Comparison of the APC cofactor activity of wild-type rPS and PS/FII chimeras in purified systems. (A) The capacity of each PS species to act as an APC cofactor in FVa inactivation was quantified by determining the rate of FXa-catalyzed prothrombin activation in a reaction mixture containing 2 nM FV/FVa, 0.1 nM FXa, 100 nM prothrombin, and 10 μM PC/PS vesicles, as described in “Materials and methods.” In the absence of wild-type rPS, thrombin generation was measured in the presence (*) or absence (×) of 0.5 nM APC. APC cofactor activity of wild-type rPS (▪), Gla_FII-ΔTSR-PS (□), and Gla_FII-PS (♦) in the FVa inactivation assay was determined by measuring thrombin generation after the simultaneous addition of 100 nM PS to 0.5 nM APC. Experimental conditions were designed to optimize detection of PS APC cofactor activity rather than prothrombinase activity; visible lag phase presumably originated from FV activation during kinetics. (B) FX activation by tenase complex (FIXa, FVIIIa, and phospholipids) was monitored in the absence of APC and PS (×) or in the presence of APC alone (final concentration, 4 nM) (*), as described in “Materials and methods.” APC cofactor activity of wild-type rPS (▪), Gla_FII-ΔTSR-PS (□), and Gla_FII-PS (♦) in the FVIIIa inactivation assay was determined by measuring FXa generation after the simultaneous addition of 100 nM PS to 4 nM APC.