Deletion of pyruvate dehydrogenase kinases reduces susceptibility to deep vein thrombosis in mice.

Neutrophils contribute to deep vein thrombosis (DVT) by releasing prothrombotic neutrophil extracellular traps (NETs). NETs formation (known as NETosis) is an energy-intensive process that requires an increased rate of aerobic glycolysis. The metabolic enzymes pyruvate dehydrogenase kinases (PDKs) inhibit the pyruvate dehydrogenase (PDH) complex to divert the pyruvate flux from oxidative phosphorylation towards aerobic glycolysis. Herein, we identified that the combined deletion of PDK2 and PDK4 (PDK2/4-/-) renders mice less susceptible to DVT (measured by thrombus incidence, weight, and length) in the inferior vena cava (IVC)-stenosis model at day 2 post-surgery. Compared to wild-type (WT) mice, the venous thrombus obtained from PDK2/4-/- mice exhibited reduced citrullinated histone content, a known marker of NETs. In line with in vivo observations, phorbol 12-myristate 13-acetate (PMA)-stimulated PDK2/4-/- neutrophils displayed reduced NETosis and secretion of cathepsin G & elastase compared to PMA-stimulated WT neutrophils. The formation of platelet aggregates mediated by PMA-stimulated PDK2/4-/- neutrophils were significantly reduced compared to PMA-stimulated WT neutrophils. Finally, PDK2/4-/- neutrophils exhibited reduced levels of intracellular Ca2+ concentration, Erk1/2 phosphorylation, and glycoPER (a measure of aerobic glycolysis), known to facilitate NETosis. Together, these findings elucidate for the first time the fundamental role of PDK2/4 in regulating NETosis and acute DVT.

Platelet Metabolic Profiling Reveals Glycolytic and 1-Carbon Metabolites Are Essential for GP VI-Stimulated Human Platelets-Brief Report.

BACKGROUND: Evolving evidence suggests that besides signaling pathways, platelet activation involves a complex interplay between metabolic pathways to support thrombus growth. Selective targeting of metabolic checkpoints may inhibit platelet activation and provide a novel antiplatelet strategy. We, therefore, examined global metabolic changes that occur during the transition of human platelets from resting to an activated state to identify metabolites and associated pathways that contribute to platelet activation. METHODS: We performed metabolic profiling of resting and convulxin-stimulated human platelet samples. The differential levels, pathway analysis, and PCA (principal component analysis) were performed using Metaboanalyst. Metascape was used for metabolite network construction. RESULTS: Of the 401 metabolites identified, 202 metabolites were significantly upregulated, and 2 metabolites were downregulated in activated platelets. Of all the metabolites, lipids scored highly and constituted ≈50% of the identification. During activation, aerobic glycolysis supports energy demand and provides glycolytic intermediates required by metabolic pathways. Consistent with this, an important category of metabolites was carbohydrates, particularly the glycolysis intermediates that were significantly upregulated compared with resting platelets. We found that lysophospholipids such as 1-palmitoyl-GPA (glycero-3-phosphatidic acid), 1-stearoyl-GPS (glycero-3-phosphoserine), 1-palmitoyl-GPI (glycerophosphoinositol), 1-stearoyl-GPI, and 1-oleoyl-GPI were upregulated in activated platelets. We speculated that platelet activation could be linked to 1-carbon metabolism, a set of biochemical pathways that involve the transfer and use of 1-carbon units from amino acids, for cellular processes, including nucleotide and lysophospholipid synthesis. In alignment, based on pathway enrichment and network-based prioritization, the metabolites from amino acid metabolism, including serine, glutamate, and branched-chain amino acid pathway were upregulated in activated platelets, which might be supplemented by the high levels of glycolytic intermediates. CONCLUSIONS: Metabolic analysis of resting and activated platelets revealed that glycolysis and 1-carbon metabolism are necessary to support platelet activation.

Metabolic targeting of platelets to combat thrombosis: dawn of a new paradigm?

Current antithrombotic therapies used in clinical settings target either the coagulation pathways or platelet activation receptors (P2Y12 or GPIIb/IIIa), as well as the cyclooxygenase (COX) enzyme through aspirin. However, they are associated with bleeding risk and are not suitable for long-term use. Thus, novel strategies which provide broad protection against platelet activation with minimal bleeding risks are required. Regardless of the nature of agonist stimulation, platelet activation is an energy-intensive and ATP-driven process characterized by metabolic switching toward a high rate of aerobic glycolysis, relative to oxidative phosphorylation (OXPHOS). Consequently, there has been considerable interest in recent years in investigating whether targeting metabolic pathways in platelets, especially aerobic glycolysis and OXPHOS, can modulate their activation, thereby preventing thrombosis. This review briefly discusses the choices of metabolic substrates available to platelets that drive their metabolic flexibility. We have comprehensively elucidated the relevance of aerobic glycolysis in facilitating platelet activation and the underlying molecular mechanisms that trigger this switch from OXPHOS. We have provided a detailed account of the antiplatelet effects of targeting vital metabolic checkpoints such as pyruvate dehydrogenase kinases (PDKs) and pyruvate kinase M2 (PKM2) that preferentially drive the pyruvate flux to aerobic glycolysis. Furthermore, we discuss the role of fatty acids and glutamine oxidation in mitochondria and their subsequent role in driving OXPHOS and platelet activation. While the approach of targeting metabolic regulatory mechanisms in platelets to prevent their activation is still in a nascent stage, accumulating evidence highlights its beneficial effects as a potentially novel antithrombotic strategy.

Characterization of bleeding symptoms in Ehlers-Danlos syndrome.

BACKGROUND: Easy bruising is included as a major or minor criterion for the classification of multiple types of Ehlers-Danlos syndrome (EDS). Despite a longstanding recognition of the association between EDS and bleeding, we still lack a definitive understanding of the frequency, severity, and types of bleeding complications in patients with EDS. OBJECTIVES: To evaluate hemorrhagic symptoms using the International Society of Thrombosis and Haemostasis bleeding assessment tool (ISTH-BAT) in a cohort of patients with defined types of EDS. METHODS: We utilized the ISTH-BAT to characterize hemorrhagic symptoms and their severity in a cohort of 52 patients with classical, classical-like, hypermobile, or vascular EDS and a matched group of 52 healthy control subjects. RESULTS: The mean ISTH-BAT score was 0.1 for healthy subjects and 9.1 for patients with EDS (p < .0001). An abnormal ISTH-BAT score was observed in 32 of 52 (62%) patients with EDS and 0 of 52 healthy controls (p < .0001). The most frequent bleeding symptoms were bruising, muscle hematomas, menorrhagia, epistaxis, bleeding from the oral cavity, and bleeding after tooth extraction. Menorrhagia that was life-threatening or required surgery was reported in 7 of 52 (14%) patients with EDS. CONCLUSION: Patients with multiple types of EDS exhibit a wide range of bleeding symptoms ranging from mild to life-threatening episodes.

Mitochondrial calcium uniporter b deletion inhibits platelet function and reduces susceptibility to arterial thrombosis.

BACKGROUND: Mitochondrial calcium uniporter b (MCUb) is a negative regulator of the mitochondrial calcium uniporter (MCU) and is known to limit mitochondrial calcium ion (Ca2+) uptake. The role of MCUb in platelet function remains unclear. OBJECTIVES: Utilizing MCUb-/- mice, we examined the role of MCUb in regulating platelet function and thrombosis. METHODS: Platelet activation was evaluated in agonist-induced standardized in vitro assays. Susceptibility to arterial thrombosis was evaluated in FeCl3 injury-induced carotid artery and laser injury-induced mesenteric artery thrombosis models. The glycolytic proton efflux rate and oxygen consumption rate were measured to evaluate aerobic glycolysis. RESULTS: Upon stimulation, MCUb-/- platelets exhibited reduced cytoplasmic Ca2+ responses concomitant with increased mitochondrial Ca2+ uptake. MCUb-/- platelets displayed reduced agonist-induced platelet aggregation and spreading on fibrinogen and decreased α and dense-granule secretion and clot retraction. MCUb-/- mice were less susceptible to arterial thrombosis in FeCl3 injury-induced carotid and laser injury-induced mesenteric thrombosis models with unaltered tail bleeding time. In adoptive transfer experiments, thrombocytopenic hIL-4Rα/GPIbα-transgenic mice transfused with MCUb-/- platelets were less susceptible to FeCl3 injury-induced carotid thrombosis compared with hIL-4Rα/GPIbα-Tg mice transfused with wild type platelets, suggesting a platelet-specific role of MCUb in thrombosis. MCUb-/- stimulated platelets exhibited reduced glucose uptake, decreased glycolytic rate, and lowered pyruvate dehydrogenase phosphorylation, suggesting that mitochondrial Ca2+ mediates bioenergetic changes in platelets. CONCLUSION: Our findings suggest that mitochondrial Ca2+ signaling and glucose oxidation are functionally linked in activated platelets and reveal a novel role of MCUb in platelet activation and arterial thrombosis.

Mitochondrial pyruvate dehydrogenase kinases contribute to platelet function and thrombosis in mice by regulating aerobic glycolysis.

Resting platelets rely on oxidative phosphorylation (OXPHOS) and aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen) for their energy requirements. In contrast, platelet activation exhibits an increased rate of aerobic glycolysis relative to OXPHOS. Mitochondrial enzymes pyruvate dehydrogenase kinases (PDKs) phosphorylate the pyruvate dehydrogenase (PDH) complex to inhibit its activity, thereby diverting the pyruvate flux from OXPHOS to aerobic glycolysis upon platelet activation. Of 4 PDK isoforms, PDK2 and PDK4 (PDK2/4) are predominantly associated with metabolic diseases. Herein, we report that the combined deletion of PDK2/4 inhibits agonist-induced platelet functions, including aggregation, integrin αIIbß3 activation, degranulation, spreading, and clot retraction. In addition, collagen-mediated PLCγ2 phosphorylation and calcium mobilization were significantly reduced in PDK2/4-/- platelets, suggesting impaired GPVI signaling. The PDK2/4-/- mice were less susceptible to FeCl3-induced carotid and laser-induced mesenteric artery thrombosis without any effect on hemostasis. In adoptive transfer experiments, thrombocytopenic hIL-4Rα/GPIbα-transgenic mice transfused with PDK2/4-/- platelets exhibited less susceptibility to FeCl3 injury-induced carotid thrombosis compared with hIL-4Rα/GPIbα-Tg mice transfused with WT platelets, suggesting a platelet-specific role of PDK2/4 in thrombosis. Mechanistically, the inhibitory effects of PDK2/4 deletion on platelet function were associated with reduced PDH phosphorylation and glycoPER in activated platelets, suggesting that PDK2/4 regulates aerobic glycolysis. Finally, using PDK2 or PDK4 single KO mice, we identified that PDK4 plays a more prominent role in regulating platelet secretion and thrombosis compared with PDK2. This study identifies the fundamental role of PDK2/4 in regulating platelet functions and identifies the PDK/PDH axis as a potentially novel antithrombotic target.

PKM2 promotes neutrophil activation and cerebral thromboinflammation: therapeutic implications for ischemic stroke.

There is a critical need for cerebro-protective interventions to improve the suboptimal outcomes of patients with ischemic stroke who have been treated with reperfusion strategies. We found that nuclear pyruvate kinase muscle 2 (PKM2), a modulator of systemic inflammation, was upregulated in neutrophils after the onset of ischemic stroke in both humans and mice. Therefore, we determined the role of PKM2 in stroke pathogenesis by using murine models with preexisting comorbidities. We generated novel myeloid cell-specific PKM2-/- mice on wild-type (PKM2fl/flLysMCre+) and hyperlipidemic background (PKM2fl/flLysMCre+Apoe-/-). Controls were littermate PKM2fl/flLysMCre- or PKM2fl/flLysMCre-Apoe-/- mice. Genetic deletion of PKM2 in myeloid cells limited inflammatory response in peripheral neutrophils and reduced neutrophil extracellular traps after cerebral ischemia and reperfusion, suggesting that PKM2 promotes neutrophil hyperactivation in the setting of stroke. In the filament and autologous clot and recombinant tissue plasminogen activator stroke models, irrespective of sex, deletion of PKM2 in myeloid cells in either wild-type or hyperlipidemic mice reduced infarcts and enhanced long-term sensorimotor recovery. Laser speckle imaging revealed improved regional cerebral blood flow in myeloid cell-specific PKM2-deficient mice that was concomitant with reduced post-ischemic cerebral thrombo-inflammation (intracerebral fibrinogen, platelet [CD41+] deposition, neutrophil infiltration, and inflammatory cytokines). Mechanistically, PKM2 regulates post-ischemic inflammation in peripheral neutrophils by promoting STAT3 phosphorylation. To enhance the translational significance, we inhibited PKM2 nuclear translocation using a small molecule and found significantly reduced neutrophil hyperactivation and improved short-term and long-term functional outcomes after stroke. Collectively, these findings identify PKM2 as a novel therapeutic target to improve brain salvage and recovery after reperfusion.

Structural, functional, and mechanistic insights uncover the fundamental role of orphan connexin-62 in platelets.

Connexins oligomerise to form hexameric hemichannels in the plasma membrane that can further dock together on adjacent cells to form gap junctions and facilitate intercellular trafficking of molecules. In this study, we report the expression and function of an orphan connexin, connexin-62 (Cx62), in human and mouse (Cx57, mouse homolog) platelets. A novel mimetic peptide (62Gap27) was developed to target the second extracellular loop of Cx62, and 3-dimensional structural models predicted its interference with gap junction and hemichannel function. The ability of 62Gap27 to regulate both gap junction and hemichannel-mediated intercellular communication was observed using fluorescence recovery after photobleaching analysis and flow cytometry. Cx62 inhibition by 62Gap27 suppressed a range of agonist-stimulated platelet functions and impaired thrombosis and hemostasis. This was associated with elevated protein kinase A-dependent signaling in a cyclic adenosine monophosphate-independent manner and was not observed in Cx57-deficient mouse platelets (in which the selectivity of 62Gap27 for this connexin was also confirmed). Notably, Cx62 hemichannels were observed to function independently of Cx37 and Cx40 hemichannels. Together, our data reveal a fundamental role for a hitherto uncharacterized connexin in regulating the function of circulating cells.

The metabolic enzyme pyruvate kinase M2 regulates platelet function and arterial thrombosis.

Very little is known about the role of metabolic regulatory mechanisms in platelet activation and thrombosis. Dimeric pyruvate kinase M2 (PKM2) is a crucial regulator of aerobic glycolysis that facilitates the production of lactate and metabolic reprogramming. Herein, we report that limiting PKM2 dimer formation, using the small molecule inhibitor ML265, negatively regulates lactate production and glucose uptake in human and murine stimulated platelets. Furthermore, limiting PKM2 dimer formation reduced agonist-induced platelet activation, aggregation, clot retraction, and thrombus formation under arterial shear stress in vitro in both human and murine platelets. Mechanistically, limiting PKM2 dimerization downregulated phosphatidylinositol 3-kinase (PI3K)-mediated protein kinase B or serine/threonine-specific protein kinase (Akt)/glycogen synthase kinase 3 (GSK3) signaling in human and murine platelets. To provide further evidence for the role of PKM2 in platelet function, we generated a megakaryocyte or platelet-specific PKM2-/- mutant strain (PKM2fl/flPF4Cre+). Platelet-specific PKM2-deficient mice exhibited impaired agonist-induced platelet activation, aggregation, clot retraction, and PI3K-mediated Akt/GSK3 signaling and were less susceptible to arterial thrombosis in FeCl3 injury-induced carotid- and laser injury-induced mesenteric artery thrombosis models, without altering hemostasis. Wild-type mice treated with ML265 were less susceptible to arterial thrombosis with unaltered tail bleeding times. These findings reveal a major role for PKM2 in coordinating multiple aspects of platelet function, from metabolism to cellular signaling to thrombosis, and implicate PKM2 as a potential target for antithrombotic therapeutic intervention.

Targeting myeloid-cell specific integrin α9ß1 inhibits arterial thrombosis in mice.

Evidence suggests that neutrophils contribute to thrombosis via several mechanisms, including neutrophil extracellular traps (NETs) formation. Integrin α9ß1 is highly expressed on neutrophils when compared with monocytes. It undergoes affinity upregulation on neutrophil activation, and stabilizes adhesion to the activated endothelium. The role of integrin α9 in arterial thrombosis remains unexplored. We generated novel myeloid cell-specific integrin α9-/- mice (α9fl/flLysMCre+) to study the role of integrin α9 in arterial thrombosis. α9fl/fl littermates were used as controls. We report that α9fl/flLysMCre+ mice were less susceptible to arterial thrombosis in ferric chloride (FeCl3) and laser injury-induced thrombosis models with unaltered hemostasis. Neutrophil elastase-positive cells were significantly reduced in α9fl/flLysMCre+ mice concomitant with reduction in neutrophil count, myeloperoxidase levels, and red blood cells in the FeCl3 injury-induced carotid thrombus. The percentage of cells releasing NETs was significantly reduced in α9fl/flLysMCre+ mouse neutrophils stimulated with thrombin-activated platelets. Furthermore, we found a significant decrease in neutrophil-mediated platelet aggregation and cathepsin-G secretion in α9fl/flLysMCre+ mice. Transfusion of α9fl/fl neutrophils in α9fl/flLysMCre+ mice restored thrombosis similar to α9fl/fl mice. Treatment of wild-type mice with anti-integrin α9 antibody inhibited arterial thrombosis. This study identifies the potential role of integrin α9 in modulating arterial thrombosis.

Non-genomic effects of the Pregnane X Receptor negatively regulate platelet functions, thrombosis and haemostasis.

The pregnane X receptor (PXR) is a nuclear receptor (NR), involved in the detoxification of xenobiotic compounds. Recently, its presence was reported in the human vasculature and its ligands were proposed to exhibit anti-atherosclerotic effects. Since platelets contribute towards the development of atherosclerosis and possess numerous NRs, we investigated the expression of PXR in platelets along with the ability of its ligands to modulate platelet activation. The expression of PXR in human platelets was confirmed using immunoprecipitation analysis. Treatment with PXR ligands was found to inhibit platelet functions stimulated by a range of agonists, with platelet aggregation, granule secretion, adhesion and spreading on fibrinogen all attenuated along with a reduction in thrombus formation (both in vitro and in vivo). The effects of PXR ligands were observed in a species-specific manner, and the human-specific ligand, SR12813, was observed to attenuate thrombus formation in vivo in humanised PXR transgenic mice. PXR ligand-mediated inhibition of platelet function was found to be associated with the inhibition of Src-family kinases (SFKs). This study identifies acute, non-genomic regulatory effects of PXR ligands on platelet function and thrombus formation. In combination with the emerging anti-atherosclerotic properties of PXR ligands, these anti-thrombotic effects may provide additional cardio-protective benefits.

A Brief Review of Cardiovascular Diseases, Associated Risk Factors and Current Treatment Regimes.

Cardiovascular diseases (CVDs) are the leading cause of premature death and disability in humans and their incidence is on the rise globally. Given their substantial contribution towards the escalating costs of health care, CVDs also generate a high socio-economic burden in the general population. The underlying pathogenesis and progression associated with nearly all CVDs are predominantly of atherosclerotic origin that leads to the development of coronary artery disease, cerebrovascular disease, venous thromboembolism and, peripheral vascular disease, subsequently causing myocardial infarction, cardiac arrhythmias or stroke. The aetiological risk factors leading to the onset of CVDs are well recognized and include hyperlipidaemia, hypertension, diabetes, obesity, smoking and, lack of physical activity. They collectively represent more than 90% of the CVD risks in all epidemiological studies. Despite high fatality rate of CVDs, the identification and careful prevention of the underlying risk factors can significantly reduce the global epidemic of CVDs. Beside making favorable lifestyle modifications, primary regimes for the prevention and treatment of CVDs include lipid-lowering drugs, antihypertensives, antiplatelet and anticoagulation therapies. Despite their effectiveness, significant gaps in the treatment of CVDs remain. In this review, we discuss the epidemiology and pathology of the major CVDs that are prevalent globally. We also determine the contribution of well-recognized risk factors towards the development of CVDs and the prevention strategies. In the end, therapies for the control and treatment of CVDs are discussed.

Non-genomic effects of nuclear receptors: insights from the anucleate platelet.

Nuclear receptors (NRs) have the ability to elicit two different kinds of responses, genomic and non-genomic. Although genomic responses control gene expression by influencing the rate of transcription, non-genomic effects occur rapidly and independently of transcriptional regulation. Due to their anucleate nature and mechanistically well-characterized and rapid responses, platelets provide a model system for the study of any non-genomic effects of the NRs. Several NRs have been found to be present in human platelets, and multiple NR agonists have been shown to elicit anti-platelet effects by a variety of mechanisms. The non-genomic functions of NRs vary, including the regulation of kinase and phosphatase activity, ion channel function, intracellular calcium levels, and production of second messengers. Recently, the characterization of mechanisms and identification of novel binding partners of NRs have further strengthened the prospects of developing their ligands into potential therapeutics that offer cardio-protective properties in addition to their other defined genomic effects.

RXR Ligands Negatively Regulate Thrombosis and Hemostasis.

OBJECTIVE: Platelets have been found to express intracellular nuclear receptors including the retinoid X receptors (RXRα and RXRß). Treatment of platelets with ligands of RXR has been shown to inhibit platelet responses to ADP and thromboxane A2; however, the effects on responses to other platelet agonists and the underlying mechanism have not been fully characterized. APPROACH AND RESULTS: The effect of 9-cis-retinoic acid, docosahexaenoic acid and methoprene acid on collagen receptor (glycoprotein VI [GPVI]) agonists and thrombin-stimulated platelet function; including aggregation, granule secretion, integrin activation, calcium mobilization, integrin αIIbß3 outside-in signaling and thrombus formation in vitro and in vivo were determined. Treatment of platelets with RXR ligands resulted in attenuation of platelet functional responses after stimulation by GPVI agonists or thrombin and inhibition of integrin αIIbß3 outside-in signaling. Treatment with 9-cis-retinoic acid caused inhibition of thrombus formation in vitro and an impairment of thrombosis and hemostasis in vivo. Both RXR ligands stimulated protein kinase A activation, measured by VASP S157 phosphorylation, that was found to be dependent on both cAMP and nuclear factor κ-light-chain-enhancer of activated B cell activity. CONCLUSIONS: This study identifies a widespread, negative regulatory role for RXR in the regulation of platelet functional responses and thrombus formation and describes novel events that lead to the upregulation of protein kinase A, a known negative regulator of many aspects of platelet function. This mechanism may offer a possible explanation for the cardioprotective effects described in vivo after treatment with RXR ligands.

Farnesoid X Receptor and Its Ligands Inhibit the Function of Platelets.

OBJECTIVE: Although initially seemingly paradoxical because of the lack of nucleus, platelets possess many transcription factors that regulate their function through DNA-independent mechanisms. These include the farnesoid X receptor (FXR), a member of the superfamily of ligand-activated transcription factors, that has been identified as a bile acid receptor. In this study, we show that FXR is present in human platelets and FXR ligands, GW4064 and 6α-ethyl-chenodeoxycholic acid, modulate platelet activation nongenomically. APPROACH AND RESULTS: FXR ligands inhibited the activation of platelets in response to stimulation of collagen or thrombin receptors, resulting in diminished intracellular calcium mobilization, secretion, fibrinogen binding, and aggregation. Exposure to FXR ligands also reduced integrin αIIbß3 outside-in signaling and thereby reduced the ability of platelets to spread and to stimulate clot retraction. FXR function in platelets was found to be associated with the modulation of cyclic guanosine monophosphate levels in platelets and associated downstream inhibitory signaling. Platelets from FXR-deficient mice were refractory to the actions of FXR agonists on platelet function and cyclic nucleotide signaling, firmly linking the nongenomic actions of these ligands to the FXR. CONCLUSIONS: This study provides support for the ability of FXR ligands to modulate platelet activation. The atheroprotective effects of GW4064, with its novel antiplatelet effects, indicate FXR as a potential target for the prevention of atherothrombotic disease.

Gap junctions and connexin hemichannels in the regulation of haemostasis and thrombosis.

Platelets are involved in the maintenance of haemostasis but their inappropriate activation leads to thrombosis, a principal trigger for heart attack and ischaemic stroke. Although platelets circulate in isolation, upon activation they accumulate or aggregate together to form a thrombus, where they function in a co-ordinated manner to prevent loss of blood and control wound repair. Previous report (1) indicates that the stability and functions of a thrombus are maintained through sustained, contact-dependent signalling between platelets. Given the role of gap junctions in the co-ordination of tissue responses, it was hypothesized that gap junctions may be present within a thrombus and mediate intercellular communication between platelets. Therefore studies were performed to explore the presence and functions of connexins in platelets. In this brief review, the roles of hemichannels and gap junctions in the control of thrombosis and haemostasis and the future directions for this research will be discussed.

Pharmacological actions of nobiletin in the modulation of platelet function.

BACKGROUND AND PURPOSE: The discovery that flavonoids are capable of inhibiting platelet function has led to their investigation as potential antithrombotic agents. However, despite the range of studies on the antiplatelet properties of flavonoids, little is known about the mechanisms by which flavonoids inhibit platelet function. In this study, we aimed to explore the pharmacological effects of a polymethoxy flavonoid, nobiletin, in the modulation of platelet function. EXPERIMENTAL APPROACH: The ability of nobiletin to modulate platelet function was explored by using a range of in vitro and in vivo experimental approaches. Aggregation, dense granule secretion and spreading assays were performed using washed platelets. Fibrinogen binding, α-granule secretion and calcium mobilization assays were performed using platelet-rich plasma and whole blood was used in impedance aggregometry and thrombus formation experiments. The effect of nobiletin in vivo was assessed by measuring tail bleeding time using C57BL/6 mice. KEY RESULTS: Nobiletin was shown to suppress a range of well-established activatory mechanisms, including platelet aggregation, granule secretion, integrin modulation, calcium mobilization and thrombus formation. Nobiletin extended bleeding time in mice and reduced the phosphorylation of PKB (Akt) and PLCγ2 within the collagen receptor (glycoprotein VI)-stimulated pathway, in addition to increasing the levels of cGMP and phosphorylation of vasodilator-stimulated phosphoprotein, a protein whose activity is associated with inhibitory cyclic nucleotide signalling. CONCLUSIONS AND IMPLICATIONS: This study provides insight into the underlying molecular mechanisms through which nobiletin modulates haemostasis and thrombus formation. Therefore, nobiletin may represent a potential antithrombotic agent of dietary origins.