ND indicates not determined.

The capacity of mAb14C12 to interfere with the binding of FVIII to VWF or PL was evaluated in enzyme-linked immunosorbent assay (ELISA). Microtitration plates were coated with VWF or PL as described in “Materials and methods.” FVIII (1 IU/mL) was preincubated with buffer alone, or buffer containing 1.25 μg/mL of either mAb14C12 or mAbBO2C11 as a control, and the mixtures were added to VWF- or PL-coated plates. The residual binding of FVIII was evaluated by sequential addition of a biotin-labeled FVIII heavy chain–specific mAb (mAb15) and avidin-peroxidase. Assays were made in duplicates. Results are shown as percentages of residual FVIII binding, taking preincubation with buffer alone as 100%.

The effect of mAb14C12 on FVIII clearance was measured by injecting 1 IU FVIII intravenously in C57BL/6 FVIII^-/- mice, alone or 15 minutes after intravenous injection of a single dose of 10 μg mAb14C12. Bleeding was carried out at 180 minutes, namely the expected FVIII t_1/2 in this animal model,¹⁷  and the residual FVIII activity was measured in a chromogenic assay (described in “Materials and methods”). The 0.34 IU/mL measured in mice pretreated with mAb14C12 compare with the 0.32 IU/mL measured in mice injected with FVIII alone.