Binding of fVIII to fibrin. (A) Various concentrations of fVIII-fluor were incubated with SF immobilized on antifibrinogen Superose beads. After 10 minutes, bound fVIII was evaluated by flow cytometry. Displayed results represent mean ± range of duplicates. Saturable binding of fVIII-fluor was observed. (B) Various concentrations of VWF were incubated with 4 nM fVIII-fluor for 15 minutes prior to mixing with immobilized fibrin–anti-fibrinogen Superose beads. VWF inhibited fVIII binding to fibrin. (C) Various concentrations of fVIII-C2 were incubated with fVIII-fluor prior to mixing with fibrin–antifibrinogen Superose beads. Experiments were performed in tris-buffered saline containing 0.02 M NaCl. fVIII-C2 competed with fVIII-fluor for binding to immobilized fibrin. Displayed data are corrected for measured background fluorescence with control beads lacking fibrin. Displayed results are mean ± SEM for 3 such experiments; also representative of 6 additional experiments with slightly different conditions (A), 2 experiments (B), and 2 experiments (C).