Time-resolved characterization of cAMP/PKA-dependent signaling reveals that platelet inhibition is a concerted process involving multiple signaling pathways

Beck, Florian; Geiger, Jörg; Gambaryan, Stepan; Veit, J.; Vaudel, Marc; Nollau, Peter; Kohlbacher, Oliver; Martens, Lennart; Walter, Ulrich; Sickmann, Albert; Zahedi, René P.

doi:10.1182/blood-2013-07-512384

Cited by 86 publications

(117 citation statements)

References 49 publications

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“…For example, endotheliumderived PGI 2 inhibits platelet aggregation by activating a G-protein-coupled receptor in platelets, increasing their intracellular cAMP levels through adenylate cyclase, with subsequent cAMP/cAMP-dependent protein kinase signaling inhibiting virtually all platelet-activating mechanisms (44,45). In this context, intracellular cAMP levels in platelets play a key role in maintaining hemostasis.…”

Section: Discussionmentioning

confidence: 99%

Glucagon-Like Peptide 1 Receptor Activation Attenuates Platelet Aggregation and Thrombosis

et al. 2016

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Short-term studies in subjects with diabetes receiving glucagon-like peptide 1 (GLP-1)-targeted therapies have suggested a reduced number of cardiovascular events. The mechanisms underlying this unexpectedly rapid effect are not known. We cloned full-length GLP-1 receptor (GLP-1R) mRNA from a human megakaryocyte cell line (MEG-01), and found expression levels of GLP-1Rs in MEG-01 cells to be higher than those in the human lung but lower than in the human pancreas. Incubation with GLP-1 and the GLP-1R agonist exenatide elicited a cAMP response in MEG-01 cells, and exenatide significantly inhibited thrombin-, ADP-, and collagen-induced platelet aggregation. Incubation with exenatide also inhibited thrombus formation under flow conditions in ex vivo perfusion chambers using human and mouse whole blood. In a mouse cremaster artery laser injury model, a single intravenous injection of exenatide inhibited thrombus formation in normoglycemic and hyperglycemic mice in vivo. Thrombus formation was greater in mice transplanted with bone marrow lacking a functional GLP-1R (Glp1r 2/2 ), compared with those receiving wild-type bone marrow. Although antithrombotic effects of exenatide were partly lost in mice transplanted with bone marrow from Glp1r 2/2 mice, they were undetectable in mice with a genetic deficiency of endothelial nitric oxide synthase. The inhibition of platelet function and the prevention of thrombus formation by GLP-1R agonists represent potential mechanisms for reduced atherothrombotic events.Type 2 diabetes (T2D) is associated with a number of risk factors that contribute to an increased risk of atherothrombotic events, including hypertension, dyslipidemia, obesity, and chronic inflammation, as well as endothelial and platelet dysfunction (1). Platelets are small, versatile, anucleate cells in the circulation that play critical roles in both early and late stages of atherothrombosis, contributing also to cell-based thrombin generation and blood coagulation (2). Subjects with T2D exhibit a prothrombotic state, including increased production of coagulation factors; decreased production of fibrinolytic factors; and a propensity to platelet activation, aggregation, and adhesion (1,3,4). Compounding the latter, subjects with T2D show reduced sensitivity to antiplatelet drugs, such as aspirin and clopidogrel (5,6), and manifest a higher incidence of cardiovascular events (1,6,7). Although the currently available antidiabetic agents have been effective at lowering blood glucose levels and preventing microvascular disease, until the recent EMPA-REG study (8), it had been exceedingly difficult to demonstrate the beneficial effects of normalizing blood glucose

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Section: Discussionmentioning

confidence: 99%

Glucagon-Like Peptide 1 Receptor Activation Attenuates Platelet Aggregation and Thrombosis

et al. 2016

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“…143 To gain an improved insight into the inhibitory cAMP/ PKA pathway, we used quantitative MS and monitored time-resolved changes of phosphorylation patterns in human platelets treated with different concentrations of the stable prostacyclin analog, iloprost. 144 Quantifying >2700 phosphorylation sites over 4 different time points, we could identify ≈300 unique proteins that are differentially phosphorylated on platelet inhibition. Our data indicate that platelet inhibition is not merely mediated by PKA but involves the interaction with other signaling pathways.…”

Section: Ptm and Signaling Proteomics In Plateletsmentioning

confidence: 99%

What Can Proteomics Tell Us About Platelets?

Burkhart

Gambaryan

Watson

et al. 2014

Circulation Research

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102

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More than 130 years ago, it was recognized that platelets are key mediators of hemostasis. Nowadays, it is established that platelets participate in additional physiological processes and contribute to the genesis and progression of cardiovascular diseases. Recent data indicate that the platelet proteome, defined as the complete set of expressed proteins, comprises >5000 proteins and is highly similar between different healthy individuals. Owing to their anucleate nature, platelets have limited protein synthesis. By implication, in patients experiencing platelet disorders, platelet (dys)function is almost completely attributable to alterations in protein expression and dynamic differences in post-translational modifications. Modern platelet proteomics approaches can reveal (1) quantitative changes in the abundance of thousands of proteins, (2) post-translational modifications, (3) protein–protein interactions, and (4) protein localization, while requiring only small blood donations in the range of a few milliliters. Consequently, platelet proteomics will represent an invaluable tool for characterizing the fundamental processes that affect platelet homeostasis and thus determine the roles of platelets in health and disease. In this article we provide a critical overview on the achievements, the current possibilities, and the future perspectives of platelet proteomics to study patients experiencing cardiovascular, inflammatory, and bleeding disorders.

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“…5 cAMP and cGMP elevation results in the activation of cyclic nucleotide-dependent protein kinases (cAMP-dependent protein kinase [PKA] and cGMP-dependent protein kinase [PKG], respectively), which phosphorylate a broad panel of substrate proteins. 6 The rise in platelet cyclic nucleotide levels results in a downregulation of activating signaling pathways that in turn inhibits platelet cytoskeleton rearrangement, fibrinogen receptor activation, secretion processes, and procoagulant activity. 2 Common substrates of these kinases include signaling regulators such as vasodilator-stimulated phosphoprotein (VASP), one of the major PKA and PKG substrates that is an important regulator of actin dynamics 2 .…”

Section: Introductionmentioning

confidence: 99%

Impaired platelet activation and cAMP homeostasis in MRP4-deficient mice

Decouture

Dreano

Belleville-Rolland

et al. 2015

Blood

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Key Points• In vivo and in vitro thrombus formation is altered in MRP4-deficient mice.• MRP4 modulates the cAMPprotein kinase A platelet signaling pathway.Molecules that reduce the level of cyclic adenosine 59-monophosphate (cAMP) in the platelet cytosol, such as adenosine 59-diphosphate (ADP) secreted from dense granules, trigger platelet activation. Therefore, any change in the distribution and/or availability of cyclic nucleotides or ADP may interfere with platelet reactivity. In this study, we evaluated the role of multidrug resistance protein 4 (MRP4, or ABCC4), a nucleotide transporter, in platelet functions in vivo and in vitro by investigating MRP4-deficient mice. MRP4 deletion resulted in a slight increase in platelet count but had no impact on platelet ultrastructure. In MRP4-deficient mice, the arterial occlusion was delayed and the tail bleeding time was prolonged. In a model of platelet depletion and transfusion mimicking a platelet-specific knockout, mice injected with MRP4 2/2 platelets also showed a significant increase in blood loss compared with mice injected with wild-type platelets.Defective thrombus formation and platelet activation were confirmed in vitro by studying platelet adhesion to collagen in flow conditions, integrin aIIbb3 activation, washed platelet secretion, and aggregation induced by low concentrations of proteinase-activated receptor 4-activating peptide, U46619, or ADP. We found no role of MRP4 in ADP dense-granule storage, but MRP4 redistributed cAMP from the cytosol to dense granules, as confirmed by increased vasodilator-stimulated phosphoprotein phosphorylation in MRP4-deficient platelets. These data suggest that MRP4 promotes platelet aggregation by modulating the cAMP-protein kinase A signaling pathway, suggesting that MRP4 might serve as a target for novel antiplatelet agents. (Blood. 2015;126(15):1823-1830

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Time-resolved characterization of cAMP/PKA-dependent signaling reveals that platelet inhibition is a concerted process involving multiple signaling pathways

Cited by 86 publications

References 49 publications

Glucagon-Like Peptide 1 Receptor Activation Attenuates Platelet Aggregation and Thrombosis

Glucagon-Like Peptide 1 Receptor Activation Attenuates Platelet Aggregation and Thrombosis

What Can Proteomics Tell Us About Platelets?

Impaired platelet activation and cAMP homeostasis in MRP4-deficient mice

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