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Platelet aggregation is a complicated process mediated by different signaling pathways. As the process is highly complex and apparently redundant, the relationships between these pathways are not yet fully known. The aim of this project was to study the interconnections among seven different aggregation pathways in a group of 53 generally healthy volunteers aged 20 to 66 years. Platelet aggregation was induced with thrombin receptor activating peptide 6 (TRAP), arachidonic acid (AA), platelet activating factor 16 (PAF), ADP, collagen, thromboxane A 2 analogue U46619 or ristocetin (platelet agglutination) ex vivo in fasting blood samples according to standardized timetable protocol. Additionally, some samples were pre-treated with known clinically used antiplatelet drugs (vorapaxar, ticagrelor or acetylsalicylic acid (ASA)). Significant correlations among all used inducers were detected (Pearson correlation coefficients (r P ): 0.3 to 0.85). Of all the triggers, AA showed to be the best predictor of the response to other inducers with r P ranging from 0.66 to 0.85. Interestingly, the antiplatelet response to ticagrelor strongly predicted the response to unrelated drug vorapaxar (r P  = 0.71). Our results indicate that a response to one inducer can predict the response for other triggers or even to an antiplatelet drug. These data are useful for future testing but should be also confirmed in patients.

Background and Aims: It is well known that elevated cholesterol is associated with enhanced platelet aggregation and patients suffering from familial hypercholesterolemia (FH) have a high risk of thrombotic cardiovascular events. Although decreasing cholesterol level is associated with attenuation of platelet hyperactivity, there are currently no data on the effect of convertase subtilisin/kexin type 9 monoclonal antibodies (PCSK9ab) on platelet reactivity in FH. The aim of the study was to analyse the impact of different therapies including PCSK9ab on platelet aggregation in FH., Methods: This study enrolled all 15 patients treated in the University Hospital Hradec Králové for FH. PCSK9ab have been administered in 12 of 15 patients while 8 patients were also undergoing lipid apheresis. Blood samples from all patients including pre- and post-apheresis period were tested for platelet aggregation triggered by 7 inducers, and the effect of 3 clinically used drugs (acetylsalicylic acid, ticagrelor and vorapaxar) was compared as well., Results: Although apheresis decreased the reactivity of platelets in general, platelet responses were not different between non-apheresis patients treated with PCSK9ab and apheresis patients (post-apheresis values) with the exception of ristocetin. However, when compared to age-matched healthy population, FH patients had significantly lower platelet aggregation responses to 4 out of 7 used inducers and higher profit from 2 out of 3 used antiplatelet drugs even after exclusion of FH patients regularly receiving conventional antiplatelet treatment., Conclusion: This study showed for the first time the suitability of PCSK9ab treatment for reduction of platelet reactivity in FH patients., (© 2023. The Author(s).)

Benzoic acids, which are commonly found in food, are also produced by human microbiota from other dietary phenolics. The aim was to investigate the interactions of 8 food-related benzoic acids with the physiological metals iron and copper under different (patho)physiologically relevant pH conditions in terms of chelation, reduction, impact on the metal-based Fenton chemistry, and copper-based hemolysis. Only 3,4-dihydroxybenzoic acid behaved as a protective substance under all conditions. It chelated iron, reduced both iron and copper, and protected against the iron and copper-based Fenton reaction. Conversely, 2,4,6-trihydroxybenzoic acid did not chelate iron and copper, reduced both metals, potentiated the Fenton reaction, and worsened copper-based hemolysis of rat red blood cells. The other tested compounds showed variable effects on the Fenton reaction. Interestingly, prooxidative benzoic acids mildly protected human erythrocytes against Cu-induced lysis. In conclusion, 3,4-dihydroxybenzoic acid seems to have a protective effect against copper and iron-based toxicity under different conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The process of platelet aggregation is often influenced by several factors including sex and age. A literature review confirmed the existence of sex-related differences in platelet aggregation. Although 68 out of 78 papers found such differences, there are still some controversies regarding these differences, which can be due to multiple factors (age, trigger, concomitant disease, sample handling, etc.). These outcomes are discussed in line with novel results obtained from a local study, in which blood samples from a total of 53 overall healthy women and men with ages ranging from 20 to 66 years were collected. Aggregation was induced with seven different triggers (ristocetin, thrombin receptor activating peptide 6 [TRAP-6], arachidonic acid [AA], platelet-activating factor 16 [PAF-16], ADP, collagen, or thromboxane A 2 analog U-46619) ex vivo. In addition, three FDA-approved antiplatelet drugs (vorapaxar, ticagrelor, or acetylsalicylic acid [ASA]) were also tested. In general, women had higher aggregation responses to some agonists (ADP, TRAP), as well as lower benefit from inhibitors (ASA, vorapaxar). The aggregatory responses to AA and TRAP decreased with age in both sexes, while responses to ADP, U-46619, and PAF were affected by age only in women. In conclusion, more studies are needed to decipher the biological importance of sex-related differences in platelet aggregation in part to enable personalized antiplatelet treatment., Competing Interests: None declared., (Thieme. All rights reserved.)

Elevated low-density lipoprotein (LDL) cholesterol levels lead to atherosclerosis and platelet hyperaggregability, both of which are known culprits of arterial thrombosis. Normalization of LDL cholesterol in familial hypercholesterolemia (FH) is not an easy task and frequently requires specific treatment, such as regularly performed lipid apheresis and/or novel drugs such as proprotein convertase subtilisin kexin 9 monoclonal antibodies (PCSK9Ab). Moreover, a high resistance rate to the first-line antiplatelet drug acetylsalicylic acid (ASA) stimulated research of novel antiplatelet drugs. 4-methylcatechol (4-MC), a known metabolite of several dietary flavonoids, may be a suitable candidate. The aim of this study was to analyse the antiplatelet effect of 4-MC in FH patients and to compare its impact on two FH treatment modalities via whole-blood impedance aggregometry. When compared to age-matched, generally healthy controls, the antiplatelet effect of 4-MC against collagen-induced aggregation was higher in FH patients. Apheresis itself improved the effect of 4-MC on platelet aggregation and blood from patients treated with this procedure and pretreated with 4-MC had lower platelet aggregability when compared to those solely treated with PCKS9Ab. Although this study had some inherent limitations, e.g., a low number of patients and possible impact of administered drugs, it confirmed the suitability of 4-MC as a promising antiplatelet agent and also demonstrated the effect of 4-MC in patients with a genetic metabolic disease for the first time.

Flavonoids are considered beneficial, but they may exhibit pro-oxidative effects likely due to metal reducing properties. For the first time, 24 structurally related flavonoids were compared for copper reduction, and modulation of the copper-triggered Fenton reaction and lysis of erythrocytes. The vast majority of flavonoids reduced cupric ions; their behaviour ranged from progressive gradual reduction through bell-shaped, neutral, to a blockade of spontaneous reduction. Similarly, different behaviours were observed with the Fenton reaction. Flavone was the only flavonoid that potentiated copper-triggered haemolysis (155 ± 81 % at twice the amount of Cu 2+ ), while 18 flavonoids were at least partly protective in some concentrations. Only 5-hydroxyflavone did not reduce Cu 2+ and behaved as an antioxidant in both assays (reduction of 60 ± 10 % and 88 ± 1%, respectively, at an equimolar ratio with Cu 2+ ). In conclusion, relatively subtle structural differences resulted in very different anti/prooxidant behaviour depending on the model., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

A polyphenol-rich diet has beneficial effects on cardiovascular health. However, dietary polyphenols generally have low bioavailability and reach low plasma concentrations. Small phenolic metabolites of these compounds formed by human microbiota are much more easily absorbable and could be responsible for this effect. One of these metabolites, 4-methylcatechol (4-MC), was suggested to be a potent anti-platelet compound. The effect of 4-MC was tested ex vivo in a group of 53 generally healthy donors using impedance blood aggregometry. The mechanism of action of this compound was also investigated by employing various aggregation inducers/inhibitors and a combination of aggregometry and enzyme linked immunosorbent assay (ELISA) methods. 4-MC was confirmed to be more potent than acetylsalicylic acid on both arachidonic acid and collagen-triggered platelet aggregation. Its clinically relevant effect was found even at a concentration of 10 μM. Mechanistic studies showed that 4-MC is able to block platelet aggregation caused by the stimulation of different pathways (receptors for the von Willebrand factor and platelet-activating factor, glycoprotein IIb/IIIa, protein kinase C, intracellular calcium elevation). The major mechanism was defined as interference with cyclooxygenase-thromboxane synthase coupling. This study confirmed the strong antiplatelet potential of 4-MC in a group of healthy donors and defined its mechanism of action.

Isoquinoline alkaloids have multiple biological activities, which might be associated with positive pharmacological effects as well as negative adverse reactions. As bleeding was suggested to be a side effect of the isoquinoline alkaloid berberine, we decided to ascertain if different isoquinoline alkaloids could influence hemocoagulation through the inhibition of either platelet aggregation or blood coagulation. Initially, a total of 14 compounds were screened for antiplatelet activity in whole human blood by impedance aggregometry. Eight of them demonstrated an antiplatelet effect against arachidonic acid-induced aggregation. Papaverine and bulbocapnine were the most potent compounds with biologically relevant IC 50 values of 26.9 ± 12.2 μM and 30.7 ± 5.4 μM, respectively. Further testing with the same approach confirmed their antiplatelet effects by employing the most physiologically relevant inducer of platelet aggregation, collagen, and demonstrated that bulbocapnine acted at the level of thromboxane receptors. None of the alkaloids tested had an effect on blood coagulation measured by a mechanical coagulometer. In conclusion, the observed antiplatelet effects of isoquinoline alkaloids were found mostly at quite high concentrations, which means that their clinical impact is most likely low. Bulbocapnine was an exception. It proved to be a promising antiplatelet molecule, which may have biologically relevant effects.

Objectives: Both pyridine and pyrano derivatives have been previously shown to possess biologically relevant activity. In this study, we report the incorporation of these two scaffolds into one molecule., Methods: The designed 3,3-dimethyl-6-oxopyrano[3,4-c]pyridines were synthesized by the acylation of enamine under Stork conditions followed by condensation of formed β-diketones with 2-cyanoacetamide. The structures of these compounds were confirmed by using a wide spectrum of physico-chemical methods. Their antiplatelet, anticoagulant and vasodilatory activity together with toxicity were evaluated., Key Findings: A series of 6-oxopyrano[3,4-c]pyridines 3a-j was obtained. Four of these compounds were reported for the first time. None of the tested compounds demonstrated anticoagulant effect but 8-methyl derivative (3a) was a potent antiplatelet compound with IC50 numerically twice as low as the clinically used acetylsalicylic acid. A series of further mechanistic tests showed that 3a interferes with calcium signaling. The compound is also not toxic and in addition possesses vasodilatory activity as well., Conclusions: Compound 3a is a promising inhibitor of platelet aggregation, whose mechanism of action should be studied in detail., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Flavonoids are associated with positive cardiovascular effects. However, due to their low bioavailability, metabolites are likely responsible for these properties. Recently, one of these metabolites, 4-methylcatechol, was described to be a very potent antiplatelet compound. This study aimed to compare its activity with its 22 close derivatives both of natural or synthetic origin in order to elucidate a potential structure-antiplatelet activity relationship. Blood from human volunteers was induced to aggregate by arachidonic acid (AA), collagen or thrombin, and plasma coagulation was also studied. Potential toxicity was tested on human erythrocytes as well as on a cancer cell line. Our results indicated that 17 out of the 22 compounds were very active at a concentration of 40 μM and, importantly, seven of them had an IC 50 on AA-triggered aggregation below 3 μM. The effects of the most active compounds were confirmed on collagen-triggered aggregation too. None of the tested compounds was toxic toward erythrocytes at 50 μM and four compounds partly inhibited proliferation of breast cancer cell line at 100 μM but not at 10 μM. Additionally, none of the compounds had a significant effect on blood coagulation or thrombin-triggered aggregation. This study hence reports four phenol derivatives (4-ethylcatechol, 4-fluorocatechol, 2-methoxy-4-ethylphenol and 3-methylcatechol) suitable for future in vivo testing.

Background: Antiplatelet drugs represent the keystone in the treatment and prevention of diseases of ischemic origin, including coronary artery disease. The current palette of drugs represents efficient modalities in most cases, but their effect can be limited in certain situations or associated with specific side effects. In this study, representatives of compounds selected from series having scaffolds with known or potential antiplatelet activity were tested. These compounds were previously synthetized by us, but their biological effects have not yet been reported., Objective: The aim of this study was to examine the antiplatelet and anticoagulation properties of selected compounds and determine their mechanism of action., Methods: Antiplatelet activity of compounds and their mechanisms of action were evaluated using human blood by impedance aggregometry and various aggregation inducers and inhibitors and compared to appropriate standards. Cytotoxicity was tested using breast adenocarcinoma cell cultures and potential anticoagulation activity was also determined., Results: In total, four of 34 compounds tested were equally or more active than the standard antiplatelet drug Acetylsalicylic Acid (ASA). In contrast to ASA, all 4 active compounds decreased platelet aggregation triggered not only by collagen, but also partly by ADP. The major mechanism of action is based on antagonism at thromboxane receptors. In higher concentrations, inhibition of thromboxane synthase was also noted. In contrast to ASA, the tested compounds did not block cyclooxygenase- 1., Conclusion: The most active compound, 2-amino-4-(1H-indol-3-yl)-6-nitro-4H-chromene-3- carbonitrile (2-N), which is 4-5x times more potent than ASA, is a promising compound for the development of novel antiplatelet drugs., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Quercetin, a common flavonoid from human diet, is extensively metabolized. Its two metabolites with the preserved flavonoid core were tested in detail for their interactions with transition metals, iron and copper. Both compounds chelated both metals; however, there were some significant differences between them notwithstanding that the major chelation site (3-hydroxy-4-keto) was the same. The complex stoichiometries were also determined under different pH conditions and in both oxidation states. Mostly, complexes 2:1, flavonoid to metal, were observed. Both compounds reduced iron and copper in a bell-shaped manner with tamarixetin being less potent in general. Both metabolites potentiated the Fenton reaction triggered by iron, while they were able to decrease the copper-based Fenton reaction under acidic conditions. In cellular experiments, both metabolites attenuated the copper-triggered hemolysis with isorhamnetin being more potent. In conclusion, there are differences between methylated metabolites of quercetin in relation to their interactions with biologically relevant transition metals.

Silymarin is a traditional drug and food supplement employed for numerous liver disorders. The available studies indicate that its activities may be broader, in particular due to claimed benefits in some cardiovascular diseases, but the contributions of individual silymarin components are unclear. Therefore, we tested silymarin flavonolignans as pure diastereomers as well as their sulfated metabolites for potential vasorelaxant and antiplatelet effects in isolated rat aorta and in human blood, respectively. Eleven compounds from a panel of 17 tested exhibited a vasorelaxant effect, with half maximal effective concentrations (EC 50 ) ranging from 20 to 100 µM, and some substances retained certain activity even in the range of hundreds of nM. Stereomers A were generally more potent as vasorelaxants than stereomers B. Interestingly, the most active compound was a metabolite-silychristin-19- O -sulfate. Although initial experiments showed that silybin, 2,3-dehydrosilybin, and 2,3-dehydrosilychristin were able to substantially block platelet aggregation, their effects were rapidly abolished with decreasing concentration, and were negligible at concentrations ≤100 µM. In conclusion, metabolites of silymarin flavonolignans seem to have biologically relevant vasodilatory properties, but the effect of silymarin components on platelets is low or negligible.

Background: Isoflavonoids seem to possess positive cardiovascular and other beneficial effects in humans., Hypothesis: Their low bioavailability, however, indicates that small isoflavonoid metabolites formed by human microflora can significantly contribute to these activities., Study Design: Testing antiplatelet activity ex vivo in human blood and interaction with transition metals in vitro., Methods: The effect on platelet aggregation induced by different triggers (arachidonic acid, collagen, ADP, TRAP-6), and interactions with transition metals (iron and copper chelation/reduction) were evaluated against four isoflavonoid-specific metabolites: S-equol; O-desmethylangolensin; 2-(4-hydroxyphenyl) propionic acid (HPPA); and 4-ethylphenol., Results: S-equol, 4-ethylphenol and O-desmethylangolensin blocked platelet aggregation induced by arachidonic acid and collagen. S-equol even matched the potency of acetylsalicylic acid in the case of collagen, which is the most physiological inducer of aggregation. Moreover, their effects in general seemed to be biologically relevant and attainable at achievable plasma concentrations, with the exception of HPPA which was ineffective. While only O-desmethylangolensin mildly chelated iron and copper, all four compounds markedly reduced cupric ions. Their direct free radical scavenging effects seem to have little clinical relevance., Conclusion: This study has shown that S-equol, O-desmethylangolensin and 4-ethylphenol, arising from isoflavonoid intake, can have biologically relevant effects on platelet aggregation., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Scope: Intake of flavonoids from the diet can be substantial, and epidemiological studies suggest that these compounds can decrease the incidence of cardiovascular diseases by involvement with increased platelet aggregation. Although parent flavonoids possess antiplatelet effects, the clinical importance is disputable due to their very low bioavailability. Most of them are metabolized by human colon bacteria to smaller phenolic compounds, which reach higher plasma concentrations than the parent flavonoids. In this study, a series of 29 known flavonoid metabolites is tested for antiplatelet potential., Methods and Results: Four compounds appear to have a biologically relevant antiplatelet effect using whole human blood. 4-Methylcatechol (4-MC) is clearly the most efficient being about 10× times more active than clinically used acetylsalicylic acid. This ex vivo effect is also confirmed using a potentially novel in-vivo-like ex ovo hen's egg model of thrombosis, where 4-MC significantly increases the survival of the eggs. The mechanism of action is studied and it seems that it is mainly based on the influence on intracellular calcium signaling., Conclusion: This study shows that some flavonoid metabolites formed by human microflora have a strong antiplatelet effect. This information can help to explain the antiplatelet potential of orally given flavonoids., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Some compounds without apparent chelation sites have been shown to chelate cupric ions using the hematoxylin assay. Since these compounds also have reduction potential (direct antioxidant effect), the aim of this study was to determine the possible interference of reducing agents with the hematoxylin assay. Four different known reducing agents (hydroxylamine, vitamin C, trolox - a water-soluble form of vitamin E and reduced glutathione /GSH/) were selected for the study together with oxidized glutathione (GSSG) for comparison. All tested compounds behaved as cupric chelators in the spectrophotometric mildly competitive hematoxylin assay. In-depth analysis however showed that only GSH and GSSG were able to form complexes with both cupric and cuprous ions and only GSSG partly retained copper in its complexes in the more competitive bathocuproine assay. Further experiments showed that with the exception of GSSG, all other compounds reduce Cu 2+ ions. Conclusion: Compounds reducing copper such as antioxidants can give false positive results in the hematoxylin-screening assay. GSSG is a stronger Cu chelator than GSH and does not reduce Cu, in contrast to the latter and thus may be a protective element after oxidation of GSH., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Silymarin, the standardized extract from the milk thistle (Silybum marianum), is composed mostly of flavonolignans and is approved in the EU for the adjuvant therapy of alcoholic liver disease. It is also used for other purported effects in miscellaneous nutraceuticals. Due to polyhydroxylated structures and low systemic bioavailability, these flavonolignans are likely to interact with transition metals in the gastrointestinal tract. The aim of this study was to analyze the interactions of pure silymarin flavonolignans with copper and iron. Both competitive and non-competitive methods at various physiologically relevant pH levels ranging from 4.5 to 7.5 were tested. Only 2,3‑dehydrosilybin was found to be a potent or moderately active iron and copper chelator. Silybin A, silybin B and silychristin A were less potent or inactive chelators. Both 2,3‑dehydrosilybin enantiomers (A and B) were equally active iron and copper chelators, and the preferred stoichiometries were mainly 2:1 and 3:1 (2,3‑dehydrosilybin:metal). Additional experiments showed that silychristin was the most potent iron and copper reductant. Comparison with their structural precursors taxifolin and quercetin is included as well. Based on these results, silymarin administration most probably affects the kinetics of copper and iron in the gastrointestinal tract, however, due to the different interactions of individual components of silymarin with these transition metals, the biological effects need to be evaluated in the future in a much more complex study., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cardiovascular diseases are a leading cause of morbidity and mortality in most developed countries of the world. Pharmaceuticals, illicit drugs, and toxins can significantly contribute to the overall cardiovascular burden and thus deserve attention. The present article is a systematic overview of drugs that may induce distinct cardiovascular toxicity. The compounds are classified into agents that have significant effects on the heart, blood vessels, or both. The mechanism(s) of toxic action are discussed and treatment modalities are briefly mentioned in relevant cases. Due to the large number of clinically relevant compounds discussed, this article could be of interest to a broad audience including pharmacologists and toxicologists, pharmacists, physicians, and medicinal chemists. Particular emphasis is given to clinically relevant topics including the cardiovascular toxicity of illicit sympathomimetic drugs (e.g., cocaine, amphetamines, cathinones), drugs that prolong the QT interval, antidysrhythmic drugs, digoxin and other cardioactive steroids, beta-blockers, calcium channel blockers, female hormones, nonsteroidal anti-inflammatory, and anticancer compounds encompassing anthracyclines and novel targeted therapy interfering with the HER2 or the vascular endothelial growth factor pathway., (© 2018 The Authors Medicinal Research Reviews Published by Wiley Periodicals, Inc.)

Background: Zinc is an essential trace element. Both its lack and excess are associated with pathological states. The former is more common and can ensue from the excessive treatment with clinically used iron/copper chelators., Aim and Method: The aim of this work was to prepare a reliable, rapid and cheap method for the screening of zinc chelation. Spectrophotometric assessment using a known zinc indicator dithizone was selected., Results: Initial screening performed by comparison of spectra of dithizone and its complex with zinc suggested 530 and 570 nm as suitable wavelengths for determination of zinc at pH 4.5 while 540 and 590 nm for pH 5.5-7.5. Additional research showed the lower wavelengths to be more suitable. The sensitivity of the method was always bellow 1 μM with good linearity relationship between absorbance and zinc concentration. The method suitability was confirmed by use of two known zinc chelators, ethylenediaminetetraacetic acid (EDTA) and N,N,N',N'-tetrakis(2-pyridylmethyl)-1,2-ethylenediamine (TPEN)., Conclusion: This method represents a sufficiently precise method for zinc chelation screening usable at pathophysiologically relevant pH conditions. Such method can be employed for both screening of novel zinc chelators and for testing affinity of other metal chelators for zinc., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Due to the limited array of the currently available copper chelators, research of such compounds continues to be of clinical interest. Notably, o-dihydroxycoumarins have been previously shown to be potent iron chelators under neutral conditions. Within this study, the interaction of a series of natural coumarins and their synthetic analogs with copper has been evaluated in order to obtain structure-activity relationships under different pathophysiological pH conditions. Both competitive and non-competitive methods have been employed. Analysis of cupric ion reduction has also been performed. Under mildly competitive conditions, cupric chelation was observed for o-dihydroxycoumarins, and partially for o-diacetoxycoumarin. Non-competitive studies showed that cuprous ions are not chelated at all and that the stoichiometries of the most active 6,7- and 7,8-dihydroxycoumarins to cupric ions ranged from 1:1 to 2:1 depending on pH and concentration. Interestingly, under highly competitive conditions, coumarins were not capable of chelating cupric ions, either. Reduction experiments have shown that 13 out of the 15 coumarins included in this study reduced cupric ions. However, significant differences depending on their structures were apparent in their potencies. O-dihydroxycoumarins were the most potent ones again., Conclusion: O-dihydroxycoumarins are moderately active cupric ion chelators with potent copper reducing properties., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Background: One reason for the lower incidence of cardiovascular diseases in Asian countries may be the high intake of isoflavonoids and their antiplatelet effects may be an important factor. To date, there is limited comparison of a range of isoflavonoids and knowledge of their effects at different levels of platelet aggregation., Purpose: To screen the antiplatelet effects of a number of isoflavonoids on the arachidonic acid based aggregation pathway and investigate how the antiplatelet activity might occur., Methods: The antiplatelet effects were first screened in whole human blood where platelet aggregation was induced by arachidonic acid. Further analysis was targeted at search of the mechanism of action., Results: Thirteen of the eighteen tested isoflavonoids had significant inhibitory effect on platelet aggregation in whole human blood. Genistein had the same potency as clinically used acetylsalicylic acid (ASA) while tectorigenin was clearly stronger than ASA. Further analyses showed that the effect of tectorigenin was not based on inhibition of cyclooxygenase-1 in contrast to ASA or thromboxane synthase but by competitive antagonism at thromboxane receptors., Conclusion: Tectorigenin is a more potent antiplatelet compound than ASA and thus an interesting substance for further testing., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Interaction of flavonoids with transition metals can be partially responsible for their impact on humans. Stoichiometry of the iron/copper complex with a flavonoid glycoside isoquercitrin, a frequent component of food supplements, was assessed using competitive and non-competitive methods in four (patho)physiologically-relevant pH values (4.5. 5.5, 6.8, and 7.5). Isoquercitrin chelated all tested ions (Fe 2+ , Fe 3+ , Cu 2+ , and Cu⁺) but its affinity for Cu⁺ ions proved to be very low. In general, the chelation potency dropped with pH lowering. Metal complexes of 1:1 stoichiometry were mostly formed, however, they were not stable and the stoichiometry changed depending on conditions. Isoquercitrin was able to reduce both Cu 2+ and Fe 3+ ions at low ratios, but its reducing potential was diminished at higher ratios (isoquercitrin to metal) due to the metal chelation. In conclusion, this study emphasizes the need of using multiple different methods for the assessment of chelation potential in moderately-active metal chelators, like flavonoids., Competing Interests: The authors declare no conflict of interest.

Flavonoids, important components of human diet, have been claimed to possess a significant antiplatelet potential, in particular due to their effects on the arachidonic acid cascade. Due to variable and incomplete results, this study was aimed at delivering a detailed analysis of the effects of 29 structurally relevant, mainly natural flavonoids on three consecutive steps of the arachidonic acid cascade.Only the isoflavonoids genistein and daidzein were shown to possess a marked cyclooxygenase-1 inhibitory activity, which was higher than that of acetylsalicylic acid using the isolated ovine enzyme, and physiologically relevant, although lower than acetylsalicylic acid in human platelets. None of the tested flavonoids possesses an effect on thromboxane synthase in a clinically achievable concentration. Contrarily, many flavonoids, particularly those possessing an isolated 7-hydroxyl group and/or a 4'-hydroxyl group, acted as antagonists on thromboxane receptors. Interestingly, the substitution of the free 7-hydroxyl group by glucose might not abolish the activity.In conclusion, the consumption of few flavonoids in a diet, particularly of the isoflavonoids genistein and daidzein, may positively influence platelet aggregation., (Georg Thieme Verlag KG Stuttgart · New York.)

Coumarins represent a large group of 1,2-benzopyrone derivatives which have been identified in many natural sources and synthetized as well. Several studies have shown that their antioxidant capacity is not based only on direct scavenging of reactive oxygen and nitrogen species (RONS) but other mechanisms are also involved. These include: a) the chelation of transient metals iron and copper, which are known to catalyse the Fenton reaction; and b) the inhibition of RONS-producing enzymes (e.g. xanthine oxidase, myeloperoxidase and lipoxygenase), suggesting that mechanism(s) involved on cellular level are complex and synergistic. Moreover, many factors must be taken into account when analysing structure-antioxidant capacity relationships of coumarins due to different in vitro/in vivo methodological approaches. The structural features necessary for the direct RONS scavenging and metal chelation are apparently similar and the ideal structures are 6,7-dihydroxy- or 7,8-dihydroxycoumarins. However, the clinical outcome is unknown, because these coumarins are able to reduce copper and iron, and may thus paradoxically potentiate the Fenton chemistry. The similar structural features appear to be associated with inhibition of lipoxygenase, probably due to interference with iron in its active site. Contrarily, 6,7-dihydroxycoumarin seems to be the most active coumarin in the inhibition of xanthine oxidase while its derivative bearing the 4-methyl group or 7,8-dihydroxycoumarin are less active or inactive. In addition, coumarins may hinder the induction of inducible NO-synthase and cyclooxygenase- 2. Sparse data on inhibition of myeloperoxidase do not enable any clear conclusion, but some coumarins may block it.

Isoflavones are commonly consumed in many Asian countries and have potentially positive effects on human being. Only a few and rather controversial data on their interactions with copper and iron are available to date. 13 structurally related isoflavones were tested in the competitive manner for their Cu/Fe-chelating/reducing properties. Notwithstanding the 5-hydroxy-4-keto chelation site was associated with ferric, ferrous, and cupric chelation, the chelation potential of isoflavones was low and no cuprous chelation was observed. None of isoflavones was able to substantially reduce ferric ions, but the vast majority reduced cupric ions. The most important feature for cupric reduction was the presence of an unsubstituted 4'-hydroxyl; contrarily the presence of a free 5-hydroxyl decreased or abolished the reduction due to chelation of cupric ions. The results from this study may enable additional experiments which might clarify the effects of isoflavones on human being and/or mechanisms of copper absorption.

Copper is an essential trace element involved in many physiological processes. Since disorder of copper homeostasis is observed in various pathologies, copper chelators may represent a promising therapeutic tool. This study was aimed at: 1) formation of an in vitro methodology for screening of copper chelators, and 2) detailed analysis of the interaction of copper with clinically used D-penicillamine (D-PEN), triethylenetetramine (trientine), experimentally tested 8-hydroxyquinolines, and the disodium salt of EDTA as a standard chelator. Methodology based on bathocuproinedisulfonic acid disodium salt (BCS), usable at (patho)physiologically relevant pHs (4.5-7.5), enabled assessment of both cuprous and cupric ions chelation and comparison of the relative affinities of the tested compounds for copper. In the case of potent chelators, the stoichiometry could be estimated too. Clioquinol, chloroxine and EDTA formed very stable complexes with Cu(+)/Cu(2+) at all tested pHs, while copper complexes with trientine were stable only under neutral or slightly acidic conditions. Non-substituted 8-hydroxyquinoline was a less efficient copper chelator, but still unequivocally more potent than D-PEN. Both 8-hydroxyquinoline and D-PEN chelation potencies, similarly to that of trientine, were pH-dependent and decreased with pH. Moreover, only D-PEN was able to reduce cupric ions. Conclusively, BCS assay represents a rapid, simple and precise method for copper chelation measurement. In addition, lower binding affinity of D-PEN compared with 8-hydroxyquinolines and trientine was demonstrated., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Platelets play a crucial role in physiological haemostasis. However, in coronary arteries damaged by atherosclerosis, enhanced platelet aggregation, with subsequent thrombus formation, is a precipitating factor in acute myocardial infarction. Current therapeutic approaches are able to reduce approximately one quarter of cardiovascular events, but they are associated with an increased risk of bleeding and in some resistant patients are not efficient. Some coumarins possess antiplatelet activity and, due to their additional antioxidant effects, may be promising drugs for use in combination with the present therapeutic agents. The aim of this study was to analyse a series of simple 4-methylcoumarins for their antiplatelet activity. Human plasma platelet suspensions were treated with different aggregation inducers [arachidonic acid (AA), collagen and ADP] in the presence of the 4-methylcoumarins. Complementary experiments were performed to explain the mechanism of action. 5,7-Dihydroxy-4-methylcoumarins, in particular those containing a lipophilic side chain at C-3, reached the activity of acetylsalicylic acid on AA-induced aggregation. Other tested coumarins were less active. Some of the tested compounds mildly inhibited either collagen- or ADP-induced aggregation. 5,7-Dihydroxy-4-methylcoumarins did not interfere with the function of thromboxane synthase, but were competitive antagonists of thromboxane A(2) receptors and inhibited cyclooxygenase-1 as well. 5,7-Dihydroxy-4-methylcoumarins appear to be promising candidates for the extension of the current spectrum of antiplatelet drugs., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)