Proteomic Analysis Reveals Procoagulant Properties of Cigarette Smoke-induced Extracellular Vesicles

Overview

Journal J Extracell Vesicles

Publisher Wiley

Date 2019 Mar 14

PMID 30863515

Citations 25

Authors

Birke J Benedikter

Freek G Bouwman

Alexandra C A Heinzmann

Tanja Vajen

Edwin C Mariman

Emiel F M Wouters

Paul H M Savelkoul

Rory R Koenen

Gernot G U Rohde

Rene Van Oerle

Henri M Spronk

Frank R M Stassen

Affiliations

Soon will be listed here.

Abstract

Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed to cigarette smoke extract (CSE). Getting insights into the composition of these EVs will help unravel their functions in homeostasis and smoking-induced pathology. Here, we characterized the proteomic composition of basal and CSE-induced airway epithelial EVs. BEAS-2B cells were left unexposed or exposed to 1% CSE for 24 h, followed by EV isolation using ultrafiltration and size exclusion chromatography. Isolated EVs were labelled with tandem mass tags and their proteomic composition was determined using nano-LC-MS/MS. Tissue factor (TF) activity was determined by a factor Xa generation assay, phosphatidylserine (PS) content by prothrombinase assay and thrombin generation using calibrated automated thrombogram (CAT). Nano-LC-MS/MS identified 585 EV-associated proteins with high confidence. Of these, 201 were differentially expressed in the CSE-EVs according to the moderated -test, followed by false discovery rate (FDR) adjustment with the FDR threshold set to 0.1. Functional enrichment analysis revealed that 24 proteins of the pathway haemostasis were significantly up-regulated in CSE-EVs, including TF. Increased TF expression on CSE-EVs was confirmed by bead-based flow cytometry and was associated with increased TF activity. CSE-EVs caused faster and more thrombin generation in normal human plasma than control-EVs, which was partly TF-, but also PS-dependent. In conclusion, proteomic analysis allowed us to predict procoagulant properties of CSE-EVs which were confirmed . Cigarette smoke-induced EVs may contribute to the increased cardiovascular and respiratory risk observed in smokers.

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