PDK1 governs thromboxane generation and thrombosis in platelets by regulating activation of Raf1 in the MAPK pathway

Abstract

Background Platelets are dynamic effector cells with functions that span hemostatic, thrombotic and inflammatory continua. Phosphoinositide-dependent protein kinase 1 (PDK1) regulates protease-activated receptor 4-induced platelet activation and thrombus formation through glycogen synthase kinase3β. However, whether PDK1 also signals through the ADP receptor and its functional importance in vivo remain unknown. Objective To establish the mechanism of PDK1 in ADP-induced platelet activation and thrombosis. Methods We assessed the role of PDK1 on 2MeSADP-induced platelet activation by measuring aggregation, thromboxane generation and phosphorylation events in the presence of BX-795, which inhibits PDK1, or by using platelet-specific PDK1 knockout mice and performing western blot analysis. PDK1 function in thrombus formation was assessed with an in vivo pulmonary embolism model. Results PDK1 inhibition with BX-795 reduced 2-methylthio-ADP (2MeSADP)-induced aggregation of human and murine platelets by abolishing thromboxane generation. Similar results were observed in pdk1 mice. PDK1 was also necessary for the phosphorylation of mitogen-activated protein kinase kinase 1/2 (MEK1/2), extracellular signal-regulated kinase 1/2, and cytosolic phospholipase A2, indicating that PDK1 regulates an upstream kinase in the mitogen-activated protein kinase (MAPK) pathway. We next determined that this upstream kinase is Raf-1, a serine/threonine kinase that is necessary for the phosphorylation of MEK1/2, as pharmacological inhibition and genetic ablation of PDK1 were sufficient to prevent Raf1 phosphorylation. Furthermore, in vivo inhibition or genetic ablation of PDK1 protected mice from collagen/epinephrine-induced pulmonary embolism. Conclusion PDK1 governs thromboxane generation and thrombosis in platelets that are stimulated with 2MeSADP by regulating activation of the MAPK pathway.

PDK1 governs thromboxane generation and thrombosis in platelets by regulating activation of Raf1 in the MAPK pathway: comment

“…PDK1 has gained interest in the field as a potential target for antithrombotic therapies [1]; however, the role of PDK1 downstream of the P2Y 1/12 receptors remained unknown. We therefore read with great interest a recent report published in the Journal of Thrombosis and Haemostasis by Manne et al [2] describing the role of PDK1 in regulating platelet activation and TxA 2 generation downstream of the P2Y 1/12 receptors. In their study, Manne et al used both pharmacological and genetic methods to conclusively show that targeting PDK1 can significantly attenuate pulmonary thromboembolisminduced mortality in an in vivo murine model.…”

PDK1 governs thromboxane generation and thrombosis in platelets by regulating activation of Raf1 in the MAPK pathway: reply

“…In conclusion, we appreciate the new findings provided by Patel and colleagues demonstrating that p38 is exclusively phosphorylated downstream of ASK1 in platelets and that the phosphorylation of cPLA2 is primarily regulated by p38 [1]. Based on these new and previously published findings [1][2][3][4], we propose the following model incorporating pathways regulating thromboxane generation by all three protein kinases (ERK1/2, p38, Pyk2), upon 2MesADP stimulation of platelets ( Fig. 1).…”

“…These findings indicate that p38 predominantly drives the phosphorylation of cPLA 2 in platelets. In support of this, a number of studies highlight the presence of other pathways that drive thromboxane production in a p38, ERK1/2, and Pyk2 mutually dependent manner [2] [3, 4]. …”

“…Based on these new and previously published findings [2] [3, 4] [1], we propose the following model incorporating pathways regulating thromboxane generation by all three protein kinases (ERK1/2, p38, Pyk2), upon 2MesADP stimulation of platelets.…”

“…We appreciate the letter from Dr Pravin Patel and colleagues [1] regarding our recent studies to determine how phosphoinositide 3-kinase (PDK1) regulates thromboxane generation [2], a mechanism that had not previously been known. We also examined the downstream signaling cascades, reporting that PDK1-regulated thromboxane generation is controlled through the Raf1-ERK1/2 pathway (Fig.…”