# The PPAR-Platelet Connection: Modulators of Inflammation and Potential Cardiovascular Effects

Authors: S. L. Spinelli; J. J. O'Brien; S. Bancos; G. M. Lehmann; D. L. Springer; N. Blumberg; C. W. Francis; M. B. Taubman; R. P. Phipps
Journal: PPAR Research
 (2008)

---

## Abstract

Historically, platelets were viewed as simple anucleate cells responsible for initiating thrombosis and maintaining
hemostasis, but clearly they are also key mediators of inflammation and immune cell activation. An emerging body of
evidence links platelet function and thrombosis to vascular inflammation.  peroxisome proliferator-activated receptors
(PPARs) play a major role in modulating inflammation and, interestingly, PPARs (PPARβ/δ and PPARγ) were recently
identified in platelets.  Additionally, PPAR agonists attenuate platelet activation; an important discovery for two reasons.
First, activated platelets are formidable antagonists that initiate and prolong a cascade of events that contribute to
cardiovascular disease (CVD) progression.  Dampening platelet release of proinflammatory mediators, including
CD40 ligand (CD40L, CD154), is essential to hinder this cascade.  Second, understanding the biologic importance
of platelet PPARs and the mechanism(s) by which PPARs regulate platelet activation will be imperative in designing
therapeutic strategies lacking the deleterious or unwanted side effects of current treatment options.


---

## Body

## 1. Introduction

Cardiovascular disease (CVD) is the leading cause of morbidity
and mortality world-wide. In part, this
is due to social and economic changes that lead to atherosclerosis, obesity,
hypertension, dyslipidemia, and type 2 diabetes mellitus (T2DM) [1–5]. Life-style
factors such as exercise, healthy diet, and avoidance of smoking are crucial
to prevent disease or reduce cardiovascular risk factors. While it is important to educate individuals
about healthy life-style decisions, it is also imperative to develop
therapeutic strategies to attenuate the chronic inflammatory pathways linked to
vascular disease [4–6]. Recently,
platelets have been implicated as key contributors to the chronic inflammation
that leads to CVD [5].While platelets are essential for hemostatic
regulation, new studies reveal an expanded role for platelets in thrombosis,
immune cell activation, and inflammatory processes creating an obvious link
between thrombosis and vascular inflammation.
Platelet hyperactivity is implicated in a variety of conditions
including atherosclerosis, peripheral arterial disease (PAD), T2DM, and
inflammatory bowel disease (IBD) [7–10]. Although
activated platelets release many proinflammatory mediators such as CD40 ligand
(CD40L, CD154) and thromboxane A2(TXA2), they also
release membrane vesicles and platelet microparticles (PMPs), which influence the
activities of other cell types both regionally and systemically. Since PMPs contain proteins important for
both hemostasis and inflammation, they may amplify or sustain inflammation and
thrombosis contributing to a chronic inflammatory state. Moreover, higher than normal levels of
platelet-released microparticles are present in individuals with
atherosclerosis, T2DM, stroke, and PAD [9, 11–13].Proteomic studies are beginning to reveal the
remarkable diversity of platelet proteins and have identified proteins not
known to be expressed in or released from platelets [14–16]. While
lacking a nucleus, platelets contain transcription factors, notably the
peroxisome proliferator-activated receptors (PPARS). PPARs are key regulators of metabolism and
inflammation, and thus are poised to play an important role in processes that
govern chronic inflammatory diseases [17]. Accumulating
evidence suggests that PPAR activation is beneficial in the prevention of
stroke and myocardial infarction (heart attack) [17, 18]. However, other
studies show that some PPAR activating drugs may increase the risk of
cardiovascular events [19]. Despite the
lack of definitive information on the risk and benefits of taking
PPAR-targeting drugs, it is clear that PPARs remain a promising target for
treating CVD and more importantly, that dampening unwanted platelet activation
will reduce the risk of CVD and/or improve disease outcome.


## 2. Platelets are Modulators of Inflammation and Thrombosis

Platelets are anuclear cells released from
megakaryocytes, a hematopoietic cell that differentiates and undergoes
endomitosis [20]. The
platelet’s composition is a product of specific packaging by the megakaryocyte
and the acquisition by endocytosis of blood components. Platelets contain classical cellular organelles
including mitochondria and lysosomes, a complex cytoskeleton, specific platelet
granules, and an open canalicular system, a complex structure of internal
membranes that serves as a conduit for the movement and release of platelet
contents. Despite the lack of a nucleus,
platelets contain mRNA and spliceosomal components for mRNA processing, as well
as the translational machinery for protein synthesis [21–23]. The recent discovery
of de novo synthesis by platelets of
mRNAs, including Bcl-3, interleukin-1β (IL-1β), plasminogen activator inhibitor-1 (PAI-1), and
tissue factor (TF), exemplifies the complexity of platelet signaling and
underscores their role as formidable players in regulating coagulant and
inflammatory pathways [24–29].Platelets
contain vast stores of bioactive mediators including thromboxanes,
prostaglandins, chemokines, and cytokines that promote clot formation and incite
inflammation. Upon activation,
platelets produce high levels of proinflammatory
mediators such as CD40L, intercellular adhesion molecule-1 (ICAM-1), tissue
factor, and C-reactive protein (CRP).
These mediators enhance inflammatory responses and recruitment of immune
cells. Recently, it was shown that
plasma levels of soluble CD40L (sCD40L) are high at birth and remain so
throughout childhood [30]. The reason for the developmental change is
not yet understood. In contrast, higher
than normal adult levels of sCD40L in the adult bloodstream are linked with
increased risk for ischemia, stroke, and myocardial infarcts due to thrombosis [4, 31]. Based on these studies, much interest has
been generated in CD40L as a possible biomarker and major factor in the
progression of CVD [32–34].


### 2.1. CD40L is a Major Contributor to Chronic Inflammation

A surprising and
important finding was that CD40L, a member of the tumor necrosis factor (TNF)
receptor superfamily and a key mediator of both innate and adaptive immunity [4, 5, 35, 36], is
released by activated platelets [31, 33, 35]. Shortly after platelets become activated, they
express CD40L on their surface which is subsequently enzymatically
cleaved releasing soluble bioactive CD40L into the bloodstream. This is highly significant for the following
two reasons. First, platelets contain approximately 95% of the CD40L found in
human beings, and thus are a crucial link in the regulation of the CD40/CD40L pathway,
as many cells express its receptor, CD40.
These cells include fibroblasts, endothelial, epithelial, monocytes,
neutrophils, B cells, and dendritic cells.
CD40L is found in abnormally
high levels in the blood of patients with chronic inflammatory diseases such as
diabetes, atherosclerosis, as well as some recipients of platelet transfusions
[33, 37–40]. Disruption
of CD40/CD40L pathway can blunt chronic inflammation, retard
atherosclerosis, and  transplant rejection [33, 35, 41]. Further,  recent exciting research
demonstrates that CD40L is crucial for stabilizing thrombi, for normal platelet
responses to sheer stress, and for platelet activation through the RGD domain
of sCD40L which binds to platelet αIIbβ3, a receptor critical for platelet
activation and aggregation [42, 43]. Collectively, these data strongly support the importance of CD40L as a
primary agonist for platelets and is considered a prototypical mediator with
roles in both hemostasis and inflammation (Figure 1 summarizes CD40 activation
by platelet CD40L). Therefore, the platelet is a crucial link in
the CD40/CD40L pathway and sCD40L release alone or in combination with other
proinflammatory mediators may increase the risk for cardiovascular effects promoting
atherosclerosis, hypertension, and dyslipidemia to list a few.Figure 1
Platelets promote inflammation. CD40 expressing cells, such as endothelial
cells or fibroblasts, can be activated by platelet-derived CD40L. CD40 signaling upregulates bioactive
mediators in these cells; therefore, potentiating inflammation and increasing
the risk for CVD.


### 2.2. Platelet-Released Microparticles are Elevated in Individuals with Chronic Inflammatory Disease

Platelet microparticles (PMPs) are
defined as microvesicle particles that measure less than 1μm
in diameter [44]. Platelet agonist stimulation or high shear
stress leads to the highly regulated formation and release of PMPs, which are
known to regulate a broad spectrum of physiological activities [45–47]. PMPs are an important delivery and
cell signaling system in both inflammatory and hemostatic processes. For example, a portion of platelet IL-1β is associated with PMPs and
signals endothelial cells, inducing their adhesiveness for neutrophils to
elicit an inflammatory response [25]. PMPs signal the expression of specific
adhesion molecules and stimulate the production of cytokines and mRNA in
endothelial cells and in the monocytic cell line, THP-1 [48]. Notably, a known α-granule component and
proinflammatory mediator, regulated on activation, normal
T-cell expressed and secreted (RANTES) (CCL5), is delivered to sites of arterial injury
and atherosclerotic endothelium via PMP to promote monocyte recruitment [49]. PMPs modulate cell-to-cell interactions by
increasing adhesive contacts between monocytes and endothelial cells, an
important first step in vascular inflammation [50]. It is also known that platelet-derived tissue
factor (TF) is transferred from CD62P positive PMPs to monocytes although the
procoagulant role of this particle delivery system has not been established [51]. Elevated numbers of
PMPs are present in a variety of diseases including atherosclerosis and other CVDs,
T2DM, and cancer [49, 51–54]. PPARs may have a potential role in the
regulation of platelet activation and release of platelet contents as will be
discussed further below.


## 2.1. CD40L is a Major Contributor to Chronic Inflammation

A surprising and
important finding was that CD40L, a member of the tumor necrosis factor (TNF)
receptor superfamily and a key mediator of both innate and adaptive immunity [4, 5, 35, 36], is
released by activated platelets [31, 33, 35]. Shortly after platelets become activated, they
express CD40L on their surface which is subsequently enzymatically
cleaved releasing soluble bioactive CD40L into the bloodstream. This is highly significant for the following
two reasons. First, platelets contain approximately 95% of the CD40L found in
human beings, and thus are a crucial link in the regulation of the CD40/CD40L pathway,
as many cells express its receptor, CD40.
These cells include fibroblasts, endothelial, epithelial, monocytes,
neutrophils, B cells, and dendritic cells.
CD40L is found in abnormally
high levels in the blood of patients with chronic inflammatory diseases such as
diabetes, atherosclerosis, as well as some recipients of platelet transfusions
[33, 37–40]. Disruption
of CD40/CD40L pathway can blunt chronic inflammation, retard
atherosclerosis, and  transplant rejection [33, 35, 41]. Further,  recent exciting research
demonstrates that CD40L is crucial for stabilizing thrombi, for normal platelet
responses to sheer stress, and for platelet activation through the RGD domain
of sCD40L which binds to platelet αIIbβ3, a receptor critical for platelet
activation and aggregation [42, 43]. Collectively, these data strongly support the importance of CD40L as a
primary agonist for platelets and is considered a prototypical mediator with
roles in both hemostasis and inflammation (Figure 1 summarizes CD40 activation
by platelet CD40L). Therefore, the platelet is a crucial link in
the CD40/CD40L pathway and sCD40L release alone or in combination with other
proinflammatory mediators may increase the risk for cardiovascular effects promoting
atherosclerosis, hypertension, and dyslipidemia to list a few.Figure 1
Platelets promote inflammation. CD40 expressing cells, such as endothelial
cells or fibroblasts, can be activated by platelet-derived CD40L. CD40 signaling upregulates bioactive
mediators in these cells; therefore, potentiating inflammation and increasing
the risk for CVD.


## 2.2. Platelet-Released Microparticles are Elevated in Individuals with Chronic Inflammatory Disease

Platelet microparticles (PMPs) are
defined as microvesicle particles that measure less than 1μm
in diameter [44]. Platelet agonist stimulation or high shear
stress leads to the highly regulated formation and release of PMPs, which are
known to regulate a broad spectrum of physiological activities [45–47]. PMPs are an important delivery and
cell signaling system in both inflammatory and hemostatic processes. For example, a portion of platelet IL-1β is associated with PMPs and
signals endothelial cells, inducing their adhesiveness for neutrophils to
elicit an inflammatory response [25]. PMPs signal the expression of specific
adhesion molecules and stimulate the production of cytokines and mRNA in
endothelial cells and in the monocytic cell line, THP-1 [48]. Notably, a known α-granule component and
proinflammatory mediator, regulated on activation, normal
T-cell expressed and secreted (RANTES) (CCL5), is delivered to sites of arterial injury
and atherosclerotic endothelium via PMP to promote monocyte recruitment [49]. PMPs modulate cell-to-cell interactions by
increasing adhesive contacts between monocytes and endothelial cells, an
important first step in vascular inflammation [50]. It is also known that platelet-derived tissue
factor (TF) is transferred from CD62P positive PMPs to monocytes although the
procoagulant role of this particle delivery system has not been established [51]. Elevated numbers of
PMPs are present in a variety of diseases including atherosclerosis and other CVDs,
T2DM, and cancer [49, 51–54]. PPARs may have a potential role in the
regulation of platelet activation and release of platelet contents as will be
discussed further below.


## 3. Peroxisome Proliferator-Activated Receptors (PPARs) and Platelets

PPARs are
ligand-activated transcription factors and members of the nuclear hormone
receptor superfamily. These receptors
are known to play a role in regulating metabolic risk factors for CVD, such as
the vascular inflammation and thrombosis associated with atherosclerosis [55]. There are
three PPAR subtypes, PPARα
(NR1C1), PPARβ/δ (NUC1, NR1C2), and PPAR (NR1C3), encoded by
separate genes and described in several organisms including humans. PPARs are differentially expressed in a
variety of tissues and are important in the regulation of lipid and
carbohydrate metabolism, energy homeostasis, cellular differentiation and
apoptosis, and immune and inflammatory responses [42]. PPARα is highly expressed in brown adipose tissue, liver,
kidney, heart, and skeletal muscles [61]. PPARβ/δ
has a broad tissue distribution with highest expression in the kidney, gut, and
heart [42, 62]. PPARγ is
abundant in adipose tissue, colon, retina, and in cells of the immune system [58]. Important for
this discussion are PPARβ/δ and PPARγ as they were recently found to be expressed
in human platelets, a surprising result considering platelets are anucleate [63, 64]. The impact
of this discovery was exemplified upon finding that exposure to PPAR agonists
attenuates platelet activation and associated inflammation [63, 64].Activation
of PPARs in nucleated cells occurs by optimal DNA binding to a PPAR DNA
response element following ligand binding and conformational changes that
facilitate heterodimerization with a second ligand-activated nuclear receptor, retinoic
X receptor (RXR, 9-cis retinoic acid
receptor)
[65, 66]. This heterodimer binds to a cis
acting DNA element in the promoters of target genes called the peroxisome
proliferator response element (PPRE) to induce or repress gene transcription in
a cell- and tissue-specific manner, depending on the receptor and a combination
of factors, including ligand and accessory molecule binding. The physiological functions of PPARα and PPARγ have been relatively well characterized, whereas
the function of PPARβ/δ is poorly understood. A summary of the PPAR subtypes and their potential roles in platelets is discussed below.


### 3.1. PPARα

PPARα activation affects transcriptional expression of approximately 80–100 genes,
the products of which regulate fatty acid oxidation, lipid metabolism, and
inflammation [67]. PPARα
is expressed in cells of the vasculature and immune system, but has not yet
been firmly identified in platelets [68]. The antiinflammatory properties of PPARα are of paramount interest, but there are also reports of
proinflammatory effects [69, 70]. For example, it was demonstrated that chronic
activation of PPARα
is detrimental to cardiac recovery during reperfusion following ischemia [71]. In contrast, it is known that PPARα
plays an antiinflammatory role in lung fibrosis although the mechanism is not well understood
[72, 73]. It is clear that the intricacies of PPARα function must be discerned to design effective and safe drug strategies. Current
PPARα agonists include the fibrates, which are therapeutic agents that increase
transcription of high density lipoproteins (HDL) such as ApoAI and ApoAII and
are effective at lowering triglyceride levels [74, 75]. PPARα agonists have also been reported to decrease weight gain, as obesity is a
contributing factor in atherosclerosis [75].


### 3.2. PPARβ/δ

PPARβ/δ is suggested to play
a role in basic cellular functions such as cellular proliferation and
differentiation, and fatty acid catabolism in skeletal muscle where it is most
abundant [76, 77]. This receptor has also been implicated in the
regulation of inflammation, and shown to slow plaque formation and attenuate the progression of atherosclerosis [78]. Although little is known
about the function of PPARβ/δ, especially in
platelets, prostacyclin (PGI2), an important antithrombotic and
endogenous platelet hormone, is reported to be a ligand for PPARβ/δ [79, 80]. Several studies
have revealed that PGI2 synergizes with nitric oxide (NO) to inhibit
platelet aggregation in response to a variety of platelet agonists including thrombin, collagen, ADP,
and lysophosphatidic acid (LPA) [64, 81–86]. It was
previously shown that the synergistic effects of NO and prostacyclin on
inhibition of platelet response were due to the simultaneous increase of cyclic
nucleotides cGMP and cAMP [81, 87, 88]. The recent
discovery that PPARβ/δ ligands and NO inhibit platelet aggregation via PPARβ/δ suggests an alternative signaling mechanism is operative in platelets [64]. This is
consistent with a previous study where Ali et al. demonstrated that
prostacyclin mimetics exhibited antiproliferative effects that were mediated by
PPARβ/δ and not via the prostacyclin receptor in lung
fibroblasts [89]. This
identified PPARβ/δ as a potential therapeutic target for the treatment of
pulmonary hypertension and supports the view that platelet PPARβ/δ may play an important role in thrombosis [64].


### 3.3. PPARγ

PPARγ is important in adipocyte differentiation, lipid
storage, and glucose homeostasis, and has emerged as a key target for new
antiinflammatory therapies [6, 90, 91]. There are 3 isoforms of PPARγ (PPARγ1, PPARγ2,
and PPARγ3). All are encoded by the same gene, but are the
result of differential promoter use and alternative RNA splicing [92]. PPARγ2 differs from PPARγ1 by an additional 30 amino acids at the N-terminus. PPARγ1 is present in adipose tissue, human spleen, liver, intestine, kidney, and
platelets, while PPARγ2 is abundantly expressed only in adipose tissue and liver [93]. PPARγ3 mRNA has been detected in mouse macrophage cells, however its function remains unknown [94].PPARγ is expressed in many cell types including fibroblasts, endothelial
cells, dendritic cells, macrophages, T cells, B cells, and most recently we
identified PPARγ in human platelets [59, 63, 91, 95–98]. Our
laboratory recently discovered that human platelets express PPARγ and that PPARγ ligands
attenuate platelet release of the proinflammatory and procoagulant mediators,
sCD40L and TXA2, a cyclooxygenase (COX) product that enhances
platelet activation [63]. Platelets can respond to at least two natural PPARγ ligands: lysophosphatidic acid (LPA) which they produce, and 15d-PGJ2 which has potent antiinflammatory properties and is a metabolite of PGD2 [91, 99, 100]. Additionally, there are several synthetic
ligands in development and clinical use that are specific and potent agonists
for PPARγ including the antidiabetic
thiazolidinedione drugs (TZDs) (e.g., rosiglitazone (Avandia) and pioglitazone (Actos) both in clinical use) [91, 99]. These will be discussed in greater detail in
Section 5.Interestingly,
human platelets also contain the PPARγ binding partner RXR, and PPARγ is able to bind DNA suggesting that it can
form an active PPARγ/RXR
heterodimer, and thus may be capable of biologic activity within the platelet. It is therefore possible that PPARγ agonists interact directly with
platelets to alter platelet activation and hemostatic function. While PPARγ was
first thought to be located only in the nucleus to regulate transcription, we
and others have demonstrated that PPARγ can be found in the cytoplasm of
eukaryotic cells [91, 101]. There is increasing evidence suggesting that
PPARγ binds proteins in the cytoplasm of cells separate from its
transcriptional role. For example, it was recently reported that
PPARγ ligands, via a PPARγ-dependent mechanism, block PKCα translocation to the membrane attenuating
inflammatory responses in monocytes/macrophages [101]. Additionally, cytoplasmic PPARγ can
repress the transcriptional activity of the proinflammatory mediator, nuclear
factor–κB (NF-κB), preventing
its translocation to the nucleus [92, 102]. NF-κB
is involved in regulating many aspects of cellular activity, including the
immune response and has a well established role in the pathological progression
of chronic inflammatory diseases [103]. Interestingly,
it has also been shown in platelets that the PPARγ binding partner, RXR,
signals through the Gq-protein receptor in a ligand-dependent manner to inhibit
platelet activation [104].Intriguingly,
our group has discovered that PPARγ is released in a PMP-associated form and
some PPARγ is expelled
from activated platelets as a functional PPARγ/RXR heterodimer [105]. Moreover, the released PPARγ is
taken up by a promonocytic cell line (THP-1) [105]. Thus, it is possible that other cells also take
up platelet-released PPARγ, quickly elevating PPARγ levels in recipient cells. This potential transcellular mechanism for
PPARγ would then influence the recipient cell’s susceptibility to PPARγ ligands
and may represent a novel antiinflammatory mechanism. For example, PPARγ and its
ligands are known to reduce VCAM-1 and ICAM-1 expression, and increase nitric
oxide synthase expression on endothelial cells which is important for
inhibiting platelet activation [106, 107]. These expanded antiinflammatory roles for PPARγ provide new
avenues to pursue novel drug strategies.


## 3.1. PPARα

PPARα activation affects transcriptional expression of approximately 80–100 genes,
the products of which regulate fatty acid oxidation, lipid metabolism, and
inflammation [67]. PPARα
is expressed in cells of the vasculature and immune system, but has not yet
been firmly identified in platelets [68]. The antiinflammatory properties of PPARα are of paramount interest, but there are also reports of
proinflammatory effects [69, 70]. For example, it was demonstrated that chronic
activation of PPARα
is detrimental to cardiac recovery during reperfusion following ischemia [71]. In contrast, it is known that PPARα
plays an antiinflammatory role in lung fibrosis although the mechanism is not well understood
[72, 73]. It is clear that the intricacies of PPARα function must be discerned to design effective and safe drug strategies. Current
PPARα agonists include the fibrates, which are therapeutic agents that increase
transcription of high density lipoproteins (HDL) such as ApoAI and ApoAII and
are effective at lowering triglyceride levels [74, 75]. PPARα agonists have also been reported to decrease weight gain, as obesity is a
contributing factor in atherosclerosis [75].


## 3.2. PPARβ/δ

PPARβ/δ is suggested to play
a role in basic cellular functions such as cellular proliferation and
differentiation, and fatty acid catabolism in skeletal muscle where it is most
abundant [76, 77]. This receptor has also been implicated in the
regulation of inflammation, and shown to slow plaque formation and attenuate the progression of atherosclerosis [78]. Although little is known
about the function of PPARβ/δ, especially in
platelets, prostacyclin (PGI2), an important antithrombotic and
endogenous platelet hormone, is reported to be a ligand for PPARβ/δ [79, 80]. Several studies
have revealed that PGI2 synergizes with nitric oxide (NO) to inhibit
platelet aggregation in response to a variety of platelet agonists including thrombin, collagen, ADP,
and lysophosphatidic acid (LPA) [64, 81–86]. It was
previously shown that the synergistic effects of NO and prostacyclin on
inhibition of platelet response were due to the simultaneous increase of cyclic
nucleotides cGMP and cAMP [81, 87, 88]. The recent
discovery that PPARβ/δ ligands and NO inhibit platelet aggregation via PPARβ/δ suggests an alternative signaling mechanism is operative in platelets [64]. This is
consistent with a previous study where Ali et al. demonstrated that
prostacyclin mimetics exhibited antiproliferative effects that were mediated by
PPARβ/δ and not via the prostacyclin receptor in lung
fibroblasts [89]. This
identified PPARβ/δ as a potential therapeutic target for the treatment of
pulmonary hypertension and supports the view that platelet PPARβ/δ may play an important role in thrombosis [64].


## 3.3. PPARγ

PPARγ is important in adipocyte differentiation, lipid
storage, and glucose homeostasis, and has emerged as a key target for new
antiinflammatory therapies [6, 90, 91]. There are 3 isoforms of PPARγ (PPARγ1, PPARγ2,
and PPARγ3). All are encoded by the same gene, but are the
result of differential promoter use and alternative RNA splicing [92]. PPARγ2 differs from PPARγ1 by an additional 30 amino acids at the N-terminus. PPARγ1 is present in adipose tissue, human spleen, liver, intestine, kidney, and
platelets, while PPARγ2 is abundantly expressed only in adipose tissue and liver [93]. PPARγ3 mRNA has been detected in mouse macrophage cells, however its function remains unknown [94].PPARγ is expressed in many cell types including fibroblasts, endothelial
cells, dendritic cells, macrophages, T cells, B cells, and most recently we
identified PPARγ in human platelets [59, 63, 91, 95–98]. Our
laboratory recently discovered that human platelets express PPARγ and that PPARγ ligands
attenuate platelet release of the proinflammatory and procoagulant mediators,
sCD40L and TXA2, a cyclooxygenase (COX) product that enhances
platelet activation [63]. Platelets can respond to at least two natural PPARγ ligands: lysophosphatidic acid (LPA) which they produce, and 15d-PGJ2 which has potent antiinflammatory properties and is a metabolite of PGD2 [91, 99, 100]. Additionally, there are several synthetic
ligands in development and clinical use that are specific and potent agonists
for PPARγ including the antidiabetic
thiazolidinedione drugs (TZDs) (e.g., rosiglitazone (Avandia) and pioglitazone (Actos) both in clinical use) [91, 99]. These will be discussed in greater detail in
Section 5.Interestingly,
human platelets also contain the PPARγ binding partner RXR, and PPARγ is able to bind DNA suggesting that it can
form an active PPARγ/RXR
heterodimer, and thus may be capable of biologic activity within the platelet. It is therefore possible that PPARγ agonists interact directly with
platelets to alter platelet activation and hemostatic function. While PPARγ was
first thought to be located only in the nucleus to regulate transcription, we
and others have demonstrated that PPARγ can be found in the cytoplasm of
eukaryotic cells [91, 101]. There is increasing evidence suggesting that
PPARγ binds proteins in the cytoplasm of cells separate from its
transcriptional role. For example, it was recently reported that
PPARγ ligands, via a PPARγ-dependent mechanism, block PKCα translocation to the membrane attenuating
inflammatory responses in monocytes/macrophages [101]. Additionally, cytoplasmic PPARγ can
repress the transcriptional activity of the proinflammatory mediator, nuclear
factor–κB (NF-κB), preventing
its translocation to the nucleus [92, 102]. NF-κB
is involved in regulating many aspects of cellular activity, including the
immune response and has a well established role in the pathological progression
of chronic inflammatory diseases [103]. Interestingly,
it has also been shown in platelets that the PPARγ binding partner, RXR,
signals through the Gq-protein receptor in a ligand-dependent manner to inhibit
platelet activation [104].Intriguingly,
our group has discovered that PPARγ is released in a PMP-associated form and
some PPARγ is expelled
from activated platelets as a functional PPARγ/RXR heterodimer [105]. Moreover, the released PPARγ is
taken up by a promonocytic cell line (THP-1) [105]. Thus, it is possible that other cells also take
up platelet-released PPARγ, quickly elevating PPARγ levels in recipient cells. This potential transcellular mechanism for
PPARγ would then influence the recipient cell’s susceptibility to PPARγ ligands
and may represent a novel antiinflammatory mechanism. For example, PPARγ and its
ligands are known to reduce VCAM-1 and ICAM-1 expression, and increase nitric
oxide synthase expression on endothelial cells which is important for
inhibiting platelet activation [106, 107]. These expanded antiinflammatory roles for PPARγ provide new
avenues to pursue novel drug strategies.


## 4. Platelets and Cardiovascular Disease

Cardiovascular
disease comprises a broad spectrum of illnesses, such as hypertension,
dyslipidemia, and myocardial infarction and stroke that affect the heart and the
blood vessels. These conditions have
similar causes (obesity, smoking, diabetes, sedentary lifestyle, and age) and platelets
play a complex role in CVD, triggering early events that lead to endothelial
dysfunction, to progression of vascular damage, to plaque production, and
to formation of thrombi that can result in myocardial infarcts and stroke.


### 4.1. Metabolic Syndrome

Platelets and their PPARs play putative roles in
several manifestations of the dyslipidemia-associated “metabolic syndrome” or
“syndrome X,” which includes hyperglycemia, insulin resistance, obesity,
hypertension, and atherosclerosis
[77, 108–113]. Dyslipidemia,
an increasingly common consequence of a high-fat diet, is characterized by
increased serum triglycerides, low levels of antiatherogenic high density
lipoprotein cholesterol (HDL) and prevalence of pro-atherogenic low density
lipoprotein particles (LDL). Considering
the imbalance between pro- and antiatherogenic factors, it is not surprising
that dyslipidemia is associated with a high risk of atherosclerosis in
afflicted patients [77]. HDL protects
against atherosclerosis by driving the reverse transport of cholesterol from
peripheral cells to the liver for excretion [77, 113]. The
contribution of LDL particles to the development of atherosclerosis is closely
connected to platelet function and may be modulated by PPARs, as described
below.


### 4.2. Atherosclerosis

Atherosclerosis is a chronic inflammatory disease
characterized by plaque development within the arterial intima [5, 114]. These
atherosclerotic plaques may erode or rupture over time, triggering
thrombogenesis, and possible myocardial infarction or stroke [5, 115]. Platelets
are famous for their role in clot formation during the final stages of
atherosclerosis, but it has become clear from studies in both humans and animal
models that the early stages of plaque formation are also platelet-mediated [5, 115–120]. Atherosclerosis
is initiated when inflammatory processes activate vascular endothelial cells,
resulting in platelet adhesion to the arterial wall [115, 121–123]. When
platelets adhere to the endothelial surface, they are activated, causing them
to release mediators that attract and activate other cell types, including
neutrophils, monocytes, and bone-marrow-derived progenitor cells [5, 115]. Monocytes
cross the endothelial monolayer and enter the arterial intima by extravasation [115]. There they
differentiate first into macrophages, and then, into cholesterol-laden foam
cells, a critical step in atherosclerotic plaque formation [77, 115, 118]. Platelets
regulate the differentiation of bone-marrow-derived progenitor cells and
macrophages into foam cells [5, 115, 118, 119, 124]. Studies
using fluorochrome-modified LDL have shown that platelets take up LDL and store
it in dense granules [115, 118]. These
platelets can then be internalized by macrophages, a critical step in foam cell
differentiation and plaque formation [115, 118, 125, 126].One platelet-derived mediator with a
clear link to atherogenesis is platelet factor 4 (PF4) which both inhibits LDL
degradation by the LDL receptor and promotes monocyte-to-foam cell
differentiation [115, 127]. Activated
platelets also release CD40L and interleukin-1β which further activate the vascular endothelium,
causing it to produce chemoattractants and adhesion molecules that act to
recruit neutrophils and monocytes into the arterial intima [5, 115, 118, 128, 129]. Matrix
metalloproteinases (MMPs) are also expressed by activated platelets, monocytes,
and endothelial cells in response to CD40L; these are important in foam cell
generation and the physical remodeling of the normal arterial wall to an
atherosclerotic plaque [115, 118, 130–136]. Smooth muscle
cell proliferation, promoted by platelet release of transforming growth factor-β, platelet-derived growth factor, and serotonin, is also critical to this process [115].PPARs appear to play a major role in
the regulation of atherogenesis by countering the inflammation-provoking action
of platelet adhesion and activation [5]. In vitro incubation of platelets with PPARγ agonists inhibits their
ability to express CD40L and to aggregate in response to thrombin [63, 137]. Pioglitazone, a PPARγ-specific ligand,
decreases platelet aggregation and delays arterial thrombus formation in male
LDL receptor-deficient mice [5, 138]. Other PPARγ ligands, including rosiglitazone and c9, t11-conjugated
linoleic acid, inhibit atherosclerotic progression in this model and in the
apoE−/− mouse [139, 140], possibly through their ability to inhibit platelet
deposition, monocyte recruitment, macrophage differentiation, LDL uptake, foam
cell formation, MMP expression, and vascular smooth muscle cell migration within
atherosclerotic plaques [115, 118, 137, 138, 141, 142]. Studies in
human patients with atherosclerosis have shown that certain TZD type PPARγ agonists reduce both platelet and endothelial cell
activation, inhibit plaque progression, improve flow-mediated vasodilation, and remarkably
promote regression of existing atherosclerotic plaques [5, 115, 143]. Since
phagocytosis of platelets (and their internalized LDL) by macrophages is
critical to foam cell formation and atherosclerotic progression,
platelet-derived PPARγ may be of paramount importance to the antiatherosclerotic actions of these
drugs [115, 118, 125, 126]. Packaging of
PPARγ into platelets and/or
its release in PMPs may be a convenient mechanism by which this transcription
factor is delivered to endothelial lesions where it may act to attenuate
pathological remodeling of the arterial wall.
The potential benefits of PPAR signaling are not limited to
atherosclerosis, but may extend to “metabolic syndrome” as a whole. Rosiglitazone therapy reduces the systemic
inflammation characteristic of “metabolic syndrome,” as evidenced by decreases
in serum levels of IL-6 and TNFα [5, 144]. PPARβ/δ
ligands have been shown to ameliorate dyslipidemia in both mice and
insulin-resistant obese rhesus monkeys [113, 145, 146]. Current data
suggest that PPARs will prove to be premium targets for the development of
drugs to combat both dyslipidemia and atherosclerosis.


### 4.3. Thrombosis

As was discussed
above, endothelial dysfunction in blood vessels is one of the earliest events
that contribute to disease development triggering a chain reaction, which
results in formation of atherosclerotic plaques and rupture in the blood vessel
walls. A major function of platelets is
to “plug” these holes by changing their shape, adhering to subendothelial surfaces,
secreting the contents of intracellular organelles, and aggregating
to form a thrombus in response to stimuli generated in endothelia of
damaged blood vessels [147]. Several mediators
are involved in platelet aggregation, such as thrombin, collagen,
epinephrine (exogenous to the platelet); agents such as ADP (secreted
from platelet storage granules); and thromboxane A2 (synthesized
by the platelets during activation) [148]. As was
mentioned above, the PPARγ agonists rosiglitazone and pioglitazone dampened platelet release of key
proinflamatory and proatherogenic mediators such as CD40L and TXA2 [63]. The PPARγ agonist troglitazone has also been shown to decrease
platelet aggregation in response to ADP, collagen, and arachidonic acid [149]. The
mechanism whereby the vascular endothelium defends against thrombus formation
involves the generation of the potent vasodilator nitric oxide (NO). NO interferes with platelet aggregation and is
generated from L-arginine by the enzyme nitric oxide synthase (NOS) which is
constitutively expressed in endothelium [150]. In
experiments where rats received pioglitazone, it was found that aortic cNOS and
thrombomodulin expression was upregulated and thrombus formation was delayed [151]. Pioglitazone
had similar effects in the human monocyte/macrophage cell line (THP-1) where
dose-dependently upregulated thrombomodulin expression was seen [152]. Other PPARγ
ligands, such as rosiglitazone, also upregulate cNOS gene expression [153, 154].


### 4.4. Myocardial Infarction and Stroke

Myocardial infarction occurs when the blood supply to
the heart is interrupted causing damage and possible death of the heart
tissue. One of the major causes of
myocardial infarction is rupture of the atherosclerotic plaque and formation of
a platelet-rich thrombus. PPARγ is present in heart tissue, but there is limited
data about its function there. The PPARγ activator rosiglitazone does inhibit TNF-α gene expression in cultured myocytes [155]. Additionally, Rosiglitazone treatment of male Lewis rats
following myocardial ischemia and reperfusion injury showed a dramatic protection
against myocardial infarction, and also improved cardiac function [156]. Ischemia/reperfusion injury is characterized by an inflammatory response. Activated neutrophils release a variety of
cytotoxic substances, such as oxygen-derived free radicals and
proteases and activated monocytes/macrophages
synthesize inflammatory cytokines [157]. Activated
platelets can upregulate these responses in neutrophils and
monocytes/macrophages. Together, these
mediators directly participate in the amplification of an inflammatory response
and, therefore, in vascular endothelial dysfunction that can lead to
myocardial injury. PPARγ is present in monocytes/macrophages, neutrophils, and platelets, which suggests a role for PPARγ in negatively regulating expression of
proinflammatory genes and thus, myocardial infarction [158].Thrombus can also form in the
cerebral arteries blocking the normal blood flow and causing a cerebrovascular
accident (stroke). Stroke is a complex
process in which several pathways are involved and successful prevention of a
stroke will require drugs with pleiotropic effects. Resveratrol, found in the seeds and skin of
grapes, was found to have neuroprotective effects [159] and shown to be a dual PPARα/γ
activator [18]. Experiments
in a rat model have shown that pretreatment with fenofibrate and/or Wy-14643,
which are PPARα
activators, and resveratrol reduced brain infarct size after permanent focal
cerebral ischemia [18]. PPARβ/δ
is found in numerous brain areas whereas PPARα and PPARγ have a more localized expression. Inflammation and oxidative stress induce
apoptotic and necrotic neuronal death and NF-κB is one of the culprits [160]. It is
thought that PPARs have a neuroprotective function due to their interaction
with NF-κB. For example, PPARγ binds to NF-κB
complexes and facilitates its translocation out of the nucleus [102]. Due to their wide distribution in
the neurovascular-glial compartments and their complex function, PPAR agonists
offer hope in the prevention of stroke [161]. It will be
of major importance to dampen platelet activity in the case of both myocardial
infarction and stroke as ultimately, hyperactive platelets will be the major
culprits in the occlusion or rupture of an artery.


### 4.5. Diabetes Mellitus

Type 2 diabetes mellitus (T2DM), primarily characterized by hyperglycemia and insulin resistance, is often part
of a “metabolic syndrome” which comprises hypertension, dyslipidemia, decreased
fibrinolysis, and increased procoagulant factors (discussed above) [162]. Thrombocytopathia
(any qualitative modification of platelets) in diabetes includes: increased
platelet aggregation and adhesiveness, increased platelet number, and enhanced
expression of activation-dependent adhesion molecules [10]. Platelet
hyperaggregability and adhesiveness in diabetes has several causes. Prostacyclin and the endothelium-derived
relaxing factor nitric oxide (NO) are released by intact vascular
endothelium and antagonize the effects of proaggregants so that
thrombi do not form in blood vessels [163]. Platelets
from diabetic patients produce less prostacyclin and NO and, in addition, they
are less sensitive to PGI2 and nitric oxides inhibitory effects [164–166]. Insulin can
target platelets directly through the platelet insulin receptor, which binds
insulin and undergoes autophosphorylation [167]. Insulin
reduces platelet responses to the agonists ADP, collagen, thrombin,
arachidonate, and platelet-activating factor [168]. However, in T2DM
platelets express fewer insulin receptors and a decreased affinity for insulin [169]. Insulin has
a direct effect on platelets and is important for maintaining platelet PGI2 sensitivity by increasing the PGI2 binding sites and as a
consequence, augments cAMP response to PGI2 [170]. Numerous
studies support the fact that there is an association between
diabetes and oxidative stress [171]. A higher
production of reactive oxygen species is thought to play an
important role in diabetes complications and has been attributed to protein
glycation and/or autoxidation caused by a hyperglycemic environment, and lipid peroxidation of cellular structures [172].Oxidative defense is
provided by vitamins, such as vitamin E, and by a number of enzymes, such as
glutathione peroxidases. Platelets
contain two glutathione peroxidases: cytosolic glutathione peroxidase (cGPx)
and phospholipid hydroperoxide glutathione peroxidase (PHGPx). CGPx is involved
in oxidative stress protection and in formation of eicosanoids [173, 174]. Vitamin E is
decreased in plasma of type 1 and type 2 diabetic patients [175]. In type 2
diabetics, platelet cGPx activities were found to be lower and can lead to a
relative accumulation of 12-hydroperoxy-eicosatetraenoic acid
(12-HpETE), the main hydroperoxide formed from arachidonic acid [175]. Thus, increase
in 12-HpETE could activate signal transduction pathways leading to
arachidonic acid release, and amplification of platelet activation [176]. Platelet PHGPx activity was also measured for the first time in diabetic
patients and was decreased in type 2 diabetics [175]. Thus, in
diabetes there is an increase in free radical production and a decrease in
mechanisms responsible for antioxidant defense which give rise to an
environment that favors generation of radical species. Type 1 and 2 diabetic patients exhibit
increased expression of activation-dependent adhesion molecules, such
as activated αIIbβ3,
lysosomal Gp53, thrombospondin, and P-selectin (CD62P) [177]. The
increased expression of αIIbβ3 is consistent with the enhanced fibrinogen binding
and aggregability seen in platelets from diabetic subjects [178]. Arachidonic acid metabolism, which leads to TXA2 production, is increased in diabetes and may cause platelet sensitivity [179, 180]. Because
diabetes is accompanied by CVD development, drugs that can reduce hyperglycemia
and inhibit the progression of cardiovascular complications are desirable. PPARα/γ/β pan agonists may offer new options for treatment of
diabetic complications. The blood of
both type 1 and 2 diabetics shows elevated levels of CD40L [39]. PPARγ ligands can reduce platelet activation and
thrombosis by reducing CD40L from platelets.
Treatment of diabetic patients with TZD-type drugs decreased circulating
CD40L blood levels [181].


### 4.6. Obesity

Obesity represents a major health threat and, in
recent years, it has become clear that obesity and inflammation are linked [109–111, 182]. Obese
individuals show persistent platelet activation and subsequent increased plasma
levels of several proinflamatory cytokines [183]. TNFα, adiponectin, leptin, and monocyte chemoattractant
protein-1, all can originate from fat, have immunomodulating functions and show an altered profile during obesity [184]. Furthermore,
PPARβ/δ has been linked to the development of obesity. Its activation decreases adipose mass in mouse
and increases fatty acid oxidation in the heart, improving muscle contraction [76]. Thus
dampening platelet activation may be a means of reducing an inflammatory
cascade that leads to further vascular damage and CVD.


## 4.1. Metabolic Syndrome

Platelets and their PPARs play putative roles in
several manifestations of the dyslipidemia-associated “metabolic syndrome” or
“syndrome X,” which includes hyperglycemia, insulin resistance, obesity,
hypertension, and atherosclerosis
[77, 108–113]. Dyslipidemia,
an increasingly common consequence of a high-fat diet, is characterized by
increased serum triglycerides, low levels of antiatherogenic high density
lipoprotein cholesterol (HDL) and prevalence of pro-atherogenic low density
lipoprotein particles (LDL). Considering
the imbalance between pro- and antiatherogenic factors, it is not surprising
that dyslipidemia is associated with a high risk of atherosclerosis in
afflicted patients [77]. HDL protects
against atherosclerosis by driving the reverse transport of cholesterol from
peripheral cells to the liver for excretion [77, 113]. The
contribution of LDL particles to the development of atherosclerosis is closely
connected to platelet function and may be modulated by PPARs, as described
below.


## 4.2. Atherosclerosis

Atherosclerosis is a chronic inflammatory disease
characterized by plaque development within the arterial intima [5, 114]. These
atherosclerotic plaques may erode or rupture over time, triggering
thrombogenesis, and possible myocardial infarction or stroke [5, 115]. Platelets
are famous for their role in clot formation during the final stages of
atherosclerosis, but it has become clear from studies in both humans and animal
models that the early stages of plaque formation are also platelet-mediated [5, 115–120]. Atherosclerosis
is initiated when inflammatory processes activate vascular endothelial cells,
resulting in platelet adhesion to the arterial wall [115, 121–123]. When
platelets adhere to the endothelial surface, they are activated, causing them
to release mediators that attract and activate other cell types, including
neutrophils, monocytes, and bone-marrow-derived progenitor cells [5, 115]. Monocytes
cross the endothelial monolayer and enter the arterial intima by extravasation [115]. There they
differentiate first into macrophages, and then, into cholesterol-laden foam
cells, a critical step in atherosclerotic plaque formation [77, 115, 118]. Platelets
regulate the differentiation of bone-marrow-derived progenitor cells and
macrophages into foam cells [5, 115, 118, 119, 124]. Studies
using fluorochrome-modified LDL have shown that platelets take up LDL and store
it in dense granules [115, 118]. These
platelets can then be internalized by macrophages, a critical step in foam cell
differentiation and plaque formation [115, 118, 125, 126].One platelet-derived mediator with a
clear link to atherogenesis is platelet factor 4 (PF4) which both inhibits LDL
degradation by the LDL receptor and promotes monocyte-to-foam cell
differentiation [115, 127]. Activated
platelets also release CD40L and interleukin-1β which further activate the vascular endothelium,
causing it to produce chemoattractants and adhesion molecules that act to
recruit neutrophils and monocytes into the arterial intima [5, 115, 118, 128, 129]. Matrix
metalloproteinases (MMPs) are also expressed by activated platelets, monocytes,
and endothelial cells in response to CD40L; these are important in foam cell
generation and the physical remodeling of the normal arterial wall to an
atherosclerotic plaque [115, 118, 130–136]. Smooth muscle
cell proliferation, promoted by platelet release of transforming growth factor-β, platelet-derived growth factor, and serotonin, is also critical to this process [115].PPARs appear to play a major role in
the regulation of atherogenesis by countering the inflammation-provoking action
of platelet adhesion and activation [5]. In vitro incubation of platelets with PPARγ agonists inhibits their
ability to express CD40L and to aggregate in response to thrombin [63, 137]. Pioglitazone, a PPARγ-specific ligand,
decreases platelet aggregation and delays arterial thrombus formation in male
LDL receptor-deficient mice [5, 138]. Other PPARγ ligands, including rosiglitazone and c9, t11-conjugated
linoleic acid, inhibit atherosclerotic progression in this model and in the
apoE−/− mouse [139, 140], possibly through their ability to inhibit platelet
deposition, monocyte recruitment, macrophage differentiation, LDL uptake, foam
cell formation, MMP expression, and vascular smooth muscle cell migration within
atherosclerotic plaques [115, 118, 137, 138, 141, 142]. Studies in
human patients with atherosclerosis have shown that certain TZD type PPARγ agonists reduce both platelet and endothelial cell
activation, inhibit plaque progression, improve flow-mediated vasodilation, and remarkably
promote regression of existing atherosclerotic plaques [5, 115, 143]. Since
phagocytosis of platelets (and their internalized LDL) by macrophages is
critical to foam cell formation and atherosclerotic progression,
platelet-derived PPARγ may be of paramount importance to the antiatherosclerotic actions of these
drugs [115, 118, 125, 126]. Packaging of
PPARγ into platelets and/or
its release in PMPs may be a convenient mechanism by which this transcription
factor is delivered to endothelial lesions where it may act to attenuate
pathological remodeling of the arterial wall.
The potential benefits of PPAR signaling are not limited to
atherosclerosis, but may extend to “metabolic syndrome” as a whole. Rosiglitazone therapy reduces the systemic
inflammation characteristic of “metabolic syndrome,” as evidenced by decreases
in serum levels of IL-6 and TNFα [5, 144]. PPARβ/δ
ligands have been shown to ameliorate dyslipidemia in both mice and
insulin-resistant obese rhesus monkeys [113, 145, 146]. Current data
suggest that PPARs will prove to be premium targets for the development of
drugs to combat both dyslipidemia and atherosclerosis.


## 4.3. Thrombosis

As was discussed
above, endothelial dysfunction in blood vessels is one of the earliest events
that contribute to disease development triggering a chain reaction, which
results in formation of atherosclerotic plaques and rupture in the blood vessel
walls. A major function of platelets is
to “plug” these holes by changing their shape, adhering to subendothelial surfaces,
secreting the contents of intracellular organelles, and aggregating
to form a thrombus in response to stimuli generated in endothelia of
damaged blood vessels [147]. Several mediators
are involved in platelet aggregation, such as thrombin, collagen,
epinephrine (exogenous to the platelet); agents such as ADP (secreted
from platelet storage granules); and thromboxane A2 (synthesized
by the platelets during activation) [148]. As was
mentioned above, the PPARγ agonists rosiglitazone and pioglitazone dampened platelet release of key
proinflamatory and proatherogenic mediators such as CD40L and TXA2 [63]. The PPARγ agonist troglitazone has also been shown to decrease
platelet aggregation in response to ADP, collagen, and arachidonic acid [149]. The
mechanism whereby the vascular endothelium defends against thrombus formation
involves the generation of the potent vasodilator nitric oxide (NO). NO interferes with platelet aggregation and is
generated from L-arginine by the enzyme nitric oxide synthase (NOS) which is
constitutively expressed in endothelium [150]. In
experiments where rats received pioglitazone, it was found that aortic cNOS and
thrombomodulin expression was upregulated and thrombus formation was delayed [151]. Pioglitazone
had similar effects in the human monocyte/macrophage cell line (THP-1) where
dose-dependently upregulated thrombomodulin expression was seen [152]. Other PPARγ
ligands, such as rosiglitazone, also upregulate cNOS gene expression [153, 154].


## 4.4. Myocardial Infarction and Stroke

Myocardial infarction occurs when the blood supply to
the heart is interrupted causing damage and possible death of the heart
tissue. One of the major causes of
myocardial infarction is rupture of the atherosclerotic plaque and formation of
a platelet-rich thrombus. PPARγ is present in heart tissue, but there is limited
data about its function there. The PPARγ activator rosiglitazone does inhibit TNF-α gene expression in cultured myocytes [155]. Additionally, Rosiglitazone treatment of male Lewis rats
following myocardial ischemia and reperfusion injury showed a dramatic protection
against myocardial infarction, and also improved cardiac function [156]. Ischemia/reperfusion injury is characterized by an inflammatory response. Activated neutrophils release a variety of
cytotoxic substances, such as oxygen-derived free radicals and
proteases and activated monocytes/macrophages
synthesize inflammatory cytokines [157]. Activated
platelets can upregulate these responses in neutrophils and
monocytes/macrophages. Together, these
mediators directly participate in the amplification of an inflammatory response
and, therefore, in vascular endothelial dysfunction that can lead to
myocardial injury. PPARγ is present in monocytes/macrophages, neutrophils, and platelets, which suggests a role for PPARγ in negatively regulating expression of
proinflammatory genes and thus, myocardial infarction [158].Thrombus can also form in the
cerebral arteries blocking the normal blood flow and causing a cerebrovascular
accident (stroke). Stroke is a complex
process in which several pathways are involved and successful prevention of a
stroke will require drugs with pleiotropic effects. Resveratrol, found in the seeds and skin of
grapes, was found to have neuroprotective effects [159] and shown to be a dual PPARα/γ
activator [18]. Experiments
in a rat model have shown that pretreatment with fenofibrate and/or Wy-14643,
which are PPARα
activators, and resveratrol reduced brain infarct size after permanent focal
cerebral ischemia [18]. PPARβ/δ
is found in numerous brain areas whereas PPARα and PPARγ have a more localized expression. Inflammation and oxidative stress induce
apoptotic and necrotic neuronal death and NF-κB is one of the culprits [160]. It is
thought that PPARs have a neuroprotective function due to their interaction
with NF-κB. For example, PPARγ binds to NF-κB
complexes and facilitates its translocation out of the nucleus [102]. Due to their wide distribution in
the neurovascular-glial compartments and their complex function, PPAR agonists
offer hope in the prevention of stroke [161]. It will be
of major importance to dampen platelet activity in the case of both myocardial
infarction and stroke as ultimately, hyperactive platelets will be the major
culprits in the occlusion or rupture of an artery.


## 4.5. Diabetes Mellitus

Type 2 diabetes mellitus (T2DM), primarily characterized by hyperglycemia and insulin resistance, is often part
of a “metabolic syndrome” which comprises hypertension, dyslipidemia, decreased
fibrinolysis, and increased procoagulant factors (discussed above) [162]. Thrombocytopathia
(any qualitative modification of platelets) in diabetes includes: increased
platelet aggregation and adhesiveness, increased platelet number, and enhanced
expression of activation-dependent adhesion molecules [10]. Platelet
hyperaggregability and adhesiveness in diabetes has several causes. Prostacyclin and the endothelium-derived
relaxing factor nitric oxide (NO) are released by intact vascular
endothelium and antagonize the effects of proaggregants so that
thrombi do not form in blood vessels [163]. Platelets
from diabetic patients produce less prostacyclin and NO and, in addition, they
are less sensitive to PGI2 and nitric oxides inhibitory effects [164–166]. Insulin can
target platelets directly through the platelet insulin receptor, which binds
insulin and undergoes autophosphorylation [167]. Insulin
reduces platelet responses to the agonists ADP, collagen, thrombin,
arachidonate, and platelet-activating factor [168]. However, in T2DM
platelets express fewer insulin receptors and a decreased affinity for insulin [169]. Insulin has
a direct effect on platelets and is important for maintaining platelet PGI2 sensitivity by increasing the PGI2 binding sites and as a
consequence, augments cAMP response to PGI2 [170]. Numerous
studies support the fact that there is an association between
diabetes and oxidative stress [171]. A higher
production of reactive oxygen species is thought to play an
important role in diabetes complications and has been attributed to protein
glycation and/or autoxidation caused by a hyperglycemic environment, and lipid peroxidation of cellular structures [172].Oxidative defense is
provided by vitamins, such as vitamin E, and by a number of enzymes, such as
glutathione peroxidases. Platelets
contain two glutathione peroxidases: cytosolic glutathione peroxidase (cGPx)
and phospholipid hydroperoxide glutathione peroxidase (PHGPx). CGPx is involved
in oxidative stress protection and in formation of eicosanoids [173, 174]. Vitamin E is
decreased in plasma of type 1 and type 2 diabetic patients [175]. In type 2
diabetics, platelet cGPx activities were found to be lower and can lead to a
relative accumulation of 12-hydroperoxy-eicosatetraenoic acid
(12-HpETE), the main hydroperoxide formed from arachidonic acid [175]. Thus, increase
in 12-HpETE could activate signal transduction pathways leading to
arachidonic acid release, and amplification of platelet activation [176]. Platelet PHGPx activity was also measured for the first time in diabetic
patients and was decreased in type 2 diabetics [175]. Thus, in
diabetes there is an increase in free radical production and a decrease in
mechanisms responsible for antioxidant defense which give rise to an
environment that favors generation of radical species. Type 1 and 2 diabetic patients exhibit
increased expression of activation-dependent adhesion molecules, such
as activated αIIbβ3,
lysosomal Gp53, thrombospondin, and P-selectin (CD62P) [177]. The
increased expression of αIIbβ3 is consistent with the enhanced fibrinogen binding
and aggregability seen in platelets from diabetic subjects [178]. Arachidonic acid metabolism, which leads to TXA2 production, is increased in diabetes and may cause platelet sensitivity [179, 180]. Because
diabetes is accompanied by CVD development, drugs that can reduce hyperglycemia
and inhibit the progression of cardiovascular complications are desirable. PPARα/γ/β pan agonists may offer new options for treatment of
diabetic complications. The blood of
both type 1 and 2 diabetics shows elevated levels of CD40L [39]. PPARγ ligands can reduce platelet activation and
thrombosis by reducing CD40L from platelets.
Treatment of diabetic patients with TZD-type drugs decreased circulating
CD40L blood levels [181].


## 4.6. Obesity

Obesity represents a major health threat and, in
recent years, it has become clear that obesity and inflammation are linked [109–111, 182]. Obese
individuals show persistent platelet activation and subsequent increased plasma
levels of several proinflamatory cytokines [183]. TNFα, adiponectin, leptin, and monocyte chemoattractant
protein-1, all can originate from fat, have immunomodulating functions and show an altered profile during obesity [184]. Furthermore,
PPARβ/δ has been linked to the development of obesity. Its activation decreases adipose mass in mouse
and increases fatty acid oxidation in the heart, improving muscle contraction [76]. Thus
dampening platelet activation may be a means of reducing an inflammatory
cascade that leads to further vascular damage and CVD.


## 5. PPAR Agonists as Platelet Therapeutics

Platelets are an important pharmacological target
because the thrombi developed during CVD that lead to morbidity and mortality
are platelet-rich in content. Nonsteroidal
antiinflammatory drugs, including aspirin, are among the most widely used
drugs around the world [185]. Aspirin’s
primary action is to inhibit arachidonate-cyclooxygenase activity in platelets
and ultimately, TXA2 release thereby, attenuating thrombus formation. Recent reports show that a subset of patients
is aspirin-resistant and that aspirin may not be as effective in women. This, coupled with the fact that the cyclooxygenase
pathway plays only a minor role in the action of many platelet agonists, has
lead to the development of new antiplatelet therapies that complement
aspirin’s therapeutic effects [186–189].There are two groups of
antiplatelet agents used in conjunction with aspirin: the thienopyridines
(ticlopidine and clopidogrel) and the glycoprotein (GP) IIb/IIIa (αIIbβ3) receptor antagonists
(abciximab and eptifibatide). The
thienopyridines are adenosine 5′-diphosphate (ADP) receptor antagonists which
block ADP from binding, thereby, inhibiting platelet activation, aggregation,
and degranulation. While for the most
part, thienopyridines are efficacious for reducing ischemic events, it is
unclear as to whether or not clopidogrel and aspirin together are more
effective than aspirin alone [190, 191]. In rare
cases, thienopyridines may cause neutropenia or thrombotic thrombocytopenia
purpura [192, 193].αIIbβ3 is the most important
platelet membrane receptor for aggregation because it is found in high
concentrations on the cell surface and binds both fibrinogen and von Willebrand
factor. Blocking this receptor reduces
thrombotic risks associated with acute coronary syndromes and diabetes. Unfortunately, αIIbβ3 receptor antagonists have
to be administered intravenously because oral therapy causes excessive bleeding [194]. Moreover, a
meta analysis of four αIIbβ3 receptor antagonist trials showed an overall increase in mortality with drug
use [195].Clearly, there is a need to develop
new therapeutics that are easily administered and can dampen platelet function
with fewer adverse side effects. Adding
complexity to function, platelets activate and release many proinflammatory
mediators and interact with not only each other, but also with many other cell-types as
described in previous sections.
Targeting this action of platelets could be effective in not only
reducing platelet aggregation and thrombus formation, but also in attenuating
chronic inflammation and, therefore, slowing disease progression.PPAR agonists are a class of potential
antiplatelet drugs that are easily administered and have the ability to impact
this new physiology of platelet function.
Even though PPAR agonists are primarily
prescribed for the treatment of metabolic disorders, some possess the secondary
benefit of inhibiting cardiovascular complications associated with
hyperlipidemia and hyperglycemia. PPARα agonists, fibrates, are prescribed for
hyperlipidemia. They potently diminish
blood cholesterol and triglyceride levels while raising plasma HDL levels
(platelet agonists that dampen platelet activation are summarized in Figure 2).Figure 2
Possible role of PPAR agonists in
dampening inflammation and reducing cardiovascular events. PPAR agonists may reduce
the risk for thrombosis. Besides playing
a role in adipogenesis, lipid metabolism, and insulin sensitivity, PPARs may
dampen inflammation by attenuating platelet activation.The effect of PPARα agonists on cardiovascular risk during clinical
studies show mixed results. The Veterans Affair High-Density Lipoprotein Cholesterol Intervention
Trial study (VA-HIT) demonstrated that the fibrate, gemfibrozil, significantly reduced
nonfatal myocardial infarction and death in men with coronary cardiopathy [196]. Disappointingly,
results from the recent Fenofibrate Intervention and Event Lowering in
Diabetes (FIELD) trial showed no reduction in risk for the primary end-point (coronary
heart disease death and nonfatatal myocardial infarction) in coronary events
with fenofibrate therapy [197]. There are
many explanations for these results, including the use of a low cardiovascular
risk diabetic population, but it is clear that more investigation is needed to
understand the clinical relevance of fibrates for treating CVD. Since platelets may lack PPARα, these drugs may not have a direct effect on
platelet function, but may be useful in conjunction with other PPAR agonists to
target multiple pathways involved in cardiovascular pathophysiology (see below).Perhaps more promising is the use of
PPARγ TZD agonists as
antiplatelet agents. TZDs are mainly
used in the treatment of T2DM because they improve insulin sensitivity by
decreasing TNF-α
and IL-6 expression and increasing adiponectin expression [198, 199]. Troglitazone
was the first PPARγ
agonist marketed, but was withdrawn in 2000 for causing hepatotoxicity [200, 201].
Rosiglitazone and pioglitazone are the current TZDs prescribed in T2DM
and have been shown to reduce the risk of myocardial infarction and stroke [202]. As was discussed
in Section 3, our laboratory demonstrated that rosiglitazone attenuates CD40L
surface expression and sCD40L release from thrombin-activated platelets [63].
Downregulating the CD40/CD40L system would likely provide great
clinical benefit for patients with CVD.
Furthermore, 15d-PGJ2 was found to attenuate TXA2 and CD40L from thrombin-activated platelets, and prevent ATP release and
ADP-induced aggregation [63]. This
correlates with data from a mouse model of atherosclerosis showing that
pioglitazone decreases platelet activation and delays arterial thrombus
formation [138]. The PROspective pioglitAzone Clinical Trial
(PROACTIVE) demonstrated that
pioglitazone is protective against macrovascular events in diabetic patients [203]. Rosiglitazone
was also shown to reduce serum levels of matrix metalloproteinase-9 (MMP-9),
implicated in atherosclerotic plaque rupture, and the proinflammatory marker CRP
in patients with T2DM [204]. Conversely, some
recent studies, A Diabetes Outcome Progression Trial (ADOPT) and
Diabetes Reduction Assessment ramipril and Rosiglitazone Medication (DREAM), demonstrated
that rosiglitazone was associated with an increase in cardiovascular risks when
compared with placebo [205, 206]. As a
consequence of these recent reports that rosiglitazone may increase the
incidence of myocardial infarction, a randomized, prospective, open-label trial
(RECORD) was performed to assess the effects of rosiglitazone on CVD [207]. The results
of this study showed a significant increase in the risk of congestive heart
failure in patients taking rosiglitazone, but no significant differences in
cardiovascular-related hospitalization or death. There are many limitations to the recent
studies on the cardiovascular effects of TZDs, such as small sample sizes and
short trials, which clearly need to be resolved before an accurate interpretation
of the data can be made. In the short
term, it appears that the use of rosiglitazone and pioglitazone in patients
that are not at high risk for congestive heart failure is warranted [19]. However, a
better understanding of the biological effects of PPARs and the cogent design
of selective therapeutics without adverse effects are imperative.One alternative may lie in a promising new class of PPARγ
ligands known as selective PPAR modulators (SPPARMs) that have been designed as
partial PPARγ agonists, retaining insulin sensitization but lacking the
fat-accumulating properties of the classical TZD PPARγ ligands [208, 209]. Given the
success with SPPARMs in targeting insulin resistance, one can speculate that
other properties of PPARγ could be targeted for partial agonist design in the
future to have specific antiinflammatory activity without interference of
normal thrombotic benefits or risk of potential negative cardiac effects.There are also many other PPAR
candidate drugs under investigation for the treatment of metabolic syndrome. PPAR dual agonists and PPAR pan agonists are
new classes of drugs that target multiple PPAR isoforms at once to produce
synergistic antidiabetic and cardioprotective effects. These drugs have the potential to improve
insulin sensitivity and lower triglycerides while reducing the unwanted
side effects of weight gain and edema associated with the administration of
fibrates and TZDs. A novel group of dual
agonists have been discovered that appear to be potent agonists of both PPARα
and PPARγ. These compounds known as
alkoxybenzylglycines are synthetic tertiary amino acids, one of which has been demonstrated
to have beneficial oral antidiabetic and antidyslipidemic efficacy in vivo [210, 211]. However, the
therapeutic efficacy of dual and pan agonists in diabetes-associated cardiovascular
risks is unknown.PPARβ/δ agonists
are being developed for their ability to treat hyperlipidemia and they have the
potential to exert antithrombotic effects.
It was recently published that platelets express PPARβ/δ a
putative receptor for PGI2 whose activation inhibits platelet
aggregation [64, 212–214]. Clearly,
further studies are needed to address the effects that all PPAR agonists have
on not only cardiovascular risks, but also on platelet activity. It appears that TZDs have potentially
beneficial effects on overall cardiovascular risk. Understanding how targeting PPAR with
pharmacological agents influences platelet biology will provide insight into
the function of PPARs in platelets and help in designing drugs with better
specificity and fewer adverse side effects.


## 6. Conclusion

The studies described herein illustrate a connection between PPARs and
platelets that is significant in the pathophysiology of CVD. Platelets are emerging as potent immune and
inflammatory mediators that both initiate early responses in the vasculature
and elicit protracted responses that lead to the development of chronic
inflammatory disease. Platelets contain
PPARβ/δ and PPARγ, nuclear receptors with known antiinflammatory
functions. Thus, platelets are important
contributors to CVD processes and PPARs have the ability to attenuate these
processes. Platelet-derived PPARs are
likely to play an important role in controlling the magnitude of a
platelet-driven inflammatory response. Treatment
of platelets with PPAR agonists dampens the risk of thrombus formation and
attenuates increased blood levels of proinflammatory mediators such as CD40L
and TXA2. These functions of
PPARs can be exploited for the development of drugs to combat such prevalent
and devastating conditions as dyslipidemia, atherosclerosis, and diabetes. Understanding the specific role of
platelet-derived PPARs in the process of platelet activation attenuation is
essential for intelligent prevention and management of these disease states.



---

*Source: 328172-2007-12-24.xml*