Phosphoinositide 3-kinase (PI3K) plays an important role in platelet function and contributes to platelet hyperreactivity induced by elevated levels of circulating peptide hormones, including thrombopoietin (TPO). Previous work established an important role for the PI3K isoform; p110β in platelet function, however the role of p110α is still largely unexplored. Here we sought to investigate the role of p110α in TPO-mediated hyperactivity by using a conditional p110α knockout (KO) murine model in conjunction with platelet functional assays. We found that TPO-mediated enhancement of collagen-related peptide (CRP-XL)-induced platelet aggregation and adenosine triphosphate (ATP) secretion were significantly increased in p110α KO platelets. Furthermore, TPO-mediated enhancement of thrombus formation by p110α KO platelets was elevated over wild-type (WT) platelets, suggesting that p110α negatively regulates TPO-mediated priming of platelet function. The enhancements were not due to increased flow through the PI3K pathway as phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃) formation and phosphorylation of Akt and glycogen synthase kinase 3 (GSK3) were comparable between WT and p110α KO platelets. In contrast, extracellular responsive kinase (ERK) phosphorylation and thromboxane (TxA₂) formation were significantly enhanced in p110α KO platelets, both of which were blocked by the MEK inhibitor PD184352, whereas the p38 MAPK inhibitor VX-702 and p110α inhibitor PIK-75 had no effect. Acetylsalicylic acid (ASA) blocked the enhancement of thrombus formation by TPO in both WT and p110α KO mice. Together, these results demonstrate that p110α negatively regulates TPO-mediated enhancement of platelet function by restricting ERK phosphorylation and TxA₂ synthesis in a manner independent of its kinase activity.

磷酸肌醇 3-激酶 (PI3K) 在血小板功能中起重要作用，并有助于由包括血小板生成素 (TPO) 在内的循环肽激素水平升高引起的血小板高反应性。以前的工作确立了 PI3K 异构体的重要作用。p110β 在血小板功能中的作用，然而 p110α 的作用在很大程度上仍未被探索。在这里，我们试图通过使用条件性 p110α 敲除 (KO) 小鼠模型结合血小板功能测定来研究 p110α 在 TPO 介导的过度活跃中的作用。我们发现 TPO 介导的胶原相关肽 (CRP-XL) 诱导的血小板聚集和三磷酸腺苷 (ATP) 分泌的增强在 p110α KO 血小板中显着增加。此外，TPO 介导的 p110α KO 血小板对血栓形成的增强作用高于野生型 (WT) 血小板，这表明 p110α 负向调节 TPO 介导的血小板功能启动。增强不是由于通过 PI3K 途径的流量增加，因为磷脂酰肌醇 3,4,5-三磷酸 (PI(3,4,5)P₃ ) Akt 和糖原合酶激酶 3 (GSK3) 的形成和磷酸化在 WT 和 p110α KO 血小板之间具有可比性。相比之下，p110α KO 血小板中的细胞外反应激酶 (ERK) 磷酸化和血栓素 (TxA ₂ ) 形成显着增强，这两者都被 MEK 抑制剂 PD184352 阻断，而 p38 MAPK 抑制剂 VX-702 和 p110α 抑制剂 PIK-75没有效果。乙酰水杨酸 (ASA) 在 WT 和 p110α KO 小鼠中阻断了 TPO 对血栓形成的增强作用。总之，这些结果表明 p110α 通过以独立于其激酶活性的方式限制 ERK 磷酸化和 TxA _{2合成来负调节 TPO 介导的血小板功能增强。}