Abstract
Phosphatidylinositol trisphosphate (PIP3) has been implicated in many platelet functions however many of the mechanisms 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 their effects on activation on washed human platelets. Addition of either DiC8- or DiC16-PIP3 to human platelets induced aggregation in the presence of extracellular Ca(2+). This was reduced by the presence of indomethacin, the phospholipase C inhibitor U73122 and apyrase. DiC8-PIP3 induced the phosphorylation of Akt-Ser(473) which was reduced by the Akt inhibitor IV, wortmannin and EGTA (suggesting a dependence on Ca(2+) entry). In Fura2 loaded platelets DiC8-PIP3 was effective at increasing intracellular Ca(2+) in a distinct and transient manner that was reduced in the presence of indomethacin, U73122 and 2-aminoethyl diphenylborinate (2APB). Ca(2+) elevation was reduced by the non-SOCE inhibitor LOE908 and also by the SOCE inhibitor BTP2. DiC8-PIP3 induced the release of Ca(2+) from stores which was not affected by the proton dissipating agent bafilomycin A1 and was more potent than the two-pore channel agonist DiC8-PI[3,5]P2 suggesting release from an endoplasmic reticulum type store. DiC8-PIP3 weakly induced the tyrosine phosphorylation of Syk but not of PLCγ2. Finally like thrombin DiC8-PIP3 induced the formation of thromboxane B2 that was inhibited by the Akt inhibitor IV. These studies suggest that PIP3 via Ca(2+) elevation and Akt phosphorylation forms a central role in thromboxane A2 formation and the amplification of platelet activation.