Association of protein disulfide isomerase family A, member 4, and inflammation in people living with HIV.

OBJECTIVES: Protein disulfide isomerase (PDI) family members are specific endoplasmic reticulum proteins associated with inflammation, obesity, and cancer. In HIV infection, the role of PDI family A, member 4 (PDIA4), is unclear. This study aimed to clarify the association between plasma PDIA4 levels and inflammation in people living with HIV (PLWH). METHODS: In this study, 287 PLWH and 74 healthy participants were enrolled. The plasma PDIA4 values, demographic data, laboratory data, and other inflammatory markers were recorded. The association between PDIA4 level and inflammatory extent was analyzed using logistic regression and Spearman rank-order correlations. Other results were analyzed using Student's t-test or chi-square test. RESULTS: In PLWH, the PDIA4 levels were positively associated with the inflammatory markers, interleukin 6 (r = 0.209, p = 0.001), and tumor necrosis factor-α (r = 0.162, p = 0.01) levels, but not with high-sensitivity C-reactive protein levels. Moreover, the plasma PDIA4 level of PLWH decreased after anti-viral treatment (p = 0.0001). CONCLUSION: Plasma PDIA4 levels are closely associated with inflammation in PLWH and have a positive correlation with the viral load during anti-viral therapy.

Circulating protein disulfide isomerase family member 4 is associated with type 2 diabetes mellitus, insulin sensitivity, and obesity.

AIMS: Endoplasmic reticulum (ER) stress is associated with obesity and type 2 diabetes mellitus (T2DM) and increasing evidence demonstrates that some ER stress markers can represent the severity of metabolic dysfunction in either cellular or animal models. However, no appropriate molecule has been identified to demonstrate these relationships in clinical practice. METHODS: To determine whether the serum level of the ER chaperone, protein disulfide isomerase family A, member 4 (PDIA4), is associated with type 2 diabetes mellitus, obesity, and insulin sensitivity, we conducted a cross-sectional study for which a total of 553 adults, including 159 with normal glucose tolerance (NGT), 169 with prediabetes (Pre-DM), and 225 with newly diagnosed T2DM, were recruited. RESULTS: Serum PDIA4 levels were significantly higher in patients with T2DM than in those with NGT (P < 0.001), even after adjustment for potential confounders. These levels correlated positively with fasting plasma glucose, BMI, waist circumference as well as high-sensitivity C-reactive protein levels, and negatively and strongly correlated with insulin sensitivity. In a multivariate logistic regression analysis, higher serum PDIA4 concentration was observed to be significantly associated with an increased risk of T2DM. CONCLUSIONS: Our findings provide new mechanistic insights linking ER stress, T2DM, insulin sensitivity, and obesity, which may, in part, account for the ER chaperone properties associated with PDIA4. The results suggest that PDIA4 may serve as a potential instigator of and a putative therapeutic target for T2DM.

Circulating Protein Disulfide Isomerase Is Associated with Increased Risk of Thrombosis in JAK2-Mutated Myeloproliferative Neoplasms.

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 with healthy controls. Plasma PDI was measured at enrollment and subjects followed prospectively for development of TE. RESULTS: A subset of patients, primarily those with JAK2-mutated MPN, had significantly elevated plasma PDI levels as compared with 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.

Endothelial microparticle-associated protein disulfide isomerase increases platelet activation in diabetic coronary heart disease.

BACKGROUND: Endothelial microparticles (EMPs) carrying the protein disulfide isomerase (PDI) might play a key role in promoting platelet activation in diabetes. This study aimed to examine the activation of platelets, the amounts of MPs, PMPs, and EMPs, and the concentration and activity of PDI in patients with diabetic coronary heart disease (CHD) and non-diabetic CHD. METHODS: Patients with CHD (n=223) were divided as non-diabetic CHD (n=121) and diabetic CHD (n=102). Platelet activation biomarkers, circulating microparticles (MPs), the concentration of protein disulfide isomerase (PDI), and MP-PDI activity were determined. The effect of EMPs on platelet activation was investigated in vitro. Allosteric GIIb/IIIa receptors that bind to PDI were detected by a proximity ligation assay (PLA). RESULTS: Platelet activation, platelet-leukocyte aggregates, circulating MPs, EMPs, PDI, and MP-PDI activity in the diabetic CHD group were significantly higher than in the non-diabetic CHD group (P<0.05). Diabetes (P=0.006) and heart rate <60 bpm (P=0.047) were associated with elevated EMPs. EMPs from diabetes increased CD62p on the surface of the platelets compared with the controls (P<0.01), which could be inhibited by the PDI inhibitor RL90 (P<0.05). PLA detected the allosteric GIIb/IIIa receptors caused by EMP-PDI, which was also inhibited by RL90. CONCLUSIONS: In diabetic patients with CHD, platelet activation was significantly high. Diabetes and heart rate <60 bpm were associated with elevated EMPs and simultaneously increased PDI activity on EMP, activating platelets through the allosteric GPIIb/IIIa receptors.

Proteomics analysis of plasma protein changes in patent ductus arteriosus patients.

OBJECTIVE: Patent ductus arteriosus (PDA) is a congenital heart defect with an unclear etiology that occurs commonly among newborns. Adequately understanding the molecular pathogenesis of PDA can contribute to improved treatment and prevention. Plasma proteins may provide evidence to explore the molecular mechanisms of abnormal cardiac development. METHODS: Isobaric tags for relative and absolute quantitation (iTRAQ) proteomics technology was used to measure different plasma proteins in PDA patients (n = 4) and controls (n = 4). The candidate protein was validated by ELISA and Western blot (WB) assays in a larger sample. Validation of the location and expression of this protein was performed in mouse heart sections. RESULTS: There were three downregulated proteins and eight upregulated proteins identified in the iTRAQ proteomics data. Among these, protein disulfide-isomerase A6 (PDIA6) was further analyzed for validation. The plasma PDIA6 concentrations (3.2 ± 0.7 ng/ml) in PDA patients were significantly lower than those in normal controls (5.8 ± 1.2 ng/ml). In addition, a WB assay also supported these results. PDIA6 was widely expressed in mouse heart outflow tract on embryonic day 14.5. CONCLUSION: Plasma proteomics profiles suggested novel candidate molecular markers for PDA. The findings may allow development of a new strategy to investigate the mechanism and etiology of PDA.

Maternal serum TXNDC5 levels and thiol/disulfide homeostasis in preeclamptic pregnancies.

Objective: To investigate thiol/disulfide homeostasis (TDH) and thioredoxin domain-containing 5 (TXNDC5) level in early and late-onset preeclampsia.Material and methods: In this cross-sectional study, 24 pregnant women with early-onset preeclampsia and 26 pregnant women with late-onset preeclampsia were compared with 30 pregnant women with no obstetric complications. The serum TXNDC5 levels and thiol/disulfide homeostasis were measured.Results: Serum TXNDC5 levels were significantly higher in the early-onset and late-onset preeclampsia groups compared with the control group (p < .05). Native thiol and total thiol levels were significantly lower in the early-onset and late-onset preeclampsia groups than control group. The disulfide levels were found as significantly high in early preeclamptic patients compared to control group (p < .05). The highest levels of TXNDC5 and the lowest levels of native thiol and total thiol were found in early-onset preeclampsia group. No significant difference was found between the patients with early onset and late onset preeclampsia regarding TXNDC5 levels and thiol/disulfide homeostasis (p > .05).Conclusion: Serum TXNDC5 levels were significantly higher in patients with early-onset and late-onset preeclampsia. The dynamic thiol/disulfide homeostasis was impaired in favor of the oxidized state in patients with preeclampsia.

Identification of Key Genes Potentially Related to Intervertebral Disk Degeneration by Microarray Analysis.

Aims: This study was designed to investigate differentially expressed genes (DEGs) in the annulus fibrosus (AF), nucleus pulposus (NP), and whole blood (WB) of intervertebral disk degeneration (IDD) patients. Materials and Methods: We retrieved microarray data set GSE70362, which contains the gene expression profiles of 24 AF and 24 NP samples from the Gene Expression Omnibus and identified DEGs in degenerative AF (AF-DEGs) and NP (NP-DEGs) samples compared with nondegenerative samples. We also examined gene expression profiles in WB from patients with IDD and healthy volunteers to identify DEGs in WB (WB-DEGs). We performed functional analyses on the DEGs common to AF-DEGs, NP-DEGs, and WB-DEGs. Expression of the common DEGs was partially validated by quantitative real-time-polymerase chain reaction (QRT-PCR). Results: In total, 846 AF-DEGs, 902 NP-DEGs, and 862 WB-DEGs were identified, and 22 DEGs were common among the three groups. Functional analyses showed that the common DEGs were enriched in 33 biological processes, 16 cellular components, 4 molecular functions, and 9 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways; 13 of the common DEGs were included in the protein-protein interaction (PPI) network and superoxide dismutase 2 (SOD2) was identified as a hub gene in the PPI network. The QRT-PCR results for the expression of the genes protein disulfide isomerase family A member 4, FKBP prolyl isomerase 11, ectonucleotide pyrophosphatase/phosphodiesterase 4, SOD2, and actin binding LIM protein 1, were consistent with the gene chip hybridization results. Conclusions: This study identified key genes for future investigations of the underlying molecular mechanisms of IDD. These genes may provide future targets for the clinical treatment and diagnosis of IDD.

Protein disulfide isomerase plasma levels in healthy humans reveal proteomic signatures involved in contrasting endothelial phenotypes.

Redox-related plasma proteins are candidate reporters of protein signatures associated with endothelial structure/function. Thiol-proteins from protein disulfide isomerase (PDI) family are unexplored in this context. Here, we investigate the occurrence and physiological significance of a circulating pool of PDI in healthy humans. We validated an assay for detecting PDI in plasma of healthy individuals. Our results indicate high inter-individual (median = 330 pg/mL) but low intra-individual variability over time and repeated measurements. Remarkably, plasma PDI levels could discriminate between distinct plasma proteome signatures, with PDI-rich (>median) plasma differentially expressing proteins related to cell differentiation, protein processing, housekeeping functions and others, while PDI-poor plasma differentially displayed proteins associated with coagulation, inflammatory responses and immunoactivation. Platelet function was similar among individuals with PDI-rich vs. PDI-poor plasma. Remarkably, such protein signatures closely correlated with endothelial function and phenotype, since cultured endothelial cells incubated with PDI-poor or PDI-rich plasma recapitulated gene expression and secretome patterns in line with their corresponding plasma signatures. Furthermore, such signatures translated into functional responses, with PDI-poor plasma promoting impairment of endothelial adhesion to fibronectin and a disturbed pattern of wound-associated migration and recovery area. Patients with cardiovascular events had lower PDI levels vs. healthy individuals. This is the first study describing PDI levels as reporters of specific plasma proteome signatures directly promoting contrasting endothelial phenotypes and functional responses.

Tongue Cancer Patients Can be Distinguished from Healthy Controls by Specific N-Glycopeptides Found in Serum.

PURPOSE: There are no blood biomarkers to detect early-stage oral cavity squamous cell carcinoma (OSCC) prior to clinical signs. Most OSCC incidence is associated with significant morbidity and poor survival. The authors aimed to use mass-spectrometry (MS) technology to find specific N-glycopeptides potentially serving as serum biomarkers for preclinical OSCC screening. EXPERIMENTAL DESIGN: Serum samples from 14 patients treated for OSCC (stage I or stage IV) with 12 age- and sex-matched controls are collected. Quantitative label-free N-glycoproteomics is performed, with MS/MS analysis of the statistically significantly different N-glycopeptides. RESULTS: Combined with a database search using web-based software (GlycopeptideID), MS/MS provided detailed N-glycopeptide information, including glycosylation site, glycan composition, and proposed structures. Thirty-eight tryptic N-glycopeptides are identified, having 19 unique N-glycosylation sites representing 14 glycoproteins. OSCC patients, including stage I tumors, can be differentiated from healthy controls based on the expression levels of these glycoforms. N-glycopeptides of IgG1, IgG4, haptoglobin, and transferrin have statistically significant different abundances between cases and controls. CONCLUSIONS AND CLINICAL RELEVANCE: The authors are the first to suggest specific N-glycopeptides to serve as potential serum biomarkers to detect preclinical OSCC in patients. These N-glycopeptides are the lead candidates for validation as future diagnostic modalities of OSCC as early as stage I.

Protein disulfide isomerase enhances tissue factor-dependent thrombin generation.

Protein disulfide isomerase (PDI) plays an important role in fibrin generation in vivo, but the underlying mechanism remains largely unknown. In this study, using thrombin generation assay (TGA), we investigated whether PDI contributes to tissue factor (TF)-mediated thrombin generation. Human peripheral blood mononuclear cells (PBMCs) were treated with 100 ng/ml lipopolysaccharide (LPS), the expression of TF on cell surface was analyzed by flow cytometry. After incubation with an inhibitory anti-TF antibody, recombinant PDI protein or a PDI inhibitor PACMA31, LPS-stimulated human PBMCs were incubated with human plasma, and thrombin generation was assessed by Ceveron Alpha TGA and a fluorescent thrombin substrate. Bone marrow mononuclear cells isolated from PDI-knockout and wild-type mice were stimulated by LPS, followed by measurement of thrombin generation. LPS stimulation increased expression of TF on PBMCs, and thrombin generation. Inhibitory anti-TF antibody almost completely suppressed thrombin generation of LPS-stimulated PBMCs, suggesting that thrombin generation was TF-dependent. Recombinant PDI protein increased thrombin generation, while PACMA31 attenuated thrombin generation. Compared with control cells, PDI-deficient marrow mononuclear cells had less capacity in thrombin generation. Taken together, these data suggest that PDI enhances TF-dependent thrombin generation.

The intersection of protein disulfide isomerase and cancer associated thrombosis.

The mechanisms underlying the hypercoagulability of cancer are complex and include the upregulation coagulation factors or procoagulant proteins, shedding of microparticles, and direct activation of vascular cells. Protein disulfide isomerase (PDI) is a thiol isomerase secreted from activated platelets and endothelial cells and plays a critical role in both platelet aggregation and fibrin generation. A number of potential intravascular targets of PDI have been identified including cell surface receptors (e.g. ß-integrins and glycoprotein Ib), receptor ligands (e.g. fibrinogen and von Willebrand factor), serine proteases (e.g. cathepsin G and kallekrein-14), and coagulation factors (e.g. factor XI and factor V). Recent clinical studies demonstrated that a small molecule inhibitor of PDI, isoquercetin, decreases platelet-dependent thrombin generation and PDI activity in plasma following oral administration. This review explores the mechanistic overlap between the molecular drivers of cancer associated thrombosis and the potential roles PDI plays in mediating thrombosis. These molecular insights provide rationale for clinical trials targeting PDI to prevent thrombosis in cancer patients.

A humanized monoclonal antibody that inhibits platelet-surface ERp72 reveals a role for ERp72 in thrombosis.

Essentials ERp72 is a thiol isomerase enzyme. ERp72 levels increase at the platelet surface during platelet activation. We generated a humanized monoclonal antibody which blocks ERp72 enzyme activity (anti-ERp72). Anti-ERp72 inhibits platelet functional responses and thrombosis. SUMMARY: Background Within the endoplasmic reticulum, thiol isomerase enzymes modulate the formation and rearrangement of disulfide bonds in newly folded proteins entering the secretory pathway to ensure correct protein folding. In addition to their intracellular importance, thiol isomerases have been recently identified to be present on the surface of a number of cell types where they are important for cell function. Several thiol isomerases are known to be present on the resting platelet surface, including PDI, ERp5 and ERp57, and levels are increased following platelet activation. Inhibition of the catalytic activity of these enzymes results in diminished platelet function and thrombosis. Aim We previously determined that ERp72 is present at the resting platelet surface and levels increase upon platelet activation; however, its functional role on the cell surface was unclear. We aimed to investigate the role of ERp72 in platelet function and its role in thrombosis. Methods Using HuCAL technology, fully humanized Fc-null anti-ERp72 antibodies were generated. Eleven antibodies were screened for their ability to inhibit ERp72 activity and the most potent inhibitory antibody (anti-ERp72) selected for further testing in platelet functional assays. Results and conclusions Anti-ERp72 inhibited platelet aggregation, granule secretion, calcium mobilisation and integrin activation, revealing an important role for extracellular ERp72 in the regulation of platelet activation. Consistent with this, infusion of anti-ERp72 into mice protected against thrombosis.

Advances in vascular thiol isomerase function.

PURPOSE OF REVIEW: The present review will provide an overview of several recent advances in the field of vascular thiol isomerase function. RECENT FINDINGS: The initial observation that protein disulfide isomerase (PDI) functions in thrombus formation occurred approximately a decade ago. At the time, there was little understanding regarding how PDI or other vascular thiol isomerases contribute to thrombosis. Although this problem is far from solved, the past few years have seen substantial progress in several areas that will be reviewed in this article. The relationship between PDI structure and its function has been investigated and applied to identify domains of PDI that are critical for thrombus formation. The mechanisms that direct thiol isomerase storage and release from platelets and endothelium have been studied. New techniques including kinetic-based trapping have identified substrates that vascular thiol isomerases modify during thrombus formation. Novel inhibitors of thiol isomerases have been developed that are useful both as tools to interrogate PDI function and as potential therapeutics. Human studies have been conducted to measure circulating PDI in disease states and evaluate the effect of oral administration of a PDI inhibitor on ex-vivo thrombin generation. SUMMARY: Current findings indicate that thiol isomerase-mediated disulfide bond modification in receptors and plasma proteins is an important layer of control of thrombosis and vascular function more generally.

A novel potential biomarker for metabolic syndrome in Chinese adults: Circulating protein disulfide isomerase family A, member 4.

BACKGROUND/OBJECTIVES: Protein disulfide isomerase (PDI) family members are specific endoplasmic reticulum proteins that are involved in the pathogenesis of numerous diseases including neurodegenerative diseases, cancer and obesity. However, the metabolic effects of PDIA4 remain unclear in humans. The aims of this study were to investigate the associations of serum PDIA4 with the metabolic syndrome (MetS) and its components in Chinese adults. SUBJECTS/METHODS: A total of 669 adults (399 men and 270 women) were recruited. Serum PDIA4 concentrations and biochemical variables were recorded. Insulin sensitivity and ß-cell function were examined by homeostasis model assessment. MetS was defined based on the modified National Cholesterol Education Program Adult Treatment Panel III criteria for Asia Pacific. RESULTS: The participants with MetS had significantly higher serum PDIA4 levels than those without MetS (P<0.001). After adjustments, the individuals with the highest PDIA4 tertile were associated with a higher risk of MetS than those with the lowest tertile (OR = 4.83, 95% CI: 2.71-8.60). The concentration of PDIA4 showed a stepwise increase with the components of MetS (P<0.001 for trend). The individuals with the highest PDIA4 tertile were significantly associated with waist circumference (OR = 2.41, 95% CI 1.34-4.32), blood pressure (OR = 2.71, 95% CI 1.57-4.67), fasting glucose concentration (OR = 3.17, 95% CI 1.80-5.57), and serum triglycerides (OR = 4.12, 95% CI 2.30-7.37) than those with the lowest tertile. At cutoff point of 15.24 ng/ml, the diagnostic sensitivity and specificity of PDIA4 for the metabolic syndrome were 67 and 72%, respectively, in male patients and 60 and 78%, respectively, in female patients. Finally, the result showed that PDIA4 had a significantly higher area under the curve compared with blood pressure to detect MetS using receiver operating characteristic analysis. CONCLUSIONS: Serum PDIA4 concentrations are closely associated to MetS and its components in Chinese adults.

Endothelial cells microparticle-associated protein disulfide isomerase promotes platelet activation in metabolic syndrome.

BACKGROUND: Metabolic syndrome (MetS) is a common challenge in the world, and the platelet activation is enhanced in MetS patients. However, the fundamental mechanism that underlies platelet activation in MetS remains incompletely understood. Endothelial cells are damaged seriously in MetS patients, then they release more endothelial microparticles (EMPs). After all, whether the EMPs participate in platelet activation is still obscure. If they were, how did they work? RESULTS: We demonstrated that the levels of EMPs, PMPs (platelet derived microparticles) and microparticle-carried-PDI activity increased in MetS patients. IR endothelial cells released more EMPs, the EMP-PDI was more activated. EMPs can enhance the activation of CD62P, GPIIb/IIIa and platelet aggregation and this process can be partly inhibited by PDI inhibitor such as RL90 and rutin. Activated platelets stimulated by EMPs expressed more PDI on cytoplasm and released more PMPs. MATERIALS AND METHODS: We obtained plasma from 23 MetS patients and 8 normal healthy controls. First we built insulin resistance (IR) model of human umbilical vein endothelial cells (HUVECs), and then we separated EMPs from HUVECs culture medium and used these EMPs to stimulate platelets. Levels of microparticles, P-selectin(CD62P), Glycoprotein IIb/IIIa (GPIIb/IIIa) were detected by flow cytometry and levels of EMPs were detected by enzyme-linked immunosorbent assay (ELISA). The protein disulfide isomerase (PDI) activity was detected by insulin transhydrogenase assay. Platelet aggregation was assessed by turbidimetry. CONCLUSION: EMPs can promote the activation of GPIIb/IIIa in platelets and platelet aggregation by the PDI which is carried on the surface of EMPs.

Elevated Levels of Protein Disulfide Isomerase and Binding Immunoglobulin Protein Implicated in Spinal Cord Injury Paraplegia Patients with Pressure Ulcers.

AIMS: To explore the associations between two endoplasmic reticulum (ER) stress proteins, protein disulfide isomerase (PDI), binding immunoglobulin protein (BIP), and the development and progression of pressure ulcers (PUs) in spinal cord injury (SCI) paraplegia patients. METHODS: ELISA kits were used to measure the levels of serum PDI and BIP in 67 SCI paraplegia patients with PUs and 61 SCI paraplegia patients without PUs. The associations between PDI and BIP, PU formation, PU staging, and pressure ulcer scale for healing (PUSH) score were analyzed. RESULTS: The patients in the PU group had higher levels of PDI and BIP than those in the non-PU group (both p < 0.05). Furthermore, the levels of PDI were positively correlated with those of BIP (r = 0.707, p < 0.0001). There were significant differences in the PDI and BIP levels among the different stages of PU (all p < 0.05). As the PU stages progressed, the levels of PDI and BIP first increased, then decreased, and finally peaked at stage III of the PUs. The PUSH scores significantly declined 7 days after debridement for the PU stage II (p < 0.01) but showed no significant difference between stages III and IV at 7 days after debridement (p > 0.05). The PUSH scores also decreased at 28 days after debridement for stages II, III, and IV (all p < 0.01). Higher PUSH scores indicated a longer time of debridement accompanied by a longer wound surface healing time (p < 0.05). CONCLUSION: ER stress proteins may be involved in the process of PU formation and healing; moreover, the levels of PDI and BIP were also associated with the severity of the PUs. Finally, we found that the PUSH scores can be used as a reference to evaluate PU severity and healing.

Intracellular Trafficking, Localization, and Mobilization of Platelet-Borne Thiol Isomerases.

OBJECTIVE: Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation, and thrombus formation. In this study, we examined the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets, and identified the cellular events responsible for their movement to the platelet surface on activation. APPROACH AND RESULTS: Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages. Immunofluorescence microscopy and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell-surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and sarco/endoplasmic reticulum calcium ATPase 3. Immunofluorescence microscopy and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin) and in the presence or absence of membrane fusion mediated by Munc13-4 (absent in platelets from Unc13d(Jinx) mice). CONCLUSIONS: Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not soluble N-ethylmaleimide-sensitive fusion protein attachment receptor/Munc13-4-dependent vesicular-plasma membrane fusion.

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.

Proteome-wide survey of the autoimmune target repertoire in autoimmune polyendocrine syndrome type 1.

Autoimmune polyendocrine syndrome type 1 (APS1) is a monogenic disorder that features multiple autoimmune disease manifestations. It is caused by mutations in the Autoimmune regulator (AIRE) gene, which promote thymic display of thousands of peripheral tissue antigens in a process critical for establishing central immune tolerance. We here used proteome arrays to perform a comprehensive study of autoimmune targets in APS1. Interrogation of established autoantigens revealed highly reliable detection of autoantibodies, and by exploring the full panel of more than 9000 proteins we further identified MAGEB2 and PDILT as novel major autoantigens in APS1. Our proteome-wide assessment revealed a marked enrichment for tissue-specific immune targets, mirroring AIRE's selectiveness for this category of genes. Our findings also suggest that only a very limited portion of the proteome becomes targeted by the immune system in APS1, which contrasts the broad defect of thymic presentation associated with AIRE-deficiency and raises novel questions what other factors are needed for break of tolerance.