Platelet Rac1 is important in promoting fibrin generation in vitro and in vivo. (A) The mean recalcification time ± SEM of human citrated PRP treated with 100 µM NSC23766 (n = 20) or vehicle control (n = 10) and of washed WT and Rac1^−/− mouse platelets (n = 5) resuspended in citrated human PPP after addition of CaCl₂ under stirring conditions. (B) The recalcification time of human citrated PRP treated with 100 µM NSC23766 or DMSO was monitored using a cone-plate rheometer under defined shear (6000 seconds⁻¹) after addition of CaCl₂ (mean ± SEM, n = 4). (C-D) Intravital microscopy was used to monitor fibrin generation (green) and platelet thrombi (red) in vivo following laser-induced cremaster arteriole wall injury in WT and platelet-specific Rac1^−/− mice. (C) The sizes of the platelet thrombus and fibrin clot were compared by calculating their respective median surface area at 60 and 240 seconds (median + interquartile range). (D) Representative images of fibrin generation (green) and platelet thrombi formation (red) and merged images. Arrows indicate direction of blood flow. Movies comparing laser-induced platelet thrombus formation and fibrin deposition in WT (supplemental Movie 3) and platelet-specific Rac1^−/− mice (supplemental Movie 4) mice, as well as the median integrated fluorescence signals of platelet thrombi and fibrin as a function of time (supplemental Figure 4), are provided. For all data, * and *** represent statistical significance of P < .05 and .001, respectively.