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

ABSTRACT: 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?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.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.EMPs can promote the activation of GPIIb/IIIa in platelets and platelet aggregation by the PDI which is carried on the surface of EMPs.

SUBMITTER: Fan GQ

PROVIDER: S-EPMC5347765 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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

Fan Guan-Qi GQ Qin Ran-Ran RR Li Yi-Hui YH Song Dai-Jun DJ Chen Tong-Shuai TS Zhang Wei W Zhong Ming M Zhang Yun Y Xing Yan-Qiu YQ Wang Zhi-Hao ZH

Oncotarget 20161201 50

<h4>Background</h4>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?<h4>Results</h4>We de ...[more]

PMID: 27825126

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Project description:BACKGROUND:Platelet-neutrophil interactions contribute to vascular occlusion and tissue damage in thromboinflammatory disease. Platelet glycoprotein Ib? (GPIb?), a key receptor for the cell-cell interaction, is believed to be constitutively active for ligand binding. Here, we established the role of platelet-derived protein disulfide isomerase (PDI) in reducing the allosteric disulfide bonds in GPIb? and enhancing the ligand-binding activity under thromboinflammatory conditions. METHODS:Bioinformatic analysis identified 2 potential allosteric disulfide bonds in GPIb?. Agglutination assays, flow cytometry, surface plasmon resonance analysis, a protein-protein docking model, proximity ligation assays, and mass spectrometry were used to demonstrate a direct interaction between PDI and GPIb? and to determine a role for PDI in regulating GPIb? function and platelet-neutrophil interactions. Also, real-time microscopy and animal disease models were used to study the pathophysiological role of PDI-GPIb? signaling under thromboinflammatory conditions. RESULTS:Deletion or inhibition of platelet PDI significantly reduced GPIb?-mediated platelet agglutination. Studies using PDI-null platelets and recombinant PDI or Anfibatide, a clinical-stage GPIb? inhibitor, revealed that the oxidoreductase activity of platelet surface-bound PDI was required for the ligand-binding function of GPIb?. PDI directly bound to the extracellular domain of GPIb? on the platelet surface and reduced the Cys4-Cys17 and Cys209-Cys248 disulfide bonds. Real-time microscopy with platelet-specific PDI conditional knockout and sickle cell disease mice demonstrated that PDI-regulated GPIb? function was essential for platelet-neutrophil interactions and vascular occlusion under thromboinflammatory conditions. Studies using a mouse model of ischemia/reperfusion-induced stroke indicated that PDI-GPIb? signaling played a crucial role in tissue damage. CONCLUSIONS:Our results demonstrate that PDI-facilitated cleavage of the allosteric disulfide bonds tightly regulates GPIb? function, promoting platelet-neutrophil interactions, vascular occlusion, and tissue damage under thromboinflammatory conditions.

Project description:BACKGROUND: Protein disulfide isomerase (PDI) is required for thrombus formation. We previously demonstrated that glycosylated quercetin flavonoids such as isoquercetin inhibit PDI activity and thrombus formation in animal models, but whether extracellular PDI represents a viable anticoagulant target in humans and how its inhibition affects blood coagulation remain unknown. METHODS: We evaluated effects of oral administration of isoquercetin on platelet-dependent thrombin generation in healthy subjects and patients with persistently elevated anti-phospholipid antibodies. RESULTS: Following oral administration of 1,000 mg isoquercetin to healthy adults, the measured peak plasma quercetin concentration (9.2 ?M) exceeded its IC50 for inhibition of PDI by isoquercetin in vitro (2.5 ± 0.4 ?M). Platelet-dependent thrombin generation decreased by 51% in the healthy volunteers compared with baseline (P = 0.0004) and by 64% in the anti-phospholipid antibody cohort (P = 0.015) following isoquercetin ingestion. To understand how PDI affects thrombin generation, we evaluated substrates of PDI identified using an unbiased mechanistic-based substrate trapping approach. These studies identified platelet factor V as a PDI substrate. Isoquercetin blocked both platelet factor Va and thrombin generation with an IC50 of ~5 ?M. Inhibition of PDI by isoquercetin ingestion resulted in a 53% decrease in the generation of platelet factor Va (P = 0.001). Isoquercetin-mediated inhibition was reversed with addition of exogenous factor Va. CONCLUSION: These studies show that oral administration of isoquercetin inhibits PDI activity in plasma and diminishes platelet-dependent thrombin generation predominantly by blocking the generation of platelet factor Va. These pharmacodynamic and mechanistic observations represent an important step in the development of a novel class of antithrombotic agents targeting PDI. TRIAL REGISTRATION: Clinicaltrials.gov (NCT01722669) FUNDING: National Heart, Lung, and Blood Institute (U54 HL112302) and Quercegen Pharma.

Project description:OBJECTIVE:Varicella zoster virus (VZV) can spread anterogradely and infect cerebral arteries causing VZV vasculopathy and arterial ischemic stroke. In this study, we tested the hypothesis that virus-infected cerebrovascular fibroblasts undergo phenotypic changes that promote vascular remodeling and facilitate virus transmission in an in vitro model of VZV vasculopathy. The aims of this project were therefore to examine the changes that virus-infected human brain adventitial vascular fibroblasts (HBVAFs) undergo in an in vitro model of VZV vasculopathy and to identify disease biomarkers relating to VZV-related vasculopathy. METHODS:HBVAFs were infected with VZV, and their ability to migrate, proliferate, transdifferentiate, and interact with endothelial cells was studied with flow cytometry. Microparticles (MPs) released from these cells were isolated and imaged with transmission electron microscopy, and their protein content was analyzed with mass spectrometry. Circulating MP profiles were also studied in children with VZV and non-VZV vasculopathy and compared with controls. RESULTS:VZV-infected HBVAFs transdifferentiated into myofibroblasts with enhanced proliferative and migratory capacity. Interaction of VZV-infected HBVAFs with endothelial cells resulted in endothelial dysfunction. These effects were, in part, mediated by the release of MPs from VZV-infected HBVAFs. These MPs contained VZV virions that could transmit VZV to neighboring cells, highlighting a novel model of VZV cell-to-cell viral dissemination. MPs positive for VZV were significantly higher in children with VZV-related vasculopathy compared to children with non-VZV vasculopathy (p = 0.01) and controls (p = 0.007). CONCLUSIONS:VZV-infected HBVAFs promote vascular remodeling and facilitate virus transmission. These effects were mediated by the release of apoptotic MPs that could transmit VZV infection to neighboring cells through a Trojan horse means of productive viral infection. VZV+ MPs may represent a disease biomarker worthy of further study.

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

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

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