GW3965 increased bleeding time and modulated platelet thrombus formation in vivo. (A) GW3965 (2 mg/kg) (n = 15) or DMSO (0.08% volume/volume) (n = 15) was administered intravenously to mice and time to cessation of bleeding, after a tail biopsy, was measured. Data represent individual mice; and horizontal lines, mean values. Statistical analysis was performed using the Mann-Whitney nonparametric T test. (B) GW3965 (2 mg/kg) or DMSO (0.08% volume/volume) was administered intravenously to mice, and platelets were fluorescently labeled by injection of Alexa488-conjugated anti-GPIb antibody. After laser-induced injury of the cremaster muscle arterioles, accumulation of platelets (shown in green) was assessed (Bi). The area covered by thrombi (Bii), maximum fluorescence value (Biii), time to peak fluorescence levels (Biv), and time elapsed between maximal and half-maximal fluorescence values (Bv) were measured. Graphs represent individual mouse data for 21 thrombi in 4 mice for each treatment. (C) Mouse whole blood mixed with the lipophilic dye 3,3-dihexyloxacarbocyanine iodide was treated with GW3965 (60μM) (Cii) or DMSO (0.1% volume/volume) (Ci) and perfused through collagen-coated (400 μg/mL) Vena8Biochip at a shear rate of 20 dyn/cm². Thrombi were recorded through a series of images in the Z-plane through their full depth every 30 seconds using a Nikon eclipse (TE2000-U) microscope, and thrombus fluorescence intensity was calculated using the Slidebook, Version 5. Numerical data (Ciii) represent sum fluorescence intensities and are the average of 3 separate experiments.