Short communicationAntithrombin is incorporated into exosomes produced by antithrombin non-expressing cells
Introduction
Extracellular vesicles carrying proteins, lipids, metabolites, RNA and DNA, are cell-secreted vesicles that are involved in intercellular communication [1]. They play crucial roles in inflammation and cancer and are regarded as ideal biomarkers for different diseases [2]. There are two main types of exovesicles: exosomes (30–150 nm) that originate in the endocytic pathway and are released after fusion of multivesicular bodies with the plasma membrane, and microvesicles (100–1000 nm) that form directly by plasma membrane budding [1,2].
Antithrombin is a plasma serpin that upon binding to heparin acts as the main inhibitor of the coagulation proteases [3]. In addition to its function in hemostasis, antithrombin has been found to exert anti-inflammatory, anti-angiogenic, anti-apoptotic, anti-viral and anti-tumor roles [4]. Proteomic studies have identified antithrombin as a component of exosomes from different cell types (www.exocarta.org). However, little is known about the origin and functional relevance of exosomal antithrombin.
In this work, we have identified serum antithrombin incorporated into exosomes from Madin-Darby canine kidney (MDCK) cells, which do not synthesize this protein. Exosomal antithrombin was found complexed with serine protease high temperature requirement A1 (HTRA1).
Section snippets
Cell culture, isolation of exosomes and western blotting
Cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) as reported previously [5] in the presence of 200 U of low molecular weight heparin (LMWH). Exosomes and microvesicles (MVs) were isolated from the conditioned medium (48 h) as previously described [5]. For more details see supplementary information. Vesicles washed in cold PBS were lysed in SDS-loading buffer, subjected to ultracentrifugation, and supernatants processed for immunoblotting (or frozen at −80 °C). For
Results and discussion
In a previous study, we identified the presence of bovine antithrombin in the proteome of exosomes released by MDCK cells [5]. This was confirmed by western blotting. Under reducing conditions, a main band of ∼55 kDa was detected corresponding to “free” antithrombin [6] together with a less abundant larger band of ∼75 kDa compatible with the formation of a covalent complex between antithrombin and an unknown protein (Fig. 1A). Interestingly, the band of ∼75 kDa corresponding to complexed
Conclusion
We show in this article that antithrombin can be found incorporated into exosomes produced by antithrombin non-expressing cells. In the case of MDCK cells, bovine antithrombin from the fetal serum used in the culture medium was internalized by the cells in the presence of heparin, likely by endocytosis through interaction with an unknown membrane receptor, and later incorporated into exosomes where it appears to interact covalently with HTRA1.
Conflict of interest
The authors declare no competing financial interest.
Acknowledgements
Ginés Luengo-Gil holds a grant from the Spanish Society of Hematology and Hemotherapy (SEHH-FEHH), Irene Martínez-Martínez holds a Miguel Servet contract from the ISCIII. Miguel Quintanilla holds a grant (SAF2017-84183-R) from the Spanish Ministry of Science, Innovation and Universities and Irene Martínez-Martínez holds grants from ISCIII (CP13/00126 & FEDER and PI17/00050 & FEDER).
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