Regular Article PLATELETS AND THROMBOPOIESIS PDK1 regulates platelet activation and arterial thrombosis

Platelets, which are derived from megakaryocytes, circulate in mammalian blood and play essential roles in hemostasis, angiogenesis, inflammation, and metastasis. Phosphoinositide 3-kinases (PI3Ks) are a conserved family of enzymes, each having both protein and lipid kinase activities. PI3K-mediated signaling affects platelet adhesion and aggregation. PI3Ks are activated downstream of several membrane proteins, including G protein–coupled receptors (GPCRs) and the multifunctional platelet receptors aIIbb3, GPIb/ IX/V, and GPVI. PI3Ks facilitate thrombus formation by enhancing aIIbb3 activation and calcium release. PI3Ks activate diverse substrates carrying the pleckstrin homology domain, which binds phosphorylated phosphatidylinositol and facilitates the recruitment of downstream effectors to the plasma membrane. A serine-threonine family kinase, protein kinase B (PKB/ Akt), which includes three isoforms, Akt1, Akt2, and Akt3, is the primary enzyme activated by PI3Ks. All of these Akt isoforms are expressed in human and mouse platelets and play critical roles in platelet activation induced by aIIbb3, GPIb/IX/V, the collagen receptor GPVI, and GPCRs. Recently, we reported that PI3K direct effector phosphatase and tensin homolog dephosphorylates PtdIns(3, 4, 5)P3 (PIP3), producing PtdIns(4, 5)P2, thereby negatively regulating Akt phosphorylation and collagen-induced platelet activation. Phosphoinositide-dependent protein kinase 1 (PDK1) is another cytoplasmic membrane–associated enzyme activated by PI3K. PDK1 plays essential roles in cell growth, metabolism, proliferation, and survival. PDK1 is activated by binding to the membrane-tethered PIP3, and the activated PDK1 phosphorylates Akt at Thr308 thereby activating its serine/threonine kinase activity. Although a study reported that integrin b3 Thr753 can be phosphorylated by PDK1 and Akt in vitro, the role of PDK1 in platelet activation and thrombus formation remains unknown. Here, we investigate the role of PDK1 in platelet activation and thrombus formation using mice with a plateletspecific PDK1 deletion and pharmacologic reagents. We found that platelet PDK1 activates Akt and inhibits Gsk3b, thereby enhancing thrombin-induced platelet aggregation, clot retraction, platelet spreading on immobilized fibrinogen (Fg), and thrombus formation.