Inhibition of intrinsic Xase complex and thrombin generation by anti-C1 mAbs. (A) BDD fVIII at a limiting concentration of 1 nM was preincubated for 2 hours at 37°C in the presence of 20 nM anti-C1 mAbs or buffer control in the absence (black bars) or presence (gray bars) of 10 µg/mL VWF. fVIII was activated by 100 nM thrombin for 30 seconds followed by inhibition of thrombin by 150 nM hirudin. fVIII is completely activated within 10 seconds at this concentration. The intrinsic Xase complex was assembled by the addition of the fVIIIa sample to factor IXa (2 nM), PCPS vesicles (20 µM), and factor X (300 nM). fVIIIa activity was determined by measurement of factor Xa generation in the intrinsic fXase assay as described in “Methods.” (B) Representative thrombin generation in fVIII-deficient plasma spiked with 1 U/mL recombinant full-length fVIII in the presence of 5 µg/mL group A mAb 2A9 and group B mAb B136. Control curves of fVIII-deficient plasma with and without fVIII are also shown. (C) ETP (black bars), peak thrombin generated (light gray bars), and lag time (dark gray bars) of fVIII-deficient plasma spiked with fVIII and anti-C1 mAbs as a percentage of control fVIII-deficient plasma spiked with fVIII. ETP, peak thrombin, and lag time of fVIII-deficient plasma alone are shown as a negative control.