Fig. 1. Synthesis of InsP₆ in mammalian cells.

(A) Inositol phosphates are derived from the cyclitol inositol (Ins). In mammalian cells the synthesis of soluble InsPs starts with inositol 1,4,5-trisphosphate (InsP₃) which is released from phosphatidylinositol 4,5-bisphosphate at the plasma membrane upon PLC activation. InsP₃ is phosphorylated to inositol 1,3,4,5,6-pentakisphosphate (InsP₅) by inositol 1,4,5-trisphosphate 3-kinase (IP3K) and inositol multikinase (IPMK), then inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP5K) performs the last phosphorylation step to generate inositol 1,2,3,4,5,6-hexakisphosphate (InsP₆), which can be phosphorylated to pyrophosphates InsP₇ and InsP₈. The negative charge increases with increasing phosphorylation (℗ symbolizes the phosphate group (insert)). (B,C) Inositol phosphates were extracted by trichloroacetic acid from lysates of washed resting (white) and washed platelets stimulated either with ADP (10 μM) and collagen I (50 μg/ml) in presence of fibrinogen (300 μg/ml) (grey) or stimulated only with 2U/ml thrombin (black) and analyzed by metal dye detection (MDD)-HPLC. Mean values ± SEM of three independent experiments are shown. P-values *<0.05, ***<0.0001. (C) MDD-HPLC chromatograms of one representative experiment out of three independent experiments are shown staggered in panel C. Chromatrogram 1: Analysis of InsPs in non-stimulated platelets, 2: Analysis of InsPs in thrombin-stimulated platelets, 3: Analysis of InsPs in the supernatant of stimulated platelets, 4: InsP standard for comparison of retention times.