Flavonoid aglycones are secondary plant metabolites that exhibit a broad spectrum of pharmacological activities, including anti-inflammatory, antioxidant, anticancer, and antiplatelet effects. However, the precise molecular mechanisms underlying their inhibitory effect on platelet activation remain poorly understood. In this study, we applied flow cytometry to analyze the effects of six flavonoid aglycones (luteolin, myricetin, quercetin, eriodictyol, kaempferol, and apigenin) on platelet activation, phosphatidylserine externalization, formation of reactive oxygen species, and intracellular esterase activity. We found that these compounds significantly inhibit thrombin-induced platelet activation and decrease formation of reactive oxygen species in activated platelets. The tested aglycones did not affect platelet viability, apoptosis induction, or procoagulant platelet formation. Notably, luteolin, myricetin, quercetin, and apigenin increased thrombin-induced thromboxane synthase activity, which was analyzed by a spectrofluorimetric method. Our results obtained from Western blot analysis and liquid chromatography–tandem mass spectrometry demonstrated that the antiplatelet properties of the studied phytochemicals are mediated by activation of cyclic nucleotide-dependent signaling pathways. Specifically, we established by using Förster resonance energy transfer that the molecular mechanisms are, at least partly, associated with the inhibition of phosphodiesterases 2 and/or 5. These findings underscore the therapeutic potential of flavonoid aglycones for clinical application as antiplatelet agents.

Curcumin is a natural bioactive component derived from the turmeric plant Curcuma longa, which exhibits a range of beneficial activities on human cells. Previously, an inhibitory effect of curcumin on platelets was demonstrated. However, it is unknown whether this inhibitory effect is due to platelet apoptosis or procoagulant platelet formation. In this study, curcumin did not activate caspase 3-dependent apoptosis of human platelets, but rather induced the formation of procoagulant platelets. Interestingly, curcumin at low concentration (5 µM) potentiated, and at high concentration (50 µM) inhibited ABT-737-induced platelet apoptosis, which was accompanied by inhibition of ABT-737-mediated thrombin generation. Platelet viability was not affected by curcumin at low concentration and was reduced by 17% at high concentration. Furthermore, curcumin-induced autophagy in human platelets via increased translocation of LC3I to LC3II, which was associated with activation of adenosine monophosphate (AMP) kinase and inhibition of protein kinase B activity. Because curcumin inhibits P-glycoprotein (P-gp) in cancer cells and contributes to overcoming multidrug resistance, we showed that curcumin similarly inhibited platelet P-gp activity. Our results revealed that the platelet inhibitory effect of curcumin is mediated by complex processes, including procoagulant platelet formation. Thus, curcumin may protect against or enhance caspase-dependent apoptosis in platelets under certain conditions.

Introduction. Analysis of the clinical and laboratory picture of the SARS-CoV-2 infection suggests the presence of microcirculation and oxygen transport disorders, hemolysis of erythrocytes, intra-alveolar fibrin formation and microthrombus formation in the patient’s pathogenesis. Accordingly, the search for potential anticoagulants, erythrocyte antiplatelet agents, membrane stabilizing drugs and mild thrombolytic drugs can prevent the development of life-threatening complications and reduce the mortality of COVID-19 patients.Aim. Isolation of formononetin-7-O-β-D-glucopyranoside from the grass of Ononis arvensis L. and identification of the molecular mechanisms of its effect on platelet activation in vitro, induced by TRAP-6 (Thrombin receptor activated peptide) and ADP (adenosine diphosphate).Materials and methods. Terrestrial parts of Ononis arvensis L. were collected in the SPCPU nursery of medicinal plants (Leningrad region, Vsevolozhsky district, Priozerskoe highway, 38 km). Isolation of formononetin-7-O-β-D-glucopyranoside was carried out by preparative high performance liquid chromatography on a Smartline device (Knauer, Germany) equipped with a spectrophotometric detector. The structure of formononetin-7-O-β-D-glucopyranoside was confirmed by one-dimensional and two-dimensional NMR spectroscopy (Bruker Avance III, 400 MHz, Germany), as well as high-resolution mass spectrometry (HR-ESI-MS) (Bruker Micromass Q-TOF, Germany). The study of the effect of formononetin- 7-O-β-D-glucopyranoside on induced platelet activation was carried out on human platelets isolated from the blood of healthy volunteers. To research the effect of formononetin-7-О-β-D-glucopyranoside on platelet aggregation flow cytofluorometry with Cyto-FLEX (Beckman-Coulter, USA) was used.Results and discussion. According to the method of fractionation and purification of the total extract of O. arvensis developed in previous studies, formononetin-7-O-β-D-glucopyranoside was isolated in an individual form for subsequent biological studies with a total yield of 30 % in comparison with its content in the original extract. In samples with formononetin-7-O-β-D-glucopyranoside and ADP, there is a pronounced inhibition of platelet activation – the percentage of active platelets ranges from 6.3–6.6 % at doses of formononetin-7-O-β-D-glucopyranoside 1 μM, 3 μM and 30 μM. The inhibitory effect of formononetin-7-O-β-D-glucopyranoside is not dose-dependent (p ≤ 0.05). In samples with formononetin-7-O-β-D-glucopyranoside and TRAP, there is also a pronounced inhibition of platelet activation. The percentage of active platelets is 8 % at 1 μM formononetin-7-O-β-D-glucopyranoside doses, 15 % at 3 μM doses, and 16 % at 30 μM doses.Conclusion. Administration of formononetin-7-O-β-D-glucopyranoside at doses of 1 μM, 3 μM, 30 μM strongly inhibits platelet activation induced by ADP and TRAP-6. For ADP, there is no dose-dependent effect, while for TRAP there is a weak dose-dependent effect, the greatest inhibition efficiency is achieved with the minimum investigated dose of 1 μM. In all cases, the results obtained are statistically significant.