Department of Internal Medicine, Molecular Medicine Program, University of Utah, Salt Lake City, UT, USA.
Department of Cardiology and Cardiovascular Medicine, University of Tübingen, Tübingen, Germany.
J Thromb Haemost. 2018 Jun;16(6):1211-1225. doi: 10.1111/jth.14005. Epub 2018 May 8.
Essentials Phosphoinositide 3-kinase and MAPK pathways crosstalk via PDK1. PDK1 is required for adenosine diphosphate-induced platelet activation and thromboxane generation. PDK1 regulates RAF proto-oncogene Ser/Thr kinase (Raf1) activation in the MAPK pathway. Genetic ablation of PDK1 protects against platelet-dependent thrombosis in vivo.
Background Platelets are dynamic effector cells with functions that span hemostatic, thrombotic and inflammatory continua. Phosphoinositide-dependent protein kinase 1 (PDK1) regulates protease-activated receptor 4-induced platelet activation and thrombus formation through glycogen synthase kinase3β. However, whether PDK1 also signals through the ADP receptor and its functional importance in vivo remain unknown. Objective To establish the mechanism of PDK1 in ADP-induced platelet activation and thrombosis. Methods We assessed the role of PDK1 on 2MeSADP-induced platelet activation by measuring aggregation, thromboxane generation and phosphorylation events in the presence of BX-795, which inhibits PDK1, or by using platelet-specific PDK1 knockout mice and performing western blot analysis. PDK1 function in thrombus formation was assessed with an in vivo pulmonary embolism model. Results PDK1 inhibition with BX-795 reduced 2-methylthio-ADP (2MeSADP)-induced aggregation of human and murine platelets by abolishing thromboxane generation. Similar results were observed in pdk1 mice. PDK1 was also necessary for the phosphorylation of mitogen-activated protein kinase kinase 1/2 (MEK1/2), extracellular signal-regulated kinase 1/2, and cytosolic phospholipase A2, indicating that PDK1 regulates an upstream kinase in the mitogen-activated protein kinase (MAPK) pathway. We next determined that this upstream kinase is Raf-1, a serine/threonine kinase that is necessary for the phosphorylation of MEK1/2, as pharmacological inhibition and genetic ablation of PDK1 were sufficient to prevent Raf1 phosphorylation. Furthermore, in vivo inhibition or genetic ablation of PDK1 protected mice from collagen/epinephrine-induced pulmonary embolism. Conclusion PDK1 governs thromboxane generation and thrombosis in platelets that are stimulated with 2MeSADP by regulating activation of the MAPK pathway.
磷脂酰肌醇 3-激酶和 MAPK 途径通过 PDK1 相互作用。PDK1 是二磷酸腺苷诱导的血小板活化和血栓素生成所必需的。PDK1 调节丝裂原活化蛋白激酶（MAPK）途径中 RAF 原癌基因丝氨酸/苏氨酸激酶（Raf1）的激活。PDK1 的基因缺失可防止体内血小板依赖性血栓形成。
血小板是具有跨越止血、血栓形成和炎症连续性的功能的动态效应细胞。磷酸肌醇依赖性蛋白激酶 1（PDK1）通过糖原合酶激酶 3β调节蛋白酶激活受体 4 诱导的血小板活化和血栓形成。然而，PDK1 是否也通过 ADP 受体信号传导及其在体内的功能重要性尚不清楚。目的是确定 PDK1 在 ADP 诱导的血小板活化和血栓形成中的作用机制。
我们通过测量在 BX-795 存在下的聚集、血栓素生成和磷酸化事件，评估了 PDK1 在 2MeSADP 诱导的血小板活化中的作用，BX-795 抑制 PDK1，或使用血小板特异性 PDK1 敲除小鼠和进行 Western blot 分析。用体内肺栓塞模型评估 PDK1 在血栓形成中的作用。
PDK1 抑制剂 BX-795 通过消除血栓素生成，减少 2-甲基硫代-ADP（2MeSADP）诱导的人源和鼠源血小板聚集。在 pdk1 小鼠中也观察到类似的结果。PDK1 对于丝裂原活化蛋白激酶激酶 1/2（MEK1/2）、细胞外信号调节激酶 1/2 和细胞质磷脂酶 A2 的磷酸化也是必需的，表明 PDK1 调节丝裂原活化蛋白激酶（MAPK）途径中的上游激酶。我们接下来确定该上游激酶是 Raf-1，一种丝氨酸/苏氨酸激酶，对于 MEK1/2 的磷酸化是必需的，因为 PDK1 的药理学抑制和基因缺失足以防止 Raf1 磷酸化。此外，体内抑制或基因缺失 PDK1 可保护小鼠免受胶原/肾上腺素诱导的肺栓塞。
PDK1 通过调节 MAPK 途径的激活来控制血小板对 2MeSADP 的刺激所引起的血栓素生成和血栓形成。
