Phosphoinositide 3-kinase p110 alpha negatively regulates thrombopoietin-mediated platelet activation and thrombus formation

摘要

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; p110 beta in platelet function, however the role of p110 alpha is still largely unexplored. Here we sought to investigate the role of p110 alpha in TPO-mediated hyperactivity by using a conditional p110 alpha 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 p110 alpha KO platelets. Furthermore, TPO-mediated enhancement of thrombus formation by p110 alpha KO platelets was elevated over wild-type (WT) platelets, suggesting that p110 alpha 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) P-3) formation and phosphorylation of Akt and glycogen synthase kinase 3 (GSK3) were comparable between WT and p110 alpha KO platelets. In contrast, extracellular responsive kinase (ERK) phosphorylation and thromboxane (TxA(2)) formation were significantly enhanced in p110 alpha KO platelets, both of which were blocked by the MEK inhibitor PD184352, whereas the p38 MAPK inhibitor VX-702 and p110 alpha inhibitor PIK-75 had no effect. Acetylsalicylic acid (ASA) blocked the enhancement of thrombus formation by TPO in both WT and p110 alpha KO mice. Together, these results demonstrate that p110 alpha negatively regulates TPO-mediated enhancement of platelet function by restricting ERK phosphorylation and TxA(2) synthesis in a manner independent of its kinase activity.