Human prothrombin

Cells were seeded in a Nunc UpCell 12 Multidish (Thermo Fisher Scientific, MA, USA) at a concentration of 0.5 × 10⁶ cells per well. To mimic the “two-hit” hypothesis, cells were stimulated with 25 ng/mL lipopolysaccharide (LPS from E. Coli O111:B4, Sigma Aldrich, MO, USA), 100 µg/mL IgG fractions isolated from serum of a patient with CAPS (aCL-, anti-β2GPI-, and aPS/PT-positive) or from sera of a HC, and 45 µg/mL of human prothrombin (Enzyme Research Laboratories, IN, USA). Final concentrations were determined after titration of the reagents.

This was performed following previously described protocol and validated method [11 (link)]. Specifically, the assays average inter- and intra-assay coefficients of variations were <3.3% and <8.2%, respectively. The diagnostic specificity for APS was 92.5% and the diagnostic sensitivity was 59.0%. The diagnostically relevant cut-off of aPS/PT was set on the 99th percentile of 222 blood donors. Briefly, phosphatidylserine was coated on polystyrene microtitre plates (medium binding, Costar, Cambridge, MA, USA) and incubated overnight at 4°C. After blocking with Tris-buffered saline (TBS) containing 1% bovine serum albumin (BSA) and 5 mM CaCl₂ (1% BSA-TBS-Ca⁺⁺) plates were washed in TBS containing 0.05% Tween-20. Human prothrombin (10 mg/L) (Enzyme Research Laboratories, UK) and patients' sera diluted 1 : 100 in 1% BSA-TBS-Ca⁺⁺ were applied to wells immediately one after the other and incubated for 1 hour at room temperature (RT). Afterwards, plates were washed and incubated with alkaline phosphatase-conjugated goat anti-human IgG or IgM (ACSC, Westbury, USA) for 30 minutes at RT. After the last wash para-nitrophenylphosphate (Sigma Chemical Company, USA) in diethanolamine buffer (pH 9.8) was applied as substrate and OD₄₀₅ was kinetically measured by microtitre plate reader (Tecan, Grödig, Austria).