Role of Rac1 in rhodocytin-induced aggregation and signaling. (Ai) Platelets were pretreated with indicated amounts of EHT 1864 and platelet aggregation was measured after addition of 3 μg/mL CRP. Addition of agonist is indicated by an arrowhead. (ii) Lysates were made from platelets pretreated with indicated amounts of EHT 1864 stimulated with or without CRP. Active Rac was pulled-down with a GST-fusion protein containing the Rac binding domain of PAK1 and detected by immunoblotting for Rac. (B) Time course of Rac activation in human platelets stimulated with 100nM rhodocytin. Platelets were stimulated with rhodocytin; at the indicated times, activation was stopped by the addition of ice-cold lysis buffer. Active Rac was pulled-down and detected by immunoblotting for Rac. (C) Lysates were made from platelets pretreated with indicated amounts of EHT 1864 and stimulated with or without rhodocytin. Active Rac was pulled-down and detected by immunoblotting for Rac. (D) Platelets pretreated with decreasing concentrations of EHT 1864 were stimulated with 300nM rhodocytin. (i) Aggregation traces are shown for control and EHT 1864–treated platelets stimulated with 300nM rhodocytin. (ii) CLEC-2 was immunoprecipitated from platelets pretreated with indicated concentrations of EHT 1864 and stimulated with vehicle or 30nM rhodocytin and blotted with an antiphosphotyrosine antibody. Membranes were subsequently stripped and reblotted with anti–CLEC-2. Data are representative of 3 independent experiments.