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.

12-lipoxigenase (12-LOX) is implicated in regulation of platelet activation processes and can be a new promising target for antiplatelet therapy. However, investigations of 12-LOX were restricted by the lack of specific and potent 12-LOX inhibitors and by controversial data concerning the role of 12-LOX metabolites in platelet functions. A novel specific 12-LOX inhibitor ML355 was shown to inhibit platelet aggregation without adverse side effects on hemostasis; however, the molecular mechanisms of its action on platelets are poorly understood. Here, we showed that ML355 inhibited platelet activation induced by thrombin or thromboxane A₂, but not by collagen-related peptide. ML355 blocked protein kinase B, phosphoinositide 3-kinase, and extracellular signal-regulated kinase, but not p38 kinase, spleen tyrosine kinase (Syk), or phospholipase Cγ2 phosphorylation in activated platelets. The main inhibitory effect of low doses of ML355 (1–20 μM) on thrombin activated platelets was mediated by the decrease in reactive oxygen species level, whereas high doses of ML355 (50 μM) caused cyclic adenosine monophosphate activation. ML355 did not affect the activity of nitric oxide-dependent soluble guanylyl cyclase, nor did it affect the relaxation of preconstricted aortic rings in mice. ML355 itself did not affect platelet viability, but at 50 μM dose blocked caspase-dependent apoptosis induced by B-cell lymphoma II inhibitor ABT-737. SIGNIFICANCE STATEMENT The current paper provides novel and original data concerning molecular mechanisms of 12-LOX inhibitor ML355 action on platelets. These data reveal antiplatelet and protective effects of ML355 on platelets and may be of importance for both antiplatelet and anticancer therapy.

The phototransduction cascade enables the photoreceptor to detect light over a wide range of intensities without saturation. The main second messenger of the cascade is cGMP and the primary regulatory mechanism is calcium feedback. However, some experimental data suggest that cAMP may also play a role in regulating the phototransduction cascade, but this would require changes in cAMP on a time scale of seconds. Currently, there is a lack of data on the dynamics of changes in intracellular cAMP levels on this timescale. This is largely due to the specificity of the sensory modality of photoreceptors, which makes it practically impossible to use conventional experimental approaches based on fluorescence methods. In this study, we employed the method of rapid cryofixation of retinal samples after light stimulation and subsequent isolation of outer segment preparations. The study employed highly sensitive metabolomics approaches to measure levels of cAMP. Additionally, PKA activity was measured in the samples using a western blot. The results indicate that when exposed to near-saturating but still moderate light, cAMP levels increase transiently within the first second and then return to pre-stimulus levels. The increase in cAMP activates PKA, resulting in the phosphorylation of PKA-specific substrates in frog retinal outer segments.

Nobiletin is a polymethoxylated flavone derived from citrus peels that possesses a wide range of beneficial effects. Specifically, nobiletin exhibits anti-inflammatory, antibacterial, and antioxidant effects. Recent research showed that nobiletin is able to diminish platelet adhesion and aggregation, indicating this compound as a potential therapeutic drug in thrombosis therapy. However, the exact mechanism of nobiletin action on platelets is unclear. Hence, the main goal of our study was to investigate the molecular mechanism of nobiletin-induced platelet inhibition. We demonstrated that nobiletin blocks induced platelet activation and does not cause platelet apoptosis. Inhibition effect of nobiletin on platelets is at least partly mediated by activation of A_2A adenosine receptor and consequent AC/cAMP/PKA signaling pathway activation.

One new compound isoembinin 1 along with ten known compounds 2-11 were isolated from the terrestrial parts of Iris lactea Pall. All of the compound structures were determined through extensive 1D and 2D NMR experiments along with HR-ESIMS analysis and comparison with literature data. Because many flavonoids exert antiplatelet and antioxidant activity we tested the effects of the isolated flavone C-glycosides 1-9 on platelet activation and reactive oxygen species (ROS) production. Platelet reactivity was assessed by activation of αIIbβ3 integrins activation and ROS production by DCF-DA fluorescence. For the analysis of whether protein kinase A or G are involved in the platelet inhibition, the activity of these kinases was analyzed by phosphorylation of their common substrate in platelets. In all experiments apigenin, which inhibit platelet activation was used as a positive control. All isolated flavone C-glycosides inhibited platelet αIIbβ3 integrins activation with IC50 in the µM range, however this inhibitory effect was found to not be mediated through the prevention of ROS formation or by the activation of cyclic nucleotide pathways. Structure-activity comparison between apigenin and compounds 1-9 shows that the presence of C-glycoside and O-glycoside residues on the aglycone apigenin diminish the degree of platelet inhibition.

Нобилетин – полиметоксилированный флавон, выделяемый из кожуры цитрусовых, который обладает множеством полезных свойств. В частности, нобилетин способен оказывать противовоспалительное, антибактериальное и антиоксидантное действие. Недавние исследования показали, что нобилетин снижает адгезию и агрегацию тромбоцитов, что делает возможным его использование в профилактике и лечении тромбообразования, однако точные механизмы его действия на тромбоциты неизвестны. В связи с этим основной целью данной работы являлось исследование молекулярных механизмов ингибирования тромбоцитов, опосредованного нобилетином. Мы показали, что нобилетин блокирует индуцированную активацию тромбоцитов, не вызывая при этом их гибель. Ингибирующий эффект нобилетина на тромбоциты по крайней мере частично обусловлен активацией рецептора A_2A к аденозину и запуском сигнальной системы АЦ/cAMP/PKA.