No platelet tissue factor expression: short or long activation. Washed platelets were activated with PAR1 (100μM) agonist peptide for 15 minutes (dotted lines) or 2 hours (black lines) at 37°C. TF expression on platelets was determined by immunostaining with (A) anti–TF-5 conjugated to AlexaFluor647 (0.5μM); (B) a 1:10 dilution of an anti-TF antibody conjugated to FITC (American Diagnostica 45-07CJ), or by equivalent concentrations of appropriate control antibodies (shaded histograms; 15 minutes activation period), in 20mM Hepes, 0.15M NaCl (pH 7.4) containing 10 μg/mL human Fc. (C) Nonstimulated (dotted line) or LPS-stimulated (1 μg/mL, 4 hours, 37°C; black line) THP-1 cells were immunostained with anti–TF-5 or an isotype-matched control antibody (shaded histogram; nonstimulated cells) as described. Platelets or THP-1 cells (10 000), identified by their forward and side scatter, were analyzed by flow cytometry using a BD LSR II Flow Cytometer. Identical results were obtained whether platelets, stained with isotype-matched control antibodies, were activated for 15 minutes or 2 hours. Similarly, LPS-stimulation of THP-1 cells was without effect on control antibody reactivity. Representative data are shown (n = 3).