PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE IS FORMED FROM PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE IN THROMBIN-STIMULATED PLATELETS

Platelets accumulate PtdIns(3,4,5)P-3 and PtdIns(3,4)P-2 in response to thrombin and thrombin-receptor-directed peptide in a GTP-dependent manner. These phosphoinositides are considered to be mediators of signalling events in a variety of cells. We have examined the metabolic route by which PtdIns(3,4,5)P-3 and PtdIns(3,4)P-2 are synthesized by briefly (10 min) incubating platelets with high activities of [P-32]P-i, followed by 20 or 60 s exposure to thrombin, and analysing the relative radioactivities of the individual phosphate groups in the resulting labelled PtdIns(3,4,5)P-3 and PtdIns(3,4)P-2. The phosphate group possessing the highest specific activity under such non-equilibrium labelling conditions indicates the last one added in a metabolic sequence. The thrombin-stimulated rate of labelling of PtdIns(3,4)P-2 was significantly slower than that of PtdIns(3,4,5)P-3. Increased labelled PtdIns3P was not detected within 60 s. The measured relative radioactivities decreased in the order 3 > 5 > 4 much greater than 1 for PtdIns(3,4,5)P-3 and 3 > 4 much greater than 1 for PtdIns(3,4)P-2. On the basis of the results of both rate-of-labelling and specific radioactivity analyses we conclude that PtdIns(3,4,5)P-3 is formed by 3-OH phosphorylation of PtdIns(4,5)P-2, whereas PtdIns(3,4)P-2, may be formed by 3-OH phosphorylation of PtdIns4P and/or dephosphorylation of PtdIns(3,4,5)P-3. These findings point to the activation of phosphoinositide 3-kinase as a critical receptor-regulated step in thrombin-stimulated platelets.