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Characterization of physical properties of tissue factor-containing microvesicles and a comparison of ultracentrifuge-based recovery procedures

Ettelaie, Camille; Collier, Mary; Maraveyas, Anthony; Ettelaie, Rammile

Authors

Rammile Ettelaie

Mary E. W. Collier

Mary E W Collier

Abstract

© 2014 Camille Ettelaie et al. Microvesicles were isolated from the conditioned media of 3 cell lines (MDA-MB-231, AsPC-1 and A375) by ultracentrifugation at a range of relative centrifugal forces, and the tissue factor (TF) protein and activity, microvesicle number, size distribution and relative density compared. Also, by expressing TF-tGFP in cells and isolating the microvesicles, the relative density of TF-containing microvesicles was established. Nanoparticle tracking analysis (NTA) indicated that the larger-diameter microvesicles ( > 200 nm) were primarily sedimented at 100,000g and possessed TF-dependent thrombin and factor Xa generation potential, while in the absence of factor VII, all microvesicles possessed some thrombin generation capacity. Immunoprecipitation of TF-containing microvesicles followed by NTA also indicated the range of these microvesicles to be 200-400 nm. Analysis of the microvesicles by gradient density centrifugation showed that lower-density ( < 1.1 g/ml) microvesicles were mainly present in the samples recovered at 100,000g and were associated with TF antigen and activity. Analysis of these fractions by NTA confirmed that these fractions were principally composed of the larger-diameter microvesicles. Similar analysis of microvesicles from healthy or patient plasma supported those obtained from conditioned media indicating that TF activity was mainly associated with lower-density microvesicles. Furthermore, centrifugation of healthy plasma, supplemented with TFtGFP- containing microvesicles, resulted in 67% retrieval of the fluorescent microvesicles at 100,000g, but only 26% could be recovered at 20,000g. Pre-centrifugation of conditioned media or plasma at 10,000g improved the speed and yield of recovered TF-containing microvesicles by subsequent centrifugation at either 20,000g or 100,000g. In conclusion, TF appears to be associated with low-density (1.03-1.08 g/ml), larger-diameter (200-350 nm) microvesicles.

Journal Article Type Article
Publication Date Jan 1, 2014
Journal Journal of extracellular vesicles
Print ISSN 2001-3078
Electronic ISSN 2001-3078
Publisher Taylor & Francis Open
Peer Reviewed Peer Reviewed
Volume 3
Issue 1
Pages 1-14
DOI https://doi.org/10.3402/jev.v3.23592
Keywords Tissue factor, Microvesicles, Ultracentrifugation, Size-distribution, Relative density, Thrombin generation activity
Publisher URL https://www.tandfonline.com/doi/full/10.3402/jev.v3.23592
Copyright Statement This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 3.0 Unported License (http://creativecommons.org/licenses/by-nc/3.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Additional Information Copy of article first published in: Journal of extracellular vesicles, 2014, v.3

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