Phosphoinositide 3-kinase p110α negatively regulates thrombopoietin-mediated platelet activation and thrombus...

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.