title: A collagen-related peptide regulates phospholipase Cgamma2 via phosphatidylinositol 3-kinase in human platelets.
abstract: The collagen receptor glycoprotein VI (GPVI) induces platelet activation through a similar pathway to that used by immune receptors. In the present study we have investigated the role of phosphatidylinositol 3-kinase (PI 3-kinase) in GPVI signalling. Our results show that collagen-related peptide {CRP: [GCP*(GPP*)(10)GCP*G](n); P*=hydroxyproline}, which is selective to GPVI, induces formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 3,4-bisphosphate [PI(3, 4)P(2)] in platelets. The increase in the two 3-phosphorylated lipids is inhibited completely by wortmannin and by LY294002, two structurally unrelated inhibitors of PI 3-kinase. The formation of inositol phosphates and phosphatidic acid (PA), two markers of phospholipase C (PLC) activation, by CRP are inhibited by between 50 and 85% in the presence of wortmannin and LY294002. This is associated with inhibition of elevation of intracellular Ca(2+) ([Ca(2+)](i)) and aggregation. Wortmannin and LY294002 also partially inhibit elevation of Ca(2+) by CRP in murine megakaryocytes. Microinjection of the pleckstrin-homology PH domain of Bruton's tyrosine kinase, which binds selectively to PI(3,4, 5)P(3), but not the R28C (Arg(28)-->Cys) mutant which binds to PI(3, 4,5)P(3) with low affinity, also inhibits elevation of [Ca(2+)](i) in megakaryocytes, suggesting that it is this lipid species which mediates the action of the PI 3-kinase pathway. Studies in platelets show that the action of wortmannin and LY294002 is not mediated through an alteration in tyrosine phosphorylation of PLCgamma2. These results demonstrate that PI 3-kinase is required for full activation of PLCgamma2 by GPVI in platelets and megakaryocytes.
authors.affiliation: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, U.K.
authors.forename: J M
authors.initials: JM
authors.lastname: Pasquet
authors.forename: R
authors.initials: R
authors.lastname: Bobe
authors.forename: B
authors.initials: B
authors.lastname: Gross
authors.forename: M P
authors.initials: MP
authors.lastname: Gratacap
authors.forename: M G
authors.initials: MG
authors.lastname: Tomlinson
authors.forename: B
authors.initials: B
authors.lastname: Payrastre
authors.forename: S P
authors.initials: SP
authors.lastname: Watson
mesh_terms: D000077329:Agammaglobulinaemia Tyrosine Kinase; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins; D002867:Chromones / Q000494:pharmacology; D006801:Humans; D016023:Integrins / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D008533:Megakaryocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051966:Phospholipase C gamma; D010766:Phosphorylation / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D038981:Receptors, Collagen; D015398:Signal Transduction / Q000187:drug effects*; D010738:Type C Phospholipases / Q000378:metabolism*; D000077191:Wortmannin
chemical_list: D000077329:Agammaglobulinaemia Tyrosine Kinase; D000730:Androstadienes; D002118:Calcium; D002352:Carrier Proteins; D002867:Chromones; D016023:Integrins; D007527:Isoenzymes; D009025:Morpholines; D010712:Phosphatidic Acids; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051966:Phospholipase C gamma; D011505:Protein-Tyrosine Kinases; D011506:Proteins; D038981:Receptors, Collagen; D010738:Type C Phospholipases; D000077191:Wortmannin; C007105:c1q-binding proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; C060980:phosphatidylinositol 3,4-diphosphate; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C000625951:Btk protein, mouse