The study of platelet aggregation using a microtiter plate reader ‒ methodological considerations.

Optical aggregometry by 96-well plate assay, the microplate method, is a fast, efficient, and readily available method for measuring the pharmacological effects of antiplatelet drugs. Even though recent years have witnessed growing interest in adopting the microplate method for widespread use, it remains in the shadow of the standard light transmission aggregometry (LTA). Regardless of the method used, the results of platelet aggregation depend on a variety of factors and often vary among laboratories worldwide. While several methodological papers have examined the microplate method, no standards have been established, most likely because the approach is not used as a diagnostic tool. Currently, the microplate method is recommended by researchers to be used in conjunction with LTA or as an adjunct to LTA. This raises the question of whether an optimal protocol exists for microplate aggregometry, and what are the key considerations in a good experimental protocol for obtaining reliable results? This article attempts to address these questions by summarizing the knowledge accumulated in this field over the last three decades.

Platelets as Potential Non-Traditional Cardiovascular Risk Factor-Analysis Performed in Healthy Donors.

Abnormal lipid profile, increased glucose level, and elevated body weight are traditional cardiometabolic risk factors; however, the role of platelets in the development of cardiovascular disease (CVD) is increasingly being highlighted. The aim of this study was to select platelet-related parameters (non-genetic molecular and routine laboratory measurements) that may be associated with increased cardiovascular risk among healthy populations. We evaluated the level of platelet indices, platelet-based inflammatory markers, platelet reactivity parameters, and platelet reactive oxygen species (ROS) generation in relation to selected cardiometabolic risk factors. We noted the association between total cholesterol and LDL cholesterol with platelet aggregation and platelet ROS generation. We found the relationship between triglycerides, glucose, and body mass index with the relatively new multi-inflammatory indices (MII-1 and MII-3). Moreover, we noticed that the mean platelet volume-to-lymphocyte ratio in healthy subjects is not a good source of information about platelets and inflammation. We also highlighted that platelet-to-HDL-cholesterol ratio may be a promising prognostic cardiometabolic indicator. The association between platelet-related (especially molecular) and cardiometabolic parameters requires further research. However, the goal of this study was to shed light on the consideration of platelets as a non-traditional cardiovascular risk factor and a crucial element in identifying individuals at high-risk of developing CVD in the future.

The Anti-Aggregative Potential of Resolvin E1 on Human Platelets.

Resolvin E1 is a metabolite of eicosapentaenoic acid (EPA) which is one of the omega-3 polyunsaturated fatty acids (omega-3 PUFAs). The antiplatelet properties of omega-3 PUFAs are well known, but the effect of resolvin E1 on platelets via the collagen receptors is extremely poorly reported. We investigated the effect of resolvin E1 on collagen-induced platelet aggregation, activation, and reactivity, and also platelet membrane fluidity. The ultimate and statistically significant results showed that resolvin E1 may inhibit platelet reactivity due to the reduction of collagen-induced platelet aggregation in platelet-rich plasma and isolated platelets, but not in whole blood. Also, resolvin E1 significantly reduced P-selectin exposure on collagen-stimulated platelets. Moreover, we demonstrated that resolvin E1 can maintain platelet membrane structure (without increasing membrane fluidity). The association between platelet reactivity and membrane fluidity, including resolvin E1 and collagen receptors requires further research. However, the goal of this study was to shed light on the molecular mechanisms behind the anti-aggregative effects of resolvin E1 on platelets, which are still not fully clarified. We also indicate an innovative research direction focused on further analysis and then use of omega-3 PUFAs metabolites as antiplatelet compounds for future applications in the treatment and prevention of cardiovascular diseases.

Platelet reactivity expressed as a novel platelet reactivity score is associated with higher inflammatory state after coronary artery bypass grafting.

Introduction: Despite therapy, patients operated using a cardiopulmonary bypass demonstrate increased platelet aggregation, which rebounds to above preoperative levels. The aim of the study was to test the interaction between platelet reactivity/activation and selected inflammatory markers in the post-operative period. Material and methods: In total, 103 patients with non-ST elevation acute coronary syndrome (NSTE-ACS) who were not eligible for percutaneous coronary interventions (PCI), and required urgent revascularization, were included. Platelet reactivity was measured using the PFA-100 platelet analyser, multiple electrode aggregometry, and was expressed as a novel platelet reactivity score (PRS). Patients were divided using their PRS scores into high platelet relativity or low platelet reactivity subgroups (HPR or LPR). Platelet basal activation was measured using immunoassays for soluble P-selectin and soluble CD40L. We measured high-sensitivity C-reactive protein (CRP), and used immunoassays for tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6) as inflammation markers. Results: Significant differences between HPR and LPR groups were found for CRP (mg/l): 81.5 vs. 44.6, p < 0.02; and TNF-α (pg/l): 3.51 vs. 2.37, p < 0.02. A significant association was found between CRP, TNF-α, IL-6 and platelet reactivity (platelet reactivity score). Cohen's k showed: CRP = 0.49, p < 0.0001, TNF-α = 0.37, p < 0.002. Perioperative myocardial infarction and rhythm disturbances occurred more frequently in the high platelet reactivity group: 7 (16.3%) vs. 2 (3.3%), p < 0.04, and 9 (20.9%) vs. 4 (6.7%), p < 0.04, respectively. Conclusions: Inflammatory parameters CRP and TNF-α are strongly associated with platelet reactivity (expressed as PRS) in cardiopulmonary bypass graft patients. Platelet hyperreactivity in the early post-operative period combined with a systemic inflammatory state correlates with a higher risk of post-operative rhythm disturbances and myocardial infarction.

Interactions of fentanyl with blood platelets and plasma proteins: platelet sensitivity to prasugrel metabolite is not affected by fentanyl under in vitro conditions.

BACKGROUND: Clinical trials indicate that fentanyl, like morphine, may impair intestinal absorption and thus decrease the efficacy of oral P2Y12 inhibitors, such as clopidogrel, ticagrelor, and prasugrel. However, the ability of fentanyl to directly negate or reduce the inhibitory effect of P2Y12 receptor antagonists on platelet function has not been established. A series of in vitro experiments was performed to investigate the ability of fentanyl to activate platelets, potentiate platelet response to ADP, and/or diminish platelet sensitivity to prasugrel metabolite (R-138727) in agonist-stimulated platelets. The selectivity and specificity of fentanyl toward major carrier proteins has been also studied. METHODS: Blood was obtained from healthy volunteers (19 women and 12 men; mean age 40 ± 13 years). Platelet function was measured in whole blood, platelet-rich plasma and in suspensions of isolated platelets by flow cytometry, impedance and optical aggregometry. Surface plasmon resonance and molecular docking were employed to determine the binding kinetics of fentanyl to human albumin, α1-acid glycoprotein, apolipoprotein A-1 and apolipoprotein B-100. RESULTS: When applied at therapeutic and supratherapeutic concentrations under various experimental conditions, fentanyl had no potential to stimulate platelet activation and aggregation, or potentiate platelet response to ADP, nor did it affect platelet susceptibility to prasugrel metabolite in ADP-stimulated platelets. In addition, fentanyl was found to interact with all the examined carrier proteins with dissociation constants in the order of 10-4 to 10-9 M. CONCLUSIONS: It does not seem that the delayed platelet responsiveness to oral P2Y12 inhibitors, such as prasugrel, in patients undergoing percutaneous coronary intervention, results from direct interactions between fentanyl and blood platelets. Apolipoproteins, similarly to albumin and α1-acid glycoprotein, appear to be important carriers of fentanyl in blood.

Systemic immune-inflammation index (SII) and neutrophil to lymphocyte ratio (NLR) are useful markers for assessing effects of anti-inflammatory diet in patients before coronary artery bypass grafting.

BACKGROUND: One of the risk factors responsible for coronary artery disease (CAD) is an inadequate diet that is frequently deficient in anti-inflammatory components, such as polyphenols and omega-3 fatty acids. The neutrophil to lymphocyte ratio (NLR) and the systemic immune-inflammation index (SII) are inflammatory markers that may reflect a diet's antiinflammatory potential. OBJECTIVE: The aim of this study was to evaluate the effects that CAD patients' nutrition patterns have on NLR and SII. MATERIAL AND METHODS: A retrospective study assessed the dietary habits and inflammatory marker levels in patients with advanced CAD before they underwent coronary artery bypass grafting (CABG) (n=101). Patients were divided into subgroups based on their NLR and SII levels. RESULTS: Subgroups with lower NLR and SII levels had consumed significantly more eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (p=0.02). The group with a lower ratio of omega-6 to omega-3 fatty acids (<4:1) also had lower NLR and SII levels (p=0.007 and p=0.01, respectively). Statistically significant negative correlations were found between EPA and DHA, as well as omega-3 intake, and both NLR and SII values. No statistically significant differences were found between the subgroups with lower and higher NLR and SII values for polyphenol intakes. CONCLUSIONS: Inflammatory markers such as NLR and SII may reflect an anti-inflammatory diet consumed by cardiac patients. A simultaneous assessment of dietary habits and inflammatory parameters is beneficial in the possible prevention of adverse cardiovascular incidents after CABG. There is also a need to establish reference values for SII and NLR.

Effects of Omega-3 Polyunsaturated Fatty Acids and Their Metabolites on Haemostasis-Current Perspectives in Cardiovascular Disease.

The beneficial effects of long-chain polyunsaturated omega-3 fatty acids (omega-3 PUFAs) in cardioprotection are widely known and generally accepted. In this literature review, we have focused on the known and postulated mechanisms of action of omega-3 PUFAs and their metabolites on various components of the haemostatic system, in particular on blood platelets and endothelium. We have also made an attempt to provide a comprehensive review of epidemiological studies with particular regard to clinical trials. Notably, the results of these studies are contradictory, and some of them failed to report the beneficial effects of taking or supplementing omega-3 PUFAs in the diet. A potential explanation, in our opinion, could be the need to use higher doses of omega-3 PUFAs and a proper ratio of omega-3 and omega-6 PUFAs. An additional problem which is difficult to solve is the use of a proper neutral placebo for interventional studies. Despite some controversies regarding the beneficial effects of supplementation of omega-3 PUFAs in cardiovascular disease, our review suggests that a promising aspect of future studies and applications is to focus on the anti-thrombotic properties of these compounds. An argument supporting this assumption is the recent use of omega-3 PUFAs as a supporting tool for the treatment of COVID-19 complications.

Dietary intake of omega fatty acids and polyphenols and its relationship with the levels of inflammatory markers in men with chronic coronary syndrome after percutaneous coronary intervention.

BACKGROUND: The platelet­to­lymphocyte ratio (PLR), neutrophil­to­lymphocyte ratio (NLR), and C­reactive protein (CRP) are useful in assessing inflammation in patients after percutaneous coronary intervention (PCI). The PLR and NLR are also independent predictors of cardiovascular mortality. Moreover, higher CRP levels increase the risk of long­term mortality in patients undergoing PCI. AIMS: We aimed to investigate the relationship between the dietary intake of omega­3 and omega­6 fatty acids and plant polyphenols and the levels of inflammatory markers in patients after PCI. METHODS: In this retrospective study, we used the validated Food Frequency Questionnaire and Aliant software to estimate the dietary intake of polyphenols and omega­3 fatty acids as well as the ratio of omega­6 to omega­3 fatty acids in patients after PCI. A total of 105 patients were divided into subgroups based on high or low dietary polyphenol intake, omega­3 fatty acid intake, and omega­6 / omega­3 fatty acid ratio. Data on complete blood count were obtained from the hospital laboratory. RESULTS: In this retrospective study, we used the validated Food Frequency Questionnaire and Aliant software to estimate the dietary intake of polyphenols and omega­3 fatty acids as well as the ratio of omega­6 to omega­3 fatty acids in patients after PCI. A total of 105 patients were divided into subgroups based on high or low dietary polyphenol intake, omega­3 fatty acid intake, and omega­6 / omega­3 fatty acid ratio. Data on complete blood count were obtained from the hospital laboratory. CONCLUSIONS: Anti­inflammatory effects of a diet should be assessed not only based on a high intake of omega­3 fatty acids but also balanced omega­6 / omega­3 ratio, which reduces PLR and CRP levels in patients with cardiovascular disease.

Polyphenol-rich diet is associated with decreased level of inflammatory biomarkers in breast cancer patients

Background: The study investigated the relationship between dietary intake of polyphenols and inflammatory markers: CRP, neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), medium platelet volume/lymphocyte ratio (MPVRL), in newly-diagnosed breast cancer patients. Objectives: The aim of this work was to verify whether diet rich in plant polyphenols affects inflammatory markers in breast cancer patients. Materials and methods: 78 patients (55.3±14.5 years) treated surgically for breast cancer were studied. A modified FFQ and authorial worksheet based on the Phenol Explorer database was used to measure the amount of plant polyphenols in a diet. Basing on the median of polyphenols intake (1780 mg/day), the group was divided into two subgroups: low- and high- dietary intake of polyphenols (LDIP and HDIP, respectively). Plasma CRP level was measured and NLR, PLR and MPVLR were calculated using results from peripheral blood morphology. Results: LDIP was associated with significantly higher CRP (elevated in 34.5% LDIP patients vs. 8.3% HDIP, p<0.003), NLR (elevated in 46.2% LDIP patients vs. 25.6% HDIP, p<0.006) and PLR level (elevated in 25.6% LDIP patients vs. 12.8% HDIP, p<0.03). MPVLR was not significantly different between both the subgroups. Conclusion: High dietary intake of polyphenols remarkably reduced process of inflammation in breast cancer patients, which has important clinical implications. The study demonstrated also an usefulness of simple, cheap and commonly available biomarkers for monitoring anti-inflammatory effects of diet.

Dual Anticoagulant/Antiplatelet Activity of Polyphenolic Grape Seeds Extract.

Because of the side-effects of commonly used anti-platelet and anticoagulant drugs, investigations into plant substances with similar activities are very common. Based on our own studies in recent years, we estimate that it is possible to use natural compounds to both inhibit coagulation pathway enzymes and to reduce blood platelets' activation. As such, in our current study we wanted to verify the anti-platelet and anticoagulant properties of grape seed extract (GSE) using in vitro models. During our analysis, the following parameters were analyzed: Coagulation times, thromboelastometry assays (coagulation time, clot formation time and maximum clot firmness), aggregation of platelets and phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Adenosine diphosphate (ADP)-induced aggregation was lower in GSE 7.5 µg/mL as well as in GSE 15.0 µg/mL. A similar dependence was observed in VASP assays for GSE 7.5 µg/mL and GSE 15 µg/mL. The effect on plasma coagulation tests was distinct only with GSE 15 µg/mL. All of the thromboelastometry variables were statistically significant with 15.0 µg/mL GSE concentration. Our results show, for the first time, the multi-potential effect of grape seed extract on coagulation systems, and clearly suggest that grape seed extract could be considered a promising nutraceutical in the prevention of cardiovascular thrombotic events caused by different mechanisms.

Differentiated mitochondrial function in mouse 3T3 fibroblasts and human epithelial or endothelial cells in response to chemical exposure.

Mouse 3T3 fibroblasts are commonly used for in vitro toxicity testing; however, their sensitivity to stimuli is not well defined. To assess the sensitivity of the 3T3 cell line, the study compared the changes in mitochondrial membrane potential (MMP) occurring after exposure to eight chemicals known to demonstrate pro-apoptotic activity (glycerol, isopropanol, ethanol, paracetamol, propranolol, cobalt chloride, formaldehyde and atropine). Five cell lines were used as follows: mouse 3T3 fibroblasts, human epithelial cells (A549, Caco-2 and HepG2) and human endothelial cells (HMEC-1). Cell sensitivity was assessed based on the total area under and over the dose-response curves (AUOC) in relation to baselines. The 3T3 fibroblasts had the highest AUOC values and were the most sensitive to the action of all the examined chemicals, with the exception of formaldehyde. Significant changes in MMP between the 3T3 cell line and other cells were observed after cell treatment with atropine (A549, Caco-2 or HMEC-1 cells vs 3T3 cells, P < 0.05), propranolol (A549 vs 3T3 cells, P < 0.01; HepG2 vs 3T3 cells, P < 0.05), cobalt chloride (A549 cells vs 3T3 cells, P < 0.01) or ethanol (HMEC-1 vs 3T3, P < 0.05). Formaldehyde appeared the most toxic compound for Caco-2 cells (Caco-2 vs 3T3 cells, P < 0.05). The surface areas (AUOC) calculated for each other chemical and obtained for HepG2, Caco-2, A549 and HMEC-1 did not differ significantly between cell lines. We postulate that mouse 3T3 fibroblasts demonstrate significantly higher relative sensitivity to many agents with toxic potential.

A new approach for the assessment of the toxicity of polyphenol-rich compounds with the use of high content screening analysis.

The toxicity of in vitro tested compounds is usually evaluated based on AC50 values calculated from dose-response curves. However, there is a large group of compounds for which a standard four-parametric sigmoid curve fitting may be inappropriate for estimating AC50. In the present study, 22 polyphenol-rich compounds were prioritized from the least to the most toxic based on the total area under and over the dose-response curves (AUOC) in relation to baselines. The studied compounds were ranked across three key cell indicators (mitochondrial membrane potential, cell membrane integrity and nuclear size) in a panel of five cell lines (HepG2, Caco-2, A549, HMEC-1, and 3T3), using a high-content screening (HCS) assay. Regarding AUOC score values, naringin (negative control) was the least toxic phenolic compound. Aronox, spent hop extract and kale leaf extract had very low cytotoxicity with regard to mitochondrial membrane potential and cell membrane integrity, as well as nuclear morphology (nuclear area). Kaempferol (positive control) exerted strong cytotoxic effects on the mitochondrial and nuclear compartments. Extracts from buckthorn bark, walnut husk and hollyhock flower were highly cytotoxic with regard to the mitochondrion and cell membrane, but not the nucleus. We propose an alternative algorithm for the screening of a large number of agents and for identifying those with adverse cellular effects at an early stage of drug discovery, using high content screening analysis. This approach should be recommended for series of compounds producing a non-sigmoidal cell response, and for agents with unknown toxicity or mechanisms of action.

Xanthohumol from hop cones (Humulus lupulus L.) prevents ADP-induced platelet reactivity.

Hop cones (Humulus lupulus L.), very rich source of phenolic compounds, possessing anticancer, antioxidant and anti-inflammatory activities, are considered as beneficial diet ingredients improving human health. In this study, the antiplatelet action of xanthohumol (XN), the principal flavonoid in hop cones, was investigated. XN significantly attenuated ADP-induced blood platelet aggregation (97.2 ± 35.7 AU for 6 µg/ml of XN vs. 120.4 ± 30.1 AU for 0.17% dimethyl sulfoxide (DMSO), p < 0.001) and significantly reduced the expression of fibrinogen receptor (activated form of GPIIbIIIa) on platelets' surface (47.6 ± 15.8 for 1.5 µg/ml XN, 44.6 ± 17.3% for 3 µg/ml XN vs. 54.5 ± 19.2% for control or 43.3 ± 18.4% for 6 µg/ml XN vs. 49.7 ± 19.4% for 0.17% DMSO, p < 0.05 or less). These findings suggest that the phenolic compounds originating from hops (XN) have a novel role as antiplatelet agents and can likely be used as dietary supplements in prophylactic approaches.

Changes in response to clopidogrel therapy in patients after percutaneous coronary interventions as assessed by different platelet function tests.

INTRODUCTION Recently, the responder status to clopidogrel therapy has been observed to change over time. OBJECTIVES The aim of the study was to investigate changes in the responder status to clopidogrel therapy over time with the use of 4 platelet function tests (light transmission aggregometry [LTA], multiple electrode aggregometry [MEA], vasodilator-stimulated phosphoprotein (VASP) phosphorylation, and INNOVANCE® PFA P2Y assays [PFA]) in patients after percutaneous coronary intervention (PCI). We also compared the results of these tests to determine the most reliable method. PATIENTS AND METHODS The study included 35 patients after PCI, receiving acetylsalicylic acid (75 mg/d) and clopidogrel (75 mg/d). The control group included 50 healthy volunteers. Platelet function was measured at 3 different time points (4 ±2 days after PCI, and then after 6 and 12 weeks).  RESULTS The responder status to clopidogrel changed in 5 patients (14%) as shown by MEA; in 7 patients (20%), by LTA and PFA; and in 13 patients (37%), by VASP. The Cohen's κ coefficient showed a moderate or poor agreement between the tests. The strongest agreement was between MEA and PFA (80%; κ = 0.46, P = 0.003), PFA and LTA (82%; κ = 0.41, P = 0.004), and MEA and LTA (80%; κ = 0.36, P = 0.008). The κ coefficient for all comparisons with VASP was less than 0.30. CONCLUSIONS Changes in the responder status over time are present for all platelet function tests, but a large discrepancy between the tests does not allow a careful assessment of this phenomenon. The tests showed only moderate agreement (in relation to one another and to time points), which significantly limits their interchangeable use in clinical practice.

Relationship between high on aspirin platelet reactivity and oxidative stress in coronary artery by-pass grafted patients.

The aim of the study was to assess the responsiveness of blood platelets to acetylsalicylic acid (ASA) in patients following coronary artery bypass grafting (CABG) surgery with relation to oxidative and antioxidative plasma status. The study included 37 patients treated with the CABG procedure. During the first 24 h after CABG patients were given 300 mg of ASA with the following dose of 150 mg daily. The blood was collected before the procedure and 10 days after. Whole blood platelet aggregation induced with arachidonic acid, collagen and adenosine diphosphate (ADP) was performed together with whole blood generation of thromboxane B2 (TxB2). Oxidative stress was measured before and 10 days after CABG with total oxidative plasma status (TOS) and total antioxidative status of the plasma (TAS). TOS/TAS index was calculated. We observed a significant increase in the TOS and TOS/TAS index and ADP-induced aggregation 10 days after CABG in comparison with its level before operation. There was a significant decrease in the arachidonic acid-induced aggregation and serum TxB2 level. Patients with ADP-induced and collagen-induced aggregation in the upper quartile had significantly higher TOS and TOS/TAS index before (ADP) and after the operation (ADP and collagen). There were 19 patients (51%) with high on aspirin platelet reactivity after CABG who had also higher TOS and TOS/TAS index and lower TAS value in comparison with aspirin responders. Despite ASA use, increased oxidative stress after CABG can overcome its antiplatelet effect and increase platelet activation through other pathways.

Does grape seed extract potentiate the inhibition of platelet reactivity in the presence of endothelial cells?

PURPOSE: Numerous studies have suggested that grape seed extract (GSE) confers vascular protection due to the direct effect of its polyphenol content on endothelial cells. The aim of the study was to determine whether GSE confers vascular protection through the direct effect of its polyphenol content on endothelial cells. MATERIAL/METHODS: After incubation with GSE-treated human umbilical vein endothelial cells (HUVECs), blood platelet reactivity was evaluated with regard to the expression of CD62P and the activated form of GPIIbIIIa in ADP-stimulated platelets. RESULTS: Lower concentrations of GSE were found to enhance the antiplatelet action of HUVECs: 1 µg/ml GSE reduced platelet reactivity by about 10%. While platelet reactivity was not altered by HUVECs incubated with higher concentrations of GSE, HUVEC proliferation was significantly reduced by GSE of up to 10 µg gallic acid equivalent/ml. CONCLUSIONS: The results of the study show that low doses of GSE potentiate the inhibitory action of HUVECs on platelet reactivity, which may account, at least partially, for the protective effects of grape products against cardiovascular diseases. In contrast, high concentrations of GSE significantly impair endothelial cell proliferation in vitro.

Biochemical and hematological changes following the 120-km open-water marathon swim.

Data on physiological effects and potential risks of a ultraendurance swimming are scarce. This report presents the unique case of a 61-year old athlete who completed a non-stop open-water 120-km ultramarathon swim on the Warta River, Poland. Pre-swimming examinations revealed favorable conditions (blood pressure, 110/70 mmHg; rest heart rate, 54 beats/minute, ejection fraction, 60%, 20.2 metabolic equivalents in a maximal exercise test). The swimming time and distance covered were 27 h 33 min and 120 km, respectively. Blood samples for hematological and biochemical parameters were collected 30 min, 4 hrs, 10 hrs and 8 days after the swim. The body temperature of the swimmer was 36.7°C before and 35.1°C after the swim. The hematological parameters remained within the reference range in the postexercise period except for leucocytes (17.5 and 10.6 x G/l noted 30 minutes and 4 hours after the swim, respectively). Serum urea, aspartate aminotransferase and C-reactive protein increased above the reference range reaching 11.3 mmol/l, 1054 nmol/l/s and 25.9 mg/l, respectively. Symptomatic hyponatremia was not observed. Although the results demonstrate that an experienced athlete is able to complete an ultra-marathon swim without negative health consequences, further studies addressing the potential risks of marathon swimming are required. Key pointsData on biochemical changes due to long-distance swimming are scarce.This report presents the unique case of a 61-year old athlete who completed a non-stop open-water 120-km ultramarathon swim.An experienced athlete is able to complete an ultra-marathon swim without serious health consequences.Regarding the growing popularity of marathon swimming further studies addressing the potential risks of such exhaustive exercise are required.

Various laboratory protocols for measuring thromboxane A2 generation to detect the effectiveness of acetylsalicylic acid therapy: a comparative study.

A reliable and simple laboratory assay for predicting clinical effectiveness of antiplatelet acetylsalicylic acid (ASA) therapy is needed. We have compared various laboratory protocols for measuring blood thromboxane A2 (TXA2) generation used to detect the effects of ASA administration. Healthy volunteers (n = 15) were given 150 mg per day ASA for 10 days, followed by ASA at 75 mg per day for 10 days. Five protocols tested for measuring TXA2 generation were: baseline TXB2 determination in plasma; static generation of TXA2 in anticoagulated blood (1 h incubation at room temperature or 37°C, respectively); dynamic generation of TXA2 in anticoagulated blood (1 h in rotary mixer); and generation of TXA2 in blood without anticoagulant (serum-generated TXA2). Platelet aggregation in whole blood was also measured using arachidonic acid (AA), collagen, and ADP as agonists. All five protocols showed significant reduction in TXB2 levels in individuals taking ASA. However, only the assay of TXA2 generation in serum was significantly different compared with the other protocols (P < 0.002). Moreover, the strongest and most significant correlation was observed between TXA2 generation in serum and AA-induced aggregation parameters (for 75 mg per day ASA).Serum TXA2 generation is the best laboratory protocol to detect the effects of ASA, based on serum markers of prostanoid metabolism.