Platelet aggregation and expression of P-selectin at cell surface upon activation. (a) The aggregation capacity of homozygous ashen mice (ash/ash) and wild-type C3H/He mice (C3H/He+/+) was tested, measuring the increasing light transmission through a platelet suspension. The agonist (thrombin at 2 U/ml) was added at t0, and the traces were recorded for 5 minutes. The experiment was repeated independently, and the results were not significantly different (see text). The 0% corresponds to the light transmission of the resting platelet suspension and the 100% to the transmission of light in suspension buffer (or totally aggregated platelets). (b) The expression of P-selectin (mainly localized in α-granule membrane) at the surface of platelets upon stimulation with PMA was used to test α-granule release capacity. Platelets in whole blood were identified with an anti–CD41 (gpIIb) Ab. The geometric mean of the population of platelets labeled with anti–P-selectin Ab was plotted. Triplicates were done for each mouse, and six mice were tested in each case. Boxes represent the 25th and 75th percentiles, and median is represented by horizontal line inside the boxes. The error bars represent the 5th and 95th percentiles. The statistical analysis was performed using an ANOVA test, and the were results found not to be significantly different. The negative controls (without anti–P-selectin Ab) showed less that 2% activated platelets.