Mass and fatty acid composition of the 3-phosphorylated phosphatidylinositol bisphosphate isomer in stimulated human platelets.

By high pressure liquid chromatography (HPLC) analysis, the occurrence of radiolabeled 3-phosphorylated phosphoinositides has been well documented in several cell systems, including agonist-stimulated platelets. The actual mass amounts and fatty acid composition of these unique lipids, however, have not been reported to date. In the present study, we report the mass and fatty acid composition of phosphatidylinositol (PI) 3,4-P2 from U46619-stimulated platelets using a thin-layer chromatographic system for the separation of PI 3,4-P2 from PI 4,5-P2. The mass of PI 3,4-P2 in the stimulated platelet was 180 +/- 9.7 pmol/1 x 10(9) platelets (mean +/- S.E., n = 4), representing 9.3% of total phosphatidylinositol bisphosphate (PIP2). Based on HPLC analysis, PI 3,4-P2 in unstimulated platelets represented < 0.5% of total PIP2 (which corresponds to < 7.0 pmol/1 x 10(9) platelets). Fatty acid analysis of this lipid revealed a composition very similar to the conventional polyphosphoinositides (stearic and arachidonic acids accounting for 44.2 and 40.4 mol %, respectively, of the fatty acids). Since PI 3,4-P2 also did not appear to be distinct from the other polyphosphoinositides, in regard to radiolabeling properties, it was concluded that this lipid is unlikely to originate from a unique precursor pool. This conclusion validates the use of HPLC analysis of radiolabeled phosphoinositides for the estimation of PI 3,4-P2 mass in agonist-stimulated platelets. The chromatographic procedure described should prove useful for the mass and fatty acid analysis of PI 3,4-P2 from other cell systems.