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.

Conflict of interest statement

J.I. Zwicker received research support from Quercegen Pharma.

Figures

Figure 1. Flow diagram of cohort enrollment and sample analysis.

Figure 2. Isoquercetin inhibits protein disulfide isomerase activity in plasma.

Levels of total plasma quercetin were determined at serial time points over 24 hours following oral administration to healthy adults of (A) 500 mg quercetin aglycone or (B) 500 mg isoquercetin. Quercetin levels were measured by HPLC after enzymatic hydrolysis. Quercetin analogs were formulated with ascorbic acid/niacin (n = 5, red) and without ascorbic acid/niacin (n = 5, blue). Error bars represent SEM. (C) Measurement of protein disulfide isomerase (PDI) inhibition using a plasma-based assay. The indicated concentrations of isoquercetin (red), quercetin-3-glucuronide (black), or quercetin-3-rutinoside (green) were incubated with recombinant PDI in human plasma and PDI activity measured using a di-eosin-GSSG–based assay. IQ, isoquercetin. Mean values of triplicates (± SEM) represent percentage of PDI activity. (D) Comparison of PDI inhibition and quercetin levels following administration of isoquercetin to healthy adults. Plasma was obtained from patients at the indicated times following ingestion of 1,000 mg isoquercetin. Inhibition of recombinant PDI by plasma (green, scale per right y axis) is presented as percentage PDI activity compared with plasma obtained prior to ingestion (time 0). Circles represent mean values of 10 patients with SEM shown as error bars. Quercetin concentration in plasma (blue, scale per left y axis) was determined using HPLC. (E) Box-and-whisker plot of peak PDI inhibition at peak quercetin concentrations above or below 4 μM. Top and bottom of box represent the interquartile range along with median values, whiskers represent 95th percentile, and outliers are shown. Mann-Whitney P = 0.002.

Figure 3. Thrombin generation is decreased in a protein disulfide isomerase–dependent manner following isoquercetin administration.

(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.

Figure 4. Inhibition of thrombin generation with isoquercetin in patients with elevated anti-phospholipid antibodies.

(A) Six subjects with anti-phospholipid antibodies received an oral dose of 1,000 mg isoquercetin. Platelet-dependent thrombin generation was assayed before (gray bars) and 4 hours following (white bars) ingestion of isoquercetin (1,000 mg). Bars represent the mean of triplicate values of individual subjects (dark circles) with error bars representing SD. (B) Box-and-whisker plot showing thrombin generation in healthy controls (n = 10) and anti-phospholipid antibody patients (n = 6) before (Baseline) and 4 hours after (4 hours) ingestion of 1,000 mg isoquercetin. Top and bottom of box represent the interquartile range along with median values, whiskers represent 95th percentiles, and outliers are shown. P values were determined using paired t tests.

Figure 5. Factor V is a substrate of protein disulfide isomerase (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.

Figure 6. Isoquercetin-mediated protein disulfide isomerase inhibition targets platelet factor Va generation.

(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.

Figure 7. Factor V activation following ingestion of isoquercetin.

(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.