Proteomic analysis reveals procoagulant properties of cigarette smoke-induced extracellular vesicles

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

doi:10.1080/20013078.2019.1585163

Proteomic analysis reveals procoagulant properties of cigarette smoke-induced extracellular vesicles

J Extracell Vesicles. 2019 Mar 5;8(1):1585163. doi: 10.1080/20013078.2019.1585163. eCollection 2019.

Authors

Affiliations

¹ Department of Medical Microbiology, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, Maastricht, The Netherlands.
² Department of Respiratory Medicine, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, Maastricht, The Netherlands.
³ Department of Human Biology, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, Maastricht, The Netherlands.
⁴ Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands.
⁵ Department of Medical Microbiology & Infection Control, VU University Medical Center, Amsterdam, The Netherlands.
⁶ Medical clinic I, Department of Respiratory Medicine, Goethe University Hospital, Frankfurt/Main, Germany.

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 t-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 in vitro. Cigarette smoke-induced EVs may contribute to the increased cardiovascular and respiratory risk observed in smokers.

Keywords: Exosomes; chronic lung disease; hypercoagulability; respiratory exposure; thrombosis.

Grants and funding

This project was supported by the Netherlands Organization for Scientific Research (NWO) under grant number 022.003.011 and by a Kootstra Talent Fellowship from the Center for Research Innovation, Support and Policy (CRISP) of Maastricht University Medical Center +, both awarded to BJB. It was partly supported by the NWO grant ZonMW VIDI under the grant number 016.126.358 awarded and by the Landsteiner Foundation for Blood Transfusion Research grant number LSBR Nr. 1638, both awarded to RRK.