Review
doi: 10.1111/joim.12296
Thrombosis formation on atherosclerotic lesions and plaque
rupture
L. Badimon1,2 & G. Vilahur1
From the1Cardiovascular Research Center, CSIC-ICCC, Hospital de la Santa Creu i Sant Pau, IIB-Sant Pau, Barcelona, Spain; and
2
Cardiovascular Research Chair, UAB, Barcelona, Spain
Abstract. Badimon L, Vilahur G (Cardiovascular
Research Center, CSIC-ICCC, Hospital de la
Santa Creu i Sant Pau, IIB-Sant Pau, Barcelona,
Spain). Thrombosis formation on atherosclerotic
lesions and plaque rupture (Review). J Intern Med
2014; doi: 10.1111/joim.12296.
Atherosclerosis is a silent chronic vascular pathol-
ogy that is the cause of the majority of cardiovas-
cular ischaemic events. The evolution of vascular
disease involves a combination of endothelial
dysfunction, extensive lipid deposition in the
intima, exacerbated innate and adaptive immune
responses, proliferation of vascular smooth mus-
cle cells and remodelling of the extracellular
matrix, resulting in the formation of an athero-
sclerotic plaque. High-risk plaques have a large
acellular lipid-rich necrotic core with an overlying
thin fibrous cap infiltrated by inflammatory cells
and diffuse calcification. The formation of new
fragile and leaky vessels that invade the expanding
intima contributes to enlarge the necrotic core
increasing the vulnerability of the plaque. In
addition, biomechanical, haemodynamic and
physical factors contribute to plaque destabiliza-
tion. Upon erosion or rupture, these high-risk
Molecular and cellular basis of atherosclerotic plaque formation
Atherosclerosis and its cardiovascular ischaemic
complications are the most common causes of
death and disability worldwide [1]. Indeed, the
World Health Organization (WHO) reported in 2010
that cardiovascular disease represents around
30% of global deaths and estimated that by 2030
more than 23.3 million persons will die annually
from cardiovascular disease [24].
Atherosclerosis is a chronic lipid-driven inflamma-
tory disease of the arterial wall characterized by the
involvement of the innate and adaptive immune
systems [5, 6]. The first step in the development of
atherosclerosis is the exposure of vascular cells to
lipid-rich vulnerable plaques expose vascular
structures or necrotic core components to the
circulation, which causes the activation of tissue
factor and the subsequent formation of a fibrin
monolayer (coagulation cascade) and, concomi-
tantly, the recruitment of circulating platelets and
inflammatory cells. The interaction between
exposed atherosclerotic plaque components, plate-
let receptors and coagulation factors eventually
leads to platelet activation, aggregation and the
subsequent formation of a superimposed throm-
bus (i.e. atherothrombosis) which may compro-
mise the arterial lumen leading to the presentation
of acute ischaemic syndromes. In this review, we
will describe the progression of the atherosclerotic
lesion along with the main morphological charac-
teristics that predispose to plaque rupture, and
discuss the multifaceted mechanisms that drive
platelet activation and subsequent thrombus
formation. Finally, we will consider the current
scientific challenges and future research
directions.
Keywords: atherosclerosis, atherosclerotic plaque,
coagulation, lipids, platelets, thrombosis.
excess lipid (LDLs) with concomitant endothelial
activation/dysfunction and the internalization and
deposition of lipids in the intima (Fig. 1). The
continuous exposure to other pathogenic factors,
such as infectious disease, chronic subclinical
inflammation, hypertension, diabetes, stress and
smoking, contributes to endothelial injury. The
dysfunctional and permeable endothelium allows
LDL particles to further infiltrate and accumulate in
the extracellular matrix (ECM) where they become
targets for oxidative and enzymatic modifications
(Fig. 1). Modified LDLs enhance a series of pro-
inflammatory reactions perpetuating the activa-
tion, recruitment and transmigration of different
innate immune cells (monocytes, mast cells, neu-
trophils, natural killer cells and dendritic cells)
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 L. Badimon & G. Vilahur Review: Thrombosis formation on atherosclerotic lesions and plaque rupture

Cardiovascular disease risk factors Healthy Fig. 1 Schematic diagram of

Elevated LDL
Free radicals (smoking)
Catecholamines (stress)
Arterial hypertension
Diabetes mellitus
Obesity
Hyperhomocysteinaemia
Hyperchoagulable state
Defecve fibrinolyc state
Infecons
Chronic subclinic inflammaon
(periodontal disease)
VSMC migraon:
ECM synthesis
Fibrous cap formaon
Fibrous cap
thinning
endothelium the aetiopathogenesis of ath-
erosclerosis and vulnerable
plaque formation. Exposure to
cardiovascular disease risk
Endothelial dysfuncon factors induces endothelial
dysfunction and increases vas-
Increased permeability
cular permeability allowing
lipid infiltration and promoting
monocyte recruitment, adhe-
sion and transmigration. Once
in the intima, monocytes differ-
Increase vascular lipid Leukocyte adhesion and entiate to macrophages and
uptake transmigraon engulf modified lipids becoming
(mainly monocytes) foamy cells. Concomitantly,
Modificaons within this first stage of the
(enzymac, oxidaon) disease, VSMCs migrate to the
intima where they synthesize
ECM and promote fibrous cap
Foam cell Innate and acquired formation. As the atheroma pro-
formaon immunity acvaon gresses, the number of VSMCs
decreases and foam cells
undergo apoptosis releasing
active metaloproteases that
degrade the fibrous cap,
Atheroma increasing the susceptibility of
Progression Progression plaque to rupture. The immune
system (innate and acquired
immunity) is actively involved
Decreased Foam cells throughout this process and
number of VSMCs apoptosis plays a key role in plaque vul-
nerability. VSMC, vascular
smooth muscle cells; ECM,
extracellular matrix.

Vulnerable plaque

although most important is the contribution of ing on the stage of atherosclerosis development.
circulating monocytes. Yet, acquired immunity, Once differentiated, macrophages exhibit high
mainly dependent on T cells [T helper (Th)1 and levels of surface pattern recognition receptors
Th2] and antibodies, is also critically involved in the which have the ability to take up modified LDLs,
progression of atherosclerosis[7, 8]. In this regard, become lipid-laden and convert into foam cells [6].
the cytokine IL-18 has been shown to orchestrate Lesion complication occurs when foam cells release
the immunological link between the innate and growth factors and cytokines which further stimu-
adaptive immune responses, enhancing athero- late vascular smooth muscle cell (VSMC) migration
sclerosis activity and progression [9]. Once mono- from the media into the intima where they divide
cytes transmigrate and reach the subendothelium and produce ECM components that contribute to
they differentiate, via macrophage colony-stimulat- the development of the fibrous cap [11]. Many of
ing factor, into macrophages. Macrophages are very these lipid-laden macrophages undergo apoptosis
versatile and, depending on the local microenviron- at early stages of atherosclerosis development and
ment, can assume different phenotypes and func- are promptly removed by M2 macrophages in a
tional characteristics, which is a reversible process process referred to as efferocytosis [12]. Neverthe-
termed polarization [10]. Distinct macrophage less, excessive intake of apoptotic cells by macro-
subtypes (M2 and M1) have been detected depend- phages may eventually stress the endoplasmic

2 2014 The Association for the Publication of the Journal of Internal Medicine
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 L. Badimon & G. Vilahur Review: Thrombosis formation on atherosclerotic lesions and plaque rupture
reticulum leading to the deterioration of efferocyto-
sis, subsequent macrophage death and release of
lipids, pro-inflammatory/pro-thrombotic media-
tors [e.g. tissue factor (TF)] and metalloproteinases
(MMPs). MMPs digest the ECM scaffold, including
the overlying fibrous cap, favouring plaque suscep-
tibility to rupture. Plaque vulnerability is also
determined by fewer VSMCs as well as formation
of immature and leaky microvessels within the
necrotic core [13].
Atherosclerotic lesion classification
The first classification of atherosclerosis was made
by the WHO in 1958 and comprised the following
sequence: fatty streak, atheroma, fibrous plaque
and complicated lesion [14]. In the mid-1990s, the
American Heart Association (AHA) recommended a
new morphological classification based on six
lesion types which denote atherosclerosis lesion
progression [15]. Virmani et al. [16] and Van der
Wal et al. [17] further redefined this classification
based on the premise that plaque erosion also
triggers coronary thrombosis. Table 1 provides a
summary of the main characteristics of each type
of atherosclerotic lesion based on the latter mod-
ification of the AHA classification.
Culprit thrombotic plaques
Post-mortem studies have revealed that coronary
luminal thrombus formation mainly arises from
plaque rupture (5565%), followed by plaque ero-
sion (3035%) and least frequently, and controver-
sially, from calcified nodules (27%) [18]. A recent
update of a worldwide survey including 1847 fatal
coronary thrombi concluded that plaque rupture is
the major cause of coronary thrombosis irrespec-
tive of age (>60 years: 77%; <60 years: 64%),
gender (women: 55%; men:76%), region (Europe:
72%; Asia: 81%; USA: 68%) and clinical presenta-
tion (myocardial infarction: 79%; sudden coronary
death: 65%) [19]. Conversely, thrombosis over
plaque erosion is more frequently detected after
coronary sudden death (35%) than after death due
to acute coronary syndrome (25%), and in women
below the age of 50 years [18].
Structural determinants of rupture-prone plaques
Numerous clinical histopathological studies of
ruptured and nonruptured plaques have been the
main source of insight into vulnerable plaques [15].
Nevertheless, accumulating evidence from in vivo
imaging modalities has allowed gaining insights
into the overall atherosclerosis burden, its rate of
progression as well as to the monitorization of
plaque stability through pharmacological and
other types of interventions before the development
of acute coronary syndromes [2022].
Intravascular ultrasound imaging studies in
humans have revealed that outward expansion of
the vessel (also known as positive remodelling) is
common at culprit lesion sites whereas inward
arterial expansion (also known as negative remod-
elling) is more frequently detected in unstable
angina [23, 24]. These observations suggest that
plaque composition rather than the degree of
stenosis mainly determines plaque rupture. In this
regard, several studies collectively demonstrate
that the key features of vulnerable plaques include
the presence of a thin fibrous cap (<65 lm), a
necrotic core that occupies more than 30% of the
total plaque, the occurrence of haemorrhage, con-
siderable infiltration of inflammatory cells (lym-
phocytes and macrophages) and low VSMC density
in the fibrous cap are (Table 1) [16]. In contrast, no
single morphological feature characterizes eroded
plaques and there is no consensus regarding the
degree of inflammation and luminal obstruction at
the eroded site [16, 17, 25]. The only pathological
characteristics are the presence of thrombus lining
over the intima (rich in VSMCs and ECM) and the
absence of the endothelial monolayer (Table 1).
Enlarged necrotic core
The vulnerable necrotic core is characterized by a
lack of supporting collagen and cells, as well as the
accumulation of free cholesterol in the centre and
the presence of a low free to esterified cholesterol
ratio at the edges, probably due to macrophage
death and ongoing inflammation [26]. The pres-
ence of haemorrhage has been shown to enlarge
the necrotic core (see below). Lipid core distribu-
tion seems to be critical for plaque instability. For
instance, eccentric distribution of the lipid core
leads to a rearrangement of circumferential stress
to the plaque shoulder regions and thus an
increase in the vulnerability of these sites to
rupture. This may partly help to explain why
almost 60% of fibrous cap fissures occur in this
region [27, 28]. The lack of collagen, reflecting the
loss of VSMCs, is also a critical feature of plaque
vulnerability. We have demonstrated that athero-
genic concentrations of LDL significantly reduce
the migratory potential of human VSMCs, reducing
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 L. Badimon & G. Vilahur Review: Thrombosis formation on atherosclerotic lesions and plaque rupture

Table 1 Modified American Heart Association (AHA) consensus classification based on morphological descriptions.

Description Characterization Thrombosis Reversible

Intimal thickening Layers of smooth muscle cells and extracellular matrix Absent Reversible
processes
Intimal xanthoma or An abundance of macrophage foam cells within the intima Absent
fatty streak rich in smooth muscle cells and proteoglycans
No necrotic core or fibrous cap
Pathological intimal Layers of smooth muscle cells in a proteoglycan-collagen Absent Irreversible
thickening matrix with an underlying lipid pool consisting of an processes
acellular area, rich in hyaluronan and proteoglycans
(mainly versican) with lipid insudation
Variable accumulation of macrophages outside the lipid
pool
Presence of microcalcification
Absence of necrosis
Fibroatheromas Well-formed acellular necrotic core containing free Absent
cholesterol and covered by a thick fibrous cap consisting
of smooth muscle cells in a proteoglycan-collagen matrix
(type I and type III)
Thin fibrous cap Intact thin (less or =65 lm) fibrous cap overlying the Absent
atheroma or necrotic core mostly made of collagen type I and
vulnerable plaque infiltrated with inflammatory cells (macrophages and
lymphocytes) with rare SMC
Plaque rupture Fibroatheroma with cap disruption Occlusive or non-
occlusive
Luminal thrombus communicates with the underlying
necrotic core
Plaque erosion Plaque same as above Thrombus mostly
Luminal thrombosis but no communication of thrombus mural and non-
with necrotic core occlusive
Calcified nodule Eruptive (shedding of) calcified nodules with an Usually non-
underlying fibrocalcific plaque with minimal or no occlusive
necrosis
Fibrocalcified plaque Collagen-rich plaque with significant stenosis usually Absent
contains large areas of calcification with few
inflammatory cells
A necrotic core may be present

their number and contributing to increase the
vulnerability of these advanced-staged plaques
[29, 30]. On the other hand, it has been shown
that apoptosis of VSMCs progresses as plaque
evolves, contributing to plaque destabilization. In
fact, a higher rate of VSMC apoptosis has been
observed in symptomatic plaques of unstable
angina patients compared to those of patients with
stable angina [31]. Moreover, apoptotic macro-
phages have been mainly detected at the sites of
plaque rupture [32]. In this regard, in vitro studies
have demonstrated the capability of macrophages
to directly stimulate apoptosis in VSMCs [33, 34].
Of note, both apoptotic VSMCs and apoptotic lipid-
laden foam cells present high TF activity, under-
lining the key role of both these cell types in

4 2014 The Association for the Publication of the Journal of Internal Medicine
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 L. Badimon & G. Vilahur Review: Thrombosis formation on atherosclerotic lesions and plaque rupture
promoting thrombus formation upon plaque rup-
ture [35]. We have demonstrated that the athero-
matous TF-rich core is the most thrombogenic
component of human atherosclerotic plaques (six-
fold greater than collagen) [36]. Interestingly,
recent data show that TF signalling is also involved
in VSMC migration, thus contributing to plaque
remodelling [37, 38].
Thin fibrous cap
The fibrous cap is located between the vascular
lumen and the necrotic core. Autopsy studies have
determined that ruptured plaques are extremely
thin (<65 lm thick), have a macrophage density of
around 26% and have a low collagen content [19].
Apoptosis of macrophages in the thin fibrous cap
derive in the continuous release of MMPs which in
turn degrade collagen in the fibrous cap [39, 40]. In
addition, loss of VSMCs (the main source of colla-
gen synthesis) also affects the thinning of the
fibrous cap, thus contributing to plaque vulnera-
bility to rupture [41].
Neovascularization
There is substantial evidence confirming a close
relationship between intralesion angiogenesis and
culprit atherosclerotic plaques [42]. Moreover, in-
traplaque neovascularization has been associated
with the occurrence of prior, and shown to predict
future cardiovascular events [43, 44]. It has been
suggested that the oxygen supply is restricted once
the wall thickness of the vessel exceeds 100 lm,
triggering hypoxia-inducible transcription factor-
1a accumulation and the subsequent expression of
vascular endothelial growth factor (VEGF) and
other angiogenic modulators. Together these
angiogenic factors promote vessel formation from
the adventitial vasa vasorum towards the intima in
order to provide oxygen and nutrients, thus allow-
ing plaque growth. Yet, in more advanced plaques,
inflammatory cell infiltration and the simultaneous
release of several pro-angiogenic cytokines result
in the induction of uncontrolled neointimal micro-
vessel formation without supporting VSMCs. These
new microvessels are weak and permeable, and
express cellular adhesion molecules favouring
local extravasation of red blood cells, plasma
proteins and inflammatory cells which conse-
quently may lead to recurrent intraplaque haem-
orrhage [13, 4548]. Moreover, released lipid-rich
erythrocyte cell membranes and free haemoglobin
enhance the inflammatory response and oxidative
stress, further expanding the lipid core and,
thereby, increasing plaque vulnerability. In fact, it
has been shown that the degree of reactivity of
glycophoryn A (a membrane sialoglycoprotein of
red blood cells) and iron deposits in the plaque
correlates with the increasing size of the necrotic
core and macrophage density, indicating that
haemorrhage per se elicits an inflammatory
response [49]. Microvessel density is much higher
in rupture-prone and ruptured plaques than in
stable plaques (2- to 4-fold increase) [23]. In
human coronary atherosclerotic lesions from
excised hearts at transplantation, we have demon-
strated that the most advanced-stage plaques,
which contain the highest neovessel content, are
associated with the highest rate of thrombotic
events [50]. Furthermore, using laser dissection
microscopy approaches, we found that new angio-
genic factors appear to contribute to plaque vas-
cularization/vulnerability [51, 52] and observed
modulation of angiogenesis through signalling of
the TF cytoplasmic domain [5355].
Calcification
Culprit plaques that account for acute coronary
events were found to be less calcified than those
associated with stable angina, indicating that the
presence of calcium provides stability [5658]. Of
note, Virmani et al. reported that calcified nodules
(calcifications with a protruding, irregular and
convex luminal surface), although rare (27%),
are a cause of thrombotic events. The findings of
the recent PROSPECT trial, [59] however, have
demonstrated that the incidence of such calcified
nodules is linked to greater plaque burden, an
increased number of thick-cap fibroatheromas and
a lower rate of events. Intravascular ultrasound
imaging studies have provided evidence that the
coronary calcification pattern determines plaque
vulnerability. As such, spotty calcifications
(defined by lesions 14 mm in length with an arc
of calcium < 90), in contrast to extensive calcified
atherosclerotic lesions, are associated with ischae-
mic events [18]. Finally, recent work has also
suggested that the presence of microcalcifications
in the fibrous cap may increase the risk of rupture
[60].
Nonplaque determinants of rupture
Local dynamic forces, including circumferential,
flexion and shear stresses, may participate in
vulnerable plaque disruption [61]. On the one
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 L. Badimon & G. Vilahur
hand, atherosclerosis usually develops at bifurca-
tions or arterial curvatures where blood flow is slow
and nonlinear [62]. Low shear stress has recently
been related to the progression of vulnerable
plaques [63]. Moreover, it has also been shown
that low shear stress upregulates inflammatory
signalling in endothelial cells and leucocytes, con-
tributing to vulnerable plaque development [62].
On the other hand, according to Laplaces law,
circumferential stress is directly related to the
luminal diameter and intraluminal pressure of a
vessel and inversely related to wall thickness [64]. It
is also generally considered that fibrous cap rupture
takes place near the shoulder region where there is a
high density of inflammatory cells. However, in an
interesting study it was revealed that rupture of the
plaque occurred in the mid-portion in those patients
who were performing intense physical exercise dur-
ing sudden coronary death whereas ruptured
occurred in the shoulder region in those who died
at rest [65]. These observations support the contri-
bution to plaque rupture of biomechanical forces
rather than an inflammatory component. It has been
shown that physical, environmental or emotional
stressors, although insufficient to cause coronary
instability per se, activate the sympathetic nervous
system and induce the release of catecholamines
leading to local vasoconstriction in addition to a rise
in cardiac rate and blood pressure. In fact, coronary
vasospasm is believed to contribute to plaque ero-
sion. Together, these events may precipitate plaque
rupture and consequent acute coronary syndrome
[66]. In addition, activation of the sympathetic
nervous system also affects haemostasis and hae-
morheology (higher platelet reactivity, coagulability
and microvascular constriction), further contribut-
ing to plaque rupture and consequently to the acute
clinical ischaemic event [67].
Atherothrombosis: a complication of the atherosclerotic plaque
Healthy endothelium: a critical feature of thromboresistance
The endothelial layer is a semi-permeable barrier
that controls the diffusion of plasma molecules,
regulates vascular tone and inflammation, and
prevents thrombus formation [68, 69]. The integrity
of the endothelial barrier is determined by the
presence of intercellular junction complexes (i.e.
occludin, claudin, junctional adhesion molecules
[70], cadherin and gap junctions) and integrin
receptors (Fig. 2). Tight and adherent junctions
maintain intercellular binding and regulate endo-
thelial cell growth and survival whereas gap junc-
tions are mainly involved in intercellular binding
6 2014 The Association for the Publication of the Journal of Internal Medicine
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Review: Thrombosis formation on atherosclerotic lesions and plaque rupture
and mediate the passage of water, ions and other
small molecules. Integrins act as receptors for
vitronectin and fibronectin and, therefore, are
responsible for adhesion of the endothelial mono-
layer to the extracellular matrix (Fig. 2) [69]. The
healthy endothelium, devoid of atherosclerotic
lesions, is also a highly thromboresistant surface
that limits thrombus formation and the occurrence
of ischaemic events. Indeed, the endothelial layer
expresses a vast array of molecules with antiplat-
elet, anticoagulant and fibrinolytic properties
(Fig. 2).
Platelets: the key triggers of haemostatic plug and thrombus
formation on damaged endothelium
Platelets are anucleated cells (2 lm in diameter)
that are devoid of genomic DNA but contain mes-
senger RNA and the capability for synthesizing
proteins. They are released into the circulation as
cytoplasmic fragments of bone marrow-derived
megakaryocytes and circulate in the blood stream
for 710 days without interacting with other blood
elements and/or the vascular wall [71]. Upon
endothelial disruption or rupture of an atheroscle-
rotic plaque, a sequence of events leads to the
development of a platelet-rich thrombus [72].
Platelets avidly adhere to dysfunctional, damaged
or disrupted endothelium.
Haemostatic plug formation, which usually occurs
at very high blood shear rates, and thrombosis on
endothelial injured vessels lead to rapid platelet
recruitment. The initial tethering is mainly mediated
by the interaction between platelet glycoprotein (GP)
Iba receptor (the main binding region of the platelet
GPIbIXV complex) and the A1 domain of von
Willebrand factor (vWF). vWF is already anchored
to collagen through its A3 domain. GPIba also
contains binding sites for Leukocyte integrin mac-
rophage antigen and P-selectin which favours fur-
ther recruitment of GPIbavWF interaction,
however, triggers a weak intracellular signal and
maintains platelets in contact with the endothelial
layer but does not mediate irreversible adhesion
[34]. Stable and strong platelet binding is brought
about by two well-established platelet collagen
receptors [GPIa/IIa (integrin a2b1) and GPVI (immu-
noglobulin)] which also induce platelet activation/
aggregation. GPVI signals through tyrosine kinases
and activates phospholipase (PL) C which mobilizes
calcium, activates the GPIIb/IIIa (integrin aIIbb3)
receptor and induces granule secretion, further
promoting platelet activation and aggregation [34,
 L. Badimon & G. Vilahur Review: Thrombosis formation on atherosclerotic lesions and plaque rupture

NON-PERMEABLE

Adherens
Cadherin Tight Occludin Claudin
juncons
juncons
JAM
Negavely CD39 CD73 Heparan sulphate Thrombomoduline
charged (EctoADPase) V -VIII

AMP adenosin X Protein S Protein C

Anthrombin III
ADP
Pla

Va -VIIIa
te
let

ANTIPLATELET
epr

Thrombin Xa ANTICOAGULANT
u lsi
on

FIBRINOLYTIC

Protacyclin Platelet NO Plasmin Fibrin

acvaon
Tissue plasminogen Endothelial cell
AA acvator
eNOS
Cox2
L-Arginine GAP
L-citruline juncons
Integrins
CD31/PECAM Vitronecn Fibronecn

Subendothelial matrix

Fig. 2 Natural thromboresistant properties of the healthy endothelium and integral components to ensure inter- and
intracellular connections and prevent endothelial permeability. Under physiological conditions, the endothelium releases
several molecules and/or expresses different transmembrane proteins which exhibit antiplatelet (blue), anticoagulant
(green) and fibrinolytic (purple) properties. The antiplatelet properties of the endothelium are due to: the presence of a
negatively charged surface that repels the negatively charged platelets; the release of prostacyclin and NO which inhibit
platelet function; and the surface expression of the ectoenzymes CD39 and CD73 that promote the degradation of
nucleotides (e.g. ADP) thus preventing platelet activation. The healthy endothelium also prevents the activation of the
coagulation cascade by expressing thrombomodulin (activates protein C) and heparan sulphate (induces antithrombin III),
which reduce the production of thrombin (the main fibrin inducer and a potent platelet activator). Finally, the endothelium
also promotes fibrinolysis through tissue plasminogen activator and consequent plasmin formation. However, the integrity
of the (nonpermeable) endothelial barrier is determined by the presence of intercellular junction complexes (i.e. occludin,
caludin, junctional adhesion molecules, cadherin and gap junctions) and integrin receptors (vitronectin and fibronectin).
Nitric oxide, NO; adenosine diphosphate, ADP; adenosine monophosphate, AMP; arachidonic acid, AA; cyclooxygenase 2,
Cox2; endothelial nitric oxide synthase, eNOS; platelet endothelial cell adhesion molecule, PECAM; junctional adhesion
molecule, JAM.

73]. GPIa/IIa supports platelet adhesion by inter-
acting with several collagen-related binding sites.
Laminin, fibronectin, thrombospondin and vitro-
nectin also contribute to platelet adhesion by bind-
ing to GPIc-IIa, GPIc-IIa, GPIV and vitronectin
receptors, respectively, although to a lesser extent.
TF plays a key role in atherothrombosis
In atherosclerotic plaques, the initial triggering is
the exposed TF that induces thrombin and the
subsequent formation of a fibrin monolayer cover-
ing the surface of the exposed vascular damage
(Fig. 3). Later thrombosis evolves with a predomi-
nance of platelets that are rapidly activated and
recruited to the growing thrombus. Exposure of the
extracellular domain of TF to flowing blood initiates
the coagulation cascade [74]. TF, mainly expressed
in foam cells and lipid-laden VSMCs, interacts with
plasma factor (F) VII/VIIa. The TF:FVIIa complex
activates both FIX and FX, and in turn, activated
FX (FXa), and its cofactor FVa, form the prothom-
binase complex which transforms prothrombin to
thrombin. Thrombin then cleaves fibrinogen to

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 L. Badimon & G. Vilahur Review: Thrombosis formation on atherosclerotic lesions and plaque rupture

fibrin and also activates transglutaminase factor

XIII thus promoting fibrin cross-linking and further
stabilization of the platelet-rich thrombus [34]. At
this stage, a nonocclusive coronary thrombus may
cause angina, or a fragment of the thrombus may
break away and be carried in the bloodstream until
it obstructs smaller vessels leading to micro-
infarctions (distal embolization).
Of interest, it has been shown that C-reactive
protein (CRP) exerts thrombotic activity [75]. In this
regard, we have previously shown that the mono-
meric form of CRP plays a role in platelet adhesion
[76]. As such, native or circulating CRP (pentamer-
ic form) does not affect platelet deposition, whereas
monomeric CRP displays a prothrombotic pheno-
type initiating platelet deposition and thrombus
growth [76]. Monomeric CRP dissociation from
pentameric CRP occurs on the surface of activated
platelets [77] in a process supported by GPIIb/IIIa
activation [78]. Moreover, microparticles [(MPs)
submicron membrane vesicles released by different
cell types upon activation or apoptosis], in addition
to acting as CRP carriers, have recently been
shown to convert native CRP to monomeric CRP
[79, 80].
Thrombus progression and growth
Platelet recruitment is initiated by several locally
accumulating mediators that are produced or
released once platelet adhesion/activation has
occurred. Amongst these mediators, thromboxane
(TX)A2 and ADP in combination with thrombin
(generated upon atherosclerotic plaque exposure to
TF) are most important (Fig. 4). TXA2 is generated
through the stimulation of PLA2 (Fig. 4). PLA2
stimulates arachidonic acid release from mem-
brane phospholipids; arachidonic acid is then
rapidly transformed by cyclooxygenase-1, first to
the endoperoxides prostaglandin G2 and H2, and
then to TXA2 by TX synthase. Released TXA2
interacts with the TX receptor (G-protein-coupled
receptor) present on the platelet surface or on other
vascular cells, thereby acting as a positive ampli-
fier in the activation and recruitment of more
platelets. TXA2 is released into the bloodstream,
where it binds to TX receptors [81] found on the
surface of neighbouring platelets and activates
PLC, and the ensuing production of diacylglycerol
(DAG) and inositol trisphosphate (IP3) (Fig. 4).
DAG and IP3 activate protein kinase C and mobi-
lize cytoplasmic Ca2+ respectively (Fig. 4) [82]. In

(a) (b)

L Platelets
Platelets L
Fibrin

V
V

Platelets Platelets
(c) L (d)

V
Fibrin V

Fig. 3 The role of tissue factor on thrombus formation. Exposure of tissue factor upon plaque disruption is considered to be
the initial trigger for thrombus formation. Immunofluorescent (a and b; red denotes platelets and green denotes fibrin) and
electron microscopy (c and d) images of thrombus formed on eroded (a and c) and severely injured (b and d) vessel wall
perfused at high shear rate (1690/s; 9200) show a layer of fibrin covering severely injured vessels (exposed vascular
media) beneath the evolving thrombus (authors unpublished data). Vessel, V; lumen, L. Black arrows denote platelets;
white arrows denote fibrin.

8 2014 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine
 L. Badimon & G. Vilahur Review: Thrombosis formation on atherosclerotic lesions and plaque rupture
contrast, ADP, which is mostly released from
intraplatelet dense granules but also from lysed
red blood cells, enhances platelet aggregation by
interacting with G-coupled-protein purinorecep-
tors (P2Y1 and P2Y12 receptors) thereby initiating
two signalling pathways: PLC-mediated increase in
cytosolic Ca2+concentration and inhibition of cAMP
production (Fig. 4). cAMP mediates protein kinase
A (PKA) phosphorylation of the vasodilator-stimu-
lated phosphoprotein which is an actin regulatory
protein and a negative modulator of GPIIb/IIIa. The
key role of ADP as a positive-feedback mediator is
highlighted by the evidence that platelet responses
to TXA2 and thrombin are diminished in the
absence of ADP receptors. Finally, not only is
thrombin the central protease in the coagulation
cascade but it is also one of the most potent platelet
activators [83] (Fig. 4). Thrombin cleaves the N-
terminus of the receptor [protease activated recep-
tor (PAR)-1 or PAR-4 in platelets] which in turn acts
as a tethered ligand activating the receptor. PAR1
and PAR4 activation initiates several G protein-
coupled signalling pathways (Gq, G12/13, Gi and G2)
which stimulate platelet shape change, the release
of the dense granule contents, generation of TXA2,
GPIIb/IIIa activation and the procoagulant
response (prothrombinase activity and thrombin
generation) (Fig. 4). Together, activation of these
three G-protein-coupled receptors (ADP-, thromb-
oxaneA2 and thrombin- receptor) enhances plate-
let shape change and subsequent release of the
granule contents, activating GPIIb/IIIa and cap-
turing more platelets to the developing thrombus
(Fig. 4). Indeed, platelet granule secretion leads to
the local release of ATP, serotonin, Ca2+, adhesion
proteins (e.g. fibrinogen, fibronectin, vWF, throm-
bospondin, vitronectin, P-selectin and integrin
aIIbb3) and coagulation factors (e.g. FV, FXI, plas-
minogen activator inhibitor type 1, plasminogen
and protein S), all of which contribute to amplify
the thrombotic response. Furthermore, activated
platelets externalize phosphatidylserine becoming
a substrate for coagulation cofactor/enzyme com-
plexes VIIa/IXa and Va/Xa, and thereby driving
procoagulant reactions [84]. Finally, platelet acti-
vation culminates in the conformational change
and consequent activation of the most abundant
platelet receptor, GPIIb/IIIa Upon activation, the
GPIIb/IIIa receptor exposes the binding sites for
the Arg-Gly-Asp-Ser (RGDS) tetrapeptide recogni-
tion sequence for fibrinogen and vWF allowing
cross-linking with neighbouring activated platelets
(platelet aggregation) (Fig. 4). Although to a lesser
extent, fibronectin and vitronectin are also ligands
for GPIIb/IIIa and therefore contribute to platelet
aggregation. Together these processes attract new
platelets and other circulating cells, including red
blood cells and leucocytes, to the site of injury
which, in combination with thrombin-mediated
fibrinogen conversion to fibrin, promotes thrombus
stabilization and enlargement [8587]. Acute
occlusive coronary thrombus growth is most fre-
quently the cause of acute coronary syndromes
and in some cases even of sudden coronary death.
Coagulation
Activation of the coagulation cascade occurs
promptly upon vascular injury. Proteins of the
coagulation cascade generally circulate in plasma
as inactive zymogens that are converted to active
coagulation factors on the surface of activated
platelets. As described above, a fibrin monolayer is
the first circulation-related response to atheroscle-
rotic plaque damage, suggesting a rapid onset of
TF-dependent thrombin formation close to the
damaged vessel wall (Fig. 3) [8891]. In this
regard, we have shown that blockade of TF by
local administration of tissue factor pathway
inhibitor (TFPI) effectively decreases arterial
thrombosis in atherosclerotic lesions [92]. Forma-
tion of the fibrin monolayer is immediately followed
by platelet deposition that may even occlude the
lumen of the vessel [36, 93]. In addition to the
expression of TF in the vessel wall, there is robust
evidence of the existence of blood-borne circulating
TF. Such circulating TF is found on monocytes and
in small microparticles (MPs) [80, 94]. Leucocytes
have been shown to transfer TF-enriched MPs to
platelets mediated by the interaction between
CD15 and platelet P-selectin. These mechanisms
may contribute to producing a higher TF concen-
tration at the site of thrombus formation. More-
over, we have demonstrated that VSMCs are also
able to release TF-enriched MPs to the ECM via an
LDL receptor-related protein-1-modified LDL-
dependent mechanism [95, 96]. The relative con-
tribution of vascular wall-derived TF versus circu-
lating TF in thrombus formation remains to be
clarified although levels of TF in the vessel wall
exceed the amount in blood [97].
New technologies are enabling better characteriza-
tion of the thrombus formed in vivo on atheroscle-
rotic plaques. Recent data obtained by thrombo-
aspiration of coronary occlusive thrombi that had
produced full coronary occlusion (ST-segment ele-
vation myocardial infarction) indicate that the
2014 The Association for the Publication of the Journal of Internal Medicine 9
Journal of Internal Medicine
 L. Badimon & G. Vilahur Review: Thrombosis formation on atherosclerotic lesions and plaque rupture

Platelet aggregaon

Fibrinogen binding Thr

omb
in
P
AD GPIIb/IIIa
P2Y1

PAR-1 Thrombin proteolyc

GPllb/llla cleavage of PAR receptor
acvaon PAR-4
q DAG
G
PKC
G-p PLC
ro PKC acvaon
ac tein m
va e
on o diated PLChydrolyzes PIP2 to
f PL PLC PIP2 PLC yield IP3 and DAG
C
GPllb/llla
acvaon
VASP IP3 G-protein mediated
Reducon PKA acvaon of PLC
PKA
acvaon PGH2
VASP-P

G
TX
PGG2

q/
sin

TXA2
Ca2+

ta
s

12
a
AC AMPc Ca2+ Ca2+

-13
Adenylyl cyclase (AC) Ca2+

Co
AMPc decrease TP

x-1
inhibion
Calcium cytosol TXA2
Gi
increase
P2Y12
AA
TXA2 generaon
PLA2
GPllb/llla
acvaon PLA2 acvaon
ADP

Fig. 4 The role of the main G-protein-coupled receptors in platelet activation. Signalling pathways involved in platelet
activation upon stimulation of the three major G-protein-coupled receptors by ADP (violet), thromboxane A2 (blue) and
thrombin (red) are shown. Platelet activation leads, in turn, to the conformational change of the platelet receptor GPIIb/IIIa
(i.e. activation) promoting plateletplatelet interaction (platelet aggregation). PLC, phospholipase C; PIP2, phosphatidylino-
sitol bisphosphate; IP3, inositol 1,4,5-trisphosphate; PLA2, phospholipase A2; TP,thromboxane receptor; TX, thromboxane;
DAG, 1,2-diacylglycerol; Cox, cyclooxygenase; AC, adenylate cyclase; AMPc, cyclic adenosine monophosphate; PKA, protein
kinase A; VASP, platelet vasodilator stimulated phosphoprotein; AA, arachidonic acid; PGG2 and PGH2, prostaglandin
endoperoxides.

recruitment of platelets is an early event as and urokinase [100]. Plasmin has great affinity for
thrombi of more than 6 h of evolution show a fibrin and, when incorporated into the clot,
reduction in the number of platelets and an degrades fibrin and thereby promotes proteolysis
increase in the level of coagulation products [98, of the thrombotic substrate. However, in stable
99]. Of interest, older intracoronary thrombi are thrombi, cross-linking of fibrin masks tPA-binding
able to recruit circulating inflammatory cells [98]. sites, thus protecting fibrin from degradation.
Moreover, tPA and urokinase are inhibited by
plasminogen activator inhibitor-1 and activated
Spontaneous fibrinolysis
by phospholipid membrane components released
Fibrinolysis is activated simultaneously with fibrin at sites of vascular injury; in addition, increased
formation. Plasminogen is released from the liver to levels of circulating a2-antiplasmin block plasmin
the circulation and converted to plasmin (active leading to enhanced thrombus resistance to prote-
form) by tissue plasminogen activator (tPA) (Fig. 2) olysis. Thrombin activatable fibrinolysis inhibitor

10 2014 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine
 L. Badimon & G. Vilahur Review: Thrombosis formation on atherosclerotic lesions and plaque rupture
(TAFI) also contributes to the overall reduction in
fibrinolysis. Thrombin may activate TAFI but the
rate of activation is increased by the thrombin
thrombomodulin complex. Once activated, TAFI
downregulates fibrinolysis by the removal of C-
terminal lysines from fibrin. As a consequence the
binding of plasminogen and t-PA to the fibrin clot is
inhibited [101].
Regulation of thrombus growth by local and systemic factors
Cardiovascular disease risk factors (such as dy-
slipidaemia, diabetes and hypertension) have been
shown to affect blood thrombogenic potential by
increasing coagulability, lowering the fibrinolytic
potential and enhancing platelet reactivity [102].
Conversely, interventions to modify cardiovascular
disease risk factors have been shown to lower the
prothrombotic risk [103]. There has been increas-
ing interest in the contribution of the cross-talk
between MPs and cells involved in atherosclerosis
progression and thrombus formation. Although MP
formation is a physiological process, it has been
demonstrated in several studies that the levels of
circulating MPs are raised in a wide range of
cardiovascular diseases, including atherosclerotic
lesion progression, thrombosis, heart failure, ar-
rhythmias and inflammation-related vascular dis-
ease, as well as in conditions in which
cardiovascular disease risk factors are not con-
trolled [104]. MPs have also been also detected in
atherosclerotic lesions, mainly originating from
macrophages, red blood cells and VSMCs, whereas
circulating MPs mainly originate from platelets. Of
note, we have recently demonstrated that statin
treatment reduces the number of circulating and
platelet-derived MPs carrying markers of activated
cells, suggesting a new mechanism of statin-
dependent protection against vascular disease
[105]. In addition, we have recently reported that
both circulating and platelet-derived MPs enhance
thrombosis on atherosclerotic plaques [106]. In
fact, it has been shown that MPs are captured by
thrombus-associated platelets through the inter-
action between MP-exposed P-selectin glycoprotein
ligand 1 and P-selectin from platelets [107].
Conclusions and future scientific challenges
Atherosclerotic lesions with the potential to rup-
ture, i.e. vulnerable plaques, are the primary cause
of luminal thrombosis and consequent clinical
episodes. Excess lipids and inflammatory reactions
(cellular and humoral) are considered the major
contributors to plaque development, and the loss of
VSMCs and increased intraplaque haemorrhage
are critical steps in necrotic core destabilization
and enlargement. In fact, reduction of apoptosis
execution has been detected in therapies
addressed towards atherosclerosis stabilization
[108] highlighting the need to assess new therapies
directed towards apoptosis modulation. Similarly,
selectively targeting new vessel formation appears
to be a promising strategy to promote plaque
stabilization. During recent years, although incon-
sistent and conflicting results have been reported,
the advent of state-of-the-art technologies has
created considerable enthusiasm for the use of
stem cell-based therapies in combination with gene
therapies (i.e. stem cell preconditioning) to prevent
and/or cause the regression of atherosclerotic
lesions [109]. In line with these approaches, the
concomitant use of molecular and conventional
imaging modalities may enable the detection of
plaque composition, activity and progression,
allowing the success of different new therapeutic
strategies for plaque stabilization to be monitored.
Conversely, taking into consideration that super-
imposition of a thrombus at the site of plaque
rupture with the consequent luminal obstruction is
the main cause of cardiovascular ischaemic events,
an in-depth understanding of platelet biology using
omic approaches as well a better knowledge of the
molecular/cellular interactions that govern plate-
let aggregation and clot formation may guide the
proper use of existing treatments and the develop-
ment of new anti-thrombotic therapies, eventually
improving patient outcomes and helping to reduce
the economic burden of cardiovascular disease.
Finally, new paradigms are emerging that
strengthen the critical role of inflammation in the
pathogenesis of atherothrombosis. As such, better
characterization of the immune-modulating role of
platelets as well as of microRNAs (endogenous
small noncoding RNAs with the ability to regulate
gene expression at transcriptional and post-
transcriptional levels) may give rise to novel
experimental strategies for the prevention and
treatment of atherosclerosis-related diseases.
Conflict of interest statement
No conflicts of interest to declare.
Acknowledgements
The work in the authors laboratory discussed in
this review was supported by PNS 2013-42962-R
2014 The Association for the Publication of the Journal of Internal Medicine 11
Journal of Internal Medicine
 L. Badimon & G. Vilahur Review: Thrombosis formation on atherosclerotic lesions and plaque rupture
(to LB), PNS 2012-40208 (to GV), from the Spanish
Ministry of Science and Innovation; Spanish Car-
diovascular Network (Red Cardiovascular) and the
Spanish Cell Therapy Network (Red de Terapia
Celular - TERCEL) from Instituto Salud Carlos III (to
LB) and a grant from lOreal-UNESCO (for Women
in Science to GV). We thank Fundacion Jesus Serra
for their continuous support. GV is a Ramon y
Cajal scientist funded by the Spanish Ministry of
Science and Innovation (MICINN).
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Correspondence: Lina Badimon, Cardiovascular Research Center,
c/Sant Antoni MClaret 167, 08025 Barcelona, Spain.
(fax: (34)-935565559; e-mail: lbadimon@csic-iccc.org).
2014 The Association for the Publication of the Journal of Internal Medicine 15
Journal of Internal Medicine