The Metabolites of the Dietary Flavonoid Quercetin Possess Potent Antithrombotic Activity, and Interact with Aspirin to Enhance Antiplatelet Effects

Fig. 1. Isorhamnetin and tamarixetin, the methylated metabolites of quercetin, inhibit collagen-stimulated platelet aggregation. Washed human platelets (2 × 10 8 cells/mL) were incubated with isorhamnetin ( A, B ), tamarixetin ( C, D ), quercetin ( E, F ), or vehicle (DMSO, 0.2% v/v) for 5 minutes (with 10 seconds stirring to mix) prior to addition of collagen (5 µg/mL). Aggregation was measured for 5 minutes at 37 °C under constant stirring (1,200 rpm) conditions in a Chrono-Log optical platelet aggregometer. ( A, C, E ) Representative traces from aggregation assays. ( B, D, F ) Four-parameter nonlinear regression curves used to estimate the IC 50 of isorhamnetin, tamarixetin, and quercetin. N = 3, data represent mean ± SEM. I, isorhamnetin; T, tamarixetin; Q, quercetin.

Fig. 2. Isorhamnetin, tamarixetin, and quercetin inhibit platelet granule secretion. Washed platelets (4 × 10 8 cells/mL) were treated with flavonoid or vehicle control (DMSO, 0.25% v/v) for 5 minutes, with 50 μL Chronolume luminescent substrate added in 2 minutes prior to stimulation. Collagen (5 μg/mL) was added to stimulate platelets and ATP secretion was monitored for 180 seconds under stirring conditions (1,200 rpm) at 37 °C. ( A, E, I ) Representative traces showing increase in luminescence upon ATP secretion and inhibition by flavonoids. ( B, F, J ) ATP secretion as a percentage of the amount secreted in the absence of isorhamnetin, tamarixetin, or quercetin (vehicle). Washed platelets (2 × 10 8 cells/mL) were incubated with isorhamnetin ( C, D ), tamarixetin ( G, H ), quercetin ( K, L ), or vehicle control (DMSO, 0.25% v/v) for 5 minutes, after which PE/Cy5 antihuman CD62P antibody was added to the sample prior to stimulation. Samples were then stimulated with CRP-XL (1 μg/mL) for 20 minutes, after which they were fixed in 0.2% paraformaldehyde. P-selectin exposure on the cell surface was then measured by flow cytometry and data normalized to the level of P-selectin exposure in…

Fig. 3. Integrin αIIbβ3 affinity and function is altered by quercetin's methylated metabolites. Washed platelets (2 × 10 8 cells/mL) were incubated with isorhamnetin ( A ), tamarixetin ( B ), quercetin ( C ), or vehicle control (DMSO, 0.25% v/v), stimulated with CRP-XL (1 µg/mL), and fibrinogen binding measured by flow cytometry. Data normalized to the level of fibrinogen binding in the absence of flavonoid (vehicle). Washed platelets (2 × 10 8 cells/mL) were incubated with isorhamnetin ( E ), tamarixetin ( F ), quercetin ( G ), or vehicle control (DMSO 0.33% v/v) for 5 minutes, then allowed to spread on fibrinogen (100 µg/mL) coated coverslips for 45 minutes at 37 °C. Cells were stained with Alexa fluor 488 phalloidin and visualized using a Nikon A1-R confocal microscope. Data represent percentage of platelets in each stage of spreading ( E–G ), mean ± SEM, with representative images in ( D ). PRP was incubated with isorhamnetin ( H ), tamarixetin ( I ), quercetin ( J ), or vehicle control (DMSO, 0.2% v/v) for 60 minutes at 30 °C, after which thrombin (1 U/mL) was added to stimulate clot formation. The assay was terminated at 120 minutes when vehicle-treated clots had retracted fully,…

Fig. 4. Isorhamnetin, tamarixetin, and quercetin inhibit calcium mobilization and signaling proximal to GPVI. Fura-2 loaded washed platelets (4 × 10 8 cells/mL) were incubated with isorhamnetin ( A, B ), tamarixetin ( C, D ), quercetin ( E, F ), or vehicle control (DMSO, 0.2% v/v) for 5 minutes at 37 °C prior to stimulation with CRP-XL (1 µg/mL). Fluorescence (excitation 340 and 380 nm, emission 510 nm) was recorded for 5 minutes using a NOVOstar plate reader, and [Ca 2+ ] i was estimated using the equation described in the Methods ( A, C, E ). Peak [Ca 2+ ] i was taken as the maximum value reached in the sample over the 5-minute period, and was normalized to peak calcium levels in the absence of flavonoid (vehicle) ( B, D, F ). N = 4, data in ( B ), ( D ), and ( F ) represent mean ± SEM, analyzed by one-way ANOVA with posthoc Dunnett's test. * p < 0.05, ** p < 0.005, *** p < 0.001, **** p < 0.0001 compared to vehicle control. Washed human platelets (4 × 10 8 cells/mL) were incubated with isorhamnetin ( I, L ), tamarixetin ( J, M ), quercetin ( K, N ), or vehicle control (DMSO, 0.4% v/v) for 5 minutes at 37 °C prior to addition of CRP-XL (1 μg/mL). Aggregation was allowed to occur for…

Fig. 5. Isorhamnetin, tamarixetin, and quercetin display antithrombotic actions in vitro, and isoquercetin inhibits thrombosis in vivo . DiOC 6 -labelled whole blood was incubated with isorhamnetin ( A ), tamarixetin ( B ), quercetin ( C ) (10 μM), or vehicle control (DMSO, 0.2% v/v) prior to perfusion through collagen-coated Vena8 biochip channels at a shear rate of 20 dyn/cm 2 , with images captured every second for 600 seconds. Mean fluorescence intensity was normalized to the level in the absence of flavonoid (vehicle) and was calculated using NIS Elements software ( A–C ), and representative images at the assay endpoint are shown in ( D ). C57BL/6 mice were dosed twice per day (9 am and 5 pm) with isoquercetin (200 mg/kg) or vehicle control (distilled water) by gavage for 48 hours prior to the experiment, with one final dose administered in the morning of the experiment. Mice were anaesthetized, platelets were labeled with DyLight 649 anti-GPIbα antibody, and laser injury of a cremaster muscle arteriole wall was performed with a Micropoint ablation laser unit. Thrombus formation was observed using an Olympus BX61W1 microscope, with images analyzed to calculate fluorescence intensity…

Fig. 6. Isorhamnetin, tamarixetin, and quercetin synergize with aspirin to enhance antiplatelet effects. Washed platelets (2 × 10 8 cells/mL) were incubated in a 96-well plate with aspirin ± isorhamnetin ( A, B ), tamarixetin ( C, D ), quercetin ( E, F ) (5 µM flavonoid shown as example), or vehicle control (0.25% ethanol + 0.5% DMSO, v/v), and aggregation stimulated by collagen (5 µg/mL). Data represent percentage aggregation normalized to levels of aggregation in the absence of flavonoid (vehicle) ( A, C, E ). ( B, D, F ) IC 50 values for aspirin upon addition of flavonoid. N = 3, data represent mean ± SEM. * p < 0.05, ** p < 0.005, *** p < 0.001, **** p < 0.0001. Blue stars indicate a significant effect of aspirin compared to vehicle control, analyzed by one-way ANOVA. Black stars indicate a significant effect of flavonoid plus aspirin compared to aspirin alone, analyzed by two-way ANOVA. “S” indicates an enhanced effect of dual treatment compared to the combined effects of individual flavonoid and aspirin concentrations ( p < 0.05), analyzed by paired t -test. Whole blood was incubated with quercetin ± aspirin, tamarixetin ± aspirin, isorhamnetin ± aspirin, aspirin alone, or vehicle…

Fig. 7. A model demonstrating the multiple platelet activation pathways inhibited by the methylated metabolites of quercetin. Isorhamnetin, tamarixetin, and quercetin inhibit multiple aspects of the GPVI activation pathway, including Syk and LAT phosphorylation, alpha and dense granule secretion, calcium mobilization, integrin αIIbβ3 inside-out signaling and, ultimately, aggregation (indicated by the red text ). In addition, this study demonstrates an inhibition of platelet aggregation downstream of ADP, thrombin, and U46619 stimulation. This multitude of inhibitory effects culminates in the potent inhibition observed upon treatment with isorhamnetin, tamarixetin, or quercetin, and results in attenuated thrombus formation. ADP, adenosine phosphate; GPCR, G-protein coupled receptor; GPVI, glycoprotein VI; I, isorhamnetin; IP3, inositol 1,4,5-trisphosphate; LAT, linker for activation of T cells; PLCγ2, phospholipase Cγ2; PIP2, phosphatidylinositol 4,5-bisphosphate; Q, quercetin; Syk, spleen tyrosine kinase; T, tamarixetin; TXA2, thromboxane A 2 .