Tyrosine kinases and phosphoinositide metabolism in thrombin-stimulated human platelets.
In this study we have examined the implication of tyrosine kinase activities in aggregation, 5-hydroxytryptamine secretion and mainly phosphoinositide metabolism in response to human platelet stimulation by thrombin. Using the potent tyrosine kinase inhibitor tyrphostin AG-213, we have observed a significant inhibition of aggregation and 5-hydroxytryptamine release; however, this percentage inhibition was lower at high thrombin concentrations. On the other hand, tyrphostin treatment of metabolically 32P-labelled platelets significantly inhibited the thrombin-dependent accumulation of PtdIns(3,4)P2, which involves at least a PtdIns 3-kinase and/or a PtdIns3P 4-kinase, whereas the synthesis of phosphatidic acid (PtdOH), a good reflection of the phospholipase C (PLC) activation in platelets, was partially blocked. Inositol phosphate production was also inhibited by about 40% when tyrphostin-treated platelets were stimulated with thrombin. In addition, we show by Western-blot analysis that PLC gamma 1, as well as the regulatory subunit (p85) of the PtdIns 3-kinase, were present in the anti-phosphotyrosine immunoprecipitate isolated from thrombin-stimulated platelets. Furthermore, tyrphostin treatment clearly decreased the PLC gamma 1 and p85 contents in such an anti-phosphotyrosine immunoprecipitate. Our results provide the first evidence for a direct or indirect regulation of PtdIns(3,4)P2 accumulation and PLC gamma 1 activity by tyrosine phosphorylation during thrombin stimulation of human platelets.
Institut National de la Santé et de la Recherche Médicale, Unité 326, Hôpital Purpan, Toulouse, France.
C
C
Guinebault
B
B
Payrastre
C
C
Sultan
G
G
Mauco
M
M
Breton
S
S
Levy-Toledano
M
M
Plantavid
H
H
Chap
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002396:Catechols / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007295:Inositol Phosphates / Q000097:blood; D007700:Kinetics; D046911:Macromolecular Substances; D009570:Nitriles / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols / Q000097:blood*; D010770:Phosphotransferases / Q000097:blood / Q000302:isolation & purification; D015539:Platelet Activation; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000097:blood*; D012701:Serotonin / Q000097:blood; D013917:Thrombin / Q000494:pharmacology*; D020032:Tyrphostins
D002396:Catechols; D007295:Inositol Phosphates; D046911:Macromolecular Substances; D009570:Nitriles; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010975:Platelet Aggregation Inhibitors; D020032:Tyrphostins; C060980:phosphatidylinositol 3,4-diphosphate; C060152:tyrphostin 47; D012701:Serotonin; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D013917:Thrombin