ACC DKI mice display enhanced primary hemostasis and thrombosis. (A) Tail bleeding times of ACC WT and ACC DKI mice in saline at 37°C. Individual values are plotted on the graph (n = 14 in each group). Bars indicate means. #P ≤ .05. Data were analyzed by the Mann-Whitney U test. (B-C) ACC WT and ACC DKI mice were subjected to in vivo FeCl₃-induced thrombosis of carotid arteries (10% FeCl₃, 5 min). Thrombus formation was monitored by analyzing exogenous carboxyfluorescein succinimidyl ester-labeled platelet accumulation by intravital microscopy and recording videos (fluorescence) of microscopic images every 2 minutes. (B) Thrombus growth kinetics was evaluated by dividing the area of the thrombus at time (t) by the area of the same thrombus at time 0, defined as the time at which the thrombus first reaches 100 µm. Thrombus growth is expressed as means ± SEM per group at different times with fitted regression lines (at least 6 mice/group). Slopes are statistically different on the basis of significant interaction (#P ≤ .05) between slope and group in a linear model, with group, time, and their interaction as covariates. (C) Representative fluorescence microscopy images at 0, 6, 12, and 18 minute after FeCl₃ application (original magnification ×10). (D-G) Whole blood from ACC WT and ACC DKI mice was perfused over collagen-coated surfaces (100 µg/mL) at a shear rate of 1000 s⁻¹. Exposure of P-selectin was evaluated by staining with CD62P Ab (D), αIIbβ3 integrin activation by JON/A Ab (E) and phosphatidylserine externalization by Annexin V (F). Thrombus formation was assessed on brightfield images taken 3.5 minutes after initial blood perfusion (G). Representative images appear on the left. Scale bars represent 20 µm. Histograms indicate quantification of surface area covered (SAC) by P-selectin (D), activated αIIbβ3 (E), Annexin V (F)–positive platelets or multilayered platelet thrombi (G). The results are expressed as means ± SEM (at least 4 mice/group). #P ≤ .05. Data were analyzed by the Mann-Whitney U test.