Innovin

TF-induced thrombin generation time was performed, as previously described10 (link)70 (link). Briefly, a 1:2 dilution of mouse plasma was incubated with ∼3 pM TF (3 μl of 1:60 dilution of stock Innovin, Siemens) and 0.42 mM Z-Gly-Gly-Arg-AMC. The reaction was initiated with the injection of 0.16 M calcium chloride, final concentration 16 mM. Substrate hydrolysis was measured on a fluorescent plate reader (NOVOstar, BMG Labtech). The thrombin generation data are expressed as an arbitrary rate of fluorescent accumulation as determined by the second derivative of the raw fluorescent values. The lag time, peak height and total area under the curve were calculated using Prism software (Graphpad, San Diego, CA).

To determine the effect of apixaban on PT and APTT levels, each sample was tested using Innovin (PT) and Actin FS (APTT) reagents (Siemens Healthcare Diagnostics). Plasma apixaban concentration was assessed using a chromogenic anti-factor Xa activity (AXA) assay, the Berichrom Heparin Assay (Dade Behring, Marburg, Germany) as this is the most reliable method to measure the pharmacodynamics of apixaban [21 (link)].

ETP was measured in platelet-poor plasma using a commercially available assay (Siemens, Marburg, Germany) in a BCS-XP System (Siemens, Marburg, Germany) according to manufacturer instructions. Coagulation activation was initiated by incubation of plasma with phospholipids, human recombinant tissue factor (Innovin; Siemens, Marburg, Germany), and calcium ions in the absence of thrombomodulin. The concentration of phospholipids and tissue factor is confidential to the manufacturer. Thrombin generation and subsequent inactivation was recorded by monitoring conversion of a specific slow reacting chromogenic substrate at a wavelength of 405 nm over time.

One vial of Innovin® (recombinant human TF: rTF) from Siemens Healthcare Diagnostics, Marburg, Germany was reconstituted with 10 mL distilled H₂O (theoretical concentration 6000 pmol/L according to the manufacturer) and then kept at − 20 °C in small aliquots. A purified phospholipid mixture (PPL, 500 µM) from Rossix, Mölndal, Sweden, was kept at 4–8 °C. Recombinant tissue-type plasminogen activator (rt-PA; Actilyse®) from Boehringer Ingelheim, Germany, was prepared (1 mg/mL) with the manufacturer’s solvent and kept in small aliquots at − 70 °C.

TF activity of EVs dervied from HUVEC stimulated with and without TNF-α was assessed as described elsewhere^{35 (link)}. Briefly, 50 µl of all centrifugation samples (S0.5, P14, S14, P100, S100) were incubated with either mouse anti-human TF antibody (American Diagnostica, Lexington, USA) or mouse IgG-control (Sigma Aldrich, St. Louis, MO, USA) for 15 min at room temperature. Subsequently, samples were incubated with 50 µl of HBSA containing 10 mM CaCl₂, 10 nM Factor VIIa (CoaChrom Diagnostica GmbH, Austria) and 150 nM Factor X (CoaChrom Diagnostica GmbH, Austria) for 2 hours at room temperature. The reaction was stopped with 25 µl HBSA containing 25 mM EDTA and samples were incubated with 25 µl chromogenic substrate (Pefachrome® FXa-8595, Pentapharm, Basel, Switzerland) for 15 min at 37 °C, and absorbance was measured at a wavelength of 405 nm. Samples were measured in duplicates and EGM-2, which was not incubated with cells, as well as PBS served as negative controls. TF activity was calculated by reference to a standard curve generated using relipidated rh TF (Innovin®, Siemens Healthcare Diagnostics, Austria). The TF-dependent FXa generation was determined by subtracting the amount of FXa generated in the presence of anti-TF antibody from the amount of FXa generated in the presence of the isotype control antibody.