Role of phosphatidylinositol-3 kinase and its association with Gab1 in thrombopoietin-mediated up-regulation of platelet function.
OBJECTIVE
Human blood platelets are easily available physiologic target cells for thrombopoietin (TPO). TPO up-regulates platelet aggregation and alpha-granule secretion induced by various agonists. We investigated the role of phosphatidylinositol 3-kinase (PI3K) and its association with Gab1 in TPO-mediated up-regulation of platelet function.


METHODS
PI3K inhibitors (wortmannin and LY294002) and a MAP/ERK-kinase (MEK) inhibitor (PD98059) were used to investigate the role of these kinases in TPO-mediated up-regulation of platelet function. To elucidate the molecules associated with PI3K, we performed immunoprecipitation and pull-down experiments followed by immunoblotting. In vitro kinase assay also was performed to detect extracellular signal-regulated kinase (ERK) kinase activity.


RESULTS
TPO up-regulated platelet alpha-granule secretion and aggregation induced by thrombin, which was dose-dependently inhibited by preincubation with wortmannin or LY294002. Immunoprecipitation and pull-down experiments revealed that regulatory subunit of PI3K, p85, was rapidly associated with tyrosine-phosphorylated Gab1 via its n- and c-terminal SH2 domains. Pretreatment of platelets with TPO dramatically augmented the thrombin-induced ERK activation, which was almost completely inhibited by LY294002. Furthermore, a MEK inhibitor, PD98059, not completely but significantly inhibited TPO-mediated up-regulation of thrombin-induced alpha-granule secretion.


CONCLUSIONS
TPO induces the association of tyrosine-phosphorylated Gab1 with p85-PI3K. In downstream signaling, ERK is PI3K-dependently activated, which plays a critical role for TPO-mediated up-regulation of platelet function.
Kojima
H
H
Division of Hematology, Institute of Clinical Medicine, University of Tsukuba, Ibaraki, Japan. hkojima@md.tsukuba.ac.jp
Shinagawa
A
A
Shimizu
S
S
Kanada
H
H
Hibi
M
M
Hirano
T
T
Nagasawa
T
T
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D048728:MAP Kinase Kinase Kinase 1; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D010750:Phosphoproteins / Q000502:physiology*; D010974:Platelet Aggregation / Q000187:drug effects*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D013917:Thrombin / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology*; D000077191:Wortmannin
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D005419:Flavonoids; D048728:MAP Kinase Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D013917:Thrombin; D013926:Thrombopoietin; D000077191:Wortmannin; C098587:GAB1 protein, human; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C482576:MAP3K1 protein, human; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one