Ginés Luengo Gil¹, Antonio Bernardino García Andreo², Carmen Ortega Sabater³, NATALIYA BOHDAN ⁴, SALVADOR ESPÍN GARCÍA⁵, JULIA PEÑAS MARTINEZ⁶, Elena Martínez Planés², Álvaro García Hernández², Vicente Vicente García⁶, Miguel Quintanilla⁷, IRENE MARTINEZ MARTINEZ⁶

(1) PATOLOGÍA MOLECULAR Y FARMACOGENÉTICA, IMIB, España
(2) Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Arrixaca, U-765, Center for Biomedical Research on Rare Diseases, España (Región de Murcia)
(3) Graduada/Colaboradora, 30110, España (Región de Murcia)
(4)
(5) FFIS, 30003, España (Región de Murcia)
(6) HEMATOLOGÍA Y ONCOLOGÍA MÉDICA CLÍNICO-EXPERIMENTAL, IMIB, España
(7) Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), España (Comunidad de Madrid)

PMID: 31445073
Referencia: Luengo-Gil G, García-Andreo AB, Ortega-Sabater C, Bohdan N, Espín S,Peñas-Martínez J, Martínez-Planes E, García-Hernández Á, Vicente V, QuintanillaM, Martínez-Martínez I. Antithrombin is incorporated into exosomes produced byantithrombin non-expressing cells. Biochimie. 2019 Oct;165:245-249. doi:10.1016/j.biochi.2019.08.010. Epub 2019 Aug 21. PubMed PMID: 31445073.
ISSN: 0300-9084
Revista: BIOCHIMIE
Factor de impacto (2017): 3.9
Cuartil: 2

Exosomes are 30-240 nm vesicles of endocytic origin which are produced by normal and tumor cells. They mediate cell-cell communication regulating processes such as inflammation and metastasis. They content proteins and different species of RNA. By proteomic analysis of exosomes from MDCK cells (canine kidney epithelial cells) it was found that antithrombin was present. Antithrombin is a serin protease inhibitor that exerts a crucial role in hemostasis as the main inhibitor of the coagulation cascade. However, it has been shown that antithrombin has other roles beyond hemostasis, such as an anti-inflammatory, anti-angiogenic, anti-apoptotic, anti-viral and anti-tumor protein.

Cell culture, western blot, exosome isolation, immunoprecipitation, peptide mass fingerprinting, protein purification, qRT-PCR.

Here we show that antithrombin present in exosomes from MDCK cells came from fetal bovine serum and heparin increased its capture. Functional antithrombin was establishing a covalent complex with a protein on the surface of the exosome. Peptide mass fingerprinting suggested that antithrombin was interacting with HTRA1, a serine protease with a variety of targets. We have demonstrated that HTRA1 is overexpressed in MDCK cells when unfolded protein response (UPR) is activated and that UPR happens when exosome secretion is stimulated. Our results support that interaction between antithrombin and HTRA1 could be established only in the context of UPR since purified proteins are unable to establish a covalent complex. The implications of the interaction between HTRA1 and antithrombin are still to be discovered, but our results suggest that addition of heparin should improve the control of the function of HTRA1.

Exosomal antithrombin is found complexed with the serine protease high temperature requirement A1 (HTRA1), whose cellular levels are increased after serum deprival, the condition used to collect exosomes. Although the biological relevance of the presence of antithrombin in exosomes remains to be investigated, our results suggest a functional interplay between antithrombin and HTRA1.