%0 Journal Article
%T Thrombin generation assay and transmission electron microscopy: a useful combination to study tissue factor-bearing microvesicles
%A Damien Gheldof
%A Julie Hardij
%A Francesca Cecchet
%A Bernard Chatelain
%J Journal of Extracellular Vesicles
%D 2013
%I Co-Action Publishing
%R 10.3402/jev.v2i0.19728
%X Introduction. Patients with cancer have a 7- to 10-fold increased risk of developing venous thromboembolism. Circulating microvesicles could be a useful predictive biomarker for venous thromboembolism in cancer. Validated and standardised techniques that could be used to determine the complete microvesicle phenotype are required. These were two-fold: a) to characterise tissue factor (TF)-bearing microvesicles released by cultured breast cancer cells MDA-MB-231 by flow cytometry (FCM), transmission electron microscopy (TEM) and thrombin generation assay (TGA); and b) to validate the sensitivity and variability intra/inter-assay of TGA as a useful method to study the procoagulant activity (PCA) of microvesicles. Methods. Cultured breast cancer cells MDA-MB-231 were incubated for 45 minutes at 37¡ãC. Samples were then centrifuged or not at 4,500 g for 15 minutes, and cells and MVs or MV-containing supernatants were used for TEM, FCM and TGA. In activity assays, microvesicles (i.e. cell-depleted supernatants) were incubated with anti-TF antibodies or with annexin V to assess the contribution of TF and phospholipids to the PCA. Alternatively, supernatants were filtered through 0.1, 0.22, 0.45 or 0.65 ¦Ìm membranes and subjected to TGA. Results. The majority of the PCA was associated with microvesicles smaller than 0.1 ¦Ìm, and the mean microvesicle size estimated by TEM after 10,000 g centrifugation was 121¡À54 nm with a majority of vesicles between 100 and 200 nm. Microvesicles derived from 5,000 MDA-MB-231cells/ml were sufficient to significantly increase the thrombin generation of normal pooled plasma. Conclusions. TEM, FCM and filtration coupled to TGA represent a useful combination to study the PCA of TF-bearing microvesicles, whatever their size. And it will be interesting to implement these techniques in patients.
%K tissue factor
%K microvesicles
%K cancer
%K thrombin generation
%U http://www.journalofextracellularvesicles.net/index.php/jev/article/view/19728/26504