Increasing the sensitivity of the human microvesicle tissue factor activity assay. Issue 182 (October 2019)

• Record Type: Journal Article
• Authors: Vallier, Loris; Bouriche, Tarik; Bonifay, Amandine; Judicone, Coralie; Bez, Jeremy; Franco, Corentin; Guervilly, Christophe; Hisada, Yohei; Mackman, Nigel; Houston, Reaves; Poncelet, Philippe; Dignat-George, Françoise; Lacroix, Romaric
• Is Part Of: Thrombosis research. Issue 182(2019)
• Page Start: 64
• Page End: 74
• Publication Date: 2019-10
• Subjects: Microvesicles -- Extracellular vesicles -- Tissue factor -- Procoagulant test; Thrombosis -- Periodicals; 616.135
• DOI: 10.1016/j.thromres.2019.07.011
• Languages: English
• ISSNs: 0049-3848

Abstract

Abstract: Introduction: The TF-FVIIa complex is the primary activator of coagulation. Elevated levels of microvesicle (MV) bearing tissue factor (TF)-dependent procoagulant activity are detectable in patients with an increased risk of thrombosis. Several methods have been described to measure MV TF activity but they are hampered by limited sensitivity and specificity. The aim of this work was to increase the sensitivity of the MV TF activity assay (called Chapel Hill assay). Material and methods: Improvements of the MV TF activity assay included i/ speed and time of centrifugation, ii/ use of a more potent inhibitory anti-TF antibody iii/ use of FVII and a fluorogenic substrate to increase specificity. Results: The specificity of the MV TF activity assay was demonstrated by the absence of activity on MV derived from a knock-out-TF cell line using an anti-human TF monoclonal antibody called SBTF-1, which shows a higher TF inhibitory effect than the anti-human TF monoclonal antibody called HTF-1. Experiments using blood from healthy individuals, stimulated or not by LPS, or plasma spiked with 3 different levels of MV, demonstrated that the new assay was more sensitive and this allowed detection of MV TF activity in platelet free plasma (PFP) samples from healthy individuals. However, the assay was limited by an inter-assay variability, mainly due to the centrifugation step. Conclusions: We have improved the sensitivity of the MV TF activity assay without losing specificity. This new assay could be used to evaluate levels of TF-positive MV as a potential biomarker of thrombotic risk in patients. Highlights: Current methods to measure MV TF activity have limited sensitivity. An improved TF-dependent FXa generation assay was developed. The updated MV TF activity assay includes a new anti-TF inhibitory antibody (SBTF-1). The MV TF activity assay improves sensitivity as compared with previous test.