Effect of protein S variants for APC cofactor activity on thrombin generation. Thrombin generation was measured in protein S–deficient plasma supplemented with 9nM APC and 60nM of protein S variants (A), 0 to 90nM WT protein S (B), 0 to 110nM protein S E36A (C), or 0 to 110nM protein S E36D (D). Typical experiments are shown (n = 3). Dose-response data from titrations with WT protein S, protein S E36A, and protein S E36D (0-90nM) in the presence of 9nM APC is shown in panel E. Data are mean ± SEM; n = 4. (F) Thrombin generation in protein S free plasma supplemented with 9nM APC and 60nM purified protein S. (Inset) Purified WT protein S and protein S E36A on a silver-stained 10% SDS-PAGE under reducing (R) and nonreducing (NR) conditions.