SDS-PAGE analysis of recombinant PC derivatives and their characterization in amidolytic, plasma-based clotting, and purified systems. (A) Recombinant PC-WT and PC-T315A (lanes 1 and 2, respectively) were fractionated on a 10% SDS-PAGE under nonreducing and reducing conditions (lanes 3 and 4, respectively). Lane 5 represents molecular mass standards in kDa. SC, single chain; HC-α, heavy chain α; HC-β, heavy chain β; LC, light chain. (B) Amidolytic activity of APC-WT (○) and APC-T315A (●) (5 nM each) toward the chromogenic substrate SpPCa. (C) Plasma clotting activity of APC-WT (○) and APC-T315A (●) were determined as a function of increasing concentrations of proteases (0-20 nM) at 37°C as described in “Materials and methods.” (D-E) FVa (2.5 nM) degradation by APC-WT (○) and APC-T315A (●) in the absence (D, 10 minutes) and presence (E, 1 minute) of protein S (110 nM) was analyzed by incubating increasing concentrations of each protease with the cofactor on PC/PS vesicles (25 μM) in TBS/Ca²⁺ in a 96-well assay plate. The remaining cofactor activity of FVa was determined by a prothrombinase (1 nM FXa and 1 μM prothrombin for 1 minute) assay as described in “Materials and methods.” (F-G) FVIIIa (20 nM) degradation by APC-WT (○) and APC-T315A (●) in the absence (F, 30 minutes) and presence (G, 5 minutes) of protein S (110 nM) was analyzed by incubating increasing concentrations of each protease with the cofactor on PC/PS vesicles (50 μM) in TBS/Ca²⁺ in a 96-well assay plate. The remaining cofactor activity of FVIIIa was determined by an intrinsic Tenase (1 nM FIXa and 200 nM FX for 2 minutes) assay as described in “Materials and methods.” Data in panels B-G are derived from 3 independent measurements (± standard deviation).