Identification of minor metabolites of phospholipid signal molecules in [³²P]P_i-labelled platelets

Author： Iversen Hege Fuglebakk Edvin Holmsen Holm Fukami Miriam H.

ISSN： 1369-1635

Source： Platelets, Vol.13, Iss.5-6, 2002-08, pp. : 279-284

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Abstract

Incubation of blood platelets with ³²P-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).[³²P]LysoPI was present in resting and activated platelets, whereas [³²P]lysoPA and [³²P]CDP-DAG were observed only upon platelet activation. The phosphoinositide cycle turns over without accumulation of [³²P]PA and [³²P]CDP-DAG in resting platelets. A large increase (as much as 40-fold) in the steady-state level of [³²P]PA is seen in thrombin-activated platelets. A slight increase in the steady-state levels of [³²P]CDP-DAG is accompanied by a similar increase in [³²P]PI and larger increases in [³²P]PIP and [³²P]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.