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

Research output: Contribution to journalArticleAcademicpeer-review

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.
Original languageEnglish
Article number1585163
JournalJournal of Extracellular Vesicles
Volume8
Issue number1
DOIs
Publication statusPublished - 2019

Cite this

Benedikter, B. J., Bouwman, F. G., Heinzmann, A. C. A., Vajen, T., Mariman, E. C., Wouters, E. F. M., ... Stassen, F. R. M. (2019). Proteomic analysis reveals procoagulant properties of cigarette smoke-induced extracellular vesicles. Journal of Extracellular Vesicles, 8(1), [1585163]. https://doi.org/10.1080/20013078.2019.1585163
Benedikter, Birke J. ; Bouwman, Freek G. ; Heinzmann, Alexandra C. A. ; Vajen, Tanja ; Mariman, Edwin C. ; Wouters, Emiel F. M. ; Savelkoul, Paul H. M. ; Koenen, Rory R. ; Rohde, Gernot G. U. ; van Oerle, Rene ; Spronk, Henri M. ; Stassen, Frank R. M. / Proteomic analysis reveals procoagulant properties of cigarette smoke-induced extracellular vesicles. In: Journal of Extracellular Vesicles. 2019 ; Vol. 8, No. 1.
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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.",
author = "Benedikter, {Birke J.} and Bouwman, {Freek G.} and Heinzmann, {Alexandra C. A.} and Tanja Vajen and Mariman, {Edwin C.} and Wouters, {Emiel F. M.} and Savelkoul, {Paul H. M.} and Koenen, {Rory R.} and Rohde, {Gernot G. U.} and {van Oerle}, Rene and Spronk, {Henri M.} and Stassen, {Frank R. M.}",
year = "2019",
doi = "10.1080/20013078.2019.1585163",
language = "English",
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journal = "Journal of Extracellular Vesicles",
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Benedikter, BJ, Bouwman, FG, Heinzmann, ACA, Vajen, T, Mariman, EC, Wouters, EFM, Savelkoul, PHM, Koenen, RR, Rohde, GGU, van Oerle, R, Spronk, HM & Stassen, FRM 2019, 'Proteomic analysis reveals procoagulant properties of cigarette smoke-induced extracellular vesicles' Journal of Extracellular Vesicles, vol. 8, no. 1, 1585163. https://doi.org/10.1080/20013078.2019.1585163

Proteomic analysis reveals procoagulant properties of cigarette smoke-induced extracellular vesicles. / Benedikter, Birke J.; Bouwman, Freek G.; Heinzmann, Alexandra C. A.; Vajen, Tanja; Mariman, Edwin C.; Wouters, Emiel F. M.; Savelkoul, Paul H. M.; Koenen, Rory R.; Rohde, Gernot G. U.; van Oerle, Rene; Spronk, Henri M.; Stassen, Frank R. M.

In: Journal of Extracellular Vesicles, Vol. 8, No. 1, 1585163, 2019.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Proteomic analysis reveals procoagulant properties of cigarette smoke-induced extracellular vesicles

AU - Benedikter, Birke J.

AU - Bouwman, Freek G.

AU - Heinzmann, Alexandra C. A.

AU - Vajen, Tanja

AU - Mariman, Edwin C.

AU - Wouters, Emiel F. M.

AU - Savelkoul, Paul H. M.

AU - Koenen, Rory R.

AU - Rohde, Gernot G. U.

AU - van Oerle, Rene

AU - Spronk, Henri M.

AU - Stassen, Frank R. M.

PY - 2019

Y1 - 2019

N2 - 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.

AB - 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.

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UR - https://www.ncbi.nlm.nih.gov/pubmed/30863515

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DO - 10.1080/20013078.2019.1585163

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