Ohtsuka Hiroko, Iguchi Tomohiro, Hayashi Moyuru, Kaneda Mizuho, Iida Kazuko, Shimonaka Motoyuki, Hara Takahiko, Arai Morio, Koike Yuichi, Yamamoto Naomasa, Kasahara Kohji
Laboratory of Biomembrane, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
Department of Chemistry, Tokyo University of Science, Tokyo, Japan.
PLoS One. 2017 Jan 10;12(1):e0169609. doi: 10.1371/journal.pone.0169609. eCollection 2017.
Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation.
基质细胞衍生因子-1α（SDF-1α）诱导的血小板聚集是通过其G蛋白偶联受体CXCR4和磷脂酰肌醇3激酶（PI3K）介导的。在此，我们证明SDF-1α可诱导人血小板中Akt在苏氨酸308和丝氨酸473位点的磷酸化。用CXCR4拮抗剂AMD3100或PI3K抑制剂LY294002预处理可抑制SDF-1α诱导的血小板聚集和Akt磷酸化。SDF-1α还可诱导PDK1在丝氨酸241位点（Akt的上游激活剂）、GSK3β在丝氨酸9位点（Akt的下游底物）以及肌球蛋白轻链在丝氨酸19位点（Akt信号通路的下游元件）的磷酸化。用Akt抑制剂MK-2206预处理可剂量依赖性地抑制SDF-1α诱导的血小板聚集。此外，用破坏脂筏的试剂甲基-β-环糊精预处理可抑制SDF-1α诱导的血小板聚集和Akt磷酸化。蔗糖密度梯度分析表明，35%的CXCR4、93%的异三聚体G蛋白Gαi-1、91%的Gαi-2、50%的Gβ和4.0%的PI3Kβ以及4.5%的Akt2定位于抗去污剂膜脂筏组分中。这些发现表明，脂筏中的SDF-1α/CXCR4信号通过PI3K依赖性的Akt磷酸化诱导血小板聚集。
