Platelet-mediated metabolism of the common dietary flavonoid, quercetin.

BACKGROUND: Flavonoid metabolites remain in blood for periods of time potentially long enough to allow interactions with cellular components of this tissue. It is well-established that flavonoids are metabolised within the intestine and liver into methylated, sulphated and glucuronidated counterparts, which inhibit platelet function. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate evidence suggesting platelets which contain metabolic enzymes, as an alternative location for flavonoid metabolism. Quercetin and a plasma metabolite of this compound, 4'-O-methyl quercetin (tamarixetin) were shown to gain access to the cytosolic compartment of platelets, using confocal microscopy. High performance liquid chromatography (HPLC) and mass spectrometry (MS) showed that quercetin was transformed into a compound with a mass identical to tamarixetin, suggesting that the flavonoid was methylated by catechol-O-methyl transferase (COMT) within platelets. CONCLUSIONS/SIGNIFICANCE: Platelets potentially mediate a third phase of flavonoid metabolism, which may impact on the regulation of the function of these cells by metabolites of these dietary compounds

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 ischemic stroke. Although platelets circulate in isolation, upon activation they accumulate or aggregate together to form a thrombus, where they function in a coordinated manner to prevent loss of blood and control wound repair. Recent reports indicate 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 coordination 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

Cobimetinib and trametinib inhibit platelet MEK but do not cause platelet dysfunction

The MEK inhibitors cobimetinib and trametinib are used in combination with BRAF inhibitors to treat metastatic melanoma but increase rates of hemorrhage relative to BRAF inhibitors alone. Platelets express several members of the MAPK signalling cascade including MEK1 and MEK2 and ERK1 and ERK2 but their role in platelet function and haemostasis is ambiguous as previous reports have been contradictory. It is therefore unclear if MEK inhibitors might be causing platelet dysfunction and contributing to increased hemorrhage. In the present study we performed pharmacological characterisation of cobimetinib and trametinib in vitro to investigate potential for MEK inhibitors to cause platelet dysfunction. We report that whilst both cobimetinib and trametinib are potent inhibitors of platelet MEK activity, treatment with trametinib did not alter platelet function. Treatment with cobimetinib results in inhibition of platelet aggregation, integrin activation, alpha-granule secretion and adhesion but only at suprapharmacological concentrations. We identified that the inhibitory effects of high concentrations of cobimetinib are associated with off-target inhibition on Akt and PKC. Neither inhibitor caused any alteration in thrombus formation on collagen under flow conditions in vitro. Our findings demonstrate that platelets are able to function normally when MEK activity is fully inhibited, indicating MEK activity is dispensable for normal platelet function. We conclude that the MEK inhibitors cobimetinib and trametinib do not induce platelet dysfunction and are therefore unlikely to contribute to increased incidence of bleeding reported during MEK inhibitor therapy

NADPH oxidase 2 (NOX2): a key target of oxidative stress-mediated platelet activation and thrombosis

Oxidative stress represents an imbalance between the production of reactive oxygen species (ROS) and the cellular antioxidant system. Increased levels of oxidative stress contribute to the development of atherosclerosis that eventually leads to thrombosis; a principle cause of heart attacks and strokes. Thrombosis is a consequence of platelet activation and aggregate formation within the circulation. Platelet ROS are mostly generated by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. NOX2 is an isoform from NADPH oxidase expressed in platelets and an important regulator of platelet activation‐associated thrombosis. The present article aims to highlight the relative contribution of NOX2 as a key target of different platelet activation pathways and antiplatelet treatment

Thiol isomerases orchestrate thrombosis and haemostasis

Since protein disulphide isomerase (PDI) was first described in 1963, researchers have shown conclusively that PDI and sibling proteins are quintessential for thrombus formation. PDI, ERp5, ERp57 and ERp72, which in most cells are located in the endoplasmic reticulum and function to assist the folding of nascent protein, are released from platelets and vascular cells and interact with integrin αIIbβ3 on the outer surface of platelets. At the cell surface they continue to influence protein folding and function, propagating thrombosis and maintaining haemostasis. TMX1, which is a transmembrane thiol isomerase, is the first family member shown to negatively regulate platelets known to date. Targets of thiol isomerases have been indentified including integrin α2β1, Von Willebrand Factor (VWF), GpIbα, Nox-1, Nox-2 and tissue factor, all of which are pro-thrombotic, and several of which are on the cell surface. In spite of this, PDI can paradoxically catalyse the delivery of nitric oxide to platelets, which inhibits their function and decreases thrombus formation. Although the overall effect of PDI is to positively regulate platelet activation, it is still unclear how thiol isomerases function in pro-thrombotic states, such as obesity, diabetes and cancer. In parallel, there has been a surge in the development of novel thiol isomerase inhibitors, which display selectivity, potency and modulate thrombosis and haemostasis. The availability of selective thiol isomerase inhibitors has culminated in clinical trials with promising outcomes for the prevention of cancer-associated thrombosis. Altogether, thiol isomerases are perceived as an orchestrating force that regulates thrombus development. In the current review we will explore the history of PDI in cardiovascular biology, detail known mechanisms of action and summarise known thiol isomerase inhibitors

Ca2+ waves coordinate purinergic receptor–evoked integrin activation and polarization

The integrin GPIIb/IIIa is highly abundant on the surface of platelets and can be activated by intracellular Ca2+ signaling in an “inside-out” manner to bind to the adhesive ligand fibrinogen. Bye et al. imaged intracellular Ca2+ signaling and fibrinogen binding events in primary rat megakaryocytes activated through the ADP-stimulated receptors P2Y1 and P2Y12. The authors found that signaling by both receptors was required for full integrin activation, which depended on P2Y1-stimulated Ca2+ signaling and P2Y12-stimulated activation of the kinase PI3K. In addition, fibrinogen binding became polarized in these cells in a manner dependent on the direction of ADP-stimulated Ca2+ waves

Platelets stop us leaking

In this issue of Blood, Welsh and colleagues determine how platelet thrombi limit the loss of plasma-borne proteins from the microvasculature. 1 The concept that platelets prevent leakage from blood vessels following a penetrating injury will come as little surprise to most readers, but exactly how platelets physically contribute to this process is not well understood. Such injuries result in the loss of blood cells and plasma into the surrounding tissues, and both forms of loss must be halted quickly. Platelets are believed to form the first line of defense in such situations, forming a thrombus that is capable of plugging the hole and supplying molecules that contribute to localized inflammation and wound healing

Conscious voiding during bladder obstruction in guinea pigs correlates with contractile activity of isolated bladders

© 2015. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/. Following 12 month embargo from date of publication (10 August 2015) in accordance with publisher copyright policy

Challenges in diagnosing and treating snakebites in a rural population of Tamil Nadu, India: the views of clinicians

Snakebites cause death, disability and economic devastation to their victims, people who live almost exclusively in rural areas. Annually an estimated two million venomous bites cause as many as 100,000 deaths worldwide as well as hundreds of thousands of deformities and amputations. Recent studies suggest that India has the highest incidence of snakebite and associated deaths worldwide. In this study, we interviewed 25 hospital-based clinicians who regularly treat snakebites in Tamil Nadu, India, in order to gauge their opinions and views on the diagnostic tools and treatment methods available at that time, the difficulties encountered in treating snakebites and improvements to snakebite management protocols they deem necessary. Clinicians identified the improvement of community education, training of medical personnel, development of standard treatment protocols and improved medication as priorities for the immediate future

The platelet-surface thiol isomerase enzyme ERp57 modulates platelet function

Background: Thiol isomerases are a family of endoplasmic reticulum enzymes which orchestrate redox-based modifications of protein disulphide bonds. Previous studies have identified important roles for the thiol isomerases PDI and ERp5 in the regulation of normal platelet function. Objectives: Recently, we demonstrated the presence of a further five thiol isomerases at the platelet surface. In this report we aim to report the role of one of these enzymes - ERp57 in the regulation of platelet function. Methods/Results: Using enzyme activity function blocking antibodies, we demonstrate a role for ERp57 in platelet aggregation, dense granule secretion, fibrinogen binding, calcium mobilisation and thrombus formation under arterial conditions. In addition to the effects of ERp57 on isolated platelets, we observe the presence of ERp57 in the developing thrombus in vivo. Furthermore the inhibition of ERp57 function was found to reduce laser-injury induced arterial thrombus formation in a murine model of thrombosis. Conclusions: These data suggest that ERp57 is important for normal platelet function and opens up the possibility that the regulation of platelet function by a range of cell surface thiol isomerases may represent a broad paradigm for the regulation of haemostasis and thrombosis