Search for TF on platelets by flow cytometry. (A) Washed platelets were activated with PAR1 (100μM) and PAR4 (500μM) agonist peptides (2 hours, 37°C). (B) Platelet-rich plasma was incubated with THP-1 cells in the presence or absence of 250 ng/mL LPS (4 hours, 37°C). For all experiments, TF expression on platelets was determined by immunostaining with an anti-TF antibody⁸  (0.5μM) in 20mM Hepes, 0.15 M NaCl (pH 7.4) containing 10 μg/mL human Fc, followed by either (A) goat anti–mouse IgG-PE (1:10 dilution) or (B) goat anti–mouse IgG–Alexa Fluor 488 (1:400 dilution) in 10% goat serum. Platelets (10 000) were analyzed by flow cytometry using a Becton Dickinson LSR II flow cytometer. The positive analyses regions were defined such that approximately 2% of the platelets stained with secondary antibodies alone were positive. The gray histograms depict anti-TF antibody staining of platelets activated with PAR peptides (A) or incubated with THP-1 cells and LPS (B). The black histograms depict immunostaining of unactivated platelets (A) or platelets incubated with THP-1 cells alone (B). The data shown are representative of 2 experiments performed in triplicate.