Elsevier

Biochemical Pharmacology

Volume 86, Issue 7, 1 October 2013, Pages 914-925
Biochemical Pharmacology

Antiplatelet effect of AMP-activated protein kinase activator and its potentiation by the phosphodiesterase inhibitor dipyridamole

https://doi.org/10.1016/j.bcp.2013.07.009Get rights and content

Abstract

AMP-activated protein kinase (AMPK) activates endothelial nitric oxide synthase (eNOS) via phosphorylation at the activating site. The eNOS-nitric oxide (NO)/soluble guanylate cyclase (sGC)-cGMP/cGMP-dependent protein kinase (PKG) signaling axis is a major antiaggregatory mechanism residing in platelets. Based on the hypothesis that direct activation of AMPK might be a potential strategy to inhibit platelet aggregation, the antiplatelet effect of AMPK activators was investigated. Treatment of isolated platelets with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) resulted in AMPK activation and a decrease in aggregation, which was abolished by pretreatment with the AMPK inhibitors compound C (CC) and ara-A. Such an AMPK-dependent antiaggregatory effect was also observed with other AMPK activators such as A-769662 and PT1. AICAR induced eNOS activation was followed by NO synthesis, cGMP production, and subsequent phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a PKG substrate. All these events were blocked by CC or ara-A pretreatment, and each event was inhibited by the eNOS inhibitor L-NAME, the sGC inhibitor ODQ, and the PKG inhibitor Rp-8-pCPT-cGMPS. Simultaneous treatment of dipyridamole, a phosphodiesterase (PDE) inhibitor, with AICAR potentiated the antiaggregatory effect by enhancing the cGMP elevation. Administration of AICAR increased platelet cGMP and prolonged FeCl3-induced arterial occlusion time in rats, which further increased in combination with dipyridamole. In conclusion, AMPK activators inhibited platelet aggregation by stimulating the eNOS-NO/sGC-cGMP/PKG signaling pathway. The antiplatelet effect of AMPK activators could be potentiated in combination with a PDE inhibitor through the common mechanism of elevating cGMP. Thus, AMPK may serve as a potential target for antiplatelet therapy.

Introduction

Platelets play a primary role in hemostasis under normal physiological conditions through aggregation. However, dysregulation of platelet activity, mainly caused by alteration in the vasculature microenvironment, is involved in atherosclerosis and inflammation by leading to thrombosis, and contributes to the development of acute coronary syndrome, stroke, and the ischemic complications of peripheral vascular disease. Therefore, platelets serve as a primary target for treating atherothrombotic disorders, and antiplatelet therapies play a key role in treating various cardiovascular diseases such as myocardial infarction, stroke, and atherosclerosis [1].

A number of antiplatelet drugs have been developed and are in trials. Current antiplatelet drugs can be classified based on their mechanisms of action. Generally, aspirin and clopidogrel have been used as gold standard antiplatelet therapies, which are a cyclooxygenase inhibitor preventing thromboxane A2 synthesis and an ADP receptor P2Y12 antagonist, respectively. However, they have critical limitations such as bleeding problems and interindividual variability in their responses. Other antiplatelet drugs include glycoprotein IIb/IIIa (GPIIb/IIIa) antagonists such as abciximab, and phosphodiesterase (PDE) inhibitors such as dipyridamole and cilostazol. Although GPIIb/IIIa antagonists show high efficacy, they require intravenous administration and are associated with a high incidence of nuisance bleeding. PDE inhibitors exert their antiplatelet effect by suppressing hydrolysis of cyclic nucleotides such as cGMP or cAMP, and thereby elevating cGMP or cAMP levels [2]. cGMP and cAMP are produced by soluble guanylate cyclase (sGC) and soluble adenylate cyclase, which are activated by nitric oxide (NO) and prostacyclin, respectively. These are critical intracellular second messengers interfering with all known platelet activation signaling pathways, which are mediated by cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase, respectively. PDE inhibitors are safe and orally available drugs, but low efficacy is a shortcoming. Thus, they are frequently used together with aspirin or clopidogrel. As mentioned above, the clinical limitations of current drugs have increased the demand for the development of novel antiplatelet drugs [1].

AMP-activated protein kinase (AMPK) acts as a sensor of cellular energy balance. AMPK is activated by low energy status such as a decrease in the cellular ATP:AMP ratio, which triggers a switch from an ATP-consuming anabolic condition to an ATP-producing catabolic condition [3]. AMPK is involved in diverse signaling pathways related to a wide range of biological activities, including cell growth, apoptosis, and aging, as well as metabolism of glucose, fatty acids, and protein [4]. AMPK is currently regarded as a potential therapeutic target for type 2 diabetes, obesity, and various cancers [5], [6]. Accordingly, a number of AMPK activators have been tested in preclinical models, although many of them have yet to reach the clinic [6].

Activating AMPK has cardiovascular protective effects in addition to improving metabolic imbalance. It stimulates a variety of signaling processes, but endothelial nitric oxide synthase (eNOS) is the main effector of AMPK-mediated vascular protection [7]. eNOS is a well-known AMPK substrate, and its activation by AMPK has been demonstrated in diverse tissues, including cardiac myocytes [8], and endothelial cells [9], [10]. The presence of AMPK in platelets and its physiological functions have been studied [11], [12]. AMPK was investigated as an intermediate molecule mediating insulin signaling. Insulin stimulates phosphatidylinositol 3-kinase which, in turn, activates AMPK. The activation of AMPK leads to stimulating eNOS followed by the generation of NO, which ultimately results in aggregation inhibition. Indeed, direct activation of AMPK by an AMPK activator, 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) reduces platelet aggregation [12]. These studies led us to postulate a concept that activating AMPK may be a potential strategy to prevent platelet aggregation; therefore, AMPK could serve as a possible target for antiplatelet drugs.

In the present study, the antiplatelet effect of an AMPK activator and its relationship with the AMPK/eNOS-NO/sGC-cGMP/PKG signaling pathway were examined by employing AICAR, an acknowledged AMPK activator. In addition to AICAR, the aggregation inhibitory effect of newly developed AMPK activators A769662 and PT1 was tested to confirm the antiaggregatory effect of activating AMPK [13], [14]. Considering the working mechanism of AMPK activators, i.e., the stimulation of cGMP production, the possible synergism was studied with dipyridamole, a PDE inhibitor that increased cGMP by interfering with cGMP degradation, to investigate therapeutic potential. In addition, the in vivo effect of an AMPK activator alone or in combination with dipyridamole was tested in an animal thrombosis model.

Section snippets

Reagents

5-Aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) was purchased from Toronto Research Chemicals (North York, Ontario, Canada). Collagen and ADP were obtained from Chrono-log Corp. (Havertown, PA, USA). NADPH, nitrate reductase, protease inhibitor cocktail, and phosphate inhibitor cocktail tablets were purchased from Roche Diagnostics (Indianapolis, IN, USA). cGMP and cAMP assay kits and bicinchoninic acid (BCA) protein assay kits were from R&D Systems (Minneapolis, MN, USA) and Pierce

Antiplatelet effect of AICAR by activating AMPK

The effects of AICAR on platelet AMPK and aggregation were examined to test our hypothesis that direct activation of AMPK could suppress platelet aggregation. AICAR treatment resulted in activation of AMPK, which was confirmed with activation-dependent phosphorylation at Thr172 (Fig. 1A). Such an effect was concentration- and treatment time-dependent at 100–1000 μM and 10–30 min, respectively. The AICAR concentration required for AMPK activation in platelets was not different from that in other

Discussion

Antiplatelet drugs are successfully used in the clinic, but they still have significant drawbacks such as limited efficacy and safety, which require further improvements in antiplatelet therapy. As substantial progress has been made in understanding the regulation of platelet function, more and more novel targets for antiplatelet drugs are being suggested, including thrombin receptors such as protease activator receptor 1 (PAR1) and PAR4, thromboxane prostanoid receptor, and cell adhesion

Acknowledgments

We thank Dr. Kang Kyu-Tae at Boston Children's Hospital/Harvard Medical School for his helpful comments on the manuscript. This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2010-0008992).

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