PI3K-C2α is critical for the regulation of basal PI3P level in platelets. (A) High-performance liquid chromatography analysis of PI3P levels in resting and stimulated (10 µg/mL CRP and 0.5 IU/mL thrombin for 3 minutes)³²P-labeled platelets (mean ± SEM; n = 4). (B) PI3P mass assay in resting and stimulated (10 µg/mL CRP and 0.5 IU/mL thrombin for 3 minutes) platelets. The assay was performed as previously described.²²  Briefly, platelet phosphatidylinositol monophosphate (PIP) fraction purified by lipid extraction and thin-layer chromatography was submitted to specific phosphorylation by recombinant PIKfyve, in the presence of [γ-³²P]ATP. PIKfyve only phosphorylates PI3P to produce [³²P]-PI(3,5)P₂, which reflects the amount of PI3P present in the sample. (mean ± SEM; n = 4; ***P < .001 vs WT, according to 1 sample t test). (C) Resting platelets were fixed and stained with simple FYVE^HRS probe coupled to 647-AlexaFluor. Representative confocal images of FYVE^HRS-647 AlexaFluor labeled platelets are shown. Scale bar: 1 µm. Staining intensity in platelets were quantified by ImageJ software (mean ± SEM; n = 3; ***P < .001 vs WT, according to 1-sample t test).