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.
View: Text | PDF
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

×

Figure 3

Thrombin generation is decreased in a protein disulfide isomerase–dependent manner following isoquercetin administration.

Options: View larger image (or click on image) Download as PowerPoint
Thrombin generation is decreased in a protein disulfide isomerase–depend...
(A) Platelet-dependent thrombin generation was evaluated in platelet-rich plasma incubated with no addition (None), 10 μM isoquercetin (IsoQ), 50 μM protein disulfide isomerase (PDI), or both (IsoQ+PDI). The reaction was initiated using 0.1 U/ml thrombin and platelet-dependent thrombin generation detected using a fluorescent thrombin-specific substrate. Triplicate results and mean values are represented as peak thrombin generated (U/ml) following initiation of reaction. P values were determined by 1-way ANOVA with Sheffe’s correction for multiplicity of comparison. (B) Ten healthy participants received an oral dose of 1,000 mg isoquercetin and blood samples were drawn just before (baseline, PDI) and 4 hours after (IsoQ, IsoQ+PDI) ingestion. Samples were subsequently incubated in the absence (baseline, IsoQ) or presence (PDI, IsoQ+PDI) of PDI and assayed for platelet-dependent thrombin generation using a fluorescent thrombin-specific substrate. Top and bottom of box represent the interquartile range along with median values, whiskers represent 95th percentiles, and outliers are shown (n = 10). P values were determined using a mixed-model ANOVA with Sheffe’s correction for multiplicity of testing.