APC degradation of factor VIIIa in the absence and presence of protein S. (A) Human factor VIIIa (1 nM) was incubated with wild-type or mutant APC derivatives (2.5 nM each) on 25 μM PC/PS vesicles in TBS containing 1 mg/mL BSA, 0.1% PEG 8000, and 5 mM Ca²⁺. At the indicated time intervals, the remaining factor VIIIa activity was determined from the rate of factor Xa generation by the intrinsic Xase complex composed of 20 nM factor IXa, 500 nM factor X, and 25 μM PC/PS as described in “Materials and methods.” (B) Procedures were the same as those described in panel A except that the inactivation assay was carried out in the presence of human protein S (250 nM). The symbols for both panels are as follows: wild type (○), Lys-Lys-Lys/Pro-Gln-Glu (•), Lys62Ala (□), Lys63Ala (▪), Arg67Ala (▵), Arg74Ala (▴), Arg75Ala (▿), and Lys78Ala (▾). The symbols for the wild-type APC (○) are masked by those of the Lys62Ala mutant (□) because of similar degradation rates for both proteases.