APC Inactivation of Factor Va Analysis

APC-mediated inactivation of FVa was analyzed in pro-thrombinase assays as described previously [12 (link)]. FVa mutants (4 nmol L⁻¹) were incubated with 2 nmol L⁻¹ wild-type recombinant human APC (Xigris®; Eli Lilly and Co., Indianapolis, IN, USA) or wild-type recombinant mouse APC [22 (link)] and 25 µmol L⁻¹ phospholipid vesicles in prothrombinase buffer, and 1-µL aliquots were removed at specified time points. Residual FVa was determined using the prothrombinase assay as described earlier. Alternatively, FV- or FVIII-deficient plasma was reconstituted with FVa (1 nmol L⁻¹), and thrombin generation was determined in ETP assays in the presence of increasing concentrations of APC. For Western blot analysis, FVa was incubated with 2 nmol L⁻¹ APC for 30 min at 37 °C. FVa inactivation fragments were resolved on 4–12% Bis-Tris gels (Invitrogen, Carlsbad, CA, USA) and detected using a polyclonal rabbit antihu-man FV heavy chain antibody (1:3500; Pierce, Rockford, IL, USA) and goat anti-rabbit-800CW secondary antibody (LI-COR; 1:10 000). Bands were visualized using the Odyssey infrared imager (LI-COR Biosciences, Lincoln, NE, USA).