Effect of fibrin on function of fVIII. (A) Various concentrations of fibrinogen were mixed with fVIII (0.5 nM), factor IXa (0.5 nM), factor X (150 nM), and PLVs (50 μM) (4:20:76 PS:PE:PC, extruded) prior to the simultaneous addition of 0.2 u/mL thrombin and 1.5 mM Ca⁺⁺. The reaction was stopped after 5 minutes by the addition of EDTA, and factor Xa was measured with chromogenic substrate S-2765. Fibrin increased activity ∼3.5-fold and the half-maximal effect was observed at ∼5 µg/mL. The smooth line represents a nonlinear least squares curve fit assuming a single, saturable fibrin-binding site (B). The experiment in (A) was repeated with PLVs of various PS content and 0.5 nM fVIII. All vesicles were extruded and had 20% PE with the balance as PC. Activity is displayed as the V_max / V₀ ratio with increasing fibrin from the respective curve fits (as in Figure 4A). Phospholipid concentrations were 1% PS (200 µM), 2% PS (100 µM), 4% PS (50 µM), and 8% and 16% PS (10 µM). Results are mean ± SD from 6 experiments (A) and from 2 experiments performed in duplicate for each vesicle type (B).