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.
(A) PDI trapping mutants react with factor V released from activated platelets. PDI variants (CGPC-PDI, CGRC-PDI, or AGHA-PDI) labeled with an N-terminal FLAG tag were either prereduced (Reduced) or preoxidized (Oxidized) prior to incubation with platelets. Washed platelets were stimulated with thrombin in the presence of 1 of the FLAG-tagged PDI variants. Platelets were separated from releasates by centrifugation and FLAG-tagged PDI constructs were pulled down using anti-FLAG antibody–coated beads. Precipitated proteins were resolved by polyacrylamide gel electrophoresis under nonreducing conditions, transferred to PVDF, and immunoblotted (IB) for both FLAG (green) and factor V (red). Overlapping bands are shown in yellow in the merge. (B) Evaluation of nonreduced samples indicates that factor V (FV) trapped by PDI variants associates with multimerin-1 (MMN-1). Pulldowns of PDI variants (CGPC-PDI, CGRC-PDI, or AGHA-PDI) were performed as described in A. Samples were resolved under nonreducing conditions using agarose electrophoresis to resolve high-molecular-weight complexes and transferred to PVDF membranes. Membranes were subsequently stained using anti-FV antibody (anti-FV) and anti-MMN1 antibody (anti-MMN1). For all panels, representative images are shown for experiments performed in triplicate.