Phosphatidylinositol-3,4,5-trisphosphate stimulates Ca2+ elevation and Akt phosphorylation to constitute a major mechanism of thromboxane A2 formation in human platelets

Nick Kassouf, Archana Ambily, Stephanie Watson, Sheila Hassock, Harmeet S. Authi, Salil Srivastava, Steve P. Watson, Kalwant S. Authi

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8 Citations (Scopus)

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 Ca2+. This was reduced by the presence of indomethacin, the phospholipase C inhibitor U73122 and apyrase. DiC8-PIP3 induced the phosphorylation of Akt-Ser473 which was reduced by the Akt inhibitor IV, wortmannin and EGTA (suggesting a dependence on Ca2 + entry). In Fura2 loaded platelets DiC8-PIP3 was effective at increasing intracellular Ca2+ in a distinct and transient manner that was reduced in the presence of indomethacin, U73122 and 2-aminoethyl diphenylborinate (2APB). Ca2+ elevation was reduced by the non-SOCE inhibitor LOE908 and also by the SOCE inhibitor BTP2. DiC8-PIP3 induced the release of Ca2+ 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 Ca2+ elevation and Akt phosphorylation forms a central role in thromboxane A2 formation and the amplification of platelet activation.
Original languageEnglish
Pages (from-to)1488-1498
Number of pages11
JournalCellular Signalling
Volume27
Issue number7
Early online date19 Mar 2015
DOIs
Publication statusPublished - Jul 2015

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