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 Research and Public Health 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 6

Isoquercetin-mediated protein disulfide isomerase inhibition targets platelet factor Va generation.

Options: View larger image (or click on image) Download as PowerPoint
Isoquercetin-mediated protein disulfide isomerase inhibition targets pla...
(A) The generation of factor Va from platelets is decreased in the presence of isoquercetin in vitro. Washed platelets were stimulated with 0.1 U/ml thrombin in the presence of varying concentrations of isoquercetin. Proteins within releasates were stained with an antibody that specifically detects factor Va (FVa) and von Willebrand factor monomer was stained as a loading control (vWF). A representative image is shown for experiments performed in triplicate. (B) Comparison of thrombin generation, FVa generation, and PDI inhibition after washed platelets were stimulated using 0.1 U/ml thrombin in the presence of the indicated concentrations of isoquercetin (IQ). Thrombin generation (squares) was evaluated using a fluorescent substrate, FVa generation (circles) using a FVa-specific antibody, and PDI inhibition (triangles) by a di-eosin-GSSG assay. Symbols represent means of triplicates of activity (± SD) compared with samples not exposed to isoquercetin. (C) The release of platelet factor 4 (PF4) from platelets is unaffected in the presence of isoquercetin. Washed platelets were incubated with 0.1 U/ml thrombin in the presence of the indicated concentrations of isoquercetin and platelet releasates isolated by centrifugation. Proteins within the releasates were resolved by gel electrophoresis and immunoblotted for PF4. Experiments were performed in triplicate and data represent band intensity compared with the no-isoquercetin control, mean ± SD. (D) Isoquercetin does not directly inhibit factor Xa. Purified factor Xa (10 μg/ml) was combined with purified FVa (7 μg/ml) and assayed for activity in the presence (black circles) or absence (white circles) of 10 μM isoquercetin using a fluorescent cleavage substrate for Xa (Va+Xa). Fluorescent substrate only (None) and FVa with substrate (Va) were included as controls. Triplicates (with lines representing means ± SD) represent activity compared with samples not exposed to isoquercetin. (E) Prothrombin activation is not inhibited by isoquercetin. Thrombin activity was measured using a fluorescent cleavage substrate after combining factor Xa (10 μg/ml), FVa (7 μg/ml), and prothrombin (50 μg/ml) (Xa-Va-II). Thrombin activity was measured after combining factors Xa and Va without prothrombin addition as a control (Xa-Va). Studies were performed in the presence (black circles) and absence (white circles) of isoquercetin. Experiments were performed in triplicate as shown (with means ± SD) and data represent activity compared with samples not exposed to isoquercetin.