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Cdc42/Rac1-dependent activation of the p21-activated kinase (PAK) regulates human platelet lamellipodia spreading: implication of the cortical-actin binding protein cortactin by Catherine Vidal, Blandine Geny, Josiane Melle, Martine Jandrot-Perrus, and Michaëla Fontenay-Roupie Blood Volume 100(13):4462-4469 December 15, 2002 ©2002 by American Society of Hematology Time course of actin polymerization and spatial reorganization in stimulated platelets.(A) After stimulation with 0.5 UI/mL THR (⋄), 10 μM TRAP (■), 250 pmol/L CVX (○), 2 μg/mL monoclonal IV.3 antibody followed by clustering of FcγRIIA with 30 μg/mL antimou... Catherine Vidal et al. Blood 2002;100:4462-4469 ©2002 by American Society of Hematology Activation of Cdc42 and Rac1 in platelets.(A) Specific interaction of Cdc42-GTP or Rac1-GTP with PBD. Platelet lysates were incubated with 100 μM GTPγS or GDP and clarified before precipitation on PBD-GST beads. Catherine Vidal et al. Blood 2002;100:4462-4469 ©2002 by American Society of Hematology Activation of PAK.(A) PAK autophosphorylation and kinase activity in stimulated platelets. Catherine Vidal et al. Blood 2002;100:4462-4469 ©2002 by American Society of Hematology Effects of LT or toxin B on Cdc42, Rac1, or PAK activation and actin reorganization induced by thrombin.Prior to stimulation with thrombin for 1 minute, platelets were incubated or not with 1 μg/mL LT or toxin B for 2 hours at 37°C. Catherine Vidal et al. Blood 2002;100:4462-4469 ©2002 by American Society of Hematology Constitutive association of cortactin with PAK in resting platelets.(A) In vitro association of PAK and cortactin. Catherine Vidal et al. Blood 2002;100:4462-4469 ©2002 by American Society of Hematology In vitro phosphorylation of cortactin by PAK.PAK was immunoprecipitated (IP) from platelets stimulated or not with THR (0.5 UI/mL) as indicated and incubated with 5 μg purified rHu cortactin in the presence of 20 μM MgCl2-ATP and 0.185MBq (5 μCi) γ[32P]-ATP. Catherine Vidal et al. Blood 2002;100:4462-4469 ©2002 by American Society of Hematology Immunolocalization of Rac1, PAK, and cortactin in thrombin-stimulated platelets.Resting or stimulated platelets (0.5 UI/mL thrombin for 1 minute at 37°C) were fixed and labeled with 1-10 μg/mL anti-Rac1 MoAb, anti-PAK1/2 rabbit polyclonal antibody, or anti-... Catherine Vidal et al. Blood 2002;100:4462-4469 ©2002 by American Society of Hematology
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