Phosphatidylinositol 3,4,5-trisphosphate is formed from phosphatidylinositol 4,5-bisphosphate in thrombin-stimulated platelets

Abstract

Platelets accumulate PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in response to thrombin and thrombin-receptor-directed peptide in a GTP-dependent manner. These phosphoinositides are considered to be mediators of signaling events in a variety of cells. We have examined the metabolic route by which PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are synthesized by briefly (10 min) incubating platelets with high activities of [32P]Pi, 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)P3 and PtdIns(3,4)P2. 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)P2 was significantly slower than that of PtdIns(3,4,5)P3. Increased labelled PtdIns3P was not detected within 60 s. The measured relative radioactivities decreased in the order 3 > 5 > 4 >> 1 for PtdIns(3,4,5)P3 and 3 > 4 >> 1 for PtdIns(3,4)P2. On the basis of the results of both rate-of-labelling and specific radioactivity analyses we conclude that PtdIns(3,4,5)Pa is formed by 3-OH phosphorylation of PtdIns(4,5)P2, whereas PtdIns(3,4)P2, may be formed by 3-OH phosphorylation of PtdIns4P and/or dephosphorylation of PtdIns(3,4,5)P3. These findings point to the activation of phosphoinositide 3-kinase as a critical receptor-regulated step in thrombin-stimulated platelets.

Thrombin stimulates wortmannin-inhibitable phosphoinositide 3-kinase and membrane blebbing in CHRF-288 cells

“…PtdIns(3,4)P # , which appears after a slight delay, may arise from hydrolysis of PtdIns(3,4,5)P $ or by PtdIns(4)P 3-kinase activation in these cells. These results are similar to those observed in thrombin-or SFLLRN-stimulated platelets [2,10,11]. Activation of PI 3-K by thrombin-receptor-directed ligand is greater than that induced by serum or LPA.…”

“…Our kinetic results for 3-PPI accumulation using SFLLRN or thrombin indicate that PtdIns(3,4,5)P $ accumulates rapidly and transiently, most likely as a result of phosphorylation of PtdIns(4,5)P # by PI 3-K, as reported for platelets [11]. PtdIns(3,4)P # , which appears after a slight delay, may arise from hydrolysis of PtdIns(3,4,5)P $ or by PtdIns(4)P 3-kinase activation in these cells.…”

“…[$#P]PtdIns(3,4,5)P $ and [$#P]PtdIns(3,4)P # were also deacylated and deglycerated, and the products were identified, respectively, as [$#P]Ins(1,3,4,5)P % and [$#P]Ins(1,3,4)P $ as described previously [11].…”

Purification and Characterization of Gβγ-responsive Phosphoinositide 3-Kinases from Pig Platelet Cytosol

“…Binding of the SH2 domains to phosphotyrosine residues within the sequence context, YMXM, which occurs in a wide range of activated growth factor receptors and adaptor proteins, causes the translocation and activation of the catalytic subunit (2, 15-16). More recently, a number of distinct p85 and p110 subunit isoforms have been identified (11,14,17), but the functional significance of this heterogeneity is not yet clear (18).Heterotrimeric G-protein-regulated forms of PI 3-kinase have also been identified following the observations that activation of G-protein-coupled receptors in neutrophils and platelets caused a rapid accumulation of PtdIns(3,4,[5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. Stephens et al (22) partially purified a G-protein ␤␥ subunit (G␤␥)-responsive PI 3-kinase from a myeloid cell line (U937).…”

“…Heterotrimeric G-protein-regulated forms of PI 3-kinase have also been identified following the observations that activation of G-protein-coupled receptors in neutrophils and platelets caused a rapid accumulation of PtdIns(3,4,[5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. Stephens et al (22) partially purified a G-protein ␤␥ subunit (G␤␥)-responsive PI 3-kinase from a myeloid cell line (U937).…”