Abstract Number: PB0224
Meeting: ISTH 2020 Congress
Background: Most TF activity assays are mainly been based on FX activation by TF in the presence of FVII/FVIIa, a system dependent on 1-2 h incubation followed by measurement of formed FXa in a chromogenic assay. As FVIIa in the presence of phospholipids also has the ability to active FX, the long incubation-times may allow expression of non-TF dependent activities which are not blocked by anti-TF antibodies.
Aims: The aim of this work was to develop a specific and sensitive TF activity assay based on short incubation times of purified clotting factors to avoid non-specific TF activities.
Methods: The TF activity was measured in a two-stage amidolytic assay based on the ability of TF to accelerate the activation of FX by FVIIa, followed by the conversion of prothrombin to thrombin by FXa in the presence of FV/Va and phospholipids. Purified factors, FVIIa, FX, FV/Va, prothrombin, phospholipids and the test sample along with Ca2+ were incubated for 4 min at 37 oC. Thrombin generation was quantified by a chromogenic end point assay.
Results: Except for a small background caused by traces of FXa in the FX reagent, the TF activity assay proved high specificity assessed by neutralization of all TF activity by anti-TF antibody. However, the background activity increased with increasing incubaton times. The specificity was further demonstrated by the lack of TF activity in EVs from a knock-out-TF cell line, HAP1/F3, as EVs had no more activity than the control. In contrast, EVs derived from HAP1 cells showed high concentration of TF activity that was totally blocked by anti-TF antibodies. The sensitivity of the assay was in the low fM range.
Conclusions: Our TF activity assay, based on short incubation and thrombin generation, provides a fast, sensitive and specific measurement of TF activity in cell-derived EVs with applicability for laboratory- and clinical research.
To cite this abstract in AMA style:Østerud B, Latysheva N, Snir O, Hansen J-. A Sensitive and Specific Assay to Measure TF Activity in Cell-derived Extracellular Vesicles Based on Thrombin Generation [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/a-sensitive-and-specific-assay-to-measure-tf-activity-in-cell-derived-extracellular-vesicles-based-on-thrombin-generation/. Accessed March 3, 2021.
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