Determination of protein S enhancement of anticoagulant activity of APC variants compared with wt-APC. (A) APC variants dose response prolongation of clotting time using normal human plasma (solid line) and protein S–depleted plasma (dashed line) in FXa-1–stage assays. wt-APC (●) was compared with GED-APC (▲) and APC-del/5 (□). (B) The anticoagulant activity of wt-APC and APC variants was determined in normal plasma (solid bars) and in protein S–depleted plasma (open bars) using FXa-1–stage assays where percent activity was calculated based on wt-APC having 100% activity. (C) The ability of protein S to enhance APC anticoagulant activity was studied using FXa-1–stage assays and using protein S–depleted plasma supplemented with varying concentrations of purified plasma-derived protein S. (D) Mutant APCs or wt-APC were incubated with wt-EPCR/SEAP-wt-PAR-1 cells or wt-EPCR/SEAP-R41Q-PAR-1 cells in Hanks balanced salt solution supplemented with 1.3 mM CaCl₂, 0.6 mM MgCl₂, and 0.1% bovine serum albumin. After 60 minutes, SEAP release was determined using 1-step p-nitrophenyl phosphate. After correction for background activity in the absence of protease, values were expressed as percentage of the total SEAP activity present on the cells.