title: Phosphatidylinositol-3,4,5-trisphosphate stimulates Ca(2+) elevation and Akt phosphorylation to constitute a major mechanism of thromboxane A2 formation in human platelets.
abstract: Phosphatidylinositol trisphosphate (PIP3) has been implicated in many platelet functions however many of the mechanisms need clarification. We have used cell permeable analogues of PIP3,1-O-(1,2-di-palmitoyl-sn-glyero-3-O-phosphoryl)-D-myo-inositol-3,4,5-trisphosphate (DiC16-PIP3) or 1-O-(1,2-di-octanoyl-sn-glyero-3-O-phosphoryl)-D-myo-inositol-3,4,5-trisphosphate (DiC8-PIP3) to study their effects on activation on washed human platelets. Addition of either DiC8- or DiC16-PIP3 to human platelets induced aggregation in the presence of extracellular Ca(2+). This was reduced by the presence of indomethacin, the phospholipase C inhibitor U73122 and apyrase. DiC8-PIP3 induced the phosphorylation of Akt-Ser(473) which was reduced by the Akt inhibitor IV, wortmannin and EGTA (suggesting a dependence on Ca(2+) entry). In Fura2 loaded platelets DiC8-PIP3 was effective at increasing intracellular Ca(2+) in a distinct and transient manner that was reduced in the presence of indomethacin, U73122 and 2-aminoethyl diphenylborinate (2APB). Ca(2+) elevation was reduced by the non-SOCE inhibitor LOE908 and also by the SOCE inhibitor BTP2. DiC8-PIP3 induced the release of Ca(2+) from stores which was not affected by the proton dissipating agent bafilomycin A1 and was more potent than the two-pore channel agonist DiC8-PI[3,5]P2 suggesting release from an endoplasmic reticulum type store. DiC8-PIP3 weakly induced the tyrosine phosphorylation of Syk but not of PLCγ2. Finally like thrombin DiC8-PIP3 induced the formation of thromboxane B2 that was inhibited by the Akt inhibitor IV. These studies suggest that PIP3 via Ca(2+) elevation and Akt phosphorylation forms a central role in thromboxane A2 formation and the amplification of platelet activation.
authors: Cardiovascular Division, BHF Centre for Research Excellence, King's College London, Franklin Wilkins Building, Stamford Street, London SE1 9NH, United Kingdom.
authors: Nick
authors: N
authors: Kassouf
authors: Cardiovascular Division, BHF Centre for Research Excellence, King's College London, Franklin Wilkins Building, Stamford Street, London SE1 9NH, United Kingdom.
authors: Archana
authors: A
authors: Ambily
authors: Centre for Cardiovascular Sciences, Institute of Biomedical Research, The Medical School, University of Birmingham, Birmingham, UK.
authors: Stephanie
authors: S
authors: Watson
authors: Cardiovascular Division, BHF Centre for Research Excellence, King's College London, Franklin Wilkins Building, Stamford Street, London SE1 9NH, United Kingdom.
authors: Sheila
authors: S
authors: Hassock
authors: Cardiovascular Division, BHF Centre for Research Excellence, King's College London, Franklin Wilkins Building, Stamford Street, London SE1 9NH, United Kingdom.
authors: Harmeet S
authors: HS
authors: Authi
authors: Cardiovascular Division, BHF Centre for Research Excellence, King's College London, Franklin Wilkins Building, Stamford Street, London SE1 9NH, United Kingdom.
authors: Salil
authors: S
authors: Srivastava
authors: Centre for Cardiovascular Sciences, Institute of Biomedical Research, The Medical School, University of Birmingham, Birmingham, UK.
authors: Steve P
authors: SP
authors: Watson
authors: Cardiovascular Division, BHF Centre for Research Excellence, King's College London, Franklin Wilkins Building, Stamford Street, London SE1 9NH, United Kingdom. Electronic address: Kalwant.authi@kcl.ac.uk.
authors: Kalwant S
authors: KS
authors: Authi
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D016257:Fura-2 / Q000737:chemistry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000494:pharmacology*; D051966:Phospholipase C gamma / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000072377:Syk Kinase; D013928:Thromboxane A2 / Q000032:analysis / Q000378:metabolism*; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D047908:Intracellular Signaling Peptides and Proteins; D018129:Phosphatidylinositol Phosphates; D047428:Protein Kinase Inhibitors; C060974:phosphatidylinositol 3,4,5-triphosphate; D004533:Egtazic Acid; D013928:Thromboxane A2; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D051057:Proto-Oncogene Proteins c-akt; D051966:Phospholipase C gamma; D002118:Calcium; D016257:Fura-2; D000077191:Wortmannin
keywords: Akt; Ca(2+) elevation; PIP(3); Platelet activation; Thromboxane A(2)