Human platelets show robust PtdIns(3,4,5)P₃generation and associated AKT pathway phosphorylation (p) in response to PAR and GPVI receptor activation. Washed human platelets were preincubated with dimethyl sulfoxide (DMSO) or 100 nM Wortmannin (WTM) for 10 minutes at 37°C before stimulation for 2 minutes with vehicle (Veh) (HEPES-Tyrode’s buffer), 0.2 U/mL thrombin (αT), or 5 μg/mL CRP. Each sample was divided in 2 for western blotting of class I PI3K pathway components (A-C) and parallel lipid extraction and measurement of C38:4 PtdIns(3,4,5)P₃ by lipidomic MS (D). Quantified data represents the mean of 3 independent donors + standard error of the mean, with representative blotting presented for 1 of the 3 donors. PtdIns(3,4,5)P₃ is normalized to C38:4 PtdIns, with each normalized to its own synthetic internal standard, as detailed in the supplemental Methods. Statistical analyses were performed by using 2-way analysis of variance with Bonferroni post-tests. ***P = .0001; ****P < .0001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.