Effect of KM33_scFv on factor VIII binding to and function on platelets. (A) Fluorescein maleimide–labeled factor VIII was preincubated with KM33_scFv for 30 minutes at room temperature. Platelets were stimulated with 10 μM TRAP or A23187 for 10 minutes and then incubated with factor VIII-fluorescein (10 nM) at room temperature. Bound factor VIII was measured after 10 minutes by flow cytometry. Values were corrected for the quantity of fluorescence from factor VIII ± KM33_scFv in the presence of resting platelets (21-33 fluorescence units). Bars represent the mean ± SEM of 3 experiments. (B) Factor VIII (5 U/mL) was incubated with KM33_scFv for 30 minutes at room temperature. Factor VIII-antibody complex was added to platelets activated for 10 minutes at room temperature with either 10 μM TRAP or A23187. Factor IXa, factor X, and Ca²⁺ were added to start the reaction. After 5 minutes, the reaction was stopped with ethylenediaminetetraacetic acid, and the generation of factor Xa was measured by monitoring the rate of cleavage of chromogenic substrate S2765. Values represent the average of 3 experiments and are normalized for comparison of the extent of inhibition. Maximum activity ranged from 8 to 18 nM FXa generated for TRAP-stimulated platelets and from 60 to 157 nM FXa generated for A23187-stimulated platelets.