Btk-/-/Tec-/- platelets do not show surface expression of P-selectin after stimulation by CRP, in contrast to wild type. Washed platelets in the presence of lotrafiban (10 μM) and at a concentration of 1 × 107/mL were incubated with vehicle or CRP (10 μg/mL) for 10 minutes before dilution to 1 × 106/mL. Basal or stimulated platelets were stained for 30 minutes with an FITC-conjugated anti-P-selectin antibody. The relative fluorescence of the stained platelets was investigated by flow cytometry and is displayed as a histogram of counts versus fluorescence intensity. Graphs show that in wild-type and Tec-/- platelets there is a significant shift of the population to higher intensities of fluorescence, indicating P-selectin expression on the platelet surface. This shift is not seen with Btk-/- and Btk-/-/Tec-/- platelets, which show no change in distribution on stimulation. This experiment was conducted 5 times, and the histograms shown are representative of these.
