Identification of minor metabolites of phospholipid signal molecules in [(32)P]P(i)-labelled platelets.

Incubation of blood platelets with P-32-labelled inorganic phosphate for 60 min leads to incorporation of radioisotope mainly into phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP) and phosphatidyl-4,5-bisphosphate (PIP2) in resting platelets and into phosphatidic acid (PA) in activated platelets. Small amounts of other important phosphoinositide isomers also become labelled following platelet activation, among them the 3-phosphorylated derivatives. In addition, several other faintly labelled spots are visible on TLC separations. Three of these lipids have now been identified as lysophosphatidylinositol (lysoPI), lysophosphatidic acid (lysoPA) and CDP-diacylglcerol (CDP-DAG).[P-32]LysoPI was present in resting and activated platelets, whereas [P-32]lysoPA and [P-32]CDP-DAG were observed only upon platelet activation. The phosphoinositide cycle turns over without accumulation of [P-32]PA and [P-32]CDP-DAG in resting platelets. A large increase (as much as 40-fold) in the steady-state level of [P-32]PA is seen in thrombin-activated platelets. A slight increase in the steady-state levels of [P-32]CDP-DAG is accompanied by a similar increase in [P-32]PI and larger increases in [P-32]PIP and [P-32]PIP2 (about 50%), which is indicative of a general increase in flux in the PPI cycle. Elevation of CDP-DAG levels is probably only a reflection of increased flux, whereas lysoPA and lysoPI have been reported to have diverse signalling functions in various cells.