Phosphatidylinositol-3,4,5-trisphosphate (PIP3) is an important second messenger formed from phosphatidylinositol-4,5,-bisphosphate by the action of PI-3kinase. It has been implicated in many cellular functions but its mechanism of action requires further study. In platelets it has been suggested to be important in amplifying the activation Rap1b , in integrin activation, PLC activation, enhancement of Ca2+ signalling and thrombus formation. However the mechanisms associated with these effects need clarification. We have used cell permeable analogues of PIP3,1-O-(1,2-di-palmitoyl-sn-glyero-3-O-phosphoryl)-D-myo-inositol-3,4,5-trisphosphate (DiC16-PIP3) or 1-O-(1,2-di-octanoyl-sn-glyero-3-O-phosphoryl)-D-myo-inositol-3,4,5-trisphosphate (DiC8-PIP3) to study effect of PIP3 on freshly prepared human platelets. We examined the effects of these analogues on platelet aggregation, Akt phosphorylation, Ca2+ elevation, PLC and syk activation and thromboxane A2 formation using standard platelet technologies.Results; Addition of either DiC8- or DiC16-PIP3 analogues induced platelet aggregation in the presence of extracellular Ca2+ . This was inhibited by a range of inhibitors including indomethacin, U73122 and reagents that inhibit Ca2+ elevation such as 2APB. Akt activation was monitored by measuring the phosphorylation of Ser473. DiC16-PIP3 induced Akt phosphorylation was reduced by Akt inhibitor IV and wortmanin and if EGTA was included in the medium. In Fura2 loaded platelets DiC8-PIP3 was effective in increasing intracellular Ca2+ in a rapid, distinct and transient manner that was reduced in the presence of wortmanin, indomethacin, U73122 and 2APB. It was only marginally affected by the SOC inhibitor BTP2 or the non-SOCE inhibitor LOE908. DiC8-PIP3 induced the release of Ca2+ from stores. The presence of bafilomycin A1 did not affect the Ca2+ transient and the cell permeable analogue of DiC8-PI-3,5-P2 was not as effective as DiC8-PIP3 in inducing Ca2+ elevation. DiC8-PIP3 did effect the phosphorylation of the kinase syk but not of PLCγ2. Importantly DiC8-PIP3 was potent at inducing the formation of thromboxane B2 that was reduced by the Akt inhibitor IV.Conclusion; These studies suggest that PIP3 plays an important role as a stimulus of Ca2+ transients, Akt phosphorylation and thromboxane A2 formation in platelets. The distinct Ca2+ mobilisation seen by PIP3 may represent an important mechanism for thromboxane generation in human platelets.