NF-κB signaling pathway as target for antiplatelet activity

    NF-κB signaling pathway as target for antiplatelet activity Eduardo Fuentes, Armando Rojas, Iv´an Palomo PII: DOI: Reference:

S0268-960X(16)00013-8 doi: 10.1016/j.blre.2016.03.002 YBLRE 427

To appear in:

Blood Reviews

Please cite this article as: Fuentes Eduardo, Rojas Armando, Palomo Iv´an, NFκB signaling pathway as target for antiplatelet activity, Blood Reviews (2016), doi: 10.1016/j.blre.2016.03.002

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NF-κB signaling pathway as target for antiplatelet activity

Laboratory of Hematology and Immunology, Department of Clinical Biochemistry and

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Eduardo Fuentes1,2*, Armando Rojas3, Iván Palomo1,2*

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Immunohematology, Faculty of Health Sciences, Interdisciplinary Excellence Research Program on Healthy Aging (PIEI-ES), Universidad de Talca, Talca, Chile 2

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Centro de Estudios en Alimentos Procesados (CEAP), CONICYT-Regional, Gore Maule,

R09I2001, Talca, Chile

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3

Biomedical Research Laboratories, Medicine Faculty, Catholic University of Maule, Talca,

* Correspondence to:

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Eduardo Fuentes, PhD

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Chile

Immunology and Haematology Laboratory, Faculty of Health Sciences, Universidad de Talca, Talca, Chile

Tel.: +56-71-200493 Fax: +56-71-20048

E-mail: [email protected]

Iván Palomo, PhD Immunology and Haematology Laboratory, Faculty of Health Sciences, Universidad de Talca, Talca, Chile Tel.: +56-71-200493 Fax: +56-71-20048 E-mail: [email protected]

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ABSTRACT

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In different nucleated cells, NF-κB has long been considered a prototypical proinflammatory signaling pathway with the expression of proinflammatory genes.

In platelet activation NF-κB

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involved in activated platelets, such as NF-κB.

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Although platelets lack a nucleus, a number of functional transcription factors are

regulation events include IKKβ phosphorylation, IκBα degradation, and p65

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phosphorylation. Multiple pathways contribute to platelet activation and NF-κB is a

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common pathway in this activation. Therefore, in platelet activation the modulation of NF-κB pathway could be a potential new target in the treatment of inflammation-

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related vascular disease therapy (antiplatelet and antithrombotic activities).

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Keywords: Platelet activation, NF-κB, Thrombin, sCD40L/CD40L, Toll-like receptors, RAGE, PPARs.

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INTRODUCTION

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Platelets represent an important link between inflammation and thrombosis [1-5]. Activated platelets stimulate thrombus formation in response to a rupture of the

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atherosclerotic plaque, promoting cardiovascular diseases (CVD) [6]. Multiple

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pathways contribute to platelet activation, including those triggered by thrombin, arachidonic acid, adenosine diphosphate (ADP) and collagen, among others [7, 8].

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As a result, platelets release different inflammatory mediators such as soluble P-

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selectin (sP-selectin, CD62P), soluble CD40 ligand (sCD40L), interleukin (IL)-1β, transforming growth factor-β1 (TGF-β1), chemokine (C-C motif) ligand 5 (CCL5),

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matrix metalloproteinases, tumor necrosis factor alpha (TNF-α) and IL-6 [9-13].

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These molecules participate in the development of atherosclerosis, from early

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lesion development to vulnerable plaque formation [14, 15]. Atherosclerosis is a chronic inflammatory disease [16, 17]. Many inflammatory pathways that contribute to the initiation and progression of atherosclerosis are regulated by the nuclear factor (NF)-κB; regulator of innate and adaptive immune responses [18-20]. Activated NF-κB has been identified in human atherosclerotic plaques and has been enhanced in unstable coronary plaques [21, 22]. In different cells, NF-κB has long been considered a prototypical proinflammatory signaling pathway with the expression of proinflammatory genes, such as cytokines, chemokines and adhesion molecules [23, 24]. Therefore, the inhibition of NF-κB may have a great impact for the treatment of various inflammatory diseases [25]. may have a great impact when these types of drugs are considered for the treatment of cancer and various inflammatory diseases.

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Different inflammatory mediators regulate the expression of cellular genes through

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NF-κB [26-28]. The pleiotropic NF-κB normally exists as an inactive cytoplasmic

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complex; its predominant form is a heterodimer composed of p50 and p65

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subunits. These subunits are tightly bound to inhibitory proteins of the IκB family. The activation of NF-κB is when IκBα is phosphorylated by the IKK complex. It

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starts dissociating of IκBα from NF-κB subunits, and then IκBα is ubiquitinated and

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rapidly degraded by the proteasome [29-32].

Although platelets lack a nucleus, a number of functional transcription factors

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including signal transducer and activator of transcription 3 (STAT3) and NF-κB are

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involved in activated platelets [33, 34]. NF-κB activation is another signaling

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pathway involved part of classical agonist-mediated platelet activation [34-38]. Yet, the mechanisms by which the NF-κB pathway may contribute to platelet activation are yet to be fully elucidated. In this article we explore the potential impact of inhibiting NF-κB function in platelet activation. NF-κB AND PLATELET ACTIVATION NF-κB is a redox-sensitive transcription factor that regulates inflammation and plays a critical role in the vascular response to injury [39]. Activated NF-κB is detected in human atherosclerotic and restenotic lesions of smooth muscle cells, monocytes, endothelial cells and platelets, among others [21]. NF-κB may have a function independent of gene regulation in platelets. Three IKK family members (α, β, and γ) are expressed in platelets, with the β form being the most strongly expressed [34].

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There is recent evidence about alternative pathways of NF-κB-dependent

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regulation of platelet function. Thus NF-κB has an important dual regulatory role on platelet function. Whereas NF-κB activation induces platelet activation, it also

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seems to be essential for shedding surface glycoprotein (GP) Ibα by activated

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platelets and protein kinase Ac (PKAc) activation (platelet inhibitory pathway) [36, 40].

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Platelet-monocyte interactions via extracellular matrix metalloproteinase inducer

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(EMMPRIN) stimulate NF-κB-driven inflammatory pathways in monocytes, such as matrix metalloproteinases and cytokine induction, thus representing another

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potential drug target to inhibit NF-κB platelet-monocyte interactions [41]. Platelets

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are activated on increase of cytosolic Ca2+ activity, accomplished by store-operated

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Ca2+ entry (SOCE) involving the pore-forming ion channel subunit Orai1. In this context, recent observations unravel serum- and glucocorticoid-inducible kinase 1 (SGK1) as novel regulator of platelet function, effective at least in part by NF-κBdependent transcriptional up-regulation of Orai1 in megakaryocytes and increasing platelet SOCE [42].

NF-κB activation during the late stage of inflammation is associated with the resolution of inflammation and anti-inflammatory gene expression [40]. Thus NF-κB activation limits platelet-leucocyte interaction by promoting a disintegrin and metalloprotease domain 17 (ADAM17)-mediated GPIbα shedding [40]. In platelet activation NF-κB signaling events included IKKβ phosphorylation, IκBα degradation and p65 phosphorylation [43]. IKKβ phosphorylation has been

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proposed as a major upstream regulator for IκBα phosphorylation leading to NF-κB

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activation during platelet activation [34, 37]. Platelets contain all three members of the SNAP-23/25/29 gene family and its

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phosphorylation provides a critical link between activation and secretory

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processes. SNAP-23 is the most highly enriched of these proteins in platelets and is required for exocytosis from platelet alpha, dense, and lysosomal granules [44,

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45]. IKKβ, in response to platelet activation, phosphorylates SNAP-23 resulting in

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enhanced SNARE complex formation, enhanced membrane fusion and granule release [38]. Meanwhile inhibition of IKKβ blocked SNAP-23 phosphorylation and

platelets

agonists/receptors

modulate

NF-κB

pathway,

such

as

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Several

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platelet secretion.

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thrombin/PAR4, sCD40L/CD40L, receptor for advanced glycation end products (RAGE) axis, toll-like receptors (TLRs) and peroxisome proliferator-activated receptors (PPARs) (figure 1). Thrombin/PAR4. Activation via thrombin/PAR4 is involved in Ca2+-dependent release of platelet granules, activation of GPIIb/IIIa, adhesion, aggregation and thrombus formation [46]. Also thrombin induces the release of platelet inflammatory mediators such as sP-selectin, sCD40L, IL-1β, TGF-β1 and CCL5 [9-11]. In platelet activation, the binding of thrombin to PAR4 triggers the activation of sphingomyelinase (nSMase) with increased of C24:0-ceramide level and NF-κB activation [47]. Thrombin not only causes platelet activation but also appears to fine-tune this response by initiating downstream NF-κB-dependent PKA activation, as a novel feedback inhibitory signaling mechanism for preventing undesired

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platelet activation [36]. The PKA activation phosphorylate multiple target proteins in

maintaining circulating platelets in a resting state [48].

The ligand CD40L is similarly expressed in the plasma

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sCD40L/CD40L.

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numerous platelet inhibitory pathways that have a very important role in

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membranes of endothelial cells, T-lymphocytes and platelets [49, 50]. Platelets

atherosclerosis progression [51, 52].

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constitute the major source of sCD40L, and trigger endothelial cell activation and

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Platelet sCD40L binds to GPIIb/IIIa improving platelet activation and aggregation [53, 54]. Furthermore, sCD40L induces platelet activation through CD40 [55].

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sCD40L significantly increases platelet activation and aggregation through CD40-

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dependent TRAF-2/Rac1/p38 MAPK signaling, while a blockade of this pathway

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with anti-CD40L antibodies can prevent or delay atherosclerosis progression [14, 56]. Another signaling pathway downstream of platelet CD40 in response to sCD40L is the activation of NF-κB signaling pathway; with the phosphorylation of IκBα on Ser32/36 [57]. However, this activation of NF-κB is not the direct target of the p38 MAPK pathway, suggesting that these pathways regulate different aspects of CD40-mediated platelet activation: (i) p38 MAPK pathway may be involved in platelet spreading and adhesion [58], and (ii) NF-κB pathway may regulate de novo protein synthesis through its interaction with microRNAs [59, 60]. Receptor for advanced glycation end products (RAGE) axis. Reducing sugars such as glucose can react non-enzymatically with the amino groups of proteins to form complex structures called advanced glycation end-products (AGEs) [61, 62]. AGEs may exert their pathogenic effects not only by changing the physicochemical

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and functional properties of molecules, but also by their binding capacity to cellular

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receptors, where the receptor for AGEs (RAGE) plays an important role in the cardiovascular burden of diabetes [63]. RAGE is expressed in diverse tissues such

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as lung, heart, kidney, brain and skeletal muscle, and in a variety of cells including

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endothelial cells, macrophages/monocytes, neutrophils and lymphocytes [64-66]. Recently, RAGE has been identified on human platelets [67] and its expression

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has been shown to be induced in the presence of AGEs [67]. In this context,

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activation of RAGE produces a marked increase in P-selectin expression, up to 7.1-fold at the platelet surface membrane [67].

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There is no direct experimental evidence of NF-kB activation triggered by RAGE in

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platelets. However, key components on canonical signaling cascade downstream

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RAGE activation in many cell types have been functionally described in platelets such as: nicotinamide adenine dinucleotide phosphate-oxidase (NADPH-oxidase) [68, 69], Src [70], rac and cdc42 [71], erk and p38 MAP kinases [72] and as well as the activation of NF-κB [34]. So a possible activation of RAGE in platelet could activate NF-κB signaling pathway [73]. Toll-like receptors (TLRs). TLRs are the main regulators of the adaptive and innate immune responses [74], and play important roles in the pathogenesis of atherosclerosis [75]. TLR-2, -4 and -9 are prominently expressed in human platelets, and their expression levels are doubled in activated platelets [76, 77]. It has been shown that lipopolysaccharide (LPS) stimulates platelet secretion and potentiates platelet aggregation via TLR4/MyD88 and the cyclic guanosine monophosphate (cGMP)-dependent protein kinase pathway [78, 79]. In addition,

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TLR4 activation increases platelet release of sCD40L and platelet-activating factor

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4 (PAF4) [80]. Meanwhile, stimulation of TLR2 with Pam3CSK4, a synthetic agonist of TLR2/TLR1, activates GPIIb/IIIa and augments the expression of P-

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selectin on the platelet surface [81, 82].

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NF-κB is a downstream signal of the TLRs in nucleated cells [83, 84]. In anucleate platelets, TLR2 and 4 agonists trigger platelet activation responses through NF-κB.

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Thus, platelet stimulation with Pam3CSK4 or LPS resulted in IκBα degradation and

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p65 phosphorylation. In fact, platelet activation is inhibited in platelets treated with the NF-κB inhibitors BAY 11-7082 or Ro 106-9920 [85, 86].

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Histones are highly alkaline proteins and can be released by inflammatory cells

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[87]. Histone-mediated fibrinogen binding, P-selectin and phosphatidylserine

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exposure and the formation of mixed aggregates were potentiated by thrombin. Functional platelet responses induced by histones were partially mediated through interaction with TLR -2 and -4 and the activation of NF-κB [88]. Peroxisome proliferator-activated receptors (PPARs). PPARs are involved in many biological processes, including lipid and energy metabolism inflammation responses, and atherosclerotic plaque formation [89-91]. Selective agonists through their action on nuclear receptors (e.g. PPARs) regulate platelet function despite the absence of a nucleus in platelets [92-94]. PPARs activation decreases platelet aggregation and delays intra-arterial thrombus formation in rats, at least partially, by an increase in the expression of nitric oxide synthase (NOS), thrombomodulin, and decreases the release procoagulant

mediators

(sCD40L

and

of

platelet proinflammatory and

thromboxane

A2

[TXA2])

[93-98].

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Additionally, cytoplasmic PPARs can repress the transcriptional activity of the

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proinflammatory mediator, such as NF-κB, preventing its translocation in macrophages [99]. In platelets, the mechanism of NF-κB inhibition could be by

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direct activation of PPARs/PKG with inhibition of p38 MAPK pathway (upstream

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signal transduction of NF-κB pathway) [100, 101]. However, at present there is no direct evidences supporting a relation between PPARs and NF-κB in platelets and

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further studies are required.

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NF-ΚB INHIBITION AND ANTIPLATELET STRATEGIES Multiple pathways contribute to platelet activation and NF-κB is a common pathway

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in this activation. Pre-treatment with an NF-κB inhibitor has been found to prevent

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multiple platelet activities, including platelets activation, adhesion, secretion and

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aggregation. Therefore NF-κB is an attractive target and an emerging theme in antiplatelet activity [35, 85].

Bortezomib is an inhibitor of NF-κB activity with remarkable preclinical and clinical antitumor activity in multiple myeloma patients [102]. In addition, bortezomib showed an inhibitory effect on platelet aggregation induced by ADP and adenosine triphosphate (ATP)-release reaction induced by collagen in human platelets [103]. These findings indicate that bortezomib may be an antiplatelet agent and its effects may be related to NF-κB inhibition. Among non-steroidal anti-inflammatory drugs (NSAIDs), acetaminophen inhibits the binding of NF-κB to deoxyribonucleic acid (DNA) in LPS- and interferon-γ– treated macrophages and thus precludes expression of inducible nitric oxide synthase (NOS) [104]. Meanwhile, sulindac, other NSAIDs, decreases IKKβ kinase

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activity and consequently inhibits NF-κB activation. Given the inhibitory activity of NF-κB, both acetaminophen and sulindac could be antiplatelet agents [105, 106].

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BAY 11-7082 and Ro 106-9920, two chemically unrelated NF-κB inhibitors,

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suppressed different platelet responses, such as suppressed platelet aggregation

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induced by ADP, epinephrine, collagen or thrombin. But BAY 11-7082 and Ro 1069920 failed to impair the AA-induced response [35]. Although these inhibitors did

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not affect AA-induced aggregation, it was determined that they blocked the

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phosphorylation of ERK, which regulates the phosphorylation of secreted phospholipases A2, the main enzyme responsible for the release of AA [35].

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Meanwhile, anti-inflammatory properties of aspirin and salicylate are mediated in

in

the

pathogenesis of

the

inflammatory response.

At

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genes involved

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part by their specific inhibition of IKKβ, thereby preventing activation by NF-κB of

concentrations measured in the serum of patients treated with aspirin and salicylate for chronic inflammatory conditions, both aspirin and salicylate inhibit activation of the NF-κB pathway and platelet aggregation [107-111]. Exposure of platelets to nifedipine significantly increased the PPAR-β/γ activity in activated human platelets. Treatment with nifedipine reduced collagen-induced NFκB pathway activation, which were markedly attenuated by GSK0660, a PPAR-β antagonist, or GW9662, a PPAR-γ antagonist [112]. In addition, nifedipine is capable of inhibiting NF-κB activity through direct interaction of PPAR-β/-γ with NFκB [113]. The thiazolidinediones (TZDs; rosiglitazone, pioglitazone and troglitazone) are a class of oral antidiabetic drugs that exert effects through a mechanism that

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involves an activation of PPAR-γ [114]. Troglitazone has a potent inhibitory effect

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on platelet aggregation via the suppression of the thrombin-induced activation of phosphoinositide signaling in human platelets [115]. Based on the function of other

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cells (adipocyte cells), the mode of action is likely related to the elevation of the

anti-inflammatory

activity

of

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intracellular cAMP level by PPAR-γ activation [116]. In addition, this is because the troglitazone

antagonizes

TNF-α

-induced

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regulatory functions of NF-κB [117].

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reprogramming of adipocyte gene expression by inhibiting the transcriptional

Bockade of NF-κB by NF-κB decoy oligodeoxynucleotides attenuated restenotic

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changes (neointimal formation) and thrombosis in treated patients on day 3 after

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the percutaneous coronary intervention associated with reduced NF-κB–dependent

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genes like monocyte chemoattractant protein-1 (MCP-1) [118, 119]. In addition, NF-κB suppression might be relevant in platelet aggregation inhibition [35]. IMPACT OF NF-ΚB INHIBITION ON OTHERS CELLS The activation of the NF-κB pathway may play a key role in a number of diseases (cancer, asthma cardiovascular, autoimmune and Alzheimer’s diseases, among others) that have an inflammatory component involved in their pathogenesis. Thus NF-κB activation in endothelial, smooth muscle, macrophages and lymphocytes cells is involved in chronic inflammatory and fibroproliferative process [120-123]. Given the diverse processes involved in activating the NF-κB pathway, it is not surprising that a number of different inhibitors can prevent activation of this pathway with protective effects in different cells. In this manner the inhibition of the NF-κB pathway prevents transendothelial migration of neutrophils in endothelial

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cells [107], and has antiinflammatory effect in macrophages [124], vascular smooth

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muscle cell [125, 126] and fibroblasts [127], among others. Therefore NF-κB could be use as a possible target for chronic inhibition.

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PLATELETS AND INFLAMMATION

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As well as initiating thrombus formation at the site of a ruptured atherosclerotic plaque, platelets play a key role in vascular inflammation, through the release of

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their own pro-inflammatory mediators and interactions with other relevant cell types

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(endothelial cells, leukocytes and smooth muscle cells) [128-130]. Thus platelets can be directly involved in the unstable plaque through the production and release

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of pro-inflammatory molecules, including a variety of cytokines, such as TGF-β, IL-

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1β and sCD40L, and chemokines, such as CCL5 [131, 132].

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Activated platelets induce endothelial secretion of IL-8 via membrane-associated IL-1β activity [133]. A portion of the IL-1β is shed in its mature form in membrane microvesicles and induces adhesiveness of human endothelial cells for neutrophils [134]. Also activated platelets modulate intercellular adhesion molecule (ICAM-1) properties of endothelial cells via an NF-κB-dependent mechanism [135]. PLATELETS AND THROMBOSIS Thrombus formation in response to tissue trauma initiates with platelet activation [136, 137]. Analysis of blood flow dynamics has revealed that discoid platelets preferentially adhere to low-shear zones at the downstream face of forming thrombi, with stabilization of aggregates dependent on the dynamic restructuring of membrane tethers [138].

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In response to vascular injury, platelets develop a sequence of events including

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platelet adhesion, activation, secretion and aggregation [139, 140]. The process of platelet adhesion involves the interaction of receptors on the platelet membrane

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(GPVI, GPIbα and GPIIb/IIIa, among others) with its ligands in the subendothelial

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matrix (collagen and Von Willebrand factor (vWF), among others) [141]. Platelet activation triggers the synthesis and release of several autocrine and paracrine

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mediators, including ADP, thrombin, epinephrine, and TXA2, among others [142,

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143]. Secreted products from activated platelets act to recruit further platelets into the growing aggregate, as well as having strong inflammatory effects on the

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endothelium [140]. Therefore, multiple pathways are involved in platelet activation.

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CONCLUSION

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In this article we explored the relative contribution of NF-κB function in platelet activation. Multiple pathways contribute to platelet activation and NF-κB is a common pathway in this activation. Therefore drugs that modulate NF-κB activation may have potential in the treatment of inflammation-related vascular disease therapy, as antiplatelet activity. However, additional experimental studies must be done in order to understand NF-κB signaling in platelet function and the novelty of this signaling pathway as a new antiplatelet target.

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PRACTICE POINTS - Platelets represent an important link between inflammation and thrombosis.

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Multiple pathways contribute to platelet activation and NF-κB is a common pathway

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in this activation.

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- Bortezomib, inhibitor with preclinical and clinical antitumor activity, may be an antiplatelet agent and its effects may be related to NF-κB inhibition.

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- Given the diverse processes involved in activating the NF-κB pathway a number

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of different inhibitors can prevent activation of this pathway with anti-inflammatory effects in different cells.

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RESEARCH AGENDA

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platelet function.

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- Experimental studies must be done in order to understand NF-κB signaling in

- Clinical value of platelets NF-κB inhibitors. - Relationship between PPARs and NF-κB in platelets.

CONFLICT OF INTEREST

The authors have no conflicts of interest to disclose.

ACKNOWLEDGEMENTS This work was funded by the CONICYT REGIONAL / GORE MAULE / CEAP / R09I2001, Interdisciplinary Excellence Research Program on Healthy Aging (PIEIES), and supported by grant no. 1130216 (I.P., M.G., R.M., M.A., J.C.) from

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Fondecyt, Chile. Also Eduardo Fuentes thanks FONDECYT (FONDECYT Initiation

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N° 11140142).

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Figure Legend

Figure 1. Main regulatory mechanisms of NF-κB (p50 and p65) signaling pathway

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in platelets. ATP, adenosine triphosphate; cAC, adenylate cyclase; cGC, guanylate

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cyclase; GP, glycoprotein; Gq, G protein-coupled receptors; GTP, guanosine triphosphate; nSMase, sphingomyelinase; PKA, protein kinase A; PKG, protein

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kinase G; PPARs, Peroxisome proliferator-activated receptors; RAGE, receptor for

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AGEs; sCD40L, soluble CD40 ligand; SM, sphingomyelin; TLRs, toll like receptors.

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NF-κB is p50 and p65. Continuous lines: activation and dotted line: inhibition.

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