Function of Lys 2092 and Phe 2093 in binding to platelets and platelet-dependent activity. (A) fVIII_YFP (solid columns) or fVIII_{YFP 2092/93} (open columns) (10 nM) were incubated with platelets stimulated with TRAP or A23187. After 10 minutes of incubation at room temperature, bound factor VIII was determined by flow cytometry. Values were corrected for the quantity of fluorescence from factor VIII in the presence of resting platelets (7-11 fluorescence units). Bars represent the mean ± SEM of at least 4 experiments. (B) Platelets were activated with TRAP or A23187 for 10 minutes at room temperature and added to factor VIII, factor IXa, factor X, and Ca²⁺. After 5 minutes, the reaction was stopped by adding 16 mM ethylenediaminetetraacetic acid. The generation of factor Xa was measured by monitoring the rate of cleavage of chromogenic substrate S2765. Solid columns represent fVIII_YFP; open columns, fVIII_{YFP 2092/93}. Data represent the mean ± SEM for 4 experiments. Maximum activity for TRAP-stimulated platelets ranged from 0.5 to 5.5 nM (fVIII_YFP) and 0 to 1.0 nM (fVIII_{YFP 2092/93}) FXa generated and from 37 to 61 nM (fVIII_YFP) and 9 to 21 nM (fVIII_{YFP 2092/93}) FXa generated for A23187-stimulated platelets.