Vascular Pharmacology 59 (2013) 6775

Contents lists available at ScienceDirect

Vascular Pharmacology
journal homepage: www.elsevier.com/locate/vph

Review

Antiplatelet properties of natural products

Gemma Vilahur a,, Lina Badimon a,b
a
b

Centro de Investigacin Cardiovascular, CSIC-ICCC, Hospital de la Santa Creu i Sant Pau and IIB-Santpau, Spain
Ctedra de Investigacin Cardiovascular, UAB-HSCSP-Fundacin Jess Serra, Barcelona, Spain

a r t i c l e

i n f o

Article history:
Received 16 August 2013
Received in revised form 19 August 2013
Accepted 20 August 2013
Chemical compounds studied in this article:
Arachidonic acid (PubMed CID: 444899)
Thromboxane (PubChem CID: 114873)
Adenosine diphosphate (PubChem CID: 6022)
Omega-3 fatty acids (PubChem CID: 56842239)
Onion (PubChem CID: 53472027)
Garlic (PubChem CID: 6850761)
Lycopene (PubChem CID: 446925)
Resveratrol (PubChem CID: 445154)
Flavonol (PubChem CID: 11349)
Ethanol (PubChem CID: 702)

a b s t r a c t
Cardiovascular diseases (CVD) and its main underlying cause, atherothrombosis, are the major culprits of morbidity
and mortality worldwide. Apart from the treatment of cardiovascular risk factors and the use of antithrombotic
agents there is considerable interest in the role of natural food products and their bioactive components in the prevention and treatment of cardiovascular disorders. The consumption of healthy diets rich in functional foods, such as
the Mediterranean diet, has shown to exert profound cardioprotective effects in the primary and secondary prevention of CVD. Moreover, accumulating data have attributed these benecial effects, at least in part, to the modulation
of key players in the pathogenesis of atherosclerosis, including amelioration in the lipid prole and vascular function
and a decrease in oxidative stress and inammation. Although with a much less clear picture, natural dietary compounds have also demonstrated to exert antiplatelet activities, further contributing to reduce the thrombotic risk.
This article provides a brief overview of the atherothrombotic process to further provide an up-to-date review of
the antiplatelet properties exerted by natural products and/or food-derived bioactive constituents including -3
PUFA, olive oil, garlic and onions, tomatoes, mushrooms, polyphenol-rich beverages, and avonol-rich cocoa as
well as to describe the mechanisms underlying these antiplatelet activities.
2013 Elsevier Inc. All rights reserved.

Keywords:
Atherothrombosis
Healthy diet
Natural foods
Antiplatelet activity

Contents
1.
2.

Introduction . . . . . . . . . . . . . . . . . .
Atherothrombosis . . . . . . . . . . . . . . .
2.1.
Atherosclerosis . . . . . . . . . . . . .
2.2.
Role of platelets in thrombus formation . .
3.
Antiplatelet properties of natural products . . . .
3.1.
-3 polyunsaturated fatty acids (PUFA) . .
3.2.
Olive oil . . . . . . . . . . . . . . . .
3.3.
Onion and garlic (ajoene) . . . . . . . . .
3.4.
Tomatoes . . . . . . . . . . . . . . . .
3.5.
Mushrooms . . . . . . . . . . . . . . .
3.6.
Polyphenol-rich beverages (wine and beer)
3.7.
Flavonol-rich cocoa . . . . . . . . . . .
4.
Summary and conclusions . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . .

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Corresponding author at: Cardiovascular Research Center, C/Sant Antoni M Claret 167, 08025 Barcelona, Spain. Tel.: +34 93 5537100; fax: +34 93 556 55 59.
E-mail address: gvilahur@csic-iccc.org (G. Vilahur).
1537-1891/$ see front matter 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.vph.2013.08.002

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G. Vilahur, L. Badimon / Vascular Pharmacology 59 (2013) 6775

1. Introduction
According to a World Health Organization fact sheet (EURO/03/06)
CVD is the number one killer in Europe, with heart disease and stroke
being the major cause of death in all 56 member states. Atherosclerosis
and its thrombotic complications (i.e., atherothrombosis) is the major
underlying cause of CVD [1]. Despite all the improved knowledge
on the etiopathogenesis and treatment of atherothrombosis, it is estimated that the socio-economic impact of this disease will increase,
rather than decrease, in the near future. It is predicted that coronary
heart disease will be the dominant cause of mortality worldwide by
2020.
The pioneering contributions from the Framingham Heart Study
launched in 1948 brought into mind the connection between cardiovascular risk factors (especially hypercholesterolemia) and CVD [2].
However, it was not until the 1960s when the Seven Countries study
established the rst link between dietary patterns and CVD [3]. In this
seminal study, Ancel Keys and colleagues evidenced that regular dietary
saturated fat intake was signicantly associated with serum cholesterol
levels and the risk of coronary heart disease [3]. Moreover, the outcome
of the study brought up the concept of the cardioprotective properties
of the dietary habits within the Mediterranean regions (low in saturated fat, rich in monounsaturated and -3 polyunsaturated fatty
acids and abundant in fruits and vegetables as well as a moderate
wine consumption) in comparison with other regions of the Western
world (high in saturated fats and simple sugars and low in -3 polyunsaturated fatty acids, bers and antioxidants) [4]. This initial observation was further supported by many other epidemiological studies
and several intervention trials. Among these, the recent PREDIMED
(Prevencin con Dieta Mediterranea) trial [5] and the Lyon Diet Heart
Study [6], which have robustly demonstrated the protective effects associated to the adherence of a Mediterranean type of diet in primary
and secondary prevention of CVD, respectively, deserve special attention. Recent analyses suggest that, together with regular physical activity and smoking cessation, over 80% of coronary heart disease and 70%
of stroke could be avoided by healthy food choices that are consistent
with the traditional Mediterranean Diet [7]. These studies have also
raised an enormous interest in identifying the food components and
the mechanisms involved in the protection against CVD. So far, compiling data from experimental, epidemiological and clinical studies have
supported that dietary Mediterranean components provide cardiovascular health benets, at least in part, by modulating key players in the
pathogenesis of atherosclerosis (e.g., amelioration in the lipid prole,
improvement of vascular function, oxidative stress diminishment
and anti-inammatory properties) [4,8]. In addition, although a much
less clear picture exists as to their antithrombotic potential, several
Mediterranean-related foods have also demonstrated to exert antiplatelet
activities, leading to a lesser pro-atherothrombotic prole.
In this mini-review we will rst provide a brief overview of the
atherothrombotic process in order to further outline some relevant
functional foods (whole foods and their bioactive ingredients) which
have been shown to modulate platelet function, and then describe the
mechanisms by which this is thought to occur.
2. Atherothrombosis
2.1. Atherosclerosis
Atherosclerosis is an inammatory disease that involves the arterial
wall, and is characterized by the progressive accumulation of lipids and
inammatory cells within the intima of large arteries [9,10]. The rst
step is the internalization of lipids (low density lipoproteins or LDLs)
in the intima, with the concomitant endothelial activation/dysfunction
(Fig. 1). The vascular endothelium is a semi-permeable barrier that
controls the diffusion of plasma molecules and regulates vascular
tone, inammation and prevents thrombus formation [11,12].

However, dysfunctional/activated endothelium is characterized by

the loss of such properties (Fig. 1). Consequently, LDL particles further inltrate and accumulate in the extracellular matrix (ECM); circulating monocytes are then recruited and attach through the exposure of
endothelial adhesion molecules. Once attached, they transmigrate into
the subendothelial space where they transform into macrophages.
Moreover, the injury in the endothelial-related antithrombotic properties facilitates platelet adhesion and activation in the dysfunctional area.
Adhered platelets, in concert with dysfunctional endothelial cells, secrete chemotactic and growth factors which stimulate migration, accumulation and proliferation of vascular smooth muscle cells (VSMC) and
leukocytes in the intimal layer, promoting plaque progression (Fig. 1)
[13,14]. LDLs retained in the ECM mainly by proteoglycans become
targets for oxidative and enzymatic modications. Oxidized LDLs enhance a series of pro-inammatory reactions via different mediators
(TNF-alpha, IL-1, MCSF, etc), perpetuating the activation, recruitment
and transmigration of monocytes and other inammatory cells across
the endothelial layer into the intima. On the other hand, the attracted
macrophages scavenge modied/oxidized LDLs, become lipid-laden,
and convert into foam cells (Fig. 1) [9]. In the early stages of atherosclerosis, the accumulation of foam cells evolves into fatty streak. Lesion
complication occurs when foam cells release growth factors and cytokines, which further stimulate VSMC migration from the media
into the intima where they divide and produce ECM components
such as collagen, and contribute to the formation of a brous cap
(Fig. 1) [15]. If the pathological triggering process persists and macrophages fail to remove accumulated cholesterol from the vessel,
they become apoptotic, releasing cholesterol to the vessel wall and,
more importantly, prothrombotic molecules (e.g., tissue factor) and
metalloproteinases (enzymes able to digest the ECM scaffold) [16].
Progression and complication of atherosclerotic plaques are also
characterized by a decreased number of VSMCs as well as the formation of immature and leaky new vessels, making atherosclerotic lesions more prone to rupture (the so-called vulnerable plaques)
[8]. Indeed, human atherectomy specimens and necropsy studies
have revealed that the main intrinsic features that characterize
plaques as vulnerable are large necrotic lipid core, increased inammatory content, reduced collagen and VSMC amount, and angiogenesis [8,17,18]. Plaque disruption and the subsequent exposure of
thrombogenic substrates initiate both platelet adhesion/activation
and aggregation on the exposed vascular surface and the activation
of the coagulation cascade, leading to thrombus formation and the
clinical manifestations of the atherosclerotic disease, acute myocardial infarction or sudden death (Fig. 1) [17].
2.2. Role of platelets in thrombus formation
Platelets, cytoplasmic fragments (25 m) originated from bonemarrow megakaryocytes, circulate within the vascular tree without
signicant interaction with the vessel wall. The endothelium inhibits
platelet reactivity by producing several local active substances, including nitric oxide (NO) and prostacyclin (prostaglandin I2; PGI2)
as well as by the presence on its surface of an ecto-ADPase (CD39)
and thrombomodulin. The endothelium also blocks the activation
of the coagulation cascade by expressing tissue factor pathway inhibitor (TFPi) and enhances brinolysis (clot dissolution) via activation of tissue plasminogen activator (tPA) (Fig. 1). However, the
clustering of cardiovascular risk factors, among which high-LDL
levels play a critical role, results in the aforementioned endothelial
dysfunction, characterized by a decrease in NO bioavailability [19].
NO is produced through the conversion of L-arginine to L-citrulline
by the enzyme endothelial NO synthase (eNOS). NO then diffuses
to the underlying vascular smooth muscle cells or circulating platelets to stimulate the production of cyclic guanosine monophosphate
(cGMP) through the activation of the enzyme guanylate cyclase
(GC)(Fig. 2). GC activation ultimately induces the activation of

G. Vilahur, L. Badimon / Vascular Pharmacology 59 (2013) 6775

69

Dysfunctional endothelium
Blood
coagulation

Platelet activation

Thrombin

Fibrinolysis

TF-FVIIa
PGI2
TFPi

TPa

Heparin sulphate
proteoglycan

NO

Thrombomodulin

Cardiovascular Risk Factors

(e.g., hypercholesterolemia)

Platelet
activation/aggregation
CXCL-2

LDL

Monocytes

MCP-1

Platelets
CXCL-2

Coagulation
cascade

vWF
MCP-1
GPIb
I-CAM

TF

V-CAM

LDL

Intima

ECM

MCP-1

Scavenger
receptors

Foam Cell

Dying
macrophage

Colony
Stimulating
factor

oxLDL
Macrophage

Media

ROS

Lipid-ladden VSMC

VSMC

Fig. 1. The following gure illustrates the complexity of events that drive the atherosclerotic process and further thrombotic complications. The rst step in atherosclerosis is the internalization of lipids in the intima that induces endothelial secretion of chemotactic substances and the expression of adhesion receptors, in turn favoring monocyte recruitment, adhesion, and
transmigration into the arterial wall. Once there, macrophages accumulate lipids, leading to foam cell formation. Foam cells aggravate the atherogenic process by releasing growth factors,
cytokines, metalloproteinases and reactive oxygen species, all of which perpetuate and amplify the vascular remodeling process and activate VSMC migration. On the other hand, the disruption of endothelial-related antithrombotic properties in concurrence with vascular damage induces platelet activation. MCP-1: monocyte chemoattractant protein-1; LDL: low density
lipoproteins; CXCL-2: Chemokine (C-X-C motif) ligand 2; vWF: Von Willebrand factor; V-CAM: Vascular cell adhesion molecule; E-CAM: endothelial cell adhesion molecule; mLDL:
modied LDL; TF: tissue factor; ROS: reactive oxygen species; TPa: tissue plasminogen activator; TFPi: tissue factor pathway inhibitor; PGI2: prostacyclin; NO: nitric oxide.

cGMP and/or cAMP-related kinases and the subsequent vasodilation

and/or inhibition of platelet aggregation, respectively [20]. In fact,
enhanced cAMP has shown to induce VASP-phosphorylation and
subsequent platelet inactivation (GPIIb/IIIa inactivation, Fig. 2).
Hence, the reduction in endothelial-related antithrombotic properties in concurrence with high reactive oxygen species generated by
atherosclerotic risk factors and the local increase in pro-thrombotic
and pro-inammatory mediators seem to contribute to platelet activation in the onset of atherosclerosis (Fig. 1) [21,22]. In advanced
disrupted plaques, platelet adhesion varies according to the shear
rate. Under low shear rate conditions platelet attachment mainly occurs through the collagen receptor that directly binds to the platelet
receptor GPIa/IIa (integrin II1) (Fig. 2). To a lesser extent, bronectin, laminin, vitronectin and thrombospondin also contribute to
platelet adhesion by binding to GPIc-IIa, GP Ic-IIa, vitronectin receptors, and to GPIV, respectively. Thereafter, the binding of platelet
GPVI receptor to collagen promotes rm platelet adhesion and mediates platelet activation and aggregation [23]. At high shear rates
platelet adhesion is mainly driven by the interaction of circulating
vWF (via its A3 domain) with exposed collagen. The binding of vWF
to collagen allows vWF A1 domain interaction with the platelet GPIb
component of the GPIb/IX/V platelet receptor complex (Fig. 2) [24].

The multimeric nature of vWF increases the local amount of active

A1 domain sites, thus reinforcing platelet-vessel wall interaction.
However, the vWF-GPIb/V/IX is known to be characterized by a fast
dissociation rate, and thus, formed bonds cannot provide stable arrest of platelets on the subendothelial matrix [25]. Again, platelet
GPVI receptor, by binding directly to collagen, induces platelet rm
adhesion and further activation and aggregation. As such, it promotes the activation of GPIIb/IIIa (integrin IIb3) and GPIa/IIa.
Both GPIIb/IIIa and GPIa/IIa act in concert to promote subsequent
rm, irreversible and stable platelet arrest on the endothelial surface, by direct binding both to collagen (GPIa/IIa) and to the vWF
C1 domain (GPIIb/IIIa; Fig. 2) [2527].
P-selectin has also been shown to mediate platelet adhesion to the
damaged vessel. P-selectin is localized in the -granules of platelets
and the WeibelPalade bodies of endothelial cells, and upon cell activation, it is translocated to the cell surface [28]. There are many putative Pselectin glycoprotein ligands that are either expressed on platelets
(sulfatides, GPIb, P-selectin glycoprotein ligand-1/PSGL-1), endothelial
cells (glycolysated cell adhesion molecule-1/GlyCAM-1, CD34, mucosal
cell adhesion molecule-1/MAdCAM-1) or leukocytes (PSGL-1) [29].
Besides platelet-collagen interaction, circulating agents such as epinephrine, serotonin, adenosine diphosphate (ADP; released from lysed

70

G. Vilahur, L. Badimon / Vascular Pharmacology 59 (2013) 6775

cGMP
PLC

AC

DAG I3P

cAMP

NITRIC
OXIDE

AMP

GC

PKA

GMP

inhibition

PKG

activation
5-HT2

SEROTONIN
Ca2+

Ca2+

VASP

-3 PUFAs
TOMATOES?
RED WINE
COCOA
TOMATOES
MUSHROOMS

GPIIb/IIIa

GARLIC
ONIONS
MUSHROOMS
RED WINE

GPIIb/IIIa

vWF

Ca2+

VASP-P

A1 A3

GARLIC (ajoene)

RGD

P2X1
PAR-1/PAR-4
DAG

EPINEPHRIN

COCOA

ATP

PKC

Fibrinogen

GARLIC
(ajoene)

PIP2

GPIIb/IIIa

PLC

OLIVE OIL
TOMATOES
MUSHROOMS
RED WINE
COCOA

VASP-P

P2Y12

PLATELET
GRANULES

AC

PLC

IP3

VASP

GPIIb/IIIa

VITRONECTIN

GPIIb/IIIa

RED WINE

PKA

Ca2+
Ca2+

cAMP

Ca2+

PI3K
MAPK

ALA?

ADP
vWF
Fgibrinogen

PGH2
PGG2

Ca2+

MAPK

TP

GARLIC
ONIONS
COCOA

TXA2
PLA2

ATP
ADP

GPIb-IX-V
vWF

PLATETL
DEGRANULATION

GPIa/IIa

AA

GPVI

-3 PUFAs
OLIVE OIL
GARLIC

COLLAGEN
A1

OLIVE OIL?

A3

RGD

RGD

vWF

COLLAGEN

OLIVE OIL
TOMATOES
MUSHROOMS
RED WINE

Fig. 2. Key agonists, their receptors and triggering signaling pathways involved in platelet activation and subsequent aggregation. In addition, the mechanisms by which natural food compounds exert antiplatelet effects are detailed. VWF: Von Willebrand factor; GP: glycoprotein; Fg: brinogen; AA: arachidonic acid; TXA2: thromboxane-A2; TP: thromboxane receptor;
ADP: adenosine diphosphate; PAR: proteinase activated receptor; TF: tissue factor, ALA: -linoleic acid.

erythrocytes at the site of injury) and thrombin (generated upon

atherosclerotic plaque exposure of tissue factor) may also activate
platelets via specic platelet surface receptors (Fig. 2). Once activated,
platelets undergo shape change, ensuing calcium translocation within
the platelet and the subsequent release of the platelet granule components (i.e., platelet degranulation). Platelet granule secretion leads
to the local release of ADP/ATP, serotonin, Ca2 +, adhesion proteins
(e.g., brinogen, bronectin, vWF, thrombospondin, vitronectin, Pselectin, GPIIb/IIIa) and coagulation factors (e.g., factor V, factor XI,
plasminogen activator inhibitor type 1, plasminogen, protein S), all of
which contribute to perpetuate and amplify the thrombotic response.
ADP released from platelet granules may exert both an autocrine effect,
promoting stable platelet aggregation by interacting with specic ADP
receptors in the membrane (P2Y1 and P2Y12; Fig. 2), and a paracrine
effect, by binding to ADP-receptors of neighboring platelets, thus
amplifying the activation process. Activated platelets are also

characterized by the externalization of phosphatidylserine in the

cell membrane, becoming a substrate for coagulation cofactor/
enzyme complexes (VIIa/IXa and Va/Xa), and thereby driving
procoagulant reactions and the eventual thrombin generation [30].
However, platelets are not only stores of several bioactive molecules,
but also generate lipid mediators, such as thromboxane A2 (TXA2).
Platelet activation induces phospholipase-A2 (PLA2) activation that
triggers arachidonic acid metabolism. Platelet cyclooxygenase 1 (COX1) catalyzes the conversion of arachidonic acid to prostaglandin G2/H2,
and the latter is converted to TXA2 (Fig. 2). TXA2 is released into the
bloodstream, where it binds to TX receptors (TP) present on the surface
of adjacent platelets were it activates phospholipase C (PLC),
resulting in the production of diacylglycerol (DAG) and inositol trisphosphate (IP3). DAG and IP3 activate protein kinase C and mobilize
cytoplasmic Ca2 +, respectively (Fig. 2) [31]. Platelets also possess
mitogen activated protein (MAP) kinases [extracellular signal-

G. Vilahur, L. Badimon / Vascular Pharmacology 59 (2013) 6775

regulated kinase 2 (ERK2), p38MAPK and c-Jun NH2-terminal kinase

1 (JNK1)], which are activated by different agonists [32]. Reports indicated that ERK2 plays a role in ADP-induced TXA2 generation [33],
whereas JNK1 and p38 MAPK are involved in collagen-inducedplatelet aggregation (Fig. 2) [34].
Regardless of the trigger, platelet aggregation is regulated in the nal
part of the pathway by activation of the platelet heterodimer GPIIb/IIIa
receptor, the most abundant protein on the platelet surface. The activation of this receptor is calcium-dependent and requires a conformational change in the 2 subunits, such that a new binding domain is
exposed. Fibrinogen (of plasma or platelet origin) is the main ligand
for the GPIIb/IIIa receptor (Fig. 2). The dimeric structure of brinogen allows its interaction with 2 platelets at the same time, thereby favoring
platelet aggregation. Hence, thrombus grows as active GPIIb/IIIa captures
new platelets by means of brinogen (Fig. 2). Other ligands for GPIIb/IIIa
that participate in platelet aggregation (although to a lesser extent)
include vWF, bronectin, and vitronectin (Fig. 2). Fibrinogen binding
to GPIIb/IIIa also triggers an in-side out signaling, causing amplication of the initial signal and further platelet activation. This leads to
further aggregation of platelets and accumulation at the site of vessel
injury; thrombus formation. In the nal phase of thrombus formation,
brinogen is converted to brin by thrombin, leading to the stabilization of the platelet aggregates, with more platelets and blood cells (leukocytes and red blood cells) thus getting trapped and contributing to
thrombus growth.
3. Antiplatelet properties of natural products
Within the following section we will provide an overview of the
antiplatelet properties exerted by natural products and/or foodderived bioactive constituents, and will describe the mechanisms
by which their antiplatelet activity may contribute to reduce the
thrombotic risk (Fig. 2).
3.1. -3 polyunsaturated fatty acids (PUFA)
Ever since a very low incidence of coronary heart disease in
Greenland Eskimos was reported, which was believed to be related to
the high intake of seafood containing -3 PUFAs [35], many large
scale studies have extensively characterized the cardioprotective effects
of sh-derived dietary -3 fatty polyunsaturated fatty acids (PUFA)
eicosapentaenoic acid (EPA; C20:5 n-3) and docosahexaenoic acid
(DHA; C22:6 n-3) [4,36]. Indeed each 20 g per day increase in sh intake has been associated with a 7% lower risk of coronary heart disease
mortality [37]. The exact mechanisms by which sh-derived -3 PUFAs
perform such protective functions are still into debate, but the main
mechanisms proposed include amelioration in the lipid prole (mainly
lowering plasma triglyceride levels), anti-inammatory responses
(decrease NF-B activation and cytokine/chemokine synthesis and
release), plaque stabilization (lower monocyte inltration) and decreased platelet aggregation [4,3841]. The antithrombotic properties of sh-derived -3 PUFAs have been well-characterized and
have been mainly attributed to the incorporation of -3 PUFAs into
the phospholipid membrane, altering the arachidonic acid metabolism
and subsequent reduction in TXA2 release (Fig. 2). As such, -3 PUFA
compete with arachidonic acid (-6 PUFA) as a substrate for COX enzyme promoting the synthesis of platelet biologically inactive eicosanoids (TXA3) rather than -6-derived platelet activator eicocainoids
(TXA2) [41]. Hence, a dominant incorporation of -3 PUFA in the cell
membrane phospholipids ultimately shifts eicosanoid production equilibrium toward an anti-thrombotic prole. Alternatively, -3 PUFA
have also demonstrated to ameliorate vascular function and enhance
NO release, thereby modulating platelet function (Fig. 2) [42]. Linolenic acid (ALA), another -3 PUFA found in vegetable oils, has
also shown to exert antiplatelet effects. Following oral intake, ALA is
partially converted (10%) into EPA and DHA, questioning whether ALA

71

conversion to EPA and DHA accounts for the detected benecial effects
or whether ALA directly exerts its own biological effects [43]. Nevertheless, an elegant recent study by Holy and colleagues has demonstrated
that ALA by itself is capable of impairing in vivo arterial thrombus formation, tissue factor expression, and platelet activation via p38MAPK
blockade (Fig. 2) [44].
3.2. Olive oil
Olive oil is the principal component of Mediterranean diet, both
by its predominance as the main energy source and its abundance
in health-promoting components [8,33]. The protective role of olive
oil against CVD can be seen in multiple benecial effects, including
an improvement in glucose metabolism and lipid prole, blood pressure
control, amelioration in endothelial function, a reduction of LDL
oxidizability, and decreased systemic inammation [33]. In addition,
olive oil has shown to ameliorate the prothrombotic environment by
favorably inuencing coagulation factors (tissue factor, TFPI, FVII,
and FXII), brinolysis (PAI-1) and platelet function [33]. We have recently reported, in asymptomatic subjects with high cardiovascular
risk, that intake (for 3 months) of traditional Mediterranean diet
supplemented with virgin olive oil actively modulates the expression
of key genes involved in vascular inammation, foam cell formation,
and thrombosis towards an anti-atherothrombotic prole [45].
With regard to platelet function, olive oil has been shown to partially
inhibit ADP-, collagen- and arachidonic acid- induced platelet aggregation, resulting in lower TXA2 release (Fig. 2) [46,47,33]. The protective properties of olive oil have been often attributed to its high
content in -9 monounsaturated fatty acids (MUFA; 7085% content), mainly in the form of oleic acid. However, olive oil, unlike
other vegetable oils, also contains high amounts of several micronutrient
constituents (e.g., phenolic compounds, such as hydroxytyrosol and
oleuropein) that have demonstrated important bioactive functions. In
fact, Karantonis and colleagues found, when comparing different sources
of oleic acid (olive oil, soya, maize, sunower and sesame oils), a
higher antiaggregating effect in the olive oil sample, supporting the
capacity of this minor components to exert antiplatelet effects [48].
In effect, olive oil phenolic compounds are thought to be responsible
for the detected reduction in TXA2 release. Finally, although controversial, some reports have been attributed to olive oil about the potential to reduce circulating vWF levels, likely impairing platelet
adhesion and aggregation (Fig. 2). Yet, more studies are needed to
support this hypothesis [49,50].
3.3. Onion and garlic (ajoene)
Plants of the genus Allium such as onion (Allium cepa) and garlic
(Allium sativum) have shown to exert multiple benecial cardiovascular effects, including lowering of blood pressure, decreasing cholesterol and triglyceride levels, and enhancement of brinolysis
and antiplatelet activities [5153]. Both raw garlic and onion have
been reported to inhibit platelet aggregation, however boiled preparations have shown little or no antiplatelet effect [54,55]. So far, several trials have demonstrated a profound effect of garlic to reduce the
ability of platelets to aggregate. Concretely, six trials have assessed
the effectiveness of garlic on platelet aggregation; of these, ve reported positive observations (decrease in platelet aggregation)
[5660] and one reported no effect [61]. No effects were observed
in brinogen levels, and inconclusive data were obtained for the brinolytic activity. Garlic and onion compounds have also shown to
exert potent inhibitory effects over TX production, likely as a result
of direct non-competitive inhibition with the COX enzyme (Fig. 2)
[54]. A study examining the effect of consuming a clove of fresh garlic
on platelet TX production reported a marked 80% reduction in serum
TXA2 levels after 26 weeks [54]. Although onions also decrease TXA2
synthesis [62] their antiplatelet activity is 13 times lower than that of

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G. Vilahur, L. Badimon / Vascular Pharmacology 59 (2013) 6775

garlic. As such, a study in rabbits reported that, while raw garlic extract
reduced serum TXB2 in rabbits, onion extract at an equivalent dose did
not [54].
Several compounds isolated from Allium species have been identied as inhibitors of platelet aggregation. These include allicin, parafnic
sulphide, and adenosine. Most of these compounds have in common a
high sulfur content. In fact, the antiplatelet activity of onions is sulfurdependent (lower sulfur content results in lower antiplatelet activity)
[63]. Allicin, and more specically, ajoene, a self-condensation product
of allicin, is thought to be the primary substance responsible for the inhibitory effect of garlic on platelet aggregation. We have reported that
ajoene inhibits the binding of brinogen and vWF to the GPIIb/IIIa receptor without affecting the binding of vWF to GPIb (Fig. 2) [64]. Ajoene
seems to diminish the number of functional GPIIb/IIIa receptor sites on
the platelet membrane [65] by reducing the viscosity in the inner part of
the lipid bilayer rather than affecting brinogen afnity to GPIIb/IIIa
receptor or GPIIb/IIIa receptor conformation [66]. The inhibitory effect of ajoene on the binding to GPIIb/IIIa supports the ndings that
ajoene inhibits the aggregation of platelets induced by all known agonists [52]. In addition, ajoene has also shown to reduce TXA2 production by altering arachidonic acid metabolism (Fig. 2) [61,66].
A parafnic sulphide component of Allium, dyallyl trisulde, has
also shown to exhibit antiplatelet functions [61]. Studies have indicated that this polysulde inhibits platelet aggregation and Ca2 +
mobilization induced by thrombin in a concentration-dependent
manner, as well as TXA2 synthesis without affecting inositol-1,4,5triphosphate formation [67].

family of mushrooms to exert anti-platelet activities by interfering

with different platelet receptors, as well as diverse signaling pathways [77]. In 2004, a methanol extract of Pleurotus orida Eger (an
edible and commercially grown mushroom) was demonstrated to almost abolish ADP-induced platelet aggregation (95% reduction) after
a 5 min incubation in washed-human platelets [77]. On the other
hand, a dry extract of Stereum hirsutum demonstrated to partly diminish platelet activation by blocking thrombin active site (Fig. 2)
[78] and an extract from Hericium erinaceus (i.e., hericenone B) to selectively impede collagen signaling from GPIIb/Ia [79]. Additionally,
recent studies by Kamruzzaman and colleagues have provided detailed insights as to the mechanisms by which certain medicinal
mushrooms may provide antiplatelet protection. They have reported
the capability of Phellinus baummii (a mushroom under the family of
Hymenochaetaceae basidiomycetes) [80] and P-terphenyl curtisian
e (an antioxidant component of the wild mushroom Paxillus curtissi)
to inhibit collagen-, thrombin- and ADPinduced platelet aggregation, suppress ATP secretion, cytoplasmic Ca2 + mobilization and brinogen binding. Interestingly, the Authors have also provide
evidence that P-terphenyl curtisian E suppresses collagen-induced
platelet GIIb/IIIa activation by interfering with the three known
platelet MAPKs (JNK, ERK2, and p38) and Akt signaling, and by
upregulating cAMP and subsequent PKA activation and VASPphosphorylation (Fig. 2) [81]. These later observations, published in
the current issue of Vascular Pharmacology, support the notion of the
power of mushrooms in interfering with platelet activation via multiple
potential targets.

3.4. Tomatoes
3.6. Polyphenol-rich beverages (wine and beer)
Both fresh and processed tomatoes (Solanum lycopersicum) have
been associated with a decrease in the prevalence of CVD through a
reduction in lipid levels, antioxidant and antiplatelet activities [68].
Tomato has shown antiaggregating activity by inhibiting ADP- and
collagen-, but not to arachidonic acid-induced platelet aggregation
in healthy subjects 3 h after ingestion [69]. The mechanism of action
by which tomatoes inhibits platelet aggregation has yet to be elucidated.
It has been suggested that adenosine and other nucleosides may be responsible for this inhibition by interacting with three platelet receptors GPVI, P2Y1 and P2Y12 (ADP-receptors; Fig. 2) [70]. As such,
bioactive tomato compounds have been suggested to interact with
intraplatelet signaling pathways downstream the activation of
these receptors, likely blocking PLC, cAMP and Akt/VASP phosphorylation. However, it remains to be identied both the tomato components possessing the antiplatelet activity (skin, pulp, seeds, etc) and
their bioactivity. Interestingly, it has been recently reported that
both green and red tomato pulp exert similar antiplatelet activity
triggered by ADP despite green tomato pulp being devoid of lycopene [71], the main natural carotenoid antioxidant present in tomatoes. Other two studies further agreed that lycopene does not seem
to contribute to modulate platelet function [72,73]. However, in contrast to these observations, Hsiao and colleagues [74] demonstrated,
in vitro and in vivo, that lycopene does exert antiplatelet activity
which may involve PLC inhibition followed by inhibition of intracellular Ca+ 2 mobilization. Moreover, lycopene also showed to activate
the formation of NO-derived cyclic GMP, resulting in the inhibition of
platelet aggregation (Fig. 2) [74].
3.5. Mushrooms
Mushrooms are an important natural source of foods and biologically
active compounds. A wide range of activities, including antitumor, antimicrobial, neuroprotective, and cardiovascular benecial (antioxidant, antihypertensive, antidiabetic, and anti-inammatory) activities have been
reported in mushrooms and fungi [75,76]. In addition, in the last
ten years several reports have revealed the capability of certain

A large number of studies have consistently found that moderate

alcohol consumption (1 to 3 drinks/day) is associated with a decreased risk of CVD [8287]. Some of the protective effects appeared
to be linked to ethanol per se; however, several studies have indicated, when comparing different beverage types, that red wine seems to
exert more protective effects than other forms of alcohol, supporting
a protective role for polyphenolic compounds [83,8589]. The benecial effects derived from polyphenols appear to be mediated via a
plethora of biochemical pathways and signaling mechanisms, acting
either independently or synergistically. Polyphenols have shown to
modulate inammation, lipid metabolism, improve antioxidant status and endothelial function, increase NO release, and protect
against platelet aggregation [33,90]. Collectively, there is emerging
evidence that resveratrol (the main polyphenol of wine found in
grape skin and seeds) has antithrombotic effects that appear to be
the result of reduced susceptibility to platelet activation and aggregation, reduced synthesis of prothrombotic mediators (eicosainoid
synthesis) and decrease gene expression of tissue factor [91,92]. Indeed,
resveratrol has shown to inhibit, in a concentration-dependent manner,
platelet aggregation induced by collagen, ADP, and thrombin (Fig. 2)
[93]. It has also demonstrated to attenuate TP-induced platelet activation by reducing PLC activity [94] and inhibit cAMP and cGMP phosphodiesterases [95], thereby increasing platelet levels of cAMP and
cGMP and the subsequent decrease in intracellular Ca2+ levels reducing
platelet activation (Fig. 2). Moreover, resveratrol, by activating eNOS
and/or inhibiting the generation of reactive oxygen species, has demonstrated to modulate the production of endothelial-related NO further
reducing platelet reactivity [96,97]. In addition, we have evidenced,
in an ex vivo perfusion experimental porcine model (the Badimon
perfusion chamber), that moderate daily intake of red wine inhibited
mural thrombosis. Such antithrombotic effects were associated with
inhibition of RhoA translocation to the platelet membrane (active
site) supporting platelet shape change regulation via calciumindependent pathways and a marked reduction in monocyte tissue
factor expression [98].

G. Vilahur, L. Badimon / Vascular Pharmacology 59 (2013) 6775

3.7. Flavonol-rich cocoa

Short-term studies have evidenced a cardioprotective effect of cocoa
and chocolate [99]. Cocoa and dark-chocolate are suggested to have
antioxidant, anti-inammatory, and antihypertensive effects, as
well as to improve vascular function and diminish platelet reactivity
[100,101]. Many benecial effects of chocolate consumption are mediated, at least in part, through naturally occurring phenolic compounds [88]. Indeed, chocolate is a rich source of polyphenolic
compounds (e.g., 41 g of chocolate contain nearly as much phenol
as 140 mL of red wine). Major phenolic compounds include quercetin, epicatechin, procyanindin, and cocoa-red (the color component of
cocoa). Human interventional studies have reported a consistent inhibition of platelet activation and aggregation upon polyphenol-rich cocoa
or dark chocolate consumption in moderate amounts, either on a
acute or a chronic basis [102]. However, the mechanism accounting
for the inhibitory effect of this food on platelet function has not yet
been claried as well as whether their high phenolic content is mainly
responsible for such antiplatelet effects. So far, cocoa and dark chocolate have shown to prevent platelet aggregation by reducing ADP-,
adrenaline- and, epinephrine- induced GPIIb/IIIa membrane activation, ADP-induced P-selectin membrane expression and PLA2 and
COX activity (Fig. 2) [93,103]. Moreover, they have also demonstrated an increase in endothelial-related NO production [104,105].
4. Summary and conclusions
CVD is the leading cause of death in the Western world and it is
foreseen to be the major cause of death worldwide in 2020. The
major reasons for the increase include the aging of the population,
the increase of certain risk factors (obesity/diabetes) especially
among the youth, and the implementation of unhealthy life-styles.
As such, unhealthy diet is considered as a main cause of increased
atherothrombotic disease in industrialized countries. Conversely,
epidemiological evidence has supported the cardinal importance of
a well-balanced diet rich in MUFAs and PUFAs, vegetables, fruits
and polyphenol-rich beverages (i.e., the Mediterranean diet) against
the development and progression of CVD. The biological actions by
which this diet inuences CVD remain to be properly demonstrated.
Yet, multiple data have evidenced that Mediterranean functional
foods display critical anti-atherosclerotic and anti-thrombotic effects.
As such, certain foods and/or their bioactive components have proven
effective in decreasing platelet activation through a variety of mechanisms, suggesting benets in attenuating the future risk of thrombosis.
Nevertheless, most data are based on in vitro experiments and only few
are based on animal studies; for this, mechanistic effects active in
humans are sometimes conjectured or extrapolated. With a few exceptions, the number of studies in humans is limited, so that, although nutritional approaches offer interesting possibilities to assist the ght
against thrombosis, further long-term randomized controlled trials are
indicated or, in the case of natural/whole foods, cohort or good quality
casecontrol studies are much awaited. These will allow to draw denitive conclusions for providing a useful preventive approach or an adjunct to current pharmacological treatments for thrombotic diseases.
Moreover, advances in the knowledge of both platelet biology and of
the biological functions of dietary foods will provide new avenues to develop pharmaceutical and/or dietary strategies aimed at promoting cardiovascular health.
Acknowledgments
This work was supported by SAF 2012-40208 (to GV), SAF 201016549 (to LB) and RD12/0019/0026 - TerCel and RD12/0042/0027Red de Investigacin Cardiovascular from Institute Carlos III (to
LB). We thank the support received by l'Oreal-UNESCO 2012 "For
women in science", Madrid (to GV) and Fundacion de Investigacin

73

Cardiovascular-Fundacin Jesus Serra, Barcelona, GV is a recipient

of a contract from the Innovation and Science Spanish Ministry
(RyC-2009-5495, MICINN, Spain).
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