Binding of fVIII-4Ala, with defective phospholipid affinity, to platelets. (A) fVIII-4Ala in complex with GMA8021-fluor were mixed with platelets stimulated as in Figure 1A. fVIII-4Ala had a 99% reduction in binding for A23187 + thrombin-stimulated platelets but a 55% reduction in binding to thrombin-stimulated platelets (B). Efficacy of fVIII or fVIII-4Ala in the factor Xase activity was evaluated in the presence of platelets stimulated with A23187 and thrombin (A23187) or by thrombin or PLVs with 4% or 20% PS content. The fVIII or fVIII-4Ala concentrations were 0.1 nM (A23187, 20% PS) or 1 nM (thrombin, 4% PS) mixed with factor IXa, 0.5 nM and factor X, 150 nM. Stimulated platelets, 1 × 10⁸/mL, or 20 μM PLVs (4:20:76 PS:PE:PC or 20:20:60 PS:PE:PC, extruded) were added with 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. fVIII-4Ala supported ∼5% of the activity on A23187 + thrombin-stimulated platelets and ∼12% residual activity on thrombin-stimulated platelets. In contrast, fVIII-4Ala supported <1% residual activity on PLVs and activity was not significantly above background. Results in (A) are mean ± standard deviation (SD) for 4 measurements with the same fVIII-4Ala or fVIII concentrations between 1 to 8 nM and for (B) are mean ± SD from 2 experiments (A23187) or 3 experiments (thrombin, 4% PS and 20% PS) each performed in duplicate. PC, phosphatidylcholine; PE, phosphatidylethanolamine.