Binding of fVIII to platelets and competition for fVIII binding sites. (A) fVIII-fluor, at various concentrations was mixed with platelets (1 × 10⁸/mL) stimulated with 10 µM A23187 + 1 u/mL thrombin for 10 minutes. The sample was diluted 10-fold and fVIII-fluor was added for 10 minutes. Samples were further diluted to 1 × 10⁶ platelets/mL and bound fVIII-fluor evaluated by flow cytometry. fVIII-fluor binding increased in a saturable manner. (B) fVIII-fluor (4 nM) was mixed with various concentrations of lactadherin prior to mixing with platelets stimulated by A23187 and thrombin. Lactadherin competed for at least 98% of binding sites indicating that PS is a critical determinant of most sites. (C) Platelets were stimulated with 1 u/mL thrombin alone for 1 minute prior to the addition of 3 u/mL hirudin. fVIII-fluor (4 nM) mixed with unlabeled fVIII or lactadherin at the indicated concentration was added to the platelets for 10 minutes prior to dilution and reading. Unlabeled fVIII competed for >95% of binding sites whereas lactadherin did not compete significantly. The fluorescence signal was corrected for the signal of unstimulated platelets with fVIII-fluor (supplemental Figure 2). Results are mean ± standard error of the mean (SEM) from 2 experiments for each panel.