Impaired activation and thrombus formation of RhoB^−/− platelets in vitro. (A-B) Platelet light transmission aggregometry. Representative aggregation curves of washed wt and RhoB^−/− platelets stimulated with different agonist concentrations under stirring conditions for 10 minutes are shown. (A) Shape change of wt and RhoB^−/− platelets in response to low concentrations of the thromboxane analog U46619. RhoA-deficient platelets displaying defective shape change under these conditions were used as a control. (B) Aggregation of RhoB^−/− platelets in vitro. (C) Platelet integrin αIIbβ3 activation (JON/A-PE binding; upper) and α-granule secretion (P-selectin-FITC; lower) in vitro were determined upon 6-minute stimulation of wt and RhoB^−/− platelets with the indicated agonists at different concentrations (n = 4). (D) In vitro thrombus formation under flow on a collagen-coated surface. Representative images of thrombus formation at an intermediate shear of 1000 s⁻¹ are shown. Upper panel: adhesion of platelets to collagen assessed by area coverage. Lower panel: formation of 3-dimensional aggregates displayed as relative thrombus formation (n = 8). (E) Induction of arterial thrombosis by mechanical injury of the abdominal aorta of wt and RhoB^−/− mice in vivo. Each data point represents 1 mouse (wt n = 7; RhoB^−/− n = 8). (F) Tail bleeding time on filter paper of wt and RhoB^−/− mice in vivo. Each data point represents 1 mouse (wt n = 15; RhoB^−/− n = 23. *P < .05; **P < .01; ***P < .001; Mann-Whitney U test, mean plus or minus SD. ADP/U466, adenosine diphosphate/U46619; CRP, collagen-related peptide; CVX, convulxin; Rest, resting; Rhd, rhodocytin; Thr, thrombin.