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 docu mented in several cell systems, including agonist-stimulated platelets . The actual mass amounts and fatty acid composition of these unique l ipids, 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 chroma tographic system for the separation of PI 3,4-P2 from PI 4,5-P2. The m ass of PI 3,4-P2 in the stimulated platelet was 180 +/- 9.7 pmol/1 x 1 0(9) platelets (mean +/- S.E., n = 4), representing 9.3% of total phos phatidylinositol bisphosphate (PIP2). Based on HPLC analysis, PI 3,4-P 2 in unstimulated platelets represented <0.5% of total PIP2 (which cor responds to <7.0 pmol/l x 10(9) platelets). Fatty acid analysis of thi s lipid revealed a composition very similar to the conventional polyph osphoinositides (stearic and arachidonic acids accounting for 44.2 and 40.4 mol %, respectively, of the fatty acids). Since PI 3,4-P2 also d id not appear to be distinct from the other polyphosphoinositides, in regard to radiolabeling properties, it was concluded that this lipid i s unlikely to originate from a unique precursor pool. This conclusion validates the use of HPLC analysis of radiolabeled phosphoinositides f or the estimation of PI 3,4-P2 mass in agonist-stimulated platelets. T he chromatographic procedure described should prove useful for the mas s and fatty acid analysis of PI 3,4-P2 from other cell systems.