Vps34 regulates stimulation-dependent PI3P production in platelets. (A) PI3P content was analyzed by mass assay in washed resting WT platelets treated for 1 hour with dimethyl sulfoxide (WT) or 1 μM VPS34-IN1 and Vps34-deficient platelets (Vps34) (mean ± SEM; n = 3-5; **P < .01 vs WT according to 1-sample Student t test). (B) PI3P content from WT platelets treated for 1 hour with dimethyl sulfoxide (WT) or 1 μM VPS34-IN1 and from Vps34-deficient platelets (Vps34) in resting or stimulated (CRP, 10 µg/mL; thrombin, 0.5 UI/mL) conditions was assessed by mass assay (mean ± SEM; n = 5; *P < .05; **P < .01 vs WT according to 2-way ANOVA). (C) HPLC analysis of the PI3P level of resting and stimulated (CRP, 10 µg/mL; thrombin, 0.5 IU/mL) 32 Pi-labeled platelets (mean ± SD; n = 2). (D) Vps34 was immunoprecipitated from resting or activated (CRP, 10 µg/mL; thrombin, 0.5 IU/mL) washed platelets and assayed for lipid kinase activity in vitro. Graph represents Vps34 activity (fold increase) normalized to the levels of immunoprecipitated kinase in each condition as assessed by immunoblot and densitometry analysis (mean ± SEM; n = 5; ***P < .001 vs resting according to 1-sample Student t test).
