Thrombin stimulation of platelets causes an increase in phosphatidylinositol 5-phosphate revealed by mass assay

Phosphatidylinositol 5-phosphate (PtdIns5P), a novel inositol lipid, has be en shown to be the major substrate for the type II PtdInsP kinases (PIPkins ) [Rameh et al. (1997) Nature 390, 192-196]. A PtdInsP fraction was prepare d from cell extracts by neomycin chromatography, using a protocol devised t o eliminate the interaction of acidic solvents with plasticware, since this was found to inhibit the enzyme. The PtdIns5P in this fraction was measure d by incubating with [gamma-P-32]ATP and recombinant PIPkin II alpha, and q uantifying the radiolabelled PtdInsP(2) formed. This assay was used on plat elets to show that during 10 min stimulation with thrombin, the mass level of PtdIns5P increases, implying the existence of an agonist-stimulated synt hetic mechanism. (C) 2000 Federation of European Biochemical Societies. Pub lished by Elsevier Science B.V. All rights reserved.