Human thrombin

APC generation assays were carried out as previously described.39 MLEC from Bambi^−/− mice and wild‐type littermates were isolated in parallel and grown to confluency. Cells were washed twice in Hanks buffered salt solutionHBSS and once in assay buffer (20 mM Tris, 100 mM NaCl, 1 mM CaCl₂, 0.1% bovine serum albumin pH 7.5) prior to addition of 100 nM recombinant human protein C (see later discussion) together with 2 nM human thrombin (Enzyme Research Laboratories, Swansea, UK). The APC activation occurred at 37 °C and reactions were stopped (0‐60 min) by addition of hirudin (100 nM; Sigma). Control cells were preincubated with 50 nM goat anti‐ thrombomodulin (R&D, Bio‐Techne) antibody for 30 min to block endogenous thrombomodulin and washed three times before addition of protein C. The APC was quantified by cleavage of the chromogenic substrate S‐2366 (0.5 mM; Quadratech Diagnostics for Chromogenix, Epsom, UK). The rate of cleavage was followed for 20 min at 37 °C using an EL x808 plate reader (Perkin Elmer) and the concentration of APC extrapolated from a standard curve of purified human APC (Cambridge Bioscience for Haematologic Technologies Inc., Cambridge, UK). Control experiments lacking thrombin or protein C were performed to demonstrate the specificity of the substrate cleavage.

Anticoagulated whole blood (EDTA 30 mmol/L pH8) was separated into plasma and cells by centrifugation (14 000g for 10 minutes). Plasma TNFα, IFN‐γ, MIF, and CCL2 were detected using separate specific ELISA kits (R&D Systems, Abingdon, UK) according to the manufacturers’ instructions. Total cholesterol, high‐density lipoprotein and low‐density lipoprotein were determined using kits from Cell Biolabs, and triglycerides with a kit from Abcam, (both Cambridge, UK) according to the manufacturer's protocol. Data were derived from triplicate analysis of each sample.
Thrombin clotting times were measured in 3.2% trisodium citrated plasma according to the protocol of Ignjatovic.16 Briefly, 100 μL mouse plasma was incubated with 2.5 U of human thrombin in a total volume of 300 μL (Enzyme Research Laboratories, Swansea, UK) at 37°C, and the time for a clot to form was measured (n=6 per group). For some experiments plasma was further centrifuged (20 000g for 10 minutes) to minimize the presence of extracellular vesicles.