Effect of platelet NO on platelet deposition onto collagen under flow. A human blood-cell suspension prepared as described in the legend to Figure 2 was perfused over immobilized fibrillar collagen type I at the shear rate of 3000 s⁻¹ for 3 minutes. When indicated, blood cells were incubated for 10 minutes at 37°C with l-Arg, with the cGMP phosphodiesterase inhibitor (Zaprinast, 1 µM)³⁷ , with the two combined or with L-NMMA at various concentrations. After 3 minutes (to allow visualization of all adhering platelets), the fluorescent dye mepacrine (8 µM) was added and the percent of surface coverage was calculated. (A) l-arginine in combination with Zaprinast inhibits platelet deposition onto collagen (*P < .01 vs control). (B) L-NMMA increases platelet deposition onto collagen (*P < .01 vs control). (C) Representative single-frame images resulting from the merging of 2 distinct grayscale images obtained at 3 and 4 minutes of perfusion. Red = DAF-FM–loaded platelets after 3 minutes of perfusion; blue = quinacrine-loaded platelets after 4 minutes of perfusion. (D) Whole blood from wild-type or eNOS^−/− mice, treated with the fluorescent dye mepacrine for platelet visualization, was perfused over immobilized fibrillar collagen type I at the shear rate of 1500 s⁻¹ for 3 minutes. eNOS^−/− mice form larger thrombi and the quantification of the surface area covered by platelets after perfusion demonstrates a significantly larger surface area in the eNOS^−/− compared with wild-type mice (*P < .01 vs wild-type).