phospholipase Cγ2 signaling pathways downstream of RANK, followed by decreased expression of nuclear factor of activated T cells c1. Notably, OC formation was efficiently restored by addition of adenosine diphosphate, a P2Y12 agonist, as well as by addition of CysLT. Furthermore, similar to montelukast, P2Y12 blockade by a pharmacological inhibitor or siRNAs suppressed OC differentiation. These data indicate the involvement of the P2Y12 receptor in the inhibitory effect of montelukast on osteoclastogenesis. Invivo, montelukast significantly inhibited inflammation‑induced osteoclastogenesis in the calvarial model. Montelukast also served a protective role in a murine ovariectomy (OVX)‑ and unloading‑induced bone loss model. Altogether, these results confirmed that the CysLTR1 antagonist exerted an inhibitory effect on OC formation invitro and invivo. It may be useful for the treatment of bone diseases associated with excessive bone resorption.

Ischemia With Preconditioning in Wistar Rats Maintains Mitochondrial Respiration, Even With Mild Hepatocellular Disturbance

IntroductionIn hepatectomy or liver transplantation, preconditioning is a procedure indicated to protect the organ from ischemia-reperfusion injury (I-R). ObjectiveEvaluate the effect of preconditioning after hepatic I-R in Wistar rats, through mitochondrial respiration, liver histology, and profile. MethodTwenty male Wistar rats, weighing on average 307.1 g, were anesthetized with sodium thiopental (25 mg/kg) intravenously and xylazine hydrochloride (30 mg/kg) intramuscularly. The animals were divided into 2 groups: the preconditioning group (PCG), which contained 10 animals, and the hepatic pedicle was isolated and submitted to clamping with microvascular clamp (10 minutes of ischemia and 10 minutes of reperfusion, followed by 30 minutes of ischemia and 30 minutes of reperfusion); and the simulated operation group (SOG), which contained 10 animals submitted to manipulation of the hepatic pedicle and observation for the same length of time, with blood collected for transaminase dosage measurements, and liver biopsy for evaluation of mitochondrial respiration and histologic liver analysis and after sacrificed under anesthesia. The project was approved by the Ethics Committee on Animal Experimentation CEEA/UNICAMP under protocol number 3905-1. ResultThe PCG mitochondria showed the same respiration level as the SOG, when stimulated with the addition of adenosine diphosphate or carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the respiratory control ratio and resting of velocity of respiration the groups behaved in a similar way. The PCG presented high aspartate and alanine transaminases (P < .03) and about 60% of sinusoidal congestion and venous congestion in the histologic analysis when compared with SOG. ConclusionWe found that ischemia with preconditioning in Wistar rats can lead to mild histologic and biochemical dysfunction without leading to impairment of mitochondrial respiration.

Mitochondrial ryanodine‐sensitive Ca2+ channels of rat liver

To examine ryanodine-sensitive Ca2+ channels in mitochondria of rat hepatocytes and their role in energy state of the cells via investigation of the ryanodine effect on mitochondrial membrane potential. Oxygen consumption was measured by polarography using the Clark electrode. The substrates of oxidation such as pyruvate (5mM), α-ketoglutarate (5mM), or succinate (5mM) were used. Oxidative phosphorylation was stimulated by the addition of adenosine diphosphate (200nM). Mitochondrial membrane potential was measured using a voltage-sensitive fluorescent probe tetramethylrhodamine-methyl-ester (0.1μM) and was analyzed by a flow cytometer. To evaluate the intact mitochondria, we used carbonil cyanide m-chlorophenyl hydrazone (CCCP, 10μM). Changes in the ionized calcium concentration in rat liver mitochondria were measured using a fluorescent probe Fluo-4 AM. Effect of ryanodine on oxygen consumption of rat liver mitochondria depends on the oxidation substrate and the incubation time. Oxidation of pyruvate in the presence of ryanodine (0.05μM) decreased the membrane potential of rat liver mitochondria by 38.4%. At higher concentrations, ryanodine (0.1μM or 1μM) led to decrease of membrane potential by 51.7% and 42.8%, respectively. In contrast, oxidation of α-ketoglutarate in the presence of ryanodine (0.05μM) increased mitochondrial membrane potential by 16.8%. However, at higher concentrations, ryanodine (0.1μM or 1μM) triggered a decreasing of membrane potential by 42.5% and 31.0%, respectively. Therefore, ryanodine at various concentrations (0.05μM, 0.1μM, or 1μM) causes differential effects on Ca2+ concentration in the mitochondria matrix under oxidation of pyruvate or α-ketoglutarate. The data suggest the presence of ryanodine receptors in mitochondrial membrane of rat hepatocytes. Their inhibition with higher concentrations of ryanodine leads to decreasing of intra-mitochondrial Ca2+ concentration and affecting the energy state of mictochondria in hepatocytes.

Calcium-Channel Blockers Attenuate the Antiplatelet Effect of Clopidogrel.

Dihydropyridine calcium-channel blockers (CCBs) inhibit cytochrome 3A4 and could therefore interfere with the conversion of clopidogrel to its active form. The impact of CCBs on the antiplatelet effect of clopidogrel has not been studied with assays directly capturing platelet activation to adenosine diphosphate (ADP), so far. We therefore sought to investigate platelet activation in response to ADP by flow cytometry in clopidogrel-treated patients without and with CCBs. Platelet surface P-selectin expression and activated glycoprotein (GP) IIb/IIIa in response to ADP were determined by flow cytometry in 302 patients on dual antiplatelet therapy with aspirin and clopidogrel after successful angioplasty with stent implantation. Ninety-two patients (30.5%) received CCBs. Patients with concomitant CCB therapy showed significantly higher platelet surface expressions of P-selectin and activated GPIIb/IIIa in response to ADP than patients without CCBs (both P≤0.03). Moreover, the fold increase of P-selectin and activated GPIIb/IIIa in response to ADP was significantly more pronounced in patients taking CCBs (both P≤0.03). The associations of ADP-inducible activated GPIIb/IIIa and fold increase of activated GPIIb/IIIa after the addition of ADP with CCB therapy remained significant after adjustment for differences in patient characteristics and factors that were previously associated with clopidogrel response by multivariate regression analyses (both P<0.05). High levels of ADP-inducible P-selectin and activated GPIIb/IIIa were seen significantly more frequent in patients with CCBs than in patients without CCB therapy (both P≤0.01). Dihydropyridine CCBs attenuate the effect of clopidogrel on ADP-inducible platelet activation in patients undergoing angioplasty and stenting for cardiovascular disease.

Interaction of Fullerene Nanoparticles With Biomembranes: From the Partition in Lipid Membranes to Effects on Mitochondrial Bioenergetics

Partition and localization of C60 and its derivative C60(OH)18-22 in lipid membranes and their impact on mitochondrial activity were studied, attempting to correlate those events with fullerene characteristics (size, surface chemistry, and surface charge). Fluorescence quenching studies suggested that C60(OH)18-22 preferentially populated the outer regions of the bilayer, whereas C60 preferred to localize in deeper regions of the bilayer. Partition coefficient values indicated that C60 exhibited higher affinity for dipalmitoylphosphatidylcholine and mitochondrial membranes than C60(OH)18-22. Both fullerenes affected the mitochondrial function, but the inhibitory effects promoted by C60 were more pronounced than those induced by C60(OH)18-22 (up to 20 nmol/mg of mitochondrial protein). State 3 and p-trifluoromethoxyphenylhydrazone-uncoupled respirations are inhibited by both fullerenes when glutamate/malate or succinate was used as substrate. Phosphorylation system and electron transport chain of mitochondria are affected by both fullerenes, but only C60 increased the inner mitochondrial membrane permeability to protons, suggesting perturbations in the structure and dynamics of that membrane. At concentrations of C60(OH)18-22 above 20 nmol/mg of mitochondrial protein, the activity of FoF1-ATP synthase was also decreased. The evaluation of transmembrane potential showed that the mitochondria phosphorylation cycle decreased upon adenosine diphosphate addition with increasing fullerenes concentration and the time of the repolarization phase increased as a function of C60(OH)18-22 concentration. Our results suggest that the balance between hydrophilicity and hydrophobicity resulting from the surface chemistry of fullerene nanoparticles, rather than the cluster size or the surface charge acquired by fullerenes in water, influences their membrane interactions and consequently their effects on mitochondrial bioenergetics.

Sampling conditions influence multiple electrode platelet aggregometry in cardiac surgery patients

Objectives. To investigate the importance of blood sampling conditions for multiple electrode platelet aggregometry (MEA) in cardiac surgery patients. Design. Eighty-one patients undergoing first time CABG surgery were included in three prospective, observational studies. MEA was used to analyze platelet aggregability after addition of adenosine-diphosphate (ADP) or thrombin activating peptide 6 (TRAP). In substudy 1, hirudin and citrate tubes were compared. In substudy 2, samples from peripheral vein, central venous catheter, and radial artery were compared and in substudy 3, the effect of surgery was investigated by analyzing pre- and postoperative samples. Results. Platelet aggregability values were 30% higher in hirudin tubes than in citrate tubes. There was a significant correlation between hirudin and citrate tubes in TRAP-induced aggregability (r = 0.84, p < 0.001) but not in ADP-induced aggregability (r = 0.25, p = 0.13). The blood sampling site did not influence platelet aggregability. Surgery reduced ADP-induced aggregability by 31% (p < 0.001) and TRAP-induced aggregability by 30% (p < 0.001) with large intraindividual variations. Conclusions. MEA results in cardiac surgery patients should not be compared between samples collected in test tubes with different anticoagulants. The choice of blood sampling site does not affect the results. The operation in itself reduces markedly mean platelet aggregability.

Analysis of a commercial dimethyl‐sulfoxide‐stabilized frozen canine platelet concentrate by turbidimetric aggregometry

To assess platelet function of a commercial dimethyl-sulfoxide (DMSO)-stabilized frozen platelet concentrate (PC) using turbidimetric aggregometry. In vitro analysis. Research laboratory in a school of veterinary medicine. Five units of frozen PC in 6% DMSO were studied. Fresh platelet-rich plasma (PRP), with and without 6% DMSO, from 6 healthy dogs were used as controls. Turbidimetric platelet aggregation was measured after initiation of platelet aggregation by addition of adenosine diphosphate (ADP), collagen, or thrombin at concentrations of 30 μM, 20μg/mL, and 0.5U/mL, respectively. Measures were performed at thaw and repeated 2 hours after thaw for the frozen PC. Compared with PRP, the frozen PC showed decreased aggregation in response to thrombin (amplitude of 84% versus 25%, P=0.01), and collagen (amplitude of 13% versus 3%, P=0.05) but not ADP (6.5% versus 18%, P=0.2). Compared with frozen PC at thaw, the frozen PC at 2 hours after thaw showed decreased aggregation in response to thrombin, collagen, and ADP (P<0.05). There was no difference in aggregation between PRP in 6% DMSO and frozen PC. These in vitro data suggest there is a decrease in platelet response to agonists associated with the freeze-thaw process in the commercially available 6% DMSO canine frozen PC.

Increased soluble urokinase plasminogen activator receptor (suPAR) is associated with thrombosis and inhibition of plasmin generation in paroxysmal nocturnal hemoglobinuria (PNH) patients

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired genetic disorder of the bone marrow that produces intravascular hemolysis, proclivity to venous thrombosis, and hematopoietic failure. Mutation in the PIG-A gene of a hematopoietic stem cell abrogates synthesis of glycosylphosphoinositol (GPI) anchors and expression of all GPI-anchored proteins on the surface of progeny erythrocytes, leukocytes, and platelets. Urokinase plasminogen activator receptor (uPAR), a GPI-linked protein expressed on neutrophils, mediates endogenous thrombolysis through a urokinase-dependent mechanism. Here we show that membrane GPI-anchored uPAR is decreased or absent on granulocytes and platelets of patients with PNH, while soluble uPAR (suPAR) levels are increased in patients' plasma. Serum suPAR concentrations correlated with the number of GPI-negative neutrophils and were highest in patients who later develop thrombosis. In vitro, suPAR is released from PNH hematopoietic cells and from platelets upon activation, suggesting that these cells are the probable source of plasma suPAR in the absence of GPI anchor synthesis and trafficking of uPAR to the cell membrane. In vitro, the addition of recombinant suPAR results in a dose-dependent decrease in the activity of single-chain urokinase. We hypothesized that suPAR, prevents the interaction of urokinase with membrane-anchored uPAR on residual normal cells.

Impact of cytochrome P450 3A4-metabolized statins on the antiplatelet effect of a 600-mg loading dose clopidogrel and on clinical outcome in patients undergoing elective coronary stent placement

Early studies suggested interactions between statins and clopidogrel. Based on the outcome and platelet data, there is now huge evidence of no interactions between statins and 75 to 300 mg clopidogrel; however, data with 600-mg loading are lacking. In a pre-specified analysis of the EXCELSIOR cohort, we investigated the interaction between statins, especially cytochrome P4503A4-metabolized atorvastatin and simvastatin, and the antiplatelet effects of a 600-mg loading dose of clopidogrel. We analyzed 1,395 patients scheduled for coronary angiography (CA). Patients received clopidogrel 600 mg at least two hours before CA and 75 mg daily thereafter in case of percutaneous coronary intervention (PCI). Statin medication on admission was continued unaltered until discharge. Platelet function was assessed by optical aggregometry and flow cytometry of adenosine diphosphate (ADP)-stimulated surface expression of CD62P, CD63 and PAC-1 before clopidogrel and immediately before CA. Residual platelet aggregation (RPA) after addition of ADP 5 muM was similar irrespective of statin treatment at baseline (p = 0.968). RPA at CA was 46.2 +/- 16.8% in patients without statin (n = 682), 45.5 +/- 17.0% in patients with atorvastatin (n = 255), 45.8 +/- 16.3% with simvastatin (n = 335), 47.3 +/- 14.9% with fluvastatin (n = 42) and 45.9 +/- 16.2% with pravastatin (n = 81; p = 0.962). Consistent results were obtained by flow cytometry. In patients with PCI (n = 553), the one-year incidence of death, myocardial infarction and target lesion reintervention did not differ between cohorts stratified according to statin co-medication (p = 0.645). Thus, peri-interventional atorvastatin and simvastatin had no effect on the antiplatelet activity of a loading dose of clopidogrel 600 mg and did not affect clinical outcome after PCI.

Platelet aggregation is affected by nitrosothiols in patients with chronic hepatitis:In vivoandin vitrostudies

To investigate the relationship among the number of platelets and plasma levels of S-nitrosothiols (S-NO), nitrite, total non-protein SH (NPSH), glutathione (GSH), cysteine (CYS), malondialdehyde (MDA), 4-hydroxininenal (4HNE), tumor necrosis factor-alpha (TNFalpha) and interleukin (IL)-6 in patients with chronic hepatitis C (CH). In vitro the aggregation of platelets derived from controls and CH patients was evaluated before and after the addition of adenosine diphosphate (ADP) and collagen, both in basal conditions and after incubation with nitrosoglutathione (GSNO). In vivo, S-NO plasma levels increased significantly in CH patients and they were significantly directly correlated with platelet numbers. Patients with platelet counts < 150000/microL, had a smaller increase in S-NO, lower levels of GSH, CYS, NPSH, TNFalpha, and IL-6, and higher levels of nitrite, MDA, and 4-HNE relative to those of patients with platelet counts > 150000/microL. In vitro, the ADP and collagen aggregation time was increased in platelets from patients and not from controls; in addition, platelets from CH patients but not from controls also showed a latency time after exposure to collagen. The incubation of platelets with GSNO improved the percentage aggregation and abolished the latency time.