BACKGROUND: Protein disulfide isomerase (PDI) is required for thrombus formation. We previously demonstrated that glycosylated quercetin flavonoids such as isoquercetin inhibit PDI activity and thrombus formation in animal models, but whether extracellular PDI represents a viable anticoagulant target in humans and how its inhibition affects blood coagulation remain unknown.
METHODS: We evaluated effects of oral administration of isoquercetin on platelet-dependent thrombin generation in healthy subjects and patients with persistently elevated anti-phospholipid antibodies.
RESULTS: Following oral administration of 1,000 mg isoquercetin to healthy adults, the measured peak plasma quercetin concentration (9.2 μM) exceeded its IC50 for inhibition of PDI by isoquercetin in vitro (2.5 ± 0.4 μM). Platelet-dependent thrombin generation decreased by 51% in the healthy volunteers compared with baseline (P = 0.0004) and by 64% in the anti-phospholipid antibody cohort (P = 0.015) following isoquercetin ingestion. To understand how PDI affects thrombin generation, we evaluated substrates of PDI identified using an unbiased mechanistic-based substrate trapping approach. These studies identified platelet factor V as a PDI substrate. Isoquercetin blocked both platelet factor Va and thrombin generation with an IC50 of ~5 μM. Inhibition of PDI by isoquercetin ingestion resulted in a 53% decrease in the generation of platelet factor Va (P = 0.001). Isoquercetin-mediated inhibition was reversed with addition of exogenous factor Va.
CONCLUSION: These studies show that oral administration of isoquercetin inhibits PDI activity in plasma and diminishes platelet-dependent thrombin generation predominantly by blocking the generation of platelet factor Va. These pharmacodynamic and mechanistic observations represent an important step in the development of a novel class of antithrombotic agents targeting PDI.
Levels of total plasma quercetin were determined at serial time points over 24 hours following oral administration to healthy adults of (A) 500 mg quercetin aglycone or (B) 500 mg isoquercetin. Quercetin levels were measured by HPLC after enzymatic hydrolysis. Quercetin analogs were formulated with ascorbic acid/niacin (n = 5, red) and without ascorbic acid/niacin (n = 5, blue). Error bars represent SEM. (C) Measurement of protein disulfide isomerase (PDI) inhibition using a plasma-based assay. The indicated concentrations of isoquercetin (red), quercetin-3-glucuronide (black), or quercetin-3-rutinoside (green) were incubated with recombinant PDI in human plasma and PDI activity measured using a di-eosin-GSSG–based assay. IQ, isoquercetin. Mean values of triplicates (± SEM) represent percentage of PDI activity. (D) Comparison of PDI inhibition and quercetin levels following administration of isoquercetin to healthy adults. Plasma was obtained from patients at the indicated times following ingestion of 1,000 mg isoquercetin. Inhibition of recombinant PDI by plasma (green, scale per right y axis) is presented as percentage PDI activity compared with plasma obtained prior to ingestion (time 0). Circles represent mean values of 10 patients with SEM shown as error bars. Quercetin concentration in plasma (blue, scale per left y axis) was determined using HPLC. (E) Box-and-whisker plot of peak PDI inhibition at peak quercetin concentrations above or below 4 μM. Top and bottom of box represent the interquartile range along with median values, whiskers represent 95th percentile, and outliers are shown. Mann-Whitney P = 0.002.