The relationship between the metabolites of the dietary flavonoid quercetin, and haemostasis and thrombosis: an integrated systems approach

Quercetin is one of the most widely consumed flavonoids worldwide, and exerts numerous effects protective against cardiovascular disease, including the inhibition of platelet function. However, quercetin is extensively metabolised, and current data is limited regarding these metabolites. In this study, the anti-platelet effects of quercetin, and two methylated metabolites, tamarixetin and isorhamnetin, were investigated, to examine how anti-platelet efficacy is altered upon metabolism. Quercetin, tamarixetin and isorhamnetin inhibited collagen-stimulated platelet aggregation, and aggregation stimulated by ADP, thrombin and the thromboxane A2 mimetic U46619, at physiologically achievable concentrations. Granule secretion, integrin aIIbb3 activation and outside-in signalling, adhesion and spreading and calcium mobilisation were inhibited in a concentrationdependent manner; metabolism of quercetin can enhance or reduce anti-platelet effect. Due to high plasma binding, anti-platelet effects in platelet rich plasma and whole blood were investigated; significant inhibitory effects were maintained, with inhibition of clot retraction and thrombus formation under arterial flow conditions. The potential pharmacological importance of quercetin intake was investigated, with in vitro anti-platelet effects of novel quercetin formulations and an in vivo anti-thrombotic effect of an isoquercetin formulation after oral administration being demonstrated for the first time, and the identification of an interaction between the methylated metabolites and aspirin. To identify and predict important flavonoid interactions and potential inhibitory mechanisms, mathematical models of platelet aggregation and thrombus formation were developed, as was a pharmacokinetic/pharmacodynamic model of the dynamic anti-thrombotic actions of quercetin, which predicted optimal dosing regimens and highlighted the potential for increased anti-thrombotic effects upon altering quercetin metabolism. In summary, this study provided evidence for the potential of quercetin and its methylated metabolites in the inhibition of platelet function and thrombus formation, and identified interactions with aspirin, highlighting the potential of quercetin as an anti-platelet dietary compound or supplement, and identified some of the mechanisms underlying these actions

Ibrutinib inhibits platelet integrin αIIbβ3 outside-in signaling and thrombus stability but not adhesion to collagen

OBJECTIVE: Ibrutinib is an irreversible Bruton tyrosine kinase inhibitor approved for treatment of Waldenstrom macroglobulinemia, chronic lymphocytic leukemia, and mantle cell lymphoma that increases the risk of bleeding among patients. Platelets from ibrutinib-treated patients exhibit deficiencies in collagen-evoked signaling in suspension; however, the significance of this observation and how it relates to bleeding risk is unclear, as platelets encounter immobile collagen in vivo. We sought to clarify the effects of ibrutinib on platelet function to better understand the mechanism underlying bleeding risk. APPROACH AND RESULTS: By comparing signaling in suspension and during adhesion to immobilized ligands, we found that the collagen signaling deficiency caused by ibrutinib is milder during adhesion to immobilized collagen. We also found that platelets in whole blood treated with ibrutinib adhered to collagen under arterial shear but formed unstable thrombi, suggesting that the collagen signaling deficiency caused by ibrutinib may not be the predominant cause of bleeding in vivo. However, clot retraction and signaling evoked by platelet adhesion to immobilized fibrinogen were also inhibited by ibrutinib, indicating that integrin αIIbβ3 outside-in signaling is also effected in addition to GPVI signaling. When ibrutinib was combined with the P2Y12 inhibitor, cangrelor, thrombus formation under arterial shear was inhibited additively. CONCLUSIONS: These findings suggest that (1) ibrutinib causes GPVI and integrin αIIbβ3 platelet signaling deficiencies that result in formation of unstable thrombi and may contribute toward bleeding observed in vivo and (2) combining ibrutinib with P2Y12 antagonists, which also inhibit thrombus stability, may have a detrimental effect on hemostasis

The metabolites of the dietary flavonoid quercetin possess potent antithrombotic activity, and interact with aspirin to enhance antiplatelet effects

Quercetin, a dietary flavonoid, has been reported to possess antiplatelet activity. However, its extensive metabolism following ingestion has resulted in difficulty elucidating precise mechanisms of action. In this study, we aimed to characterize the antiplatelet mechanisms of two methylated metabolites of quercetin—isorhamnetin and tamarixetin—and explore potential interactions with aspirin. Isorhamnetin and tamarixetin inhibited human platelet aggregation, and suppressed activatory processes including granule secretion, integrin αIIbβ3 function, calcium mobilization, and spleen tyrosine kinase (Syk)/linker for activation of T cells (LAT) phosphorylation downstream of glycoprotein VI with similar potency to quercetin. All three flavonoids attenuated thrombus formation in an in vitro microfluidic model, and isoquercetin, a 3-O-glucoside of quercetin, inhibited thrombosis in a murine laser injury model. Isorhamnetin, tamarixetin, and quercetin enhanced the antiplatelet effects of aspirin more-than-additively in a plate-based aggregometry assay, reducing aspirin IC50 values by an order of magnitude, with this synergy maintained in a whole blood test of platelet function. Our data provide mechanistic evidence for the antiplatelet activity of two quercetin metabolites, isorhamnetin and tamarixetin, and suggest a potential antithrombotic role for these flavonoids. In combination with their interactions with aspirin, this may represent a novel avenue of investigation for the development of new antithrombotic strategies and management of current therapies

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. The fibrinogen binding, α-granule secretion and calcium mobilisation 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 supress a range of well-established activatory mechanisms, including platelet aggregation, granule secretion, integrin modulation, calcium mobilisation and thrombus formation. Nobiletin was shown to extend bleeding time in mice and reduce the phosphorylation of Akt and PLCγ2 within the collagen receptor (GPVI) - stimulated pathway, in addition to increasing the levels of cGMP and phosphorylation of VASP, 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

Platelet factor XIII-A regulates platelet function and promotes clot retraction and stability.

Factor XIII (FXIII) is an important proenzyme in the hemostatic system. The plasma-derived enzyme activated FXIII cross-links fibrin fibers within thrombi to increase their mechanical strength and cross-links fibrin to fibrinolytic inhibitors, specifically α2-antiplasmin, to increase resistance to fibrinolysis. We have previously shown that cellular FXIII (factor XIII-A [FXIII-A]), which is abundant in the platelet cytoplasm, is externalized onto the activated membrane and cross-links extracellular substrates. The contribution of cellular FXIII-A to platelet activation and platelet function has not been extensively studied. This study aims to identify the role of platelet FXIII-A in platelet function. We used normal healthy platelets with a cell permeable FXIII inhibitor and platelets from FXIII-deficient patients as a FXIII-free platelet model in a range of platelet function and clotting tests. Our data demonstrate that platelet FXIII-A enhances fibrinogen binding to the platelet surface upon agonist stimulation and improves the binding of platelets to fibrinogen and aggregation under flow in a whole-blood thrombus formation assay. In the absence of FXIII-A, platelets show reduced sensitivity to agonist stimulation, including decreased P-selectin exposure and fibrinogen binding. We show that FXIII-A is involved in platelet spreading where a lack of FXIII-A reduces the ability of platelets to fully spread on fibrinogen and collagen. Our data demonstrate that platelet FXIII-A is important for clot retraction where clots formed in its absence retracted to a lesser extent. Overall, this study shows that platelet FXIII-A functions during thrombus formation by aiding platelet activation and thrombus retraction in addition to its antifibrinolytic roles

Mycolactone-dependent depletion of endothelial cell thrombomodulin is strongly associated with fibrin deposition in Buruli ulcer lesions

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin's substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells' ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone's effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tisischemia could contribute to the development of the tissue necrosis seen in BU lesions

Anti-platelet properties of Pim kinase inhibition is mediated through disruption of thromboxane A2 receptor signalling

Pim kinases are upregulated in several forms of cancer, contributing to cell survival and tumour development, but their role in platelet function and thrombotic disease has not been explored. We report for the first time that Pim-1 kinase is expressed in human and mouse platelets. Genetic deletion or pharmacological inhibition of Pim kinase results in reduced thrombus formation but is not associated with impaired haemostasis. Attenuation of thrombus formation was found to be due to inhibition of the thromboxane A2 receptor as effects on platelet function was non-additive to inhibition caused by the cyclooxygenase inhibitor indomethacin or thromboxane A2 receptor antagonist GR32191. Treatment with Pim kinase inhibitors caused reduced surface expression of the thromboxane A2 receptor and resulted in reduced responses to thromboxane A2 receptor agonists, indicating a role for Pim kinase in the regulation of thromboxane A2 receptor function. Our research identifies a novel, Pim kinase dependent regulatory mechanism for the thromboxane A2 receptor and represents a new targeting strategy that is independent of COX- 1 inhibition or direct antagonism of the thromboxane A2 receptor that whilst attenuating thrombosis does not increase bleeding

Transient calculation of a high pressure power plant conduit system

Abweichender Titel nach Übersetzung der Verfasserin/des VerfassersZusammenfassung in englischer SpracheDiese Arbeit befasst sich mit der instationären Berechnung des Triebwasserweges inklusive den vorhandenen Absperrorganen eines Hochdruck-Kraftwerks der Tiroler Wasserkraft AG. Ziel war es, ein numerisches 1-D Modell des Kraftwerks zu erstellen, mit dem es möglich ist, das Notschlussverhalten der Drosselklappe zu untersuchen. Der Umbau des Kraftwerks besteht aus einem neuen Druckstollen mit Wasserschloss, die für einen zukünftigen Ausbau dimensioniert wurden. Bis ein neuer Druckstollen gebaut wird, ist der neue Kraftabstieg mit dem bestehenden Druckstollen verbunden. Bei dieser vorübergehenden Anordnung, Drosselklappe vor dem Wasserschloss, musste das Schließverhalten der Drosselklappe näher betrachtet werden.This diploma thesis deals with the transient calculation of a high pressure power plant conduit system. The aim was to create a 1-D numerical model of the power plant which is able to investigate the closing behavior of the safety valve under different condititions. The rebuild of the power plant includes a new pressure tunnel and surge tank which are designed for the future expansion of the power plant. Until this expansion is realised the new pressure tunnel and surge tanke are connected with the existing penstock. This transitional solution, surge tank behind safety valve, needed a closer inspection regarding the closing behavior of the safety valve.7

Atomic Force Microscopy Imaging in Turbid Liquids: A Promising Tool in Nanomedicine

Tracking of biological and physiological processes on the nanoscale is a central part of the growing field of nanomedicine. Although atomic force microscopy (AFM) is one of the most appropriate techniques in this area, investigations in non-transparent fluids such as human blood are not possible with conventional AFMs due to limitations caused by the optical readout. Here, we show a promising approach based on self-sensing cantilevers (SSC) as a replacement for optical readout in biological AFM imaging. Piezo-resistors, in the form of a Wheatstone bridge, are embedded into the cantilever, whereas two of them are placed at the bending edge. This enables the deflection of the cantilever to be precisely recorded by measuring the changes in resistance. Furthermore, the conventional acoustic or magnetic vibration excitation in intermittent contact mode can be replaced by a thermal excitation using a heating loop. We show further developments of existing approaches enabling stable measurements in turbid liquids. Different readout and excitation methods are compared under various environmental conditions, ranging from dry state to human blood. To demonstrate the applicability of our laser-free bio-AFM for nanomedical research, we have selected the hemostatic process of blood coagulation as well as ultra-flat red blood cells in different turbid fluids. Furthermore, the effects on noise and scanning speed of different media are compared. The technical realization is shown (1) on a conventional optical beam deflection (OBD)-based AFM, where we replaced the optical part by a new SSC nose cone, and (2) on an all-electric AFM, which we adapted for measurements in turbid liquids