Abstract 22: Crosstalk Between Phosphotidylinositol-4,5 Bisphosphonate-3 Kinase Pathway and Mitogen-Activated Protein Kinase Pathway Regulates Platelet Activation and Protein Synthesis via Phosphoinositide-Dependent Kinase-1

Phosphoinositide-dependent protein kinase 1 (PDK1) is known to regulate PAR4 induced platelet activation and thrombus formation through GSK3β. However, whether PDK1 signaling also involves the ADP receptor and, if so, downstream functional consequences are unknown. We employed both pharmacologic (e.g. the selective PDK1 inhibitor, BX795) and genetic (platelet specific deletion of PDK1) approaches to dissect the role of PDK1 in ADP-induced platelet activation and protein synthesis. Inhibition of PDK1 with BX795 reduced 2MeSADP-induced platelet aggregation by abolishing thromboxane generation. Similar results were observed in PDK1 -/- mice (Fig A). Inhibition of PDK1 protected mice from collagen and epinephrine-induced pulmonary embolism (Fig B). PDK1 was also necessary for the phosphorylation of MEK1/2, Erk1/2 and cPLA2, indicating that PDK1 regulates an upstream kinase in MAPK pathway. We next identified that this upstream kinase is Raf1 (necessary for the phosphorylation of MEK1/2), as pharmacologic inhibition and genetic ablation of PDK1 was sufficient to prevent Raf1 phosphorylation (Fig C). Pharmacologic inhibition and genetic ablation of PDK1 blocked MAPK- and mTORC1-dependent protein synthesis in platelets through a mechanism requiring the phosphorylation of eIF4E and S6K. Concordantly, PDK1 is necessary for signal-dependent synthesis of the protein bcl3, which is under mTORC1-dependent control (Fig C). Taken together, our findings show for the first time that PDK1, a master kinase in the PI3K pathway, directly governs thromboxane generation, thrombosis, and protein synthesis in platelets through regulating MAPK and mTORC1 pathways.