Inhibition by anti-C2 MAbs of the activation of fVIII by thrombin or factor Xa. (A) FVIII (1 nM) was incubated for 2 hours at 37°C in the absence (closed symbols) or presence (open symbols) of 10 μg/mL VWF and in the absence (○) or presence of 130 nM 2–77 (group BC) (▵) or ESH8 (group C) (□). The fVIII sample was diluted 0.5 nM in 1.5 nM factor IXa/20 μM PCPS and thrombin (0.55 nM) was added for the indicated times, followed by measurement of the initial velocity of factor X activation as described in “Intrinsic fXase assays for measurement of fVIIIa activity.” (B) FVIII (4 nM) was incubated for 2 hours at 37°C in the absence (closed symbols) or presence (open symbols) of 40 μg/mL VWF and in the absence (○) or presence of 520 nM 2–77 (group BC) (▵) or ESH8 (group C) (□). The fVIII sample was diluted to 2 nM and activated with 2.5 nM factor Xa in the presence of 1.5 nM factor IXa/20 μM PCPS for the indicated times, followed by measurement of the initial velocity of factor X activation as described in “Intrinsic fXase assays for measurement of fVIIIa activity.”