Antiplatelet Effects of Flavonoid Aglycones Are Mediated by Activation of Cyclic Nucleotide-Dependent Protein Kinases.

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.

ML355 Modulates Platelet Activation and Prevents ABT-737 Induced Apoptosis in Platelets.

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.

Curcumin by activation of adenosine A2A receptor stimulates protein kinase a and potentiates inhibitory effect of cangrelor on platelets.

Curcumin is a natural polyphenol derived from the turmeric plant (Curcuma longa) which exhibits numerous beneficial effects on different cell types. Inhibition of platelet activation by curcumin is well known, however molecular mechanisms of its action on platelets are not fully defined. In this study, we used laser diffraction method for analysis of platelet aggregation and Western blot for analysis of intracellular signaling mechanisms of curcumin effects on platelets. We identified two new molecular mechanisms involved in the inhibitory effects of curcumin on platelet activation. Firstly, curcumin by activation of adenosine A2A receptor stimulated protein kinase A activation and phosphorylation of Vasodilator-stimulated phosphoprotein. Secondly, we demonstrated that curcumin even at low doses, which did not inhibit platelet aggregation, potentiated inhibitory effect of ADP receptor P2Y12 antagonist cangrelor which partly could be explained by activation of adenosine A2A receptor.

Potential and limitations of PKA/ PKG inhibitors for platelet studies.

Cyclic nucleotides (cAMP and cGMP) and corresponding protein kinases, protein kinase A (PKA) and protein kinase G (PKG), are the main intracellular mediators of endothelium-derived platelet inhibitors. Pharmacological PKA/PKG inhibitors are often used to discriminate between these two kinase activities and to analyze their underlying mechanisms. Previously we showed that all widely used PKG inhibitors (KT5823, DT3, RP isomers) either did not inhibit PKG or inhibited and even activated platelets independently from PKG. In this study, we examined several PKA inhibitors as well as inhibitors of adenylate and guanylate cyclases to reveal their effects on platelets and establish whether they are mediated by PKA/PKG. The commonly used PKA inhibitor H89 inhibited both PKA and PKG but PKA-independently inhibited thrombin-induced platelet activation. In our experiments, KT5720 did not inhibit PKA and had no effect on platelet activation. PKI inhibited PKA activity in platelets but also strongly PKA-independently activated platelets. Inhibition of adenylate and guanylate cyclases may be an alternative approach to analyze PKA/PKG function. Based on our previous and presented data, we conclude that all results where the mentioned PKA inhibitors were used for the analysis of PKA activity in intact platelets should be considered with caution.

Curcumin at Low Doses Potentiates and at High Doses Inhibits ABT-737-Induced Platelet Apoptosis.

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.

Multifaceted effects of arachidonic acid and interaction with cyclic nucleotides in human platelets.

INTRODUCTION: Arachidonic acid induced aggregation is a generally accepted test for aspirin resistance. However, doubts have been raised that arachidonic acid stimulated aggregation can be regarded as reliable testing for aspirin resistance. Arachidonic acid, in addition to platelet activation, can induce phosphatidylserine translocation on the outer surface of platelet membrane which could be mediated by apoptosis pathways or transformation of platelets to the procoagulant state. MATERIALS AND METHODS: We explored effects of arachidonic acid over a vast range of concentrations and a wide range of read-outs for human platelet activation, procoagulant activity, and platelet viability. Additionally we tested whether cAMP- or cGMP-dependent protein kinase activation can inhibit procoagulant activity or platelet viability. RESULTS: Arachidonic acid-induced washed platelet activation was detected at low micromolar concentrations during the first 2 min of stimulation. After longer incubation and/or at higher concentrations arachidonic acid triggered platelet procoagulant activity and reduced platelet viability. At the same time, arachidonic acid stimulated adenylate cyclase mediated protein phosphorylation which correlated with reduced platelet activation. Moreover, additional stimulation of cAMP- or cGMP-dependent protein kinase inhibited only platelet activation, but did not prevent pro-coagulant activity and platelet death. CONCLUSIONS: While arachidonic acid induces platelet activation at low concentrations and during short incubation time, higher concentrations and lasting incubation evokes adenylate cyclase activation and subsequent protein phosphorylation corresponding to reduced platelet activation, but also enhanced pro-coagulant activity and reduced viability. Our observations provide further proof for the complex fine tuning of platelet responses in a time and agonist concentration dependent manner.