Quantification of Procoagulant Microvesicles

MV-TF activity and MV-FXa generation was measured with an adapted method from Wang et al [24 (link)]. First, 600 μL plasma was diluted in 1 mL HBSA buffer (137 mM NaCl, 5.38 mM KCl, 5.55 mM glucose, 10 mM HEPES, 0.1% (w/v) bovine serum albumin, pH 7.4) and centrifuged at 20,000 × g for 15 minutes at 4°C in order to pellet microvesicles. The pellets were washed once in 1 mL HBSA and resuspended in 180 μL HBSA. The samples were then incubated with monoclonal mouse anti-CD142 antibody (clone HTF-1, BD Pharmingen) or control IgG from mouse serum (Sigma-Aldrich) for 15 minutes at room temperature in a 96-well plate. After incubation, 50 μL HBSA containing 10 mM CaCl_2, 73 nM FX (Enzyme Research Laboratories, South Bend, IN, USA), and 2.4 nM factor VIIa (Enzyme Research Laboratories) was added to each sample and incubated for two hours at 37°C. The reaction was stopped by addition of 25 μL HBSA containing 25 mM EDTA. Then, 25 μL of 4 mM chromogenic Pefachrome FXa 8595 (Pentapharm, Basel, Switzerland) was added to the wells and incubated at 37°C for 15 minutes. The plate was read at absorbance 405 nm on a Fluostar Optima (BMG Labtech, Ortenberg, Germany). Innovin (Siemens Healthcare, Erlangen, Germany) was used to generate a standard curve to calculate the procoagulant activity of microvesicles.