The effects of Rac1 inhibition and ROCK inhibition on p38, ERK, and MLC phosphorylation and clot retraction. (A)Washed platelets were pretreated with either 20 μM NSC23766, 20 μM ROCK inhibitor, Y27632, both NSC23766 and Y27632, or the appropriate vehicle control for 30 minutes at 25°C. Platelets were added to polystyrene dishes coated with 100 μg/mL fibrinogen to induce adhesion and spreading at 37°C for 60 minutes or kept in suspension. After washing, adherent platelets or control platelets were solubilized as described in “Immunoblot detection of p38, ERK, and MLC phosphorylation platelets.” Phosphorylation of p38, ERK, and MLC were analyzed by Western blot analysis using specific antibodies against phosphorylated p38, ERK, and MLC. A monoclonal antibody against tubulin was used to verify equal loading. (B) Densitometry measurements from results in panel A. Values were normalized with respect to resting control for each immunoblot and are expressed as relative phosphorylation (mean ± SD from 3 separate experiments). Statistical significance was determined using Student t test. (C,D) Citrated PRP was preincubated with inhibitors for 30 minutes as indicated. Coagulation was induced with 0.2 U/mL thrombin, and clots were allowed to retract at 37°C and photographed. (E) Washed platelets from wild-type and Rac1 knockout mice were solubilized, and expression of Rac1 was analyzed by immunoblot with monoclonal antibody against Rac1 (Sigma-Aldrich). (F) Washed platelets from wild-type, and Rac1 knockout mice were added to polystyrene dishes coated with 100 μg/mL fibrinogen to induce adhesion and spreading at 37°C for 60 minutes or kept in suspension. After washing, adherent platelets or control platelets were solubilized as described in “Immunoblot detection of p38, ERK, and MLC phosphorylation in platelets.” Phosphorylation of p38 and ERK were analyzed by Western blot analysis. A monoclonal antibody against tubulin was used to verify equal loading. (G) Platelets from wild-type or Rac1 knockout mice were mixed with human platelet-poor plasma at a concentration of 4 × 10⁸/mL, induced to coagulate with 0.4 U/mL thrombin, and photographed. (H-J) Extent of clot retraction in panels C, D, and G was measured using NIH ImageJ and expressed as percent retraction (mean ± SD). Statistical significance was determined using a Student t test.