Extracellular Protein Disulfide Isomerase Research Articles

Piericones A and B as Potent Antithrombotics: Nanomolar Noncompetitive Protein Disulfide Isomerase Inhibitors with an Unexpected Chemical Architecture

Extracellular protein disulfide isomerase (PDI) is a promising target for thrombotic-related diseases. Four potent PDI inhibitors with unprecedented chemical architectures, piericones A-D (1-4), were isolated from Pieris japonica. Their structures were elucidated by spectroscopic data analysis, chemical methods, quantum 13C nuclear magnetic resonance DP4+ and electronic circular dichroism calculations, and single-crystal X-ray diffraction analysis. Piericones A (1) and B (2) were nanomolar noncompetitive PDI inhibitors possessing an unprecedented 3,6,10,15-tetraoxatetracyclo[7.6.0.04,9.01,12]pentadecane motif with nine contiguous stereogenic centers. Their biosynthetic pathways were proposed to include a key intermolecular aldol reaction and an intramolecular 1,2-migration reaction. Piericone A (1) significantly inhibited in vitro platelet aggregation and fibrin formation and in vivo thrombus formation via the inhibition of extracellular PDI without increasing the bleeding risk. The molecular docking and dynamics simulation of 1 and 2 provided a novel structure basis to develop PDI inhibitors as potent antithrombotics.

Effects of Isoquercetin on Plasma Protein Disulfide Isomerase Activity and Extracellular Vesicle Tissue Factor Antigen/Activity in Sickle Cell Disease

Introduction: Sickle cell disease (SCD) results from a single mutation in the β-globin gene that causes hemoglobin polymerization, red cell sickling, vascular injury, and recurrent vaso-occlusive crises (VOC). Vascular protein disulfide isomerase (PDI) is shown to be critical for thrombus formation by facilitating neutrophil recruitment, platelet activation and vascular fibrin deposition, all processes implicated in sickle vasculopathy. Since plasma PDI is elevated in SCD and cell-surface associated PDI could promote tissue factor (TF) dependent thrombin generation, we investigated the effects of Isoquercetin (IQ) in patients with steady state SCD. Methods: RBC surface associated PDI was quantified by western blotting. Inhibitory effect of IQ on LPS-induced TF expression on sickle monocytes in vitro was determined using flow cytometry. We also measured in vivo the effect of exposure of IQ or placebo for at least 28 days on plasma PDI protein, PDI reductase activity, total plasma extracellular vesicles (EVs), total plasma TF+ EVs, EV-TF-dependent procoagulant activity and markers of coagulation (D-dimer and Thrombin-antithrombin Complexes) using previously published methods. We used plasma from steady state SCD patients participating in a randomized controlled trial (NCT04514510) obtained at baseline and 28 days after exposure to either Isoquercetin 1000mg once daily (n=22) or placebo (n=22). The patients were in steady state, at least 60 days remote from VOC or blood transfusion and on a stable dose of hydroxyurea. The Wilcoxon rank-sum test was used to perform significance testing for differences in the percent change in outcomes between the Isoquercetin and placebo groups. Results: Sickle RBCs had higher cell-surface associated PDI compared to ethnic matched healthy controls (fold change 5.09; p=0.028). In confirmatory experiments with RBC ghosts, extracellular PDI was cell-surface associated. Pretreatment with Quercetin (the active metabolite of Isoquercetin) reduced LPS-induced cell-surface TF expression on sickle monocytes in vitro in a dose dependent manner (Fig 1). Following Isoquercetin administration, there was no change in the percent reduction in PDI inhibitory activity in the IQ group (mean: 6.6, 95%CI [-21.6, 34.9]) compared to placebo group (mean; -31, 95%CI [-94, 32.6], p=0.63). The percent change from baseline in PDI antigen also did not differ between the IQ or placebo groups (IQ mean: 1.2, 95%CI [-11.3, 13.6]; placebo mean: 12, 95%CI [-6, 30.4], p=0.52) (Fig 1). There was no correlation between the percent change in PDI activity and antigen from baseline after treatment with IQ (Spearman's Rho, IQ: r2 =0.28; p=0.20 placebo: r2=0.10; p=0.67). Unexpectedly, total plasma EVs showed no difference between the IQ (mean: 38.4; 95%CI [-4.6, 81.5]) and the placebo group (mean: 9, 95%CI [-18, 37.3]; p=0.27) (Table 1) suggesting that IQ has no obvious anti-inflammatory effects. Moreover, plasma EVs were predominantly derived from RBCs and platelets as published previously by us and others, indicating no membrane stabilizing effect on sickle RBCs and platelets. Inhibiting PDI reductase activity could allosterically reduce TF procoagulant activity, but this hypothesis was untestable because the plasma EV-TF dependent procoagulant activity in this patient population was undetectable, possibly reflecting effective disease control by hydroxyurea. Conclusions: The anti-inflammatory effects of flavonoid quercetin in vitro do not appear to translate into measurable clinical benefits in vivo following short term treatment with IQ in steady state SCD. Trials of higher dose IQ and/or prolonged exposure duration could yield greater inhibition of plasma PDI activity that might reveal detectable clinical antithrombotic effects. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of Antithrombotic Natural Products Targeting the Major Substrate Binding Pocket of Protein Disulfide Isomerase.

Protein disulfide isomerase (PDI) is a vital oxidoreductase. Extracellular PDI promotes thrombus formation but does not affect physiological blood hemostasis. Inhibition of extracellular PDI has been demonstrated as a promising strategy for antithrombotic treatment. Herein, we focused on the major substrate binding site, a unique pocket in the PDI b' domain, and identified four natural products binding to PDI by combining virtual screening with tryptophan fluorescence-based assays against a customized natural product library. These hits all directly bound to the PDI-b' domain and inhibited the reductase activity of PDI. Among them, galangin showed the most prominent potency (5.9 μM) against PDI and as a broad-spectrum inhibitor for vascular thiol isomerases. In vivo studies manifested that galangin delayed the time of blood vessel occlusion in an electricity-induced mouse thrombosis model. Molecular docking and dynamics simulation further revealed that the hydroxyl-substituted benzopyrone moiety of galangin deeply inserted into the interface between the PDI-b' substrate-binding pocket and the a' domain. Together, these findings provide a potential antithrombotic drug candidate and demonstrate that the PDI b' domain is a critical domain for inhibitor development. Besides, we also report an innovative high-throughput screening method for the rapid discovery of PDI b' targeted inhibitors.

Inhibitory effects of Syzygium jambos extract on biomarkers of endothelial cell activation

BackgroundDisordered endothelial cell activation plays an important role in the pathophysiology of atherosclerosis, cancer, sepsis, viral infections, and inflammatory responses. There is interest in developing novel therapeutics to regulate endothelial cell function in atherothrombotic, metabolic, vascular, and hematological diseases. Extracts from leaves of the Syzygium jambos (L.) Alston (S. jambos) trees have been proposed to treat cardiovascular diseases and diabetes through unclear mechanisms. We investigated the effects of the S. jambos extract on biomarkers of endothelial dysfunction and immune responses in the human endothelial cell line, EA.hy926.MethodsLeaves of S. jambos were collected, concocted and lyophilized. To study the effects of S. jambos on endothelial cell activation, we used the human endothelial cell line. IL-6 levels were measured using qPCR and ELISA. PDI activity was measured using Insulin Turbidity and Di-E-GSSG assays. CM-H2DCFDA was used to study ROS levels. Migration assay was used to study S. jambos effect on ex vivo human polymorphonuclear and human mononuclear cells.ResultsOur results show that incubation of EA.hy926 cells with ET-1 led to a 6.5 ± 1.6 fold increase in IL-6 expression by qPCR, an event that was blocked by S. jambos. Also, we observed that ET-1 increased extracellular protein disulfide isomerase (PDI) activity that was likewise dose-dependently blocked by S. jambos (IC50 = 14 μg/mL). Consistent with these observations, ET-1 stimulated ex vivo human polymorphonuclear and mononuclear cell migration that also was dose-dependently blocked by S. jambos. In addition, ET-1 stimulation led to significant increases in ROS production that were sensitive to S. jambos.ConclusionOur results suggest that the S. jambos extract represents a novel cardiovascular protective pharmacological approach to regulate endothelial cell activation, IL-6 expression, and immune-cell responses.

Inhibition of Protein Disulfide Isomerase Ameliorates Vaso-Occlusion and Thrombosis in a Mouse Model of Sickle Cell Disease

IntroductionSickle cell disease (SCD) results from a single mutation in the β-globin gene. Altered erythrocyte physiology in SCD results in severe vascular complications, including vaso-occlusion (VOC) and painful crises along with various other manifestations, such as thrombosis and end organ damage. Extracellular protein disulfide isomerase (PDI) plays a critical role in the regulation of neutrophil recruitment, platelet activation and fibrin formation, processes implicated in the thrombo-inflammatory vasculopathy in SCD. We investigated the effect of a PDI inhibitor, isoquercetin, in combination with hydroxyurea, in TNFα-induced VOC and thrombosis in a mouse model of SCD. In addition, the effect of chronic PDI inhibition by isoquercetin on thrombo-inflammatory vasculopathy was also examined.MethodsHumanized TOWNES knock-in mice, containing human normal (βA/βA, control) or sickle (βS/βS, SCD) hemoglobin β chains, were used as a mouse model of SCD. PDI inhibition in the plasma was achieved by oral gavage or daily diet feeding of isoquercetin. Intravital microscopy was performed to determine vaso-occlusive events in the venules and thrombus formation in the arterioles in TNFα-stimulated mice. Plasma levels of PDI and parameters for coagulation and vascular inflammation were determined by ELISA. Neutrophil accumulation in the organs was determined as the myeloperoxidase (MPO) levels in tissue homogenates.ResultsCompared to control mice, SCD mice have elevated plasma PDI levels. Gavage of isoquercetin resulted in reduced PDI activity in the plasma of treated mice (38±10% inhibition, p <0.05). Isoquercetin ingestion increased leukocyte rolling flux fraction in TNFα-treated SCD mice as monitored using intravital microscopy, whereas the influence on leukocyte adhesion and shear rate was minimal. When a reversal agent for PDI inhibitor, b'x (the isolated substrate binding domain of PDI) was co-infused, the effect of isoquercetin on leukocyte rolling was reversed, suggesting this effect of isoquercetin is mediated by PDI inhibition. We also determined the effect of isoquercetin in combination with hydroxyurea, a treatment with established efficacy in SCD, on the vaso-occlusive events in our model. We confirmed that hydroxyurea treatment elevated the plasma nitric oxide level as determined by plasma nitrite/nitrate content (11.5±1.9 μM vs 5.0±1.9 μM, p <0.05). Consistent with previous studies, hydroxyurea decreased leukocyte adhesion while the rolling flux fraction was not changed. However, combined treatment with isoquercetin and hydroxyurea in SCD mice increased the rolling flux fraction (15±3% vs 6±1%, p <0.05), decreased leukocyte adhesion (158±13 mm-2 vs 289±24 mm-2, p <0.05), and increased vessel shear rate (302±18 s-1 vs 122±11 s-1, p <0.05). We investigated in vivo thrombus formation in the arterioles of TNFα-treated SCD mice using intravital microscopy following laser injury. Compared to the control mice, SCD mice have significantly enhanced fibrin generation but normal platelet accumulation. Both isoquercetin and hydroxyurea inhibited TNFα-induced platelet accumulation (62±36% AUC-Area Under the Curve with isoquercetin and 48±17% AUC with hydroxyurea of untreated SCD) and fibrin generation (30±6% AUC with isoquercetin and 44±9% AUC with hydroxyurea of untreated SCD). To study the effect of chronic isoquercetin treatment in SCD, control and SCD mice were fed isoquercetin for 4 weeks. Daily consumption of isoquercetin resulted in reduction of plasma PDI activity (18±5% inhibition, p <0.05). Consistent with the intravital microscopic data, chronic PDI inhibition reduced neutrophil migration and accumulation into the lung, as determined by the MPO level (481±85 ng/mg vs 829±140 ng/mg, p <0.05) in the tissue homogenate. Chronic treatment with isoquercetin also reduced plasma fibrinogen degradation products (FDP) (102±15 μg/ml vs 183±25 μg/ml, p <0.05) in SCD mice. In addition, a correlation (Pearson r=0.651) was observed between reducing plasma FDP and lung MPO levels.ConclusionsAcute and chronic PDI inhibition by isoquercetin may provide beneficial effect on vascular complications in a mouse model of SCD. PDI inhibition has the potential to ameliorate thrombo-inflammatory syndromes associated with SCD. These studies justify a longitudinal trial of PDI inhibitors in SCD. DisclosuresNo relevant conflicts of interest to declare.

Protein Disulfide Isomerase Negatively Regulates Platelet Function through Modulation of the Thromboxane A2 Receptor

Protein disulfide isomerase (PDI) is an intracellular oxidoreductase that cleaves and forms disulfide bonds in substrate proteins to facilitate their proper folding. However, platelets and endothelium release PDI upon vascular injury. This extracellular PDI serves a critical function in platelet activation and thrombus formation. A PDI antagonist is currently in phase II/III clinical trials for thrombosis prevention. Yet the mechanisms of how PDI enables platelets to respond to the local redox environment remain poorly understood. In particular, the substrates on which PDI acts to modify platelet function have yet to be elucidated.To identify platelet receptors that are controlled by PDI, we evaluated the ability of PDI to modify the effect of different platelet agonists on a platelet aggregation. Recombinant PDI (rPDI) had no effect on platelet aggregation induced by a dose curve of a PAR1 activating peptide, a PAR4 activating peptide, thrombin, collagen, or epinephrine. Unexpectedly, rPDI markedly inhibited aggregation induced by the stable thromboxane A2 analog U46619 (13.8±5.6% of control). In contrast, ERp57, a related thiol isomerase, had little effect and a mutant PDI that lacks the active site cysteines responsible for disulfide bond rearrangement failed to inhibit U46619-induced platelet aggregation, implicating disulfide bond rearrangement. rPDI also blocked U46619-induced dense granule secretion (15.0±5.5% of control), α-granule secretion (24.4±0.1% of control) and α2β3 activation (65.0±0.1% of control). Conversely, inhibition of endogenous PDI augmented U46619-mediated platelet activation. To confirm that PDI was acting upstream in the U46619 activation pathway, we evaluated proximal signaling events. rPDI significantly decreased U46619-induced p38 MAPK phosphorylation in both platelets and HEK cells overexpressing the thromboxane prostanoid (TP) receptor, indicating inhibition of Gα12/13 pathways. In contrast, there was little inhibition of Ca2+ flux, indicating lack of inhibition of Gαq-mediated signaling. These results suggest the presence of a functional disulfide bond within the TP receptor that is sensitive to PDI and required for Gα12/13 signaling.Evaluation of the amino sequence of the TP receptor demonstrates two cysteines, Cys-105 in the extracellular loop I and Cys-183 in the extracellular loop II, that form a disulfide bond and may be modified by redox environment. To determine if the platelet TP receptor is sensitive to modification of redox environment, we evaluated U46619-induced signaling in the presence of GSH and GSSG. Incubation with >0.25 mM GSH reduced U46619-induced platelet aggregation to 11.0±0.6% of control, whereas GSSG did not alter platelet aggregation in response to U46619. These results indicate a functional disulfide bond in the TP receptor that responds to changes in redox environment.Evaluation of additional agonists demonstrated that ADP-induced aggregation was sensitive to rPDI. rPDI did not affect the initial response to 3.5 μM ADP, but completely abrogated the secondary wave of aggregation. This defect in the secondary wave could be overcome by using 5 μM ADP, thus suggesting impaired endogenous ADP release. Indeed, rPDI blocked dense granule release even in response to 5 μM ADP. This result is consistent with the known reliance of dense granule release on TP stimulation and suggests that the negative regulation of PDI on ADP-induced platelet activation is secondary to the inhibition of PDI on the TP receptor.These are the first studies to our knowledge that demonstrate the ability of a thiol isomerase to modulate the function of a G-protein coupled receptor via disulfide bond rearrangement. This mechanism could provide a means to modify signaling through the TP receptor in respond to changes in redox environment. DisclosuresNo relevant conflicts of interest to declare.

Pathophysiological roles of cell surface and extracellular protein disulfide isomerase and their molecular mechanisms.

Protein disulfide isomerase (PDI) is the prototypic member of the thiol isomerase family that catalyses disulfide bond rearrangement. Initially identified in the endoplasmic reticulum as folding catalysts, PDI and other members in its family have also been widely reported to reside on the cell surface and in the extracellular matrix. Although how PDI is exported and retained on the cell surface remains a subject of debate, this unique pool of PDI is developing into an important mechanism underlying the redox regulation of protein sulfhydryls that are critical for the cellular activities under various disease conditions. This review aims to provide an overview of the pathophysiological roles of surface and extracellular PDI and their underlying molecular mechanisms. Understanding the involvement of extracellular PDI in these diseases will advance our knowledge in the molecular aetiology to facilitate the development of novel pharmacological strategies by specifically targeting PDI in extracellular compartments.

Protein Disulfide Isomerases Regulate IgE-Mediated Mast Cell Responses and Their Inhibition Confers Protective Effects During Food Allergy.

The thiol isomerase, protein disulfide isomerase (PDI), plays important intracellular roles during protein folding, maintaining cellular function and viability. Recent studies suggest novel roles for extracellular cell surface PDI in enhancing cellular activation and promoting their function. Moreover, a number of food-derived substances have been shown to regulate cellular PDI activity and alter disease progression. We hypothesized that PDI may have similar roles during mast cell-mediated allergic responses and examined its effects on IgE-induced mast cell activity during cell culture and food allergy. Mast cells were activated via IgE and antigen and the effects of PDI inhibition on mast cell activation were assessed. The effects of PDI blockade in vivo were examined by treating mice with the irreversible PDI inhibitor, PACMA-31, in an ovalbumin-induced model of food allergy. The role of dietary PDI modulators was investigated using various dietary compounds including curcumin and quercetin-3-rutinoside (rutin). PDI expression was observed on resting mast cell surfaces, intracellularly, and in the intestines of allergic mice. Furthermore, enhanced secretion of extracellular PDI was observed on mast cell membranes during IgE and antigen activation. Insulin turbidimetric assays demonstrated that curcumin is a potent PDI inhibitor and pre-treatment of mast cells with curcumin or established PDI inhibitors such as bacitracin, rutin or PACMA-31, resulted in the suppression of IgE-mediated activation and the secretion of various cytokines. This was accompanied by decreased mast cell proliferation, FcεRI expression, and mast cell degranulation. Similarly, treatment of allergic BALB/c mice with PACMA-31 attenuated the development of food allergy resulting in decreased allergic diarrhea, mast cell activation, and fewer intestinal mast cells. The production of TH2-specific cytokines was also suppressed. Our observations suggest that PDI catalytic activity is essential in the regulation of mast cell activation, and that its blockade may benefit patients with allergic inflammation.

Extracellular Protein Disulfide Isomerase Cleaves Allosteric Disulfides in Histidine-Rich Glycoprotein to Regulate Thrombus Formation

Introduction The fine-tuning of thrombus formation is influenced by multiple factors among which extracellular protein disulfide isomerase (PDI) released by activated platelets and endothelial cells plays critical roles. However, the precise mechanisms whereby PDI modulates the kinetics of thrombosis remain elusive. Using mechanism-based kinetic trapping strategy, we identified plasma histidine-rich glycoprotein (HRG) as a substrate of extracellular PDI during thrombus formation. HRG exerts both anticoagulant and procoagulant functions. On one hand, HRG inhibits the contact pathway by binding to activated factor XII (fXIIa); on the other hand, HRG attenuates the anticoagulant activity of antithrombin (AT) by competing with AT binding to endothelial heparan sulfate. Both functions are dependent on zinc ions. In this study, we characterized the effects of PDI-mediated disulfide bond cleavage on HRG functions in the context of thrombosis. Methods Recombinant PDI variant with the C-terminal catalytic Cys of the CGHC motif replaced with Ala (PDI-CA) was used to trap its redox substrates in platelet rich plasma (PRP). Dual fluorescent immunoblotting was utilized to detect the stabilized intermediate complex between PDI-CA and HRG. Differential cysteine alkylation and mass spectrometry was performed using purified plasma HRG to identify the disulfide bonds targeted by PDI. ELISA was performed to determine the effects of PDI treatment on HRG binding to heparin, an analog of endothelial heparan sulfate, and fXIIa. Cell-based ELISA, immunofluorescent imaging, and immunohistochemistry were employed to examine in vitro and in vivo binding of HRG and AT on endothelial cells. HRG-mediated inhibition of fXIIa activity was determined using the chromogenic substrate S-2302. The kinetics of HRG accumulation during thrombus formation were examined using high-speed intravital microscopy in the cremasteric arterioles. The effects of HRG on thrombus formation were examined in the laser injury thrombosis model in the presence (wild-type mice) or absence of fXII (f12-/- mice). Results The trapping mutant PDI-CA, but not variants of endoplasmic protein 57 (ERp57), a close member in the PDI family with similar domain structure, formed disulfide-linked complexes with HRG in PRP. Mass spectrometry showed that PDI cleaves three disulfide bonds, C306-C309, C390-C434 and C409-C410, in the histidine-rich region of HRG that is important for its binding to heparan sulfate and fXIIa. Compared to inert-PDI (PDI-AA), where both catalytic Cys were substituted with Ala, wild-type PDI (PDI-CC) increased HRG binding to heparin in a Zn2+-dependent manner. Plasma treated with PDI-CC had increased HRG binding but decreased AT binding to cultured endothelial cells compared to PDI-AA treated control. Further, PDI-CC increased HRG binding to fXIIa and enhanced its inhibitory effect on fXIIa activity. Following laser injury of cremaster arterioles, plasma HRG accumulates rapidly at the injury site preceding the main platelet signal. When mice were treated with Eptifibatide, an integrin αIIbβ3 antagonist that eliminates platelet deposition and Zn2+release, plasma HRG accumulation at the site of vessel injury was reduced, indicating a critical role of Zn2+ for HRG binding in vivo. Intravenous treatment with a PDI inhibitor, isoquercetin, also inhibited HRG accumulation in the growing thrombus. In addition, following FeCl3-induced carotid injury, PDI inhibition by isoquercetin was found to reduce HRG binding but sustain AT binding on the injured artery as determined by immunohistochemistry. Finally, knockdown of plasma HRG with vivo-siRNA in f12-/- mice attenuated thrombus formation compared to scramble siRNA treatment thus suggesting a procoagulant role of HRG independent of fXIIa. Conclusion PDI cleavage of allosteric disulfide bonds in HRG represents a novel regulatory mechanism that fine-tunes the kinetics of thrombus formation. Our results indicate that at the early stage of thrombosis, PDI promotes HRG binding to endothelial cells to suppress the anticoagulant activity of AT and allow the rapid initiation of thrombosis; at the later stage, PDI reduction of HRG enhances its binding to fXIIa leading to inhibition of fXIIa activity to prevent excessive clot formation. Disclosures Bowley: Pfizer: Current Employment.

Integrin-mediated cell adhesion requires extracellular disulfide exchange regulated by protein disulfide isomerase

Cell adhesion to extracellular matrix, mediated by integrin receptors, is crucial for cell survival. Receptor-ligand interaction involves conformational changes in the integrin by a mechanism not fully elucidated. In addition to several direct evidence that there is disulfide re-arrangement of integrins, we previously demonstrated a role for extracellular thiols and protein disulfide isomerase (PDI) in integrin-mediated functions using platelets as model system. Exploring the possible generality of this mechanism, we now show, using three different nucleated cells which depend on adhesion for survival, that non-penetrating blockers of free thiols inhibit α2β1 and α5β1 integrin-mediated adhesion and that disulfide exchange takes place in that process. Inhibiting extracellular PDI mimics thiol blocking. Transfection with WT or enzymatically inactive PDI increased their membrane expression and enhanced cell adhesion, suggesting that PDI level is a limiting factor and that the chaperone activity of the enzyme contributes to adhesion. Exogenously added PDI also enhanced adhesion, further supporting the limiting factor of the enzyme. These data indicate that: a) Dependence on ecto-sulfhydryls for integrin-mediated adhesion is not exclusive to the platelet; b) PDI is involved in integrin-mediated adhesion, catalyzing disulfide bond exchange; c) PDI enhances cell adhesion by both its oxidoreductase activity and as a chaperone.

OR04-5 Stimulation of Protein Disulfide Isomerase Activity by Activation of The Renin-Angiotensin System

Understanding the molecular and cellular mechanisms that are affected by activation of the renin-angiotensin system (RAS) are important for development of novel therapeutic strategies to more specifically treat hypertension and reduce cardiovascular risk. RAS regulates blood pressure (BP) in part via its effector molecule, angiotensin II (AngII), a potent vasopressor that stimulates the production of reactive oxygen species (ROS). Protein disulfide isomerase (PDI) is a multifunctional oxidoreductase that catalyzes thiol/disulfide interchange reactions within the endoplasmic reticulum and regulates ROS production. Also, PDI is present in circulation and at the cell surface of platelets, lymphocytes, erythrocytes and endothelial cells and plays a critical role in initiating thrombus formation. However, the interaction of RAS and PDI remain unclear. We hypothesized that RAS activation would lead to increased PDI levels thus contributing to oxidative stress. First, we studied the in vitro effects of AngII on EA.hy926, a human endothelial cell line, and measured PDI activity. Our results show that AngII dose and time dependently increased extracellular PDI activity (p<0.05, n=6); an event that was reduced to baseline levels by preincubation with 0.5 uM losartan, an AngII type 1 receptor antagonist (ARB). Consistent with these results, AngII stimulated: 1) a 3-fold rise of extracellular PDI levels as determined by ELISA (p<0.05, n=4); 2) a 2.5-fold increase in PDI mRNA levels by RT-PCR (p<0.05, n=4); and 3) increased PDI protein levels by western blot analyses that were blocked by losartan. We also studied the effect of AngII on extracellular PDI activity in differentiated HL-60 cells, a human polymorphonuclear neutrophil cell line, and observed significantly increased extracellular PDI activity following incubation with AngII (p<0.05, n=4). We then characterized the in vivo effects of exogenous AngII infusion on PDI in Sprague-Dawley rats, a model of increased BP, inflammation and target organ damage. We studied three conditions: (1) control; (2) AngII infused (80 ng/min x 28 days); (3) AngII + ARB (10 mg losartan/kg/day x 21 days). AngII infusion led to significant increases in plasma PDI levels that were reduced by 20% following ARB treatment (p<0.05, n=5/group). We then studied a model of naturally increased AngII and hypertension, the Otsuka Long Evans Tokushima Fatty rats (OLETF) and their strain controls that were randomly assigned to the following groups: (1) untreated strain controls; (2) untreated OLETF; (3) OLETF + ARB. OLETF rats had increased BP and significantly greater circulating PDI activity than control rats that was likewise blocked by ARB treatment (p<0.05; n=6/group). Thus, RAS activation represents a novel mechanism for increased PDI levels and activity that may contribute to the deleterious effects of disordered RAS in cardiovascular disease.

Activation of Renin‐Angiotensin Aldosterone System's Effect on Protein Disulfide Isomerase

Disordered activation of the Renin‐Angiotensin‐Aldosterone‐System (RAAS) is associated with hypertension and insulin resistance. The hormones Angiotensin II (Ang II) and Aldosterone (Aldo) are two of its principal effector molecules that have been shown to regulate sodium and volume homeostasis leading to blood pressure control. Ang II has been shown to activate endothelial cells and increase the secretion of extracellular protein disulfide isomerase (PDI). PDI is a multifunctional protein critical to thrombus formation and regulation of reactive oxygen species among other functions. Our group has recently reported that caloric restriction (CR) in rodents leads to increases in Aldo secretion from adrenal zona glomerulosa cells. However, the effects of CR on PDI are unknown. We hypothesize that CR, due to its association with activating RAAS, will lead to increases in the circulating PDI activity. To test our hypothesis, we propose to study the in vitro effects of AngII and Aldo on PDI activity. We will study the in vivo effects of CR on the circulating activity of PDI in Long‐Evans Tokushima Fatty (LETO) and Otsuka Long‐Evans Tokushima Fatty (OLETF). We use two assays to measure PDI activity. In one approach we will use insulin degradation assays. The measurement of circulating PDI activity will be further validated with a fluorescent of Di‐E‐GSSG assay. Initial studies have shown specificity of our assay using the insulin degradation assay and recombinant PDI. Controls of our fluorescent assay are currently underway. We expect that CR will be associated with increased PDI activity.Support or Funding InformationK.V. is supported by an NIH MARC grant R25HL121029 and by the Division of Medical Sciences.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Greetings from the endoplasmic reticulum (ER): escaping ER thiol isomerases regulate thrombosis

Greetings from the endoplasmic reticulum (ER): escaping ER thiol isomerases regulate thrombosis

Kinetic-based trapping by intervening sequence variants of the active sites of protein-disulfide isomerase identifies platelet protein substrates

Thiol isomerases such as protein-disulfide isomerase (PDI) direct disulfide rearrangements required for proper folding of nascent proteins synthesized in the endoplasmic reticulum. Identifying PDI substrates is challenging because PDI catalyzes conformational changes that cannot be easily monitored (e.g. compared with proteolytic cleavage or amino acid phosphorylation); PDI has multiple substrates; and it can catalyze either oxidation, reduction, or isomerization of substrates. Kinetic-based substrate trapping wherein the active site motif CGHC is modified to CGHA to stabilize a PDI-substrate intermediate is effective in identifying some substrates. A limitation of this approach, however, is that it captures only substrates that are reduced by PDI, whereas many substrates are oxidized by PDI. By manipulating the highly conserved -GH- residues in the CGHC active site of PDI, we created PDI variants with a slowed reaction rate toward substrates. The prolonged intermediate state allowed us to identify protein substrates that have biased affinities for either oxidation or reduction by PDI. Because extracellular PDI is critical for thrombus formation but its extracellular substrates are not known, we evaluated the ability of these bidirectional trapping PDI variants to trap proteins released from platelets and on the platelet surface. Trapped proteins were identified by mass spectroscopy. Of the trapped substrate proteins identified by mass spectroscopy, five proteins, cathepsin G, glutaredoxin-1, thioredoxin, GP1b, and fibrinogen, showed a bias for oxidation, whereas annexin V, heparanase, ERp57, kallekrein-14, serpin B6, tetranectin, and collagen VI showed a bias for reduction. These bidirectional trapping variants will enable more comprehensive identification of thiol isomerase substrates and better elucidation of their cellular functions.

Activation of the Renin-Angiotensin II-Aldosterone-System leads to increases in extracellular protein disulfide isomerase: role in insulin resistance

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Challenges in the evaluation of thiol-reactive inhibitors of human protein disulfide Isomerase

This paper addresses how to evaluate the efficacy of the growing inventory of thiol-reactive inhibitors of mammalian protein disulfide Isomerase (PDI) enzymes under realistic concentrations of potentially competing thiol-containing peptides and proteins. For this purpose, we introduce a variant of the widely-used reductase assay by using a commercially-available cysteine derivative (BODIPY FL L-Cystine; BD-SS) that yields a 55-fold increase in fluorescence (excitation/emission; 490/513nm) on scission of the disulfide bond. This plate reader-compatible method detects human PDI down to 5–10nM, can utilize a range of thiol substrates (including 5µM dithiothreitol, 10µM reduced RNase thiols, and 5mM glutathione; GSH), and can operate from pH 6–9.5 in a variety of buffers. PDI assays often employ low micromolar levels of substrates leading to ambiguities when thiol-directed inhibitors are evaluated. The present work utilizes 5mM GSH for both pre-incubation and assay phases to more realistically reflect the high concentration of thiols that an inhibitor would encounter intracellularly. Extracellular PDI faces a much lower concentration of potentially competing thiols; to assess reductase activity under these conditions, the pre-reduced PDI is treated with inhibitor and then fluorescence increase upon reduction of BD-SS is followed in the absence of additional competing thiols. Both assay modes were tested with four mechanistically diverse PDI inhibitors. Two reversible reagents, 3,4-methylenedioxy-β-nitrostyrene (MNS) and the arsenical APAO, were found to be strong inhibitors of PDI in the absence of competing thiols, but were ineffective in the presence of 5mM GSH. A further examination of the nitrostyrene showed that MNS not only forms facile Michael adducts with GSH, but also with the thiols of unfolded proteins (Kd values of 7 and <0.1µM, respectively) suggesting the existence of multiple potential intracellular targets for this membrane-permeant reagent. The inhibition of PDI by the irreversible alkylating agent, the chloroacetamide 16F16, was found to be only modestly attenuated by 5mM GSH. Finally, the thiol-independent flavonoid inhibitor quercetin-3-O-rutinoside was found to show equal efficacy in reoxidation and turnover assay types. This work provides a framework to evaluate inhibitors that may target the CxxC motifs of PDI and addresses some of the complexities in the interpretation of the behavior of thiol-directed reagents in vivo.

Protein disulfide isomerase externalization in endothelial cells follows classical and unconventional routes

Extracellular protein disulfide isomerase (PDIA1) pool mediates thrombosis and vascular remodeling, however its externalization mechanisms remain unclear. We performed systematic pharmacological screening of secretory pathways affecting extracellular PDIA1 in endothelial cells (EC). We identified cell-surface (csPDIA1) and secreted non-particulated PDIA1 pools in EC. Such Golgi bypass also occurred for secreted PDIA1 in EC at baseline or after PMA, thrombin or ATP stimulation. Inhibitors of Type I, II and III unconventional routes, secretory lysosomes and recycling endosomes, including syntaxin-12 deletion, did not impair EC PDIA1 externalization. This suggests predominantly Golgi-independent unconventional secretory route(s), which were GRASP55-independent. Also, these data reinforce a vesicular-type traffic for PDIA1. We further showed that PDIA1 traffic is ATP-independent, while actin or tubulin cytoskeletal disruption markedly increased EC PDIA1 secretion. Clathrin inhibition enhanced extracellular soluble PDIA1, suggesting dynamic cycling. Externalized PDIA1 represents <2% of intracellular PDIA1. PDIA1 was robustly secreted by physiological levels of arterial laminar shear in EC and supported alpha 5 integrin thiol oxidation. Such results help clarify signaling and homeostatic mechanisms involved in multiple (patho)physiological extracellular PDIA1 functions.

Extracellular Protein Disulfide Isomerase Regulates Vitronectin during the Initiation of Thrombus Formation

Protein disulfide isomerase (PDI), secreted by platelets and endothelial cells upon vascular injury, is required for thrombus formation. However, the precise mechanism by which PDI regulates thrombosis remains elusive. Using PDI variants that form stable mixed disulfide complexes with their substrates, we performed kinetic trapping experiment in platelet rich plasma and identified multiple substrate proteins for PDI, including vitronectin. Importantly, when using variants of endoplasmic reticulum protein 57 (ERp57), a thiol isomerase that has a similar domain structure as PDI and is also important for thrombus formation, the trapping mutants of ERp57 do not interact with vitronectin. This result has demonstrated the substrate specificity of PDI during our kinetic trapping experiment. Further study using polyethylene glycol (PEG)-based gel mobility shift assay combined with mass spectrometry has identified the redox reaction between PDI and vitronectin occurs on two disulfide bonds Cys 137-161 and Cys 274-453 in the hemopexin-like domains of plasma vitronectin.Vitronectin, as a substrate of extracellular PDI, has been shown to be important for thrombus formation. Vitronectin null mice have reduced platelet accumulation and fibrin deposition in the cremaster arterioles following laser injury. Vitronectin null mice also have significantly prolonged large-vessel thrombosis in the carotid artery using the ferric chloride thrombosis model. Using intravital microscopy we showed that vitronectin rapidly accumulates in a growing thrombus following vessel injury. When mice are treated with eptifibatide to eliminate platelet accumulation, we still observe significant amount of vitronectin accumulation on the vessel wall in the absence of platelet thrombus. This observation was further confirmed using confocal intravital microscopy. After 3D reconstruction of a growing thrombus in mouse cremaster arteriole, vitronectin was identified to locate primarily on the CD31 stained vessel wall. These combined studies suggest that plasma-derived vitronectin and not platelet-derived vitronectin is the primary substrate of PDI.Our study further showed that the indispensable role of vitronectin to a growing thrombus depends on extracellular PDI. Native plasma vitronectin does not bind to αvβ3 or αIIbβ3-integrins on endothelial cells and platelets. On solid phase binding assay, plasma sample pre-treated with wild-type PDI showed significantly increased binding of vitronectin to its ligand αvβ3 or αIIbβ3-integrins. However, this increase was not observed in plasma pre-treated with dead-mutant PDI or ERp57. In addition, using immunofluorescent staining, PDI treated plasma sample also showed significantly increased binding of vitronectin to activated human umbilical vein endothelial cells (HUVECs) and this binding was abrogated by RGD peptides or an αvβ3 blocking antibody. The critical role of extracellular PDI for the regulation of vitronectin in a growing thrombus was further confirmed in our in vivo studies. When mice were treated with quecetin-3-rutinoside or two different inhibitory antibodies that selectively block PDI activity, the accumulation of vitronectin and platelets was significantly reduced. These combined results demonstrate that extracellular PDI regulates vitronectin in a growing thrombus to promote platelet accumulation and fibrin generation.In summary, our studies have revealed a novel regulatory mechanism during the initiation of thrombus formation. Under normal physiologic conditions in the absence of secreted PDI, thrombus formation is suppressed and maintains a quiescent, patent vasculature. The release of PDI during vascular injury serves as a novel regulatory switch that allows activation of proteins, including vitronectin, which are critical for the following platelet accumulation and fibrin generation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PDI Inhibition Blocks Thrombin Generation in Humans By Interfering with Platelet Factor V Activation

Protein disulfide isomerase (PDI) is a ubiquitously expressed oxidoreductase that serves an essential role in protein folding in the endoplasmic reticulum by reshuffling disulfide bonds within nascent proteins. PDI can be released from vascular cells, including platelets, and inhibition or platelet-specific deletion of PDI blocks thrombus formation in vivo. However, the specific function of PDI in thrombus formation is poorly understood. Unlike the role of proteases in blood coagulation, which have been studied in depth, little is known about PDI substrates in the vasculature. Several platelet and endothelial integrins have been identified as putative substrates for PDI, but whether coagulation factors are directly targeted by extracellular PDI has not been established. We now identify platelet factor V as a principal coagulation substrate of extracellular PDI. We developed an unbiased strategy to identify novel substrates of PDI in washed platelets using PDI variants capable of trapping substrates: FLAG-tagged PDI mutants modified by a substitution of arginine or proline for histidine (CGHC → CGRC; CGHC → CGPC) in the catalytic motif of both the a and a’ domains. Whereas the AGHA-PDI variant which has no catalytic activity serves as a control. The CGRC-PDI variant co-precipitated with platelet factor V in a redox-sensitive manner while there was no platelet factor V detected with the AGHA-PDI variant, thus confirming that binding of PDI to platelet-derived factor V occurs through disulfide bond exchange. Platelet factor V associates with multimerin-1 through a disulfide bond. Trapping PDI mutants also bind to multimerin-1 in a reaction requiring disulfide bond exchange. To evaluate the effect of PDI inhibition on the activation of platelet factor V, washed platelets from healthy donors were stimulated with 0.1 U/mL of thrombin in the presence of varying concentrations of isoquercetin (0 to 50 µM), which has previously been shown to inhibit PDI function. We observed a dose-dependent reduction of factor Va following platelet activation despite the fact that isoquercetin did not inhibit platelet release of PF4 or block Xa or thrombin enzymatic activity directly. We next performed a clinical study designed to determine whether oral isoquercetin inhibits thrombin generation in human subjects via its ability to inhibit platelet Va generation. Plasma samples collected from healthy participants before and 4 hours after ingestion of 1000 mg of isoquercetin (N=17). In plasma samples, post-isoquercetin platelet-dependent thrombin generation decreased by 51% compared with pre-ingestion controls (P=0.0004). Furthermore, we observed an overall 26% reduction in FVa in non-FV depleted plasma (P<0.001), which corresponded with a 53% decrease in FVa generated from platelets (P<0.001). These data confirm a significant effect of PDI inhibition on the generation of FVa following platelet activation. Considering that isoquercetin reduces platelet FVa generation and similarly inhibits platelet-dependent thrombin generation in a PDI-dependent manner, we investigated whether the addition of FVa in vitro restored platelet-dependent thrombin generation. The pre-incubation of 7 µg/mL FVa prior to stimulation with low dose thrombin restored platelet-dependent thrombin generation to within 80% baseline of pre-treatment levels. We conclude that platelet factor V is an essential substrate in mediating PDI-dependent thrombin generation on platelets and propose that PDI cleaves a disulfide bond that links platelet factor V to multimerin-1, thereby releasing platelet factor V for activation and subsequent thrombin generation. DisclosuresZwicker:Quercegen Pharma: Research Funding.

Inhibition of Protein Disulfide Isomerase in Thrombosis.

This MiniReview addresses our current understanding of the mechanisms by which protein disulfide isomerase (PDI) mediates thrombus formation and discusses the potential of blocking thrombosis by targeting PDI. Thiol isomerases are ubiquitous oxidoreductases primarily localized to the endoplasmic reticulum (ER) where they serve a critical role in protein folding. PDI is the founding member of the thiol isomerase family. Although PDI is an essential intracellular enzyme, it can participate in pathological processes once released from cells. In particular, PDI serves a critical role in thrombus formation, the underlying cause of myocardial infarction and stroke. Both platelets and endothelial cells secrete PDI upon vascular injury. Secreted PDI appears to activate multiple extracellular substrates in the vasculature, enabling the initiation of thrombus formation. As an essential component of thrombus formation, extracellular PDI represents a new target for pharmacological inhibition of clinical thrombosis. Quercetin-3-rutinoside, a flavonol highly abundant in common foods, inhibits PDI and blocks thrombus formation both in vitro and in vivo. Such observations have prompted clinical trials targeting PDI in thrombotic diseases.