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. Author manuscript; available in PMC: 2022 Jun 6.
Published in final edited form as: Clin Cancer Res. 2021 Aug 16;27(20):5708–5717. doi: 10.1158/1078-0432.CCR-21-1140

Circulating Protein Disulfide Isomerase is associated with increased risk of Thrombosis in JAK2-mutated Myeloproliferative Neoplasms

Anish V Sharda 1,2, Thomas Bogue 1, Alexandra Barr 2, Lourdes Mendez 1, Robert Flaumenhaft 2, Jeffrey I Zwicker 1,2
PMCID: PMC9170286  NIHMSID: NIHMS1810700  PMID: 34400417

Abstract

Purpose:

Thromboembolic events (TE) are the most common complications of myeloproliferative neoplasms (MPN). Clinical parameters including patient age and mutation-status are used to risk-stratify patients with MPN, but a true biomarker of TE risk is lacking. Protein disulfide isomerase (PDI), an endoplasmic reticulum protein vital for protein folding, also possesses essential extracellular functions, including regulation of thrombus formation. Pharmacologic PDI inhibition prevents thrombus formation, but whether pathologic increases in PDI increase TE risk remains unknown.

Experimental Design:

We evaluated the association of plasma PDI levels and risk of TE in a cohort of patients with MPN with established diagnosis of polycythemia vera (PV) or essential thrombocythemia (ET), compared to healthy controls. Plasma PDI was measured at enrollment and subjects followed prospectively for development of TE.

Results:

A subset of patients, primarily JAK2-mutated MPN, had significantly elevated plasma PDI levels as compared to controls. Plasma PDI was functionally active. There was no association between PDI levels and clinical parameters typically used to risk-stratify patients with MPN. The risk of TE was 8-fold greater in those with PDI levels above 2.5 ng/ml. Circulating endothelial cells from JAK2-mutated MPN patients, but not platelets, demonstrated augmented PDI release, suggesting endothelial activation as a source of increased plasma PDI in MPN.

Conclusions:

The observed association between plasma PDI levels and increased risk of TE in patients with JAK2-mutated MPN has both prognostic and therapeutic implications.

Introduction

Myeloproliferative neoplasms (MPNs) are disorders of the bone marrow characterized by excess clonal hematopoiesis resulting in elevated peripheral blood counts. Among seven distinct clinicopathologic entities defined by the 2016 World Health Organization classification system of tumors of the hematopoietic and lymphoid tissues, polycythemia vera (PV) and essential thrombocythemia (ET) are the most common (1,2). Acquired JAK2 mutations define PV, particularly V617F mutation in exon 14 of JAK2, whereas CALR and MPL mutations are specific to JAK2-unmutated ET (1).

The most common complication associated with PV or ET is an elevated risk of thromboembolic events (TEs) (3–6). The risk of TE is about five- to seven-fold elevated compared to the general population and can involve both arterial and venous circulations (7,8). TEs are associated with increased morbidity and mortality in the context of ET and PV. While the association of significantly elevated risk of TE in these disorders is well-recognized, the pathophysiology remains poorly defined. Multiple factors, including platelet hyperreactivity, monocytic and endothelial activation, and extracellular vesicles have been reported as potential contributors to TE in MPNs (9–11).

Many clinical parameters, such as patient age, blood cell counts, such as total white blood cell count, prior history of TE, and presence of JAK2 mutations, have been found to be associated with elevated risk of TE (8,12,13). Consequently, many risk-stratification models, such as the three-step International Prognostic Score for ET (IPSET) , which takes into consideration age, prior history of TE and mutation status, have been validated and commonly used to identify those at higher risk of TE (14,15). But a biomarker that can reliably identify patients with MPN with elevated risk of TE, particularly short-term risk of TE, is lacking.

Protein disulfide isomerase (PDI), an endoplasmic reticulum protein critical for protein folding and chaperoning functions, also plays essential roles in the extracellular milieu. We and others have established a critical role of PDI in the pathophysiology of thrombus formation (16–18). PDI, released by stimulated platelets and endothelial cells, regulates thrombus formation and the mechanisms by which PDI promotes thrombosis is an area of active research. In a phase 2 clinical study, we observed that inhibitors of PDI reduced thrombin generation and prevented venous thromboembolism, and larger clinical trials are underway in patients with cancer (19,20). Given the essential role of PDI in thrombosis, we hypothesized that pathologic elevations in circulating PDI are prothrombotic. Measurable plasma PDI has been previously described but has never been prospectively evaluated in any particular disease state, particularly those associated with elevated risk of TEs (21–23). Due to high occurrence of TE, as well as associated platelet and endothelial dysfunction, we chose to explore our hypothesis in MPN.

Material and Methods

Study Approval

The protocol was approved by the Institutional Review Board at Beth Israel Deaconess Medical Center. Studies were conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all study participants prior to inclusion in the study.

Study design and patients

The study was a prospective, standard-of-care, observational cohort study conducted between 2016 and 2020. Patients were eligible if they were 18 years of age or older and had a prior diagnosis of PV (with a positive JAK2 mutation) or ET (history of platelet count > 450 K/μl with either positive JAK2/CALR/MPL mutation or characteristic bone marrow biopsy). Controls were individuals 18 years of age or older, considered to be in good health, without significant cardiac disease, disorders of hemostasis or thrombosis, active cancer, liver disease (bilirubin > 2 mg/dl) or chronic kidney disease (creatinine > 2 mg/dl). Clinical and laboratory parameters were recorded for all study subjects and PDI levels estimated at enrollment. Subsequent sample collection was performed at routine 3–6 month follow-up visit and patients were followed prospectively for development of TE.

Reagents

Antibodies used were as follows: Mouse monoclonal anti-PDI (RL77) (Abcam Cat#ab5484, RRID:AB_304927); rabbit polyclonal anti-PDI (DL-11) (Sigma-Aldrich Cat# P7122, RRID:AB_477395); rabbit polyclonal anti-phospho-STAT3 (Cell Signaling Technology Cat# 9134, RRID:AB_331589); rabbit polyclonal anti-STAT3 (Cell Signaling Technology Cat# 9132, RRID:AB_331588); rabbit polyclonal anti-JAK2 (Cell Signaling Technology Cat# 3230, RRID:AB_2128522); mouse monoclonal anti-GAPDH antibodies (Cell Signaling Technology Cat# 97166, RRID:AB_2756824); and FITC-labeled mouse monoclonal anti-PECAM1 (WM59) (Bio-Rad Cat# MCA1738T, RRID:AB_1101905). Platelet factor 4 ELISA kit was obtained from R&D Technologies. (Cat# DPF40). HUVEC and endothelial growth medium were obtained from Lonza (Cat# C2519A and CC3156, respectively). All other reagents were obtained from Sigma.

ELISA

For PDI ELISA, 96-well ELISA plates were coated with rabbit anti-PDI (DL-11) at a concentration of 1 μg/ml for 1 hr at room temperature. Plates were then blocked with 3% BSA in PBS for 30 min at room temperature, followed by incubation with PDI standards or samples (plasma samples diluted 1/4 and 1/8 with PBS) overnight at 4°C. Biotinylated mouse anti-PDI RL77 was then used as a detection antibody at a concentration of 1 μg/ml for 1 hr at RT, followed by streptavidin-HRP at a concentration of 0.1 μg/ml for 45 min. Finally, platelets were incubated with TMB substrate for 15–30 min and absorbance estimated using a plate reader. PDI ELISA was highly specific to PDI and did not detect other vascular thiol isomerases (Fig. S1). vWF ELISA was carried out as previously described (24).

Di-eosin-GSSG reductase assay

A previously described assay(25) was modified to use plasma (1/10 final dilution) as sample. Plasma samples were assayed in duplicates with and without PDI inhibitor quercetin-3-rutinoside (10 uM final concentration) to estimate PDI-dependent reductase activity of the plasma. In addition, controls with recombinant PDI were assayed with and without quercetin-3-rutinoside. The reductase activity obtained from recombinant PDI was used to normalize plasma PDI-dependent reductase activity for each sample to obtain plasma PDI reductase activity in arbitrary units.

Platelet isolation and stimulation

Platelets were isolated from whole blood, washed and stimulated with 0.1 U/ml of thrombin using a previously published protocol (26). PDI antigen in platelet releasates was measured using PDI ELISA described above.

Cell culture

Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza and cultivated using previously published protocol (26). Mycoplasma testing was not performed. Blood out-growth endothelial cells (BOECs) were cultivated as previously described (27). Mycoplasma testing was not performed. Only passages 2–4 were used for experiments described. For estimation of basal PDI release, cells were incubated with serum-free media for 6 hours followed by estimation of PDI antigen in the media using PDI ELISA described above.

Lentiviral transduction

Lentiviral plasmid pCW107-V5-JAK2(V617F) was obtained from Addgene. pCW107-V5-JAK2(WT) plasmid was obtained by mutagenesis of pCW107-V5-JAK2(V617F) plasmid using Phusion site-directed mutagenesis kit employing manufacturer’s protocol. Lentiviral particles were generated and HUVEC transduced with lentiviral particles using as previously described (28).

Immunoblotting

Cell lysates were treated with IP lysis buffer, resolved using SDS-PAGE, transferred onto nitrocellulose membranes using Trans-Blot transfer system (Bio-Rad), blocked with 3% BSA and finally immunoblotted with respective antibodies.

Flowcytometry

BOECs were detached using accutase, washed with the endothelial basal media, incubated with FITC-labeled mouse anti-PECAM-1 antibody or isotype control in PBS for 15 minutes, and then PECAM-1 expression estimated using Beckman Coulter Gallios flow cytometer.

Digital PCR

Mutation testing in the BOECs was carried out using JAK2 V617F TaqMan dPCR liquid biopsy assay in the QuantStudio 3D Digital PCR System per the manufacturer’s protocol.

Statistical analysis

Based on our preliminary data on 5 controls (mean PDI 3.6 ng/ml and SD 2.4 ng/ml) and 15 patients (mean 14.57 ng/ml and SD 13.59 ng/ml), we estimated that a cohort of 25 control individuals and 50 MPN patients would provide greater than 0.95 power to reject the null hypothesis that the PDI levels are equal based on a two-sided alpha of 0.05 using a two-sample t-test with unequal variances. These subjects were included in the final analysis.

Unpaired t-test assuming unequal SD was used to compare mean plasma PDI of control and MPN cohort. Pearson correlation was used to estimate correlation coefficient between plasma PDI antigen and activity. Log Rank was used to compare time-to-event thrombosis rates. Statistical significance was defined as p value of < 0.05.

Results

JAK2-mutated MPN patients have high circulating levels of PDI

The baseline characteristics of the study population are shown in Table 1. A total of 65 patients with MPN and 27 controls were enrolled. Among the MPN cohort, 28 had PV and 37 ET; 55 patients with JAK2 V617F mutation, 8 with CALR mutation and 2 mutation-negative (‘triple-negative’ MPN). 44 (68%) patients with MPN had high-risk disease defined as IPSET score > 3. A total of 24 (37%) patients were receiving hydroxyurea, and 53 (81%) receiving aspirin. No patients were receiving ruxolitinib. There were 17 (29%) patients with MPN with a history of thrombosis and 9 (14%) were taking therapeutic anticoagulation. By comparison, 3 (11%) control subjects had a history of thrombosis, with 9 (33%) on aspirin, but none on therapeutic anticoagulation.

Table 1.

Clinical Characteristics of the MPN and control cohorts

Characteristic Controls (27) MPN (65)
Age median (range) 64.5 (21–84) 66 (29–93)
Gender female (%) 16 (59) 31 (48)
MPN type - 28 PV
37 ET
Mutation type - 55 JAK2
8 CALR
2 ‘triple negative’
High-risk MPN (IPSET score > 3) 44 (68)
Cardiovascular Disease (%) 2 (7.4) 10 (15)
History of Cancer (%) 5 (18.5) 8 (12)
History of thrombosis (%) 3 (11.1) 19 (29)
Aspirin (%) 9 (33.3) 53 (81)
Hydroxyurea (%) - 24 (37)
Anticoagulation (%) - 9 (14)
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Plasma PDI antigen was measured for all study subjects at time of study enrollment. Plasma PDI antigen was normally distributed in the control cohort (Pearson normality test p = 0.33) with mean PDI level 2.1 ng/ml, median 2.1 ng/ml, range 1.1–3.7 ng/ml and SD 0.68 ng/ml (±2SD 1.9–2.4 ng/ml) (Fig. 1A). The mean plasma PDI antigen in the MPN cohort was 3.3 ng/ml and was significantly higher than the controls (2.1 ng/ml versus 3.3, p=0.01). The subgroup of patients with elevated plasma PDI antigen were primarily JAK2-mutated MPN (Fig. 1B). When compared to controls, the JAK2-mutated patients had significantly higher plasma PDI antigen than control (2.1 ng/ml versus 3.1 ng/ml, p = 0.03).

Figure 1. Plasma PDI is detectable and elevated in JAK2-mutated MPN. A and B).

Figure 1.

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Scatter plots showing baseline plasma PDI levels in control cohort (n=27) and MPN cohort (n=65) or JAK2-mutated (n=55) and non-JAK2-mutated (n=10) MPN patients, respectively. Bars in (A) show mean and ± 2SD; * p = 0.019. C) Comparison of plasma PDI antigen versus plasma PDI activity measured in 6 MPN patients with the highest plasma PDI antigen levels and 6 patients with low plasma PDI antigen levels (R2 = 0.66; ** p = 0.001) D) Scatter plot showing fold-change in PDI levels obtained during follow up (median follow up 3 months) in control (n=8) and MPN cohorts (n=41). (Bars show mean and ± 2 SD; p = 0.3)

Excluding one outlier (a ‘triple-negative’ patient with plasma PDI 19.37 ng/dl), patients with non-JAK2 mutated MPN had mean and median plasma PDI of 2.2 ng/dl and 2 ng/dl, respectively (Fig. 1B).

In order to confirm that circulating PDI antigen was functionally active, we measured plasma PDI-dependent reductase activity. Utilizing a fluorescence-based probe, di-eosin-GSSG, the PDI-dependent liberation of eosin moieties results in a detectable increase in fluorescence (18). We compared PDI activity from patients with the highest plasma PDI levels (N=6) with those with lower PDI levels (N=6). As shown in Figure 1C, PDI antigen was active and correlated with plasma PDI-dependent reductase activity (R2 =0.66, p = 0.001).

In order to assess whether PDI levels fluctuate over time, we measured serial PDI levels in 41 patients and 8 control subjects. PDI levels did not significantly change over time with a median follow-up period of 3 months (Fig. 1D). The mean fold-change in PDI levels in MPN group (0.2, 95% CI, −0.11 to 0.52) was not significantly different from that of the control group (−0.18, 95% CI, −1.13 to 0.75) (p = 0.32).

Plasma PDI levels and correlation with demographic or laboratory parameters

We determined whether plasma PDI levels in patients with MPN were associated with clinical, laboratory, and thrombosis risk model parameters. Plasma PDI levels did not appear to be influenced by sex or older age (above or below 60 years) (Fig. 2A–B). Plasma PDI levels were similar among patients with low/intermediate IPSET scores compared with high-risk scores (Fig. 2C) (14). Additionally, plasma PDI antigen were not significantly different in patients on aspirin versus those not on aspirin (3.2 ng/ml vs. 3.7 ng/ml; p = 0.6)

Figure 2. Lack of association between plasma PDI antigen and patient clinical characteristics or laboratory parameters.

Figure 2.

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. Scatter plots illustrating plasma PDI antigen in MPN patients according to gender (A), Age (B), High or Low/Intermediate IPSET score (C), and prior history of thromboembolism (D). Comparison of plasma PDI antigen and white blood cell count (WBC) (E), hematocrit (F), platelet count (G), and plasma platelet factor 4 (PF4) levels (H). R2 = 0 for all comparisons.

Similarly, plasma PDI levels were not significantly different among those with or without a prior history of TE. (Fig. 2D). Neither white blood cell count, hematocrit nor platelet counts correlated with plasma PDI antigen, arguing against PDI representing a surrogate marker of blood count parameters (R2 =0) (Fig. 2E–G). To evaluate the possibility that plasma PDI reflected platelet activation (either in vivo or ex vivo), we compared PDI levels with plasma platelet factor 4 (PF4) antigen, a platelet-specific protein that is commonly used as a marker of platelet activation (29). Plasma PDI antigen had poor correlation with plasma PF4 antigen in MPN cohort (R2 =0) (Fig. 2H).

High plasma PDI levels predict risk of TE in MPN

We monitored patients following enrollment for the development of venous or arterial thrombotic events. In median follow up of 11 months (range 1 to 43 months), there were 8 TEs, with a cumulative incidence of 11.4% per year. Four TEs were arterial (3 cerebrovascular accidents and 1 peripheral arterial thrombosis) and 4 venous (2 deep vein thrombosis, 1 pulmonary embolism and 1 portal vein thrombosis). Mean plasma PDI among patients who developed TE was 5.4 ng/ml, median 2.8 ng/ml (Table S1). In order to establish a PDI threshold that was most predictive of TE, we performed a receiver operating characteristic (ROC) analysis and identified 2.5 ng/ml as predictive of TE with a sensitivity of 65% and specificity of 88% for TE (Fig. S2). Alternatively, two standard deviations from the mean plasma PDI antigen in control cohort similarly resulted in a 2.5 ng/ml PDI cutoff. Applying the 2.5 ng/ml threshold to identify high versus low risk MPN, the cumulative incidence of TE at 1 year for those patients with lower PDI levels was 5.5% compared with 26.6% in those with higher PDI levels, corresponding with an 8-fold increased risk of TE (HR 8.02, 95% CI, 1.99–32.16; Log rank p = 0.01) (Fig. 3A). Specifically, in JAK2 V617F-mutated MPN, high plasma PDI levels were associated with a 7-fold increased risk of TE (n = 55; HR 7.3, 95% CI, 1.8–29.5; Log rank p = 0.02). None of the 9 patients on therapeutic anticoagulation at baseline (3 with low and 6 with high plasma PDI levels), experienced a TE during study follow up. Excluding these patients from the analysis, the risk of TE in patients with high PDI levels (≥2.5 ng/ml) was nearly 10-fold higher (HR 9.9, 95% CI, 2.42–40.4; Log rank p = 0.005).

Time-to-event analyses were also performed to compare the risk of TE using traditional risk factors, such as white blood cell (WBC) count (>15,000/μl vs. <15,000/μl), hematocrit (>45% vs. < 45%) and platelet count (> 600/μl vs. < 600/μl) (8,13). Specifically, all thrombotic events occurred in patients with WBC below the 15,000/μl threshold and none of the blood count parameters were predictive of a higher TE risk (Fig. S3A-C). Survival curves were also generated to compare a high IPSET score (>3) to low/intermediate IPSET score as a predictor of TE. Patients with a high IPSET score were at approximately 4-fold higher risk of TE than those with low scores (HR 3.45, 95% CI, 0.79–15.05), but this was not statistically significant in our cohort (p = 0.36). Adding plasma PDI antigen to patients with high IPSET score improved its predictability of TE risk (HR 6.89, 95% CI, 1.56–30.44, p = 0.03) but was less discriminating than simply applying PDI levels alone (Fig. 3B). We also measured D-dimer in the plasma samples of the MPN cohort. Patients with high PDI levels had significantly higher median D-dimer concentrations compared to patients with lower PDI levels (265 ng/ml versus 176 ng/ml, Mann-Whitney rank sum p = 0.036). These data support potential contribution of PDI in TE in MPN.

Figure 3. High plasma PDI antigen confers increased risk of thromboembolic events in MPN. A).

Figure 3.

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Kaplan Meier curves showing elevated incidence of thromboembolic events in MPN patients with high plasma PDI (>2.5 ng/ml) as compared to low plasma PDI (< 2.5 ng/ml) (HR 8 (1.99–32.16); p = 0.01) B) Kaplan Meier curve showing incidence of thrombosis stratified on IPSET score (HR 3.45, 95% CI 0.79–15.05; p = 0.36). Addition of plasma PDI level (above or below 2.5 ng/ml) to IPSET score increased the predictive specificity for thromboembolic events (HR 6.89 (1.56–30.44); p = 0.03).

Platelet and endothelial sources of PDI in MPN

Endothelium and platelets are the principal sources of vascular thiol isomerases, including PDI (30). Since platelet hyperreactivity has been reported in MPN, we first evaluated whether platelets could be the dominant source of elevated plasma PDI levels observed in JAK2-mutated MPN. Platelets from three patients with JAK2 V617F mutation on treatment with aspirin and high plasma PDI (plasma PDI levels of 3.09, 4.47 and 4.77 ng/ml) were isolated and stimulated with 0.5 U/ml thrombin. Platelet-derived PDI was measured in the releasates using ELISA. Platelet-derived PDI levels in releasates of three control subjects (plasma PDI levels of 1.64, 1.72 and 1.66 ng/ml) was also measured. There was no difference between thrombin-stimulated platelet PDI release in JAK2- mutated MPN versus controls (p = 0.7) (Fig. 4A).

Figure 4. Elevated endothelial basal PDI release in JAK2-mutated MPN.

Figure 4.

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. A) Comparison of PDI antigen measured in releasates and lysates of platelets collected from JAK2-mutated MPN patients and controls. Washed platelets were stimulated with thrombin and buffer collected as releasates. Pelleted platelets then lysed with lysis buffer. PDI antigen determined in releasates and lysates using ELISA (p = 0.7; n=3) B) Light micrograph showing characteristic endothelial cobblestone-like morphology of blood outgrowth endothelial cells (BOECs) C) BOECs were detached, washed, and then incubated with FITC-labeled anti-PECAM-1 antibody. PECAM-1 expression was then determined by flowcytometry (green, isotype control; blue, control BOEC; red, patient BOEC) D) BOEC genomic DNA was extracted using Qiagen DNeasy blood and tissue kit and digital PCR carried out using JAK2 V617F TaqMan dPCR liquid biopsy assay in the QuantStudio 3D Digital PCR System. Analysis was carried out using QuantStudio 3D Analysis Suite Cloud Software (mutation copies 1 ng/μl ± 95% CI) E) BOEC lysates were resolved by SDS-PAGE and immunoblotted with anti-phospho-STAT3, anti-STAT3 and anti-GAPDH antibodies F) Cells were grown to confluency and incubated with serum free media for 6 hours. Media was then collected and vWF Ag determined using vWF ELISA (n=2; * p = 0.04) G) Comparison of PDI antigen in releasates and lysates BOECs from JAK2-mutated MPN patients compared to controls. Cells were grown to confluency and incubated with serum free media for 6 hours. Media was then collected as releasates and cells treated with lysis buffer to prepare lysates. PDI antigen determined in releasates and lysates using ELISA (n=2; * p = 0.04)

The endothelium is thought to contribute to vascular inflammation associated with MPN and the JAK2 mutation has been implicated in endothelial dysfunction (10,31,32). We isolated blood outgrowth endothelial cells (BOECs) from two patients with JAK2-mutated MPN with high PDI levels (3.09 and 4.77 ng/ml) and one control (1.64 ng/ml). BOECs had a characteristic cobblestone-like morphology of endothelial cells and expressed endothelial-specific antigen PECAM-1 (Fig. 4B–C) (27). Digital PCR confirmed presence of JAK2 V617F mutation in BOECs from both patients (Fig. 4D). Immunoblot analysis of MPN BOECs demonstrated increased levels of phospho-STAT3, indicating augmented JAK-STAT signaling (Fig. 4E). In addition, basal release of vWF antigen was also elevated in MPN BOECs, indicating increased Weibel-Palade body release, as has previously been observed in MPN with the JAK2 V617F mutation (Fig. 4F).(32) Unlike vWF, however, endothelium is thought to store PDI in a granule distinct from Weibel-Palade bodies.(33) We therefore evaluated PDI release from (BOECs) from the two patients with JAK2-mutated MPN. Basal PDI release from MPN BOECs was 48±9.4% (p = 0.035) higher than the control BOECs (Fig. 4G). PDI antigen levels in BOEC lysates were similar between the two groups (Figure 4G).

To confirm increased basal release of PDI from endothelial cells, we expressed WT and mutant JAK2 in human umbilical vein endothelial cells (HUVECs) using lentiviral transduction. The transduced HUVECs expressing mutant JAK2 V617F had higher expression of JAK2 as compared to WT JAK2 (Fig. 5A) (32). Moreover, transduction was associated with JAK-STAT pathway activation in the resting state, as evidenced by STAT3 phosphorylation (Fig. 5B). Increased basal release of vWF Ag was also observed in the transfected HUVECs (Fig. 5C). Similar to the patient derived BOECs, JAK2 V617F expressing HUVECs had significantly elevated basal release of PDI (112%±26%; p = 0.002) as compared to WT HUVECs, but similar PDI content (Figure 5D). Treatment of these cells with 100 nM JAK1/2 inhibitor ruxolitinib (IC50 for peripheral blood mononuclear cells 60–130 nM(34,35) and plasma Cmax in adults following 15 mg twice daily administration is >600 nM(36)), significantly reduced PDI release from JAK2 V617F expressing HUVECs without affecting WT cells (p = 0.035) (Figure 5E).

Figure 5. Elevated PDI release from HUVEC expressing JAK2 V617F; effect of pharmacologic inhibition of JAK2.

Figure 5.

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A) JAK2 V617F and WT HUVEC lysates were subjected to SDS-PAGE and immunoblotted with anti-JAK2 and anti-GAPDH antibodies B) JAK2 V617F and WT HUVEC lysates were subjected to SDS-PAGE and immunoblotted with phospho-STAT3 and anti-STAT3 antibodies. C) Cells were grown to confluency and incubated with serum free media for 6 hours. Media was then collected and vWF Ag determined using vWF ELISA (n=3; ** p = 0.005) D) Cells were grown to confluency and incubated with serum free media for 6 hours. Media was then collected as releasates and cells treated with lysis buffer to prepare lysates. PDI antigen determined in releasates and lysates using PDI ELISA (n = 3; ** p = 0.002) E) Comparison of PDI antigen in releasates of HUVECs expressing JAK2 V617F mutation, as in (D), with and without 100 nM ruxolitinib, compared to HUVECs expressing WT JAK2 (n = 3; * p = 0.035)

Discussion

Our data show that plasma PDI is detectable in patients with myeloproliferative neoplasms and a subgroup of patients with MPN, which consists primarily of those harboring the JAK2 V617F mutation, have pathologic elevations in plasma PDI that predict a higher risk of developing thromboembolic events. Moreover, we show that circulating PDI antigen possesses PDI enzyme activity and remains stable for most patients during follow up. PDI antigen represents a novel, independent biomarker of thrombosis that does not correlate with standard clinical or laboratory parameters commonly utilized to stratify risk of thrombosis in MPN.

Detectable plasma PDI has been previously reported in humans. Oliveira et al., in a cohort of 35 healthy volunteers, demonstrated that plasma PDI antigen and activity was detectable, although at lower concentrations (mean ~ 540 pg/ml) than in our study (23). This difference may be secondary to the differences in the assay and antibodies used. In our hands, the commercial ELISA described above did not reliably detect recombinant PDI protein. Prior to this, Essex et al. detected plasma PDI in the 250–1000 ng/ml range in a small study of volunteers, but details relating to the sample size, subjects or assay used are not available (21). PDI has also been previously reported to be present in the plasma proteome quantified using modified aptamers, but was not found to be associated with cardiovascular disease (22).

Clinical parameters, such as age, history of thrombosis, leukocytosis, mutation-status and traditional cardiovascular risk factors are currently in use to classify MPN into low- or high-risk groups for thrombosis, and also to guide cytoreductive treatment (8,12–15). Risk-stratification models, such the IPSET score, have been devised that incorporate these variables to identify patients with essential thrombocythemia at higher risk of TE, but a specific biomarker that can reliably predict risk of TE in MPN is lacking. Moreover, these clinical scoring systems have been developed and validated using retrospective data (12,14,37). Markers of hypercoagulability (such as thrombin-antithrombin complexes, prothrombin fragment 1+2, D-dimer), markers of endothelial activation (such as von Willebrand factor, E-selectin), and elevated levels of microparticles have been described in MPN; however, the role of these biomarkers in identifying patients at increased risk of thrombosis is unclear (38–40). Finding of pathologic elevations of plasma PDI particularly in patients with JAK2 V617F-mutated MPN and demonstration of its predictability to identify those at higher risk for TEs prospectively is, therefore, a major finding of this study. Furthermore, addition of plasma PDI levels to high IPSET score improved the latter’s specificity to predict TE. JAK2 mutation is known to confer a higher risk of thrombosis in MPN over CALR and MPL mutations. CALR mutations have not been previously reported in the endothelial cells, which may be one factor. In addition, absence of a more generalized JAK/STAT stimulation in CALR mutated-MPN may account for its lower risk of thrombosis, as mutant calreticulin functions exclusively through MPL, the thrombopoietin receptor (41).

Our ex vivo and in vitro results implicate endothelial cells as the source of pathologic elevations of PDI in MPN. The endothelium has been previously implicated in vascular inflammation associated with MPN, and endothelium-specific JAK2-mutated murine models revealed significant endothelial dysfunction associated with abnormalities in coagulation (10,31,32). Our data show that endothelial exocytosis of PDI is elevated from blood outgrowth endothelial cells derived from patients with JAK2-mutated MPN with high plasma PDI levels and is increased in primary endothelial cells harboring JAK2 V617F mutation. Blood outgrowth endothelial cells are a rare population of circulating mononuclear cells that possess endothelial markers and features, and have been widely used in vascular biology, particularly in evaluation of disorders of hemostasis and thrombosis (42–47). Blood outgrowth endothelial cells from patients with JAK2-mutated MPN demonstrate augmented endothelial secretion including that of inflammatory cytokines (10). Elevated plasma PDI may therefore reflect endothelial activation known to occur in MPN thereby contributing to its prothrombotic phenotype. The premise that increased PDI release is associated with the JAK2 V617F mutation is supported by the observation that primary endothelial cells from patients with JAK2 V617F mutation, and cultured endothelial cells transduced with JAK2 V617F mutation, also demonstrate increased PDI release. In contrast, our studies indicate that the platelets, a major source of vascular thiol isomerases, do not release excessive PDI in patients with MPN patients and high circulating PDI levels. While aspirin is not known to affect thrombin-mediated platelet activation, considering the studies we performed were in patients with MPN receiving aspirin, we cannot exclude the possibility that excess PDI is released from platelets in patients with MPN not taking aspirin (48).

Although circulating PDI has never previously been linked to a prothrombotic state in humans, the critical role that PDI serves in pathophysiology of thrombosis is well-recognized (16,30). Stimulation of endothelial cells results in PDI release, which precedes platelet accumulation and fibrin generation at the site of vessel wall injury. The exact molecular mechanism by which PDI regulates thrombosis is not known, but various downstream effectors have been reported, such as αIIbβ3, GPIbα, tissue factor, vitronectin and platelet factor V (17,18). Mouse models of platelet-specific knockdown or conditional expression of PDI that lacks oxidoreductase activity demonstrate impaired thrombus formation (49,50). Inhibition of PDI with antibodies or specific inhibitors also prevents thrombus formation (20,25). As such, PDI inhibitors are being evaluated in clinical trials as novel anticoagulants with phase 2 data demonstrating efficacy in reducing thrombin generation and preventing venous thromboembolism in patients with cancer (19).

The limitations of our study include its relatively small sample size. Yet unlike many previous studies evaluating the risk of thrombosis in MPN, patients were followed prospectively for the development of TE (12,14,51,52). External validation using larger sample repositories will be required before consideration for clinical applications. We show that PDI levels stay stable over 3–6 month follow up, but we do not know whether PDI levels change more significantly over time as may occur with disease progression. This study was also underpowered to determine any statistical differences between JAK2 and non-JAK2-mutated MPN, specifically CALR mutation. This study was also underpowered to determine the effect of hematocrit, which has been previously shown in clinical trials to predict thrombosis (53). We also did not explore other prothrombotic disease states such as cancer and thus cannot conclude whether PDI elevations are unique to MPN. While PDI plays a critical role in regulating thrombus formation in vivo (16), PDI elevation may represent a state of endothelial activation associated with MPN, and additional studies, including animal models of thrombosis, will be required to more definitively establish a causal relationship between increased circulating PDI and MPN-associated hypercoagulability. Since pathologic elevations in circulating PDI are predictive of thrombosis in MPN, pharmacologic inhibition of JAK-STAT pathway to decrease PDI release, or direct therapeutic targeting of PDI by reduction of its extracellular oxidoreductase activity would be attractive approaches towards preventing thrombosis, specifically in this high-risk population.

Supplementary Material

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Translational Relevance.

While many scoring systems, which incorporate various clinical and laboratory parameters to identify patients with myeloproliferative neoplasms (MPN) at elevated risk of thromboembolic events are in clinical use, a biomarker that can identify patients with elevated risk of thromboembolic complications, particularly short-term risk, is lacking. Protein disulfide isomerase (PDI) plays a regulatory role in thrombosis and its inhibitors have been shown to prevent thromboembolic events in patients with cancer. Pathologic elevations in circulating protein PDI in a subset of patients with MPN and its association with an elevated risk of thromboembolic complications, therefore, opens an entirely new area of investigation for prognostication and prevention of patients with MPN.

Acknowledgements

This study was supported by a sponsored research grant from the Incyte Corporation (J. Zwicker), and 1U01HL143365–01 (J. Zwicker, R. Flaumenhaft) and R35 HL135775 (R. Flaumenhaft) from NHLBI.

Footnotes

Conflict of Interest: JZ reports research funding from Quercegen; consultancy: Sanofi, CSL, Parexel; honoraria/advisory boards: Pfizer/BMS, Portola, Daiichi. RF has a private equity interest in PlateletDiagnostics, and he is also a founder and consultant for that company. The interests of RF are reviewed and managed by the Beth Israel Deaconess Medical Center Office of Compliance and Business Conduct. RF is on advisory boards for Moderna, Quercegen, and Functional Therapeutics.

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Supplementary Materials

1
NIHMS1810700-supplement-1.pdf (123.6KB, pdf)
2
NIHMS1810700-supplement-2.pdf (101.4KB, pdf)
3
NIHMS1810700-supplement-3.pdf (111.2KB, pdf)
4
NIHMS1810700-supplement-4.pdf (94KB, pdf)

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