Distinctive roles of PKC delta isozyme in platelet function

Review paper

Distinctive roles of PKC delta isozyme in platelet function

Y. Zaid ^a , N. Senhaji ^b, *, Y. Darif ^c , K. Kojok ^a , M. Oudghiri ^c , A. Naya ^c

a

Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, Montreal, Quebec H1T 1C8, Canada

b

Laboratory of Genetics and Molecular Pathologies (LGPM), Faculty of Medicine and Pharmacy of Casablanca, Hassan II University, Casablanca, Morocco

c

Laboratory of Physiology and Molecular Genetics, Faculty of Sciences, Hassan II University, Casablanca, Morocco

1. Introduction

Regulation of hemostasis and thrombosis by platelets is an important aspect to the physiological state of the cardiovascular system [1–4]. Platelet activation relies on interactions between specific cell surface receptors and the vascular wall or blood components, such as tethering of the platelet glycoprotein VI (GPVI) receptor to the collagen in the sub-endothelial matrix, the binding of GPIb-IX-V complex to Von Willebrand factor (VWf), or the hydrolysis of the protease-activated receptors (PARs) bythrom- bin [1,5]. Hallmarks of platelet activation are the activation- dependent conformational changes in integrin a

IIb

b

3

through inside-out signalling, and exposure of P-selectin present in a - granules [6].

Upon stimulation, platelet adhesion and granule secretion result from signaling cascades downstream of two platelet receptors converging on phospholipase C (PLC) activation, PLC b and PLC g 2 are activated by G-protein-coupled receptors (GPCR) and receptor tyrosine kinase (RTK), respectively. PLC activation hydrolyses

membrane phosphoinositides (such as phophatidylinositol 4,5- bisohosphate [PIP

₂

]), thereby releasing inositol-1,4,5-triphosphate (IP

3

) and diacylglycerol (DAG). In turn, IP

3

induces a flux of intracellular Ca

²⁺

, while DAG activates protein kinases C (PKCs).

PKCs play a central role in regulating platelet activation responses, including release of alpha and dense granules (i.e. P-selectin, ADP), synthesis of thromboxane A

2

(TXA

2

), a

IIb

b

3

integrin activation, aggregation and spreading [1,4,7–19]. However, the role of individual PKC isoforms in platelet function remains to be elucidated. This review was therefore undertaken to examine the different role of PKC d isoform in platelet activation and aggregation.

2. Protein kinase C (PKC)

PKCs belong to serine/threonine kinases that are part of the AGC-type kinase (PKA/PKG/PKC) superfamily, and encompass 10 distinct isozymes grouped into three classes according to their lipid or second messenger requirements: the classical or conven- tional (cPKC), novel (nPKC) and atypical (aPKC) kinases. Such family plays key roles in cell proliferation and apoptosis [20]. Members of the cPKC ( a , b I, b II, and g ) require both second messengers Ca

²⁺

and DAG for activation. In contrast, nPKC ( d , e , h , and u ) activation is DAG or phorbol esters sensitive, but Ca

²⁺

- independent [20], while aPKC ( z and i / l ) activation is independent of Ca

²⁺

and DAG, but requires phosphatidyl serine [1,2,4,14].

A R T I C L E I N F O

Article history:

Received 20 February 2016 Accepted 20 May 2016 Available online xxx

Keywords:

Platelets PKC delta Thrombosis

A B S T R A C T

Platelet activation is a complex balance of positive and negative signaling pathways. Several protein kinase C (PKC) isoforms are expressed in human platelets. They are a major regulator of platelet granule secretion, activation and aggregation activity. One of those isoforms is the PKC d isozyme, it has a central yet complex role in platelets such as opposite signaling functions depending on the nature of the agonist, it concentration and pathway. In fact, it has been shown that PKC d has an overall negative influence on platelet function in response to collagen, while, following PAR stimulation, PKC d has a positive effect on platelet function. Understanding the crucial role of PKC d in platelet functions is recently emerging in the literature, therefore, further investigations should shed light into its specific role in hemostasis. In this review, we focus on the different roles of PKC d in platelet activation, aggregation and thrombus formation.

ß 2016 Elsevier Masson SAS. All rights reserved.

* Corresponding author.

E-mail addresses: [email protected] (Y. Zaid), [email protected] (N. Senhaji), [email protected] (Y. Darif), [email protected] (K. Kojok), [email protected] (M. Oudghiri), [email protected] (A. Naya).

Available online at

ScienceDirect

www.sciencedirect.com

http://dx.doi.org/10.1016/j.retram.2016.05.001

2452-3186/ß 2016 Elsevier Masson SAS. All rights reserved.

The primary structure of cPKCs constitutes a single polypeptide chain, divided into four conserved domains (C1–C4) and five variable regions (V1-V5), pseudo-substrate motif is present in regulatory domain of all PKCs as shown on Fig. 1. The NH2 terminal half of the polypeptide contains C1, C2, V1, V2 and partial V3 regions constituting the regulatory domain [21].

Specificity of PKCs interactions with regulators or substrates involved in the physiological responses of platelets is regulated through isozyme-specific anchoring proteins, termed receptors for activated C-kinase (RACKs) [1,4,14,22–24]. Upon phosphorylation, PKC regulation could occur through positive or negative feedback loops by secondary messengers, for which in turn, at high levels, are controlled by PKC isozymes themselves [25,26]. On the other hand, PKCs possess a catalytic domain binding adenosine triphosphate (ATP) or protein substrates like PKD2, whose PKC- dependent activation is required for optimal platelet-dense granule secretion and aggregation, therefore regulating thrombus formation on collagen surfaces [14,27].

Characterization of PKC isozymes in platelets, namely PKC a , b ,

d , and u , led to a better understanding of their selective role in regulating platelet functions, and shed light onto their association with some cardiovascular diseases, such as endothelial dysfunc- tion and vascular restenosis [4,14,28]. PKCs also contribute to mechanisms underlying myocardial infarction, acute ischemia- reperfusion injury or cardiac arrhythmia (PKC d and PKC e ), compensatory hypertrophy (PKC a , PKC b II, PKC d and PKC e ), heart failure (PKC a and PKC b II), and diabetic cardiomyopathy (PKC a , PKC b II, PKC d and PKC e ) [28]. Essential (patho) physiological roles of PKCs in the cardiovascular system are shaped through their regulation of various signaling cascades, among them the Erk, p38 MAPK, JNK, and Akt pathways [14].

3. PKC d isozyme

In 1988, structural determination of identified rabbit cDNA clones, coding for PKC a , b , and g isozymes, led to the identification of a fourth homologous type of cDNA clone, termed delta, by the use of these cloned cDNAs as hybridization probes within a brain PKC preparation [29]. PKC d specific expression profile is a consequence of its distinct roles in cells, such as B cell signaling and the regulation of growth, apoptosis, secretion, tumor development, and differen- tiation of a variety of cell types [30]. PKC d expression is ubiquitous in mammalian tissues, including epidermis, placenta, uterus, brain,

lung, hematopoietic system, and kidney [20]. Moreover, upon its activation, PKC- d auto-inhibitory domain undergoes a conforma- tional change in order to open the phorbol ester-binding pocket and allows interaction of C1 and C2 domains with membrane lipids, resulting in its translocation from the cytosol to subcellular membranes, including lysosomal membrane [31].

Given its overexpression resulting in inhibition of cell growth, PKC d shows a unique ability to mediate self-antigen-induced B cell tolerance [32]. Its contrasting implication in cell survival and cell death is regulated by various factors, such as its localization, the presence of oxidative stress causing DNA damage or other pro- and anti-apoptoic signaling molecules [33]. In addition, PKC d acts downstream of several tyrosine kinases to regulate the function of a plethora of transcription factors, such as Sp1, NF- k B, p300, STAT- 1 and STAT-3 [33].

Studies on lesion development in vascular disease demonstrat- ed a role for PKC d in re-endothelialization, where its deficiency in mice led to accelerated neointimal lesions and vasohibin-1 accumulation, the latter’s expression dependent of PKC d [34]. Such deficiency also correlates with intimal hyperplasia in which vascular smooth muscle cell (VSMC) migration and proliferation is accentuated to potentiate vascular restenosis [14,34]. More recent studies have also highlighted a role for PKC d as a key regulator of oxLDL-induced endoplasmic reticulum stress-medi- ated apoptosis in VSMC, which may contribute to atherosclerotic plaque instability and rupture [35]. The previous finding evokes cardiovascular conditions of thrombotic occlusion or embolization, which account for the major morbidity and mortality in most countries around the world [36].

By fostering an inflammatory environment, platelets remain a key element in the outcome of atherosclerotic lesion progression, and facilitate plaque rupture, in addition to their role in acute thrombus formation [37]. Since PKC isozymes were found to regulate main functions of platelet upon their activation, under- standing the central role of PKC d in platelet functions is recently emerging in the literature, and further investigations should bring insight into its specific role in hemostasis and thrombus formation.

3.1. PKC d in platelet activation, granule secretion and TXA

2

generation

PKC d is involved in signal transduction downstream of receptors for collagen and thrombin, GPVI/ a

2

b

1

and PAR1/4,

Fig.1.

Schematic of the the primary structure of PKCs family members.

respectively. A schematic overview of some of the PKC d predomi- nant functions during the platelet activation response is given on Fig. 2.

It was shown that PKC d is activated and tyrosine phosphory- lated in thrombin-stimulated platelets, and also shown that thrombin induced PKC d activation is independent of the a

IIb

b

3

mediated signaling pathway [38,39]. In addition, inhibition of PKC d caused a marked increase in [

³

H]5-HT released [40].

PKC d differentially regulates dense granule secretion in an agonist-dependent manner through GPVI and PARs. Indeed, PKC d could limit the release of ADP via GPVI, whereas it is thought to positively regulate dense granule secretion via PARs [4,41]. For instance, PKD2 is a substrate of PKC d , which mediates its phosphorylation on serines 744/748 downstream of PAR4, and yields dense granule secretion following activation [2,27]. In contrast, down-regulation of such GPVI-mediated secretion would implicate interactions between Lyn, PKC d , and SHIP-1, consistent with a down-regulation of a

IIb

b

3

signaling by the Lyn-SHIP-1 complex [3,4]. By using an isoform selective antagonistic RACK peptide d (V1-1)TAT, it was shown that PKC d regulates positively PARs-mediated dense granule secre- tion, while this regulation is negative following GPVI activation [22].

Intracellular Ca

²⁺

ions concentration in platelets is increased upon platelet activation, and results in the release of TXA

₂

[42]. PKC d may also differentially regulate TXA

2

(a positive- feedback mediator during platelet activation) synthesis in a positive manner through PARs and GPVI [43]. However, the generation of TXA

2

by PKC d via TXA synthase is not established since in another study, PKC d has been shown to negatively regulate TXA

2

generation through GPVI as demonstrated by PKC d gene knockdown in mice platelets [22]. Moreover, it was recently established that tyrosine phosphorylated PKC d Y311 regulates

ADP-induced thromboxane generation in platelets, independently of its catalytic activity through tyrosine kinase Lyn [44].

A novel role for PKC d in the signaling pathway downstream of GPIb-IX-V complex, a non-GCPR in platelets, has been proposed by the group of Canobbio et al. [45]. GPIb-IX-V interaction with VWF initiates transmembrane signalling events for platelet activation, including ‘‘inside-out’’ activation of the a

IIb

b

3

integrin [45]. It appears that signaling pathways through GCPR and non-GCPR converge to the activation of PKC d , the latter therefore playing a central role in regulating platelet signalling.

3.2. PKC d in platelet aggregation and thrombus formation

It is established that the PKC isoforms could regulate a plethora of signaling pathways involved in platelet aggregation and thrombus formation [46–49].

Negative regulation of filopodial formation by PKC d may reduce platelet aggregation by means of restricted thrombus formation [4]. This previous process would underlie a physical interaction between PKC d and the actin regulator vasodilator-stimulated phosphoprotein (VASP), in which PKC d acts as a negative regulator of VASP phosphorylation on Ser

¹⁵⁷

[23]. Table 1 shows the different proteins interacting with PKC d .

PKC d

^/

platelets show enhanced collagen/collagen-related peptide-induced aggregation [23]. Crosby et al. [40] have demonstrated that rottlerin (selective PKC d inhibitor) potentiates Alboaggregin-A-induced platelet aggregation, when the agonist was used at submaximal concentrations. Different studies performed in our laboratory have shown that collagen-induced platelet aggregation was completely blocked by rottlerin, whereas aggregation of platelets by thrombin remained unaffected by it [43]. However, many concerns have been raised regarding the specificity and mechanism of action of rottlerin [50].

Table1

Proteins interacting with PKC d in platelets.

Type of interaction Interacting proteins Function of interaction

Direct Protein kinase D (PKD) Dense granule secretion

Direct Multiple Src family kinases including Src, Lyn and SHIP-1 Src family kinase activity is required for activation of PKC d

Direct Fyn tyrosine kinase but not Syk, Src and Btk Reciprocal kinase regulation

Direct Vasodilator-stimulated phosphoprotein (VASP) Regulation of filopodia formation

Indirect Gp130 via STAT3 binding to the catalytic domain of PKC d Important role in Il-6 signaling PKC: protein kinase C.

Fig.2.

Diagram summarizing the different roles of protein kinase C d (PKC d ) in platelet signaling.

On the other hand, to date, only one study evaluated the role of PKC d in a mouse thrombosis model, and concluded that PKC d

deficiency seems not to be affecting the thrombus formation in vivo [22]. However, a novel selective PKC d inhibitor, CGX1037 was shown to be able to regulate negatively PAR-mediated dense granule secretion but enhanced GPVI-mediated dense granule secretion as well as the capacity to inhibit the phosphorylation of PKD2, a substrate of PKC d . Thus, further studies with CGX1037 could shed light on the implication of PKC d in thrombosis [51]. Even if little is known of the PKC d in thrombosis, suppression of PKC d was suggested as a potential therapeutic strategy to treat several pathologies such coeliac disease [52], endotoxin [53] or sepsis [54] induced lung injury and abdominal aortic aneurysm (AAA) [55]. Therefore, understanding the involvement of PKC d as an important regulatory in various pathological should bring more insights into the involvement of PKC d in thrombosis through the discovery of new targets in the PKC d signaling pathway in platelets. All in all, it is suggested that PKC d tend to negatively regulate collagen-dependent thrombus formation [48]. Further studies are expected to extend our knowledge about the implication of PKC d in thrombosis.

4. Conclusion

In platelets, the PKC family is an important signaling mediator required for activation, secretion of granule contents, and aggregation. However, the relative importance of the major platelet PKC isoforms and their downstream effectors in platelet signaling and function remain unclear. PKC d isoform plays a complex role, having effects on both positive and negative signaling, depending of the nature of the agonist. Indeed, PKC d

positively regulates TXA

2

via PARs and GPVI, however, PKC d

negatively regulates dense granule secretion via GPVI, whereas it is enhanced via PARs, therefore, PKC d plays an important role in modulating platelet function. The mechanisms that might underlie differential regulation of granule secretion by PKC d by GPVI and PAR1/4 stimulation are still unclear. Further investigations should be done in order to uncover PKC d function in platelets and it potential role in the cardiovascular diseases. Nevertheless, PKC d

might represent an important therapeutic target due to its implication in many aspects of platelet activation.

Disclosure of interest

The authors declare that they have no competing interest.

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