The Effects of Caffeine on Blood Platelets and the Cardiovascular System through Adenosine Receptors.

Caffeine is the most popular and widely consumed behaviourally active substance in the world. This review describes the influence of caffeine on the cardiovascular system, with a special focus on blood platelets. For many years, caffeine was thought to have a negative effect on the cardiovascular system mainly due to increasing blood pressure. However, more recent data suggest that habitual caffeine consumption may reduce the risk of cardiovascular disease and hypertension. This could be a significant finding as cardiovascular disease is the leading cause of death worldwide. Caffeine is known to inhibit A1 adenosine receptors, through which it is believed to modulate inter alia coronary blood flow, total peripheral resistance, diuresis, and heart rate. It has been shown that coffee possesses antiplatelet activity, but depending on the dose and the term of its use, caffeine may stimulate or inhibit platelet reactivity. Also, chronic exposure to caffeine may sensitize or upregulate the adenosine receptors in platelets causing increased cAMP accumulation and anti-aggregatory effects and decrease calcium levels elicited by AR agonists. The search for new, selective, and safe AR agonists is one of the new strategies for improving antiplatelet therapy involving targeting multiple pathways of platelet activation. Therefore, this review examines the AR-dependent impact of caffeine on blood platelets in the presence of adenosine receptor agonists.

Evaluation of a novel pyridinium cation-linked styryl-based boronate probe for the detection of selected inflammation-related oxidants.

Reactive oxygen and nitrogen species (RONS) are a range of chemical individuals produced by living cells that contribute to the proper functioning of organisms. Cells under oxidative and nitrative stress show excessive production of RONS (including hydrogen peroxide, H2O2, hypochlorous acid, HOCl, and peroxynitrite, ONOO-) which may result in a damage proteins, lipids, and genetic material. Thus, the development of probes for in vivo detection of such oxidants is an active area of research, focusing on molecular redox sensors, including boronate-caged fluorophores. Here, we report a boronate-based styryl probe with a cationic pyridinium moiety (BANEP+) for the fluorescent detection of selected biological oxidants in vitro and in vivo. We compare the chemical reactivity of the BANEP+ probe toward H2O2, HOCl, and ONOO- and examine the influence of the major intracellular non-enzymatic antioxidant molecule, glutathione (GSH). We demonstrate that, at the physiologically relevant GSH concentration, the BANEP+ probe is efficiently oxidized by peroxynitrite, forming its phenolic derivative HNEP+. GSH does not affect the fluorescence properties of the BANEP+ and HNEP+ dyes. Finally, we report the identification of a novel type of molecular marker, with the boronate moiety replaced by the iodine atom, formed from the probe in the presence of HOCl and iodide anion. We conclude that the reported chemical reactivity and structural features of the BANEP+ probe may be a basis for the development of new red fluorescent probes for in vitro and in vivo detection of ONOO-.

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.

SARS-CoV-2 Spike Protein and Neutralizing Anti-Spike Protein Antibodies Modulate Blood Platelet Function.

Several studies report elevated blood platelet activation and altered platelet count in COVID-19 patients, but the role of the SARS-CoV-2 spike protein in this process remains intriguing. Additionally, there is no data that anti-SARS-CoV-2 neutralizing antibodies (nAb) may attenuate spike protein activity toward blood platelets. Our results indicate that under in vitro conditions, the spike protein increased the collagen-stimulated aggregation of isolated platelets and induced the binding of vWF to platelets in ristocetin-treated blood. The spike protein also significantly reduced collagen- or ADP-induced aggregation or decreased GPIIbIIIa (fibrinogen receptor) activation in whole blood, depending on the presence of the anti-spike protein nAb. Our findings suggest that studies on platelet activation/reactivity in COVID-19 patients or in donors vaccinated with anti-SARS-CoV-2 and/or previously-infected COVID-19 should be supported by measurements of spike protein and IgG anti-spike protein antibody concentrations in blood.

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.

Platelet-Derived Procoagulant Microvesicles Are Elevated in Patients with Retinal Vein Occlusion (RVO).

The etiopathogenesis of retinal vein occlusion (RVO) is multifactorial, and the contribution of platelets to RVO development has not been fully elucidated. We aimed to analyze platelet function in RVO patients (n = 35) and controls (n = 35). We found a higher (p < 0.05) level of soluble P-selectin in RVO group vs. controls. Additionally, in RVO patients, the concentration of platelet-derived microvesicles was higher (p < 0.05), and the difference between groups was deeper for the fraction of platelet-derived microvesicles with the procoagulant phenotype (p < 0.0001) and for overall procoagulant microvesicles level (p < 0.0001). The results were similar for the total RVO group and for both RVO types (central- and branched-retinal vein occlusion). We did not find differences in simple platelet parameters (platelet count, mean platelet volume, platelet distribution width, platecrit, reticulated platelets) and inflammatory markers (platelet-lymphocyte ratio, neutrophil-lymphocyte ratio). Similarly, no differences were found for platelet aggregation-stimulated byadenosine diphosphate; collagen; arachidonic acid; and in multiparametric flow cytometry evaluation of P-selectin, PAC-1, and fibrinogen binding for both unstimulated and adenosine diphosphate-, collagen-, and thrombin receptor activating peptide-stimulated platelets. Our results suggest that platelets can contribute to developing RVO by enhancing procoagulant activity through providing a procoagulation surface via platelet-derived microvesicles. The direct role of platelets' hyperreactivity in developing RVO is less apparent, which is consistent with the complexity and multifactorial background of this disorder.

An evaluation of a new high-sensitivity PrestoBlue assay for measuring cell viability and drug cytotoxicity using EA.hy926 endothelial cells.

INTRODUCTION: Commercially-available resazurin-based reagents used for cell viability assessment contain varying amounts of resorufin; these may contribute to differences in autofluorescence, signal-to-background (S/B) ratio and the dynamic range of the assay. OBJECTIVES: This in vitro study compares the sensitivity of a new, high-sensitivity PrestoBlue (hs-PB) assay with standard PrestoBlue (PB) in assessing the efficacy of valinomycin and antimycin A in human vascular endothelial EA.hy926 cells, as well as cell viability. METHODS: The metabolic activity of EA.hy926 was evaluated based on resorufin fluorescence (PB assays) or formazan absorbance (MTT assay). RESULTS: The hs-PB assay demonstrated lower resorufin autofluorescence than the PB, resulting in a ≥ 1.4-fold increase in S/B ratio in hs-PB compared to PB. Valinomycin was more potent cytotoxic agent than antimycin A. The hs-PB, PB and MTT produced similar IC50 values for valinomycin. Antimycin A showed significantly higher potency in the MTT than in the resazurin-based assays. The EA.hy926 cells demonstrated higher metabolic activity in the presence of the antimycin A solvent - DMSO. CONCLUSION: All the examined methods may be used interchangeably to analyze drug cytotoxicity. Any differences in drug cytotoxicity observed between the assays may be due to relatively low drug potency and/or the influence of solvent on metabolism of assay reagent. The hs-PB assay appears to more effectively detect cell viability and produce a stronger signal than its conventional counterpart.

Dietary Intake of Polyphenols or Polyunsaturated Fatty Acids and Its Relationship with Metabolic and Inflammatory State in Patients with Type 2 Diabetes Mellitus.

BACKGROUND: The aim of the study was to evaluate the relationship between polyphenol or polyunsaturated fatty acids (PUFAs) consumption and the selected metabolic and inflammatory markers in type 2 diabetes (T2DM) patients. METHODS: The study enrolled 129 diabetics (49 men, mean age 64.1 ± 9.8 years) with different amounts of polyphenol and PUFAs consumption. RESULTS: A significant effect of polyphenol or PUFAs omega-3 consumption on fasting glucose concentration (FG) or glycated haemoglobin fraction (HbA1c) was reported. A negative association was observed between FG and total polyphenol, flavonoid, flavan-3-ol and stilbene intake. In the group with high flavonoid intake, the FG was significantly lower compared to the group characterised by low flavonoid intake. Polyphenols, except stilbenes, did not modulate HbA1c. Additionally, higher consumption of PUFAs omega-3 significantly decreased HbA1c, and the intake of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids negatively and significantly correlated with FG and HbA1c. Further analysis confirmed a significant association between EPA + DHA intake and HbA1c, with significant interactions with age and gender or with body mass index and waist-to-hip ratio. The dietary intake of polyphenols or PUFAs was independent of familial diabetes or diabetic diet application. CONCLUSIONS: Our study indicates a positive effect of high consumption of flavonoids, omega-3 PUFAs and stilbenes on the markers of carbohydrate metabolism balance and the absence of such an effect on other cardiometabolic markers and inflammatory conditions.

Adenosine Receptor Agonists Increase the Inhibition of Platelet Function by P2Y12 Antagonists in a cAMP- and Calcium-Dependent Manner.

We have shown previously that platelet activity can be lowered through the simultaneous inhibition of P2Y12 receptor and activation of adenosine receptors (AR). This work explores this concept by testing the antiplatelet potential of nine AR agonists in combination with P2Y12 receptor antagonists-cangrelor and prasugrel metabolite. A panel of in vitro methods was used to assess platelet viability, P-selectin expression, GPIIb-IIIa activation, fibrinogen binding, calcium ion mobilization, VASP-P level, and cAMP formation, utilizing whole blood or isolated platelets from healthy volunteers. The AR agonists demonstrated anti-platelet effects, but stimulated signaling pathways to varying degrees. AR agonists and P2Y12 antagonists reduced expression of both P-selectin and the activated form of GPIIb-IIIa on platelets; however, the combined systems (AR agonist + P2Y12 antagonist) demonstrated stronger effects. The antiplatelet effects of AR when combined with P2Y12 were more pronounced with regard to exogenous fibrinogen binding and calcium mobilization. The cAMP levels in both resting and ADPactivated platelets were increased by AR agonist treatment, and more so when combined with P2Y12 inhibitor. In conclusion, as AR agonists are fast-acting compounds, the methods detecting early activation events are more suitable for assessing their antiplatelet action. The exogenous fibrinogen binding, calcium mobilisation and cAMP level turned out to be sensitive markers for detecting the inhibition caused by AR agonists alone or in combination with P2Y12 receptor antagonists.

Fibrinogen Glycation and Presence of Glucose Impair Fibrin Polymerization-An In Vitro Study of Isolated Fibrinogen and Plasma from Patients with Diabetes Mellitus.

BACKGROUND: Fibrin formation and structure may be affected by a plethora of factors, including both genetic and posttranslational modifications, such as glycation, nitration or acetylation. METHODS: The present study examines the effect of fibrinogen glycation on fibrin polymerization, measured in fibrinogen concentration-standardized plasma of subjects with type 2 diabetes mellitus (T2DM) and in a solution of human fibrinogen exposed to 30 mM glucose for four days. RESULTS: The fibrin polymerization velocity (Vmax) observed in the T2DM plasma (median 0.0056; IQR 0.0049‒0.0061 AU/s) was significantly lower than in non-diabetic plasma (median 0.0063; IQR 0.0058‒0.0071 AU/s) (p < 0.05). Furthermore, significantly lower Vmax was observed for glucose-treated fibrinogen (Vmax 0.046; IQR 0.022‒0.085 AU/s) compared to control protein incubated with a pure vehicle (Vmax 0.053; IQR 0.034‒0.097 AU/s) (p < 0.05). The same tendency was observed in the fibrinogen samples supplemented with 6 mM glucose just before measurements. It is assumed that glucose may affect the ability of fibrinogen to form a stable clot in T2DM subjects, and that this impairment is likely to influences the outcomes of some diagnostic assays. As the example, the impaired clotting ability of glycated fibrinogen may considerably influence the results of the standard Clauss method, routinely used to determine fibrinogen concentration in plasma. The stoichiometric analysis demonstrated that spontaneous glycation at both the sites with high and low glycation potential clearly dominated in T2DM individuals in all fibrinogen chains.

Diabetes and Hyperglycemia Affect Platelet GPIIIa Expression. Effects on Adhesion Potential of Blood Platelets from Diabetic Patients under In Vitro Flow Conditions.

Blood platelets play a crucial role in the early stages of atherosclerosis development. The process is believed to require firm adhesion of platelets to atherosclerosis-prone sites of the artery. However, little evidence exists regarding whether the blood platelets of individuals with pathological conditions associated with atherosclerosis have higher potential for adhesion. This process is to a large extent dependent on receptors present on the platelet membrane. Therefore, the aim of the presented study was to determine whether blood platelets from diabetic patients have higher capacity of adhesion under flow conditions and how diabetes affects one of the crucial platelet receptors involved in the process of adhesion-GPIIIa. The study compares the ability of platelets from non-diabetic and diabetic humans to interact with fibrinogen and von Willebrand factor, two proteins found in abundance on an inflamed endothelium, under flow conditions. The activation and reactivity of the blood platelets were also characterized by flow cytometry. Platelets from diabetic patients did not demonstrate enhanced adhesion to either studied protein, although they presented increased basal activation and responsiveness towards low concentrations of agonists. Platelets from diabetic patients were characterized by lower expression of GPIIIa, most likely due to an enhanced formation of platelet-derived microparticles PMPs, as supported by the observation of elevated concentration of this integrin and of GPIIIa-positive PMPs in plasma. We conclude that altered functionality of blood platelets in diabetes does not increase their adhesive potential. Increased glycation and decrease in the amount of GPIIIa on platelets may be partially responsible for this effect. Therefore, higher frequency of interactions of platelets with the endothelium, which is observed in animal models of diabetes, is caused by other factors. A primary cause may be a dysfunctional vascular wall.

Bioavailability and metabolism of selected cocoa bioactive compounds: A comprehensive review.

Cocoa beans and their co-products are a rich source of beneficial compounds for health promotion, including polyphenols and methylxanthines. Knowledge of bioavailability and in vivo bioactivity of these phytochemicals is crucial to understand their role and function in human health. Therefore, many studies concerning bioavailability and bioactivity of cocoa bioactive compound have been done in both in vivo animal models and in humans. This critical review comprehensively summarizes the existing knowledge about the bioavailability and the major metabolic pathways of selected cocoa bioactive compounds (i.e. monomeric flavan-3-ols, procyanidins, anthocyanins, flavonols, phenolic acids, N-phenylpropenoyl-L-amino acids, stilbenes, and methylxanthines). The compiled results indicated that many of these compounds undergo extensive metabolism prior to absorption. Different factors have been suggested to influence the bioavailability of polyphenols and methylxanthines among them the role of gut microbiota, structure of these compounds, food matrix and occurrence of other substances were the most often considered. Aforementioned factors decided about the site where these bioactive compounds are digested and absorbed from the alimentary tract, as well as the pathway by which they are metabolized. These factors also determine of the type of transport through the intestine barrier (passive, involving specific enzymes or mediated by specific transporters) and their metabolic path and profile.

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.

The influence of Rubus idaeus and Rubus caesius leaf extracts on platelet aggregation in whole blood. Cross-talk of platelets and neutrophils.

Recently, polyphenols have gained attention as potential natural cardioprotective therapeutics, due to their antiplatelet, anti-inflammatory and anticoagulant activity. Species belonging to the genus Rubus sp. have been reported to be a source of polyphenolic compounds with antioxidative proprieties and beneficial biological activities. This study investigates the effects of leaf extracts obtained from red raspberry (Rubus idaeus L.) and European dewberry (Rubus caesius L.) on the reactivity of blood platelets. In ADP-stimulated blood, raspberry and dewberry extracts (15 µg/ml) markedly decreased platelet surface membrane expression of activated GPIIbIIIa receptor by 16% and 21%, respectively (P < 0.01) and significantly inhibited platelet aggregation (by 31-41% for raspberry and by 38-55% for dewberry, P < 0.01). In platelet-rich plasma (PRP), the extracts had no effect on ADP-induced platelet aggregation. The effectiveness of the extracts in whole blood and the lack of their activity in PRP indicate that leukocytes are likely to participate in the platelet response to the extracts. Our experiments show that the extracts significantly reduced the amount of free radicals released by activated neutrophils in whole blood (P < 0.001), as well as in suspensions of isolated neutrophils (P < 0.05). Moreover, the reduced number of neutrophils leads to the decreased efficiency of the extracts in the inhibition of platelet aggregation. In summary, our findings show that the raspberry and dewberry leaf extracts considerably modulated blood platelet reactivity in whole blood: they influenced blood platelet aggregation, possibly via the modulation of the redox status dependent on the oxidative activity of neutrophils.

Comparison of cytotoxic and anti-platelet activities of polyphenolic extracts from Arnica montana flowers and Juglans regia husks.

Polyphenolic compounds of plant origin are well known to be beneficial to human health: they exert protective effects on haemostasis and have a particular influence on blood platelets. However, the anti-platelet properties of polyphenolic compounds observed so far have not been weighed against their potential cytotoxic action against platelets. The aim of this study was to demonstrate that anti-platelet and cytotoxic effects on blood platelets may interfere and therefore, may often lead to confusion when evaluating the properties of plant extracts or other agents towards blood platelets. The anti-platelet and cytotoxic in vitro effects of plant extracts obtained from the husks of walnuts (J. regia) and flowers of arnica (A. montana) on platelet reactivity and viability were examined. Platelet function was assessed using standard methods (flow cytometry: P-selectin expression, activation of GPIIbIIIa complex, vasodilator-stimulated phosphoprotein, VASP index; turbidimetric and impedance aggregometry) and newly set assays (flow cytometric monitoring of platelet cytotoxicity). The results reveal that none of the studied plant extracts demonstrated cytotoxicity towards blood platelets. The phenolic acid-rich extract of A. montana (7.5 and 15 µg/ml) significantly reduced the ADP-induced aggregation in both whole blood and PRP, and decreased the platelet reactivity index (PRI; VASP phosphorylation) in whole blood, while showing excellent antioxidant capacity. The extract of J. regia husks significantly reduced ADP-induced platelet aggregation in whole blood when applied at 7.5 µg/ml, and only slightly decreased the PRI at 15 µg/ml. Both examined extracts suppressed platelet hyper-reactivity, and such influence did not interfere with cytotoxic effects of the extracts. Thus, its high polyphenol content, excellent antioxidant capacity and distinct anti-platelet properties, in combination with its lack of toxicity, make the extract of A. montana flowers a possible candidate as an anti-platelet agent or a compounding diet supplement.

Extract from Ribes nigrum leaves in vitro activates nitric oxide synthase (eNOS) and increases CD39 expression in human endothelial cells.

The aim of the present study was to evaluate whether blackcurrant leaf extract (BLE) modulates endothelium antithrombotic function, namely increases the expression/activity of ADPase (CD39) and augments the production of nitric oxide in human umbilical vein endothelial cells (HUVEC). It was found that BLE with proanthocyanidins (60 % of the total polyphenol content) increased the CD39-positive endothelial cell fraction (up to 10 % for 2.5 µg/ml, and up to 33 % for 15 µg/ml, p < 0.05 or less) in a concentration-dependent manner, and enhanced endothelial nitric oxide synthase (eNOS) activation (T495 phosphorylation decreased by 31 ± 6 % for 2.5 µg/ml and 48 ± 6 % for 15 µg/ml; S1177 phosphorylation increased by 13 ± 3 % for 2.5 µg/ml and 18 ± 7 % for 15 µg/ml, compared to untreated cells, p < 0.05 or less). Additionally, incubation for 24 or 48 h with BLE at a lower range of polyphenol concentrations, significantly increased cell viability with a maximal effect at 2.5 µg/ml (viability increased by 24.8 ± 1.0 % for 24 h and by 32.5 ± 2.7 % for 48-h time incubation, p < 0.0001). The increased CD39 expression and the increased eNOS activation in HUVEC can be regarded as the beneficial markers of the improvement of antiplatelet action of endothelial cells. Unexpectedly, these assumptions were not confirmed in the experimental model of platelet-endothelial cell interactions. These observations lead to the conclusion that BLE may improve endothelial cell viability at low physiological concentrations without affecting the antiplatelet action of endothelium.

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.

CD39/NTPDase-1 expression and activity in human umbilical vein endothelial cells are differentially regulated by leaf extracts from Rubus caesius and Rubus idaeus.

Many experimental studies have demonstrated the favorable biological activities of plants belonging to the genus Rubus, but little is known of the role of Rubus leaf extracts in the modulation of the surface membrane expression and activity of endothelial apyrase. The aim of this study was to assess the influence of 1-15 µg/ml Rubus extracts on CD39 expression and enzymatic activity, and on the activation (ICAM-1 expression) and viability of human umbilical vein endothelial cells (HUVEC). The polyphenolic contents and antioxidative capacities of extracts from dewberry (R. caesius L.) and raspberry (R. idaeus L.) leaves were also investigated. The techniques applied were flow cytometry (endothelial surface membrane expression of ICAM-1 and CD39), malachite green assay (CD39 activity), HPLC-DAD (quantitative analysis of polyphenolic extract), ABTS, DPPH and FRAP spectrometric assays (antioxidant capacity), and the MTT test (cell viability). Significantly increased CD39 expressions and significantly decreased ATPDase activities were found in the cells treated with 15 µg/ml of either extract compared to the results for the controls. Neither of the extracts affected cell proliferation, but both significantly augmented endothelial cell ICAM-1 expression. The overall antioxidant capacities of the examined extracts remained relatively high and corresponded well to the determined total polyphenol contents. Overall, the results indicate that under in vitro conditions dewberry and raspberry leaf extracts have unfavorable impact on endothelial cells.

Enhanced platelet-derived microparticle formation is associated with carotid atherosclerosis in convalescent stroke patients.

Platelets participate in the development and progression of atherosclerosis. During this process they interact with endothelial cells and leukocytes. Therefore, we investigated the associations between carotid atherosclerosis and platelet reactivity markers. The platelet surface expression of P-selectin (CD62P) and the activated GPIIb/IIIa receptor (corresponding to increased binding of PAC-1), as well as the fraction of platelet-derived microparticles (PMPs) prior to and after platelet stimulation with TRAP or ADP, were determined using flow cytometry in 94 subjects in the convalescent phase of ischaemic stroke and in 76 disease controls. The mean common carotid intima-media thickness (CCA(mean) IMT), maximal common carotid IMT (CCA(max) IMT) and maximal bifurcation IMT (BIF(max) IMT) were measured bilaterally using B mode, colour Doppler ultrasonography. In stroke subjects IMT within CCA and BIF were greater than in disease controls and the percentage of PMPs prior to and after ex vivo stimulation with agonists was significantly higher than in controls. Multiple regression analysis revealed that PMPs were positively and independently correlated with both CCA(mean) IMT (ß = 0.23; p < 0.01) and stroke (ß = 0.21; p<0.01), while PAC-1 binding to platelets activated with ADP was negatively and independently associated with CCA(mean) IMT (ß = -0.29; p<0.001) and atherosclerotic carotid plaque presence (ß = -0.28, p = 0.003). We found a positive association between enhanced PMP formation and atherosclerotic thickening of carotid intima-media or carotid plaque in patients after ischaemic stroke. We demonstrated that diminished expression of active GPIIb/IIIa in the ADP-activated platelets is associated with increased carotid IMT, independently of stroke.