Contribution of ADP and thrombin to the formation of the hemostatic plug core and shell. (A) Graph depicts total platelet accumulation and P-selectin expression over time following laser injury in wild-type mice before (blue lines) and after (red lines) treatment with the P2Y12 antagonist cangrelor. Values are mean area ± SEM for n = 15 injuries before and 24 injuries after cangrelor infusion in 4 wild-type mice. Representative time-lapse videos are included in supplemental Video 5. (B) A comparison of shell and core area 2 minutes post-injury. Data are normalized to mean area of the wild-type for each region and are expressed as mean ± SEM. Statistical analysis was performed by 2-way ANOVA with Bonferroni post-hoc test. (C–F) Impact of thrombin inhibition on the core and shell. Representative photomicrographs show hemostatic plugs 2 minutes post-injury before (C) and after (D) intravenous infusion of hirudin (30 µg/kg prior to injury). Platelets are labeled red, P-selectin is labeled green, and fibrin is labeled blue. Overlay of platelets and P-selectin is yellow and overlay of all 3 fluorophores is white. (E) and (F) Kinetics of hemostatic plug formation for the 2 injuries shown in (C) and (D), respectively. The red line is total platelet accumulation (CD41 area), the green line is P-selectin expression, and the blue line is fibrin accumulation. Time-lapse videos of the hemostatic plugs shown are included in supplemental Video 6. Data are representative of at least 6 injuries in 2 mice.