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PDK1 Governs Thromboxane Generation and Thrombosis in Platelets by Regulating Activation of Raf1 in the MAPK Pathway

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PDK1 Governs Thromboxane Generation and Thrombosis in Platelets by Regulating Activation of Raf1 in the MAPK Pathway Journal of Thrombosis and Haemostasis, 16: 1211–1225 DOI: 10.1111/jth.14005 ORIGINAL ARTICLE PDK1 governs thromboxane generation and thrombosis in platelets by regulating activation of Raf1 in the MAPK pathway B. K. MANNE,* P. MUN€ Z E R , † R. BADOLIA,‡ B. WALKER-ALLGAIER,† R. A. CAMPBELL,* E. MIDDLETON,* A. S. WEYRICH,* S. P. KUNAPULI,‡ O. BORST† andM. T. RONDINA*§ *Department of Internal Medicine, Molecular Medicine Program, University of Utah, Salt Lake City, UT, USA; †Department of Cardiology and Cardiovascular Medicine, University of Tubingen,€ Tubingen,€ Germany; ‡Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA; and §Department of Internal Medicine, GRECC, George E. Wahlen VAMC, Salt Lake City, UT, USA To cite this article: Manne BK, Munzer€ P, Badolia R, Walker-Allgaier B, Campbell RA, Middleton E, Weyrich AS, Kunapuli SP, Borst O, Rondina MT. PDK1 governs thromboxane generation and thrombosis in platelets by regulating activation of Raf1 in the MAPK pathway. J Thromb Hae- most 2018; 16: 1211–25. function in thrombus formation was assessed with an Essentials in vivo pulmonary embolism model. Results: PDK1 inhi- bition with BX-795 reduced 2-methylthio-ADP • Phosphoinositide 3-kinase and MAPK pathways cross- (2MeSADP)-induced aggregation of human and murine talk via PDK1. platelets by abolishing thromboxane generation. Similar • PDK1 is required for adenosine diphosphate-induced À À results were observed in pdk1 / mice. PDK1 was also platelet activation and thromboxane generation. necessary for the phosphorylation of mitogen-activated • PDK1 regulates RAF proto-oncogene Ser/Thr kinase protein kinase kinase 1/2 (MEK1/2), extracellular signal- (Raf1) activation in the MAPK pathway. regulated kinase 1/2, and cytosolic phospholipase A2, • Genetic ablation of PDK1 protects against platelet- indicating that PDK1 regulates an upstream kinase in the dependent thrombosis in vivo. mitogen-activated protein kinase (MAPK) pathway. We next determined that this upstream kinase is Raf-1, a ser- Summary. Background: Platelets are dynamic effector cells ine/threonine kinase that is necessary for the phosphoryla- with functions that span hemostatic, thrombotic and tion of MEK1/2, as pharmacological inhibition and inflammatory continua. Phosphoinositide-dependent pro- genetic ablation of PDK1 were sufficient to prevent Raf1 tein kinase 1 (PDK1) regulates protease-activated receptor phosphorylation. Furthermore, in vivo inhibition or 4-induced platelet activation and thrombus formation genetic ablation of PDK1 protected mice from collagen/ b through glycogen synthase kinase3 . However, whether epinephrine-induced pulmonary embolism. Conclusion: PDK1 also signals through the ADP receptor and its func- PDK1 governs thromboxane generation and thrombosis tional importance in vivo remain unknown. Objective: To in platelets that are stimulated with 2MeSADP by regulat- establish the mechanism of PDK1 in ADP-induced plate- ing activation of the MAPK pathway. let activation and thrombosis. Methods: We assessed the role of PDK1 on 2MeSADP-induced platelet activation Keywords: phosphoinositide dependent kinase 1; platelet by measuring aggregation, thromboxane generation and activation; purinergic receptor P2Y12; thrombosis; phosphorylation events in the presence of BX-795, which thromboxane A2. inhibits PDK1, or by using platelet-specific PDK1 knock- out mice and performing western blot analysis. PDK1 Correspondence: Matthew Rondina, Department of Internal Medi- Introduction cine, Molecular Medicine Program, University of Utah, EIHG Platelets are involved in many processes, ranging from #4220, 15 N 230 E, Salt Lake City, UT 84112, USA fighting microbial infections and triggering inflammation Tel.: +1 801 585 0950 to promoting tumor angiogenesis and metastasis [1–9]. E-mail: [email protected] Nevertheless, one of the primary physiological functions Received: 27 September 2017 of platelets is maintaining homeostasis of the circulatory Manuscript handled by: D. Mezzano system by forming hemostatic thrombi that prevent blood Final decision: P. H. Reitsma, 5 March 2018 loss and maintain vascular integrity [10–13]. When there © 2018 International Society on Thrombosis and Haemostasis 1212 B. K. Manne et al is vascular damage, exposure of the extracellular matrix signaling pathway, and prompted us to investigate the results in the recruitment and activation of platelets, thromboxane-related mechanisms whereby PDK1 regu- thereby leading to aggregation and the formation of a fib- lates ADP-induced platelet activation and thrombosis. rin-rich hemostatic plug at the injured site. Platelet activa- The mechanism by which PDK1 controls ADP-induced tion is associated with platelet shape change, secretion of platelet activation has not been examined, and its role in granule contents (ADP and serotonin), and activation of thromboxane B2 (TxB2) generation is not known. It is the fibrinogen receptor, resulting in platelet aggregation important fo fill these knowledge gaps, especially as [14–17]. Activated platelets also generate the important PDK1 has recently gained attention as a potential drug lipid mediator thromboxane A2 (TxA2), which serves to target for thrombosis and cancer [30–38], and a deeper recruit other platelets to the site of injury and reinforces understanding of the mechanism by which PDK1 acts the platelet plug [16,18–20]. Thromboxane generation may shed light on clinical outcomes when PDK1 inhibi- induced by 2-methylthio-ADP (2MeSADP) is coordinated tors are being used. by P2Y purinoceptor 1 (P2Y1), P2Y purinoceptor 12 In the present study, we demonstrate that PDK1, which (P2Y12), and integrin aIIbb3. Inhibition of either extracel- is activated downstream of the PI3K pathway, plays a lular signal-regulated kinase 1/2 (ERK1/2) or protein tyr- crucial role in 2MeSADP-induced platelet aggregation osine kinase 2 (Pyk2) activation results in abolished and thromboxane generation by regulating the mitogen- thromboxane generation in ADP-stimulated platelets. activated protein kinase (MAPK) pathway. We show that 2MeSADP-induced ERK1/2 phosphorylation occurs inde- pharmacological blockade or genetic deletion of PDK1 in pendently of outside-in signaling via mitogen-activated platelets abolishes 2MeSADP-induced thromboxane gen- protein kinase kinase 1/2 (MEK1/2), Src family kinases eration and the activation of critical MAPK pathway pro- (SFKs), and phosphoinositide 3-kinase (PI3K). teins (e.g. Raf1, MEK1/2, and ERK1/2). Moreover, Thromboxane is generated from its precursor arachi- inhibition or deletion of PDK1 reduces death from pul- donic acid through cyclooxygenase pathways, which act as monary embolism in vivo. Therefore, we conclude that a positive feedback mediator to amplify the initial platelet crosstalk between the MAPK and PI3K pathways via functional responses and reinforce the stability of the PDK1 is crucial for 2MeSADP-induced platelet activa- hemostatic plug. Previous studies have shown that ADP- tion, thromboxane generation, and thrombosis. induced thromboxane generation depends on both inside- out and outside-in pathways [16,21]. In particular, ERK1/ Materials and methods 2 activation during inside-out signaling through P2Y receptors plays a critical role in TxA generation, as 2 Reagents ERK1/2 is important for cytosolic phospholipase A2 (cPLA2) phosphorylation and activation [22–24]. Inhibi- 2MeSADP (Cat. no. 1624) was from Tocris (Minneapolis, tion of PI3Kb abolishes ERK1/2 activation when platelets MN, USA). Epinephrine, SB-216763 and apyrase (type are stimulated with ADP [24]. However, the mechanism VII) were from Sigma (St Louis, MO, USA). BX-795 was through which the PI3K pathway regulates the phosphory- from Selleckchem (Houston, TX, USA). MSC 2032964A lation and activation of ERK1/2 has not been identified. and Whatman protein nitrocellulose transfer membrane Phosphoinositide-dependent protein kinase 1 (PDK1), a were from Fisher Scientific (Pittsburg, PA, USA). LI-COR member of the protein A, G and C (AGC) family of pro- Odyssey blocking buffer was from LI-COR Biosciences teins, is a Ser/Thr protein kinase that phosphorylates and (Lincoln, NE, USA). Phospho-Raf1 (Ser338), phospho- activates other protein kinases from the AGC family, all MEK1/2 (Ser217/221), phospho-Erk1/2 (Thr202/Tyr204), of which mediate cellular responses [25–27]. Chen et al. phospho-cPLA (Ser505), phospho-PDK1 and phospho- demonstrated that aggregation of PDK1-deficient plate- GSK3b antibodies were from Cell Signaling Technology lets was reduced in response to thrombin and U46619 (Beverly, MA, USA). Phospho-myelin basic protein [28]. For thrombin signaling, this involved aIIbb3- (MBP), total MEK1/2 and b-actin antibodies were from mediated outside-in signaling. They further showed that Santa Cruz Biotechnologies (Santa Cruz, CA, USA). activation of PDK1 with thrombin inhibits glycogen syn- thase kinase 3b (GSK3b) (a major substrate of PDK1), Mice thereby enhancing thrombin-induced platelet functional responses and thrombus formation. In other studies, Dan- Platelet-specific PDK1 knockout mouse platelet samples gelmaier et al. demonstrated that inhibition of PDK1 by were provided by O. Borst (University of Tubingen).€ BX-795, a well-characterized and specific inhibitor of PDK1, impaired platelet aggregation and protein kinase Human platelet isolation B (Akt) Thr308 phosphorylation [29]. However, thrombin and ADP activate via distinct sig- Washed non-aspirinated human platelets were prepared naling pathways. These published
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