Rac1 is important for the exposure of PS during thrombus formation in vitro under shear. Washed WT and Rac1^−/− platelets were stimulated with 0.03 U/mL thrombin and subjected to shear (6000 seconds⁻¹) over a fibrinogen-coated surface. Platelets (red) were stained using anti-mouse CD41 and Alexa Fluor-546–conjugated anti-rat antibodies. Surface-exposed PS (green) was stained using Alexa Fluor-488–conjugated Annexin V. (A) Representative fluorescence microscopy images showing WT and Rac1^−/− platelet thrombi (red) and exposed PS associated with thrombi (green). Scale bar represents 10 μm. (B) Quantification of surface area of thrombi per field (mean ± SEM, n = 7). (C) Quantification of the Annexin V surface area per field (mean ± SEM, n = 7). (D) Representative 3-dimensional reconstruction of confocal Z-stack images of WT and Rac1^−/− platelet thrombi and exposed PS. Scale bar represents 10 μm. Movies of confocal Z-stack images of WT (supplemental Movie 1) and Rac1^−/− (supplemental Movie 2) platelet thrombi and exposed PS are provided. For all data, ** represents statistical significance of P < .01.