Protein disulfide isomerase inhibition blocks thrombin generation in humans by interfering with platelet factor V activation
Jack D. Stopa, … , Robert Flaumenhaft, Jeffrey I. Zwicker
Jack D. Stopa, … , Robert Flaumenhaft, Jeffrey I. Zwicker
Published January 12, 2017
Citation Information: JCI Insight. 2017;2(1):e89373. https://doi.org/10.1172/jci.insight.89373.
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Clinical Medicine Hematology

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

Abstract

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.

TRIAL REGISTRATION: Clinicaltrials.gov (NCT01722669)

FUNDING: National Heart, Lung, and Blood Institute (U54 HL112302) and Quercegen Pharma

Authors

Jack D. Stopa, Donna Neuberg, Maneka Puligandla, Bruce Furie, Robert Flaumenhaft, Jeffrey I. Zwicker

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Figure 7

Factor V activation following ingestion of isoquercetin.

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Factor V activation following ingestion of isoquercetin. (A) Platelet-ri...
(A) Platelet-rich plasma (PRP) samples and platelet-poor plasma (PPP) samples were either untreated (Control) or depleted of plasma factor V (FV-IP) using an antibody directed against FV. Samples were subsequently stimulated with 0.1 U/ml thrombin and subjected to centrifugation. Proteins in the supernatant were resolved by gel electrophoresis, transferred to PVDF, and stained for factor Va (FVa). Staining of samples using anti–factor XII antibody was performed as a loading control (FXII). A representative blot is shown for experiments run for every patient (n = 17). (B) The generation of FVa from platelets is decreased in the plasma of a subject following ingestion of isoquercetin. Plasma samples were obtained from 17 healthy participants before (baseline, gray boxes) and 4 hours after (IsoQ, white boxes) ingestion of 1,000 mg isoquercetin. Samples were either left untreated (FV replete) or depleted of FV (FV depleted) and processed as described in A. FVa staining in immunoblots was quantified by densitometry. Top and bottom of box-and-whisker plots represent the interquartile range along with median values, whiskers represent 95th percentiles, and outliers are shown. P values represent paired t test. (C) Isoquercetin-mediated reduction in platelet-dependent thrombin generation is reversed with FVa. Plasma samples were obtained from 17 healthy participants before (gray boxes) and 4 hours after (white boxes) ingestion of 1,000 mg isoquercetin. Samples were then incubated with no addition (No addition) or 7 μg/ml FVa (FVa) and evaluated for platelet-dependent thrombin generation. Top and bottom of box-and-whisker plots represent the interquartile range along with median values, whiskers represent 95th percentiles, and outliers are shown. P values represent 1-sample t test of the Baseline/IsoQ ratios with and without FVa to test the null hypothesis that these ratios are the same.