Kassouf Nick, Ambily Archana, Watson Stephanie, Hassock Sheila, Authi Harmeet S, Srivastava Salil, Watson Steve P, Authi Kalwant S
Cardiovascular Division, BHF Centre for Research Excellence, King's College London, Franklin Wilkins Building, Stamford Street, London SE1 9NH, United Kingdom.
Centre for Cardiovascular Sciences, Institute of Biomedical Research, The Medical School, University of Birmingham, Birmingham, UK.
Cell Signal. 2015 Jul;27(7):1488-98. doi: 10.1016/j.cellsig.2015.03.008. Epub 2015 Mar 19.
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.
磷脂酰肌醇三磷酸（PIP3）与多种血小板功能有关，但其许多机制仍需阐明。我们使用了PIP3的细胞可渗透类似物，1-O-(1,2-二棕榈酰-sn-甘油-3-O-磷酸基)-D-肌醇-3,4,5-三磷酸（DiC16-PIP3）或1-O-(1,2-二辛酰-sn-甘油-3-O-磷酸基)-D-肌醇-3,4,5-三磷酸（DiC8-PIP3）来研究它们对洗涤后的人血小板激活的影响。在细胞外Ca(2+)存在的情况下，向人血小板中添加DiC8-或DiC16-PIP3会诱导聚集。吲哚美辛、磷脂酶C抑制剂U73122和腺苷三磷酸双磷酸酶的存在会使其减少。DiC8-PIP3诱导Akt-Ser(473)磷酸化，Akt抑制剂IV、渥曼青霉素和乙二醇双(2-氨基乙基)醚四乙酸（EGTA）可使其减少（表明依赖于Ca(2+)内流）。在负载Fura2的血小板中，DiC8-PIP3能以一种独特且短暂的方式有效增加细胞内Ca(2+)，在吲哚美辛、U73122和2-氨基乙基二苯基硼酸盐（2APB）存在时会减少。非钙库操纵性钙内流（SOCE）抑制剂LOE908和SOCE抑制剂BTP2也会降低Ca(2+)升高。DiC8-PIP3诱导Ca(2+)从储存库释放，质子消散剂巴弗洛霉素A1对此无影响，且比双孔通道激动剂DiC8-PI[3,5]P2更有效，表明是从内质网型储存库释放。DiC8-PIP3微弱诱导Syk的酪氨酸磷酸化，但不诱导PLCγ2的酪氨酸磷酸化。最后，与凝血酶一样，DiC8-PIP3诱导血栓素B2形成，Akt抑制剂IV可抑制此过程。这些研究表明，PIP3通过Ca(2+)升高和Akt磷酸化在血栓素A2形成和血小板激活的放大过程中起核心作用。
