Thrombin-induced integrin αIIbβ3 activation, P-selectin exposure and LAMP1 exposure. (A-F) Washed platelets were stimulated with increasing thrombin concentrations and periods of time (numbers in graph legend indicate the final thrombin concentration) and analyzed by flow cytometry. Red symbols, WT; blue symbols, DKO. All graphs represent mean ± SEM for n = 3 independent experiments. Statistical analyses were performed using 2-way ANOVA, Bonferroni posttest, to compare differences between WT and DKO in similar stimulation conditions. (A) αIIbβ3 integrin activation as measured by JonA-PE labeling and represented as mean fluorescence intensity. At each time point and for all thrombin concentrations, DKO platelet JonA-PE labelings were significantly different from WT ones (P < .001), except after stimulation with 0.05 U/mL thrombin for 10 seconds, where there was no difference. (B) αIIbβ3 integrin activation in the presence (dotted lines) or absence (straight lines) of ADP (10 µM). The presence of ADP significantly increases JonA-PE labeling in DKO mice compared with absence of ADP for all conditions (P < .001). (C) P-selectin exposure at the surface of platelets. No significant difference was observed between WT and DKO platelets, except after stimulation with 0.05 U/mL thrombin for 300 seconds (P < .01). (D) P-selectin exposure in the presence (dotted lines) or absence (straight lines) of ADP (10 µM). The presence of ADP did not increase P-selectin exposure. No significant difference between WT and DKO platelets. (E, left) LAMP1 platelet surface exposure. LAMP1 exposure is significantly decreased in DKO platelets compared with WT (P < .001) except for 0.1 U/mL thrombin at 30 seconds. (F) Exogenous addition of 10 µM ADP (dotted lines) only partially rescued DKO platelet LAMP1 exposure in response to 1 U/mL thrombin.