title: Thrombin generation assay and transmission electron microscopy: a useful combination to study tissue factor-bearing microvesicles.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  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°C. 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 µm membranes and subjected to TGA.
  
  
  RESULTS
  The majority of the PCA was associated with microvesicles smaller than 0.1 µm, and the mean microvesicle size estimated by TEM after 10,000 g centrifugation was 121±54 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.
authors.affiliation: Department of Pharmacy, Namur Research Institute for LIfe Sciences, Namur Thrombosis and Hemostasis Center (NTHC), University of Namur, Belgium ; Haematology Laboratory, Namur Research Institute for LIfe Sciences, Namur Thrombosis and Hemostasis Center (NTHC), CHU Mont-Godinne, Université Catholique de Louvain, Belgium.
authors.forename: Damien
authors.initials: D
authors.lastname: Gheldof
authors.forename: Julie
authors.initials: J
authors.lastname: Hardij
authors.forename: Francesca
authors.initials: F
authors.lastname: Cecchet
authors.forename: Bernard
authors.initials: B
authors.lastname: Chatelain
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Dogné
authors.forename: François
authors.initials: F
authors.lastname: Mullier
keywords: cancer; microvesicles; thrombin generation; tissue factor