Protein disulfide isomerase inhibition blocks thrombin generation in humans by interfering with platelet factor V activation.

: 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. : We evaluated effects of oral administration of isoquercetin on platelet-dependent thrombin generation in healthy subjects and patients with persistently elevated anti-phospholipid antibodies. : Following oral administration of 1,000 mg isoquercetin to healthy adults, the measured peak plasma quercetin concentration (9.2 μM) exceeded its IC 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 ( = 0.0004) and by 64% in the anti-phospholipid antibody cohort ( = 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 IC of ~5 μM. Inhibition of PDI by isoquercetin ingestion resulted in a 53% decrease in the generation of platelet factor Va ( = 0.001). Isoquercetin-mediated inhibition was reversed with addition of exogenous factor Va. : 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. : Clinicaltrials.gov (NCT01722669) : National Heart, Lung, and Blood Institute (U54 HL112302) and Quercegen Pharma.