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doi: 10.1111/joim.12296

Thrombosis formation on atherosclerotic lesions and plaque

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
Cardiovascular Research Chair, UAB, Barcelona, Spain

Abstract. Badimon L, Vilahur G (Cardiovascular lipid-rich vulnerable plaques expose vascular

Research Center, CSIC-ICCC, Hospital de la structures or necrotic core components to the
Santa Creu i Sant Pau, IIB-Sant Pau, Barcelona, circulation, which causes the activation of tissue
Spain). Thrombosis formation on atherosclerotic factor and the subsequent formation of a fibrin
lesions and plaque rupture (Review). J Intern Med monolayer (coagulation cascade) and, concomi-
2014; doi: 10.1111/joim.12296. tantly, the recruitment of circulating platelets and
inflammatory cells. The interaction between
Atherosclerosis is a silent chronic vascular pathol- exposed atherosclerotic plaque components, plate-
ogy that is the cause of the majority of cardiovas- let receptors and coagulation factors eventually
cular ischaemic events. The evolution of vascular leads to platelet activation, aggregation and the
disease involves a combination of endothelial subsequent formation of a superimposed throm-
dysfunction, extensive lipid deposition in the bus (i.e. atherothrombosis) which may compro-
intima, exacerbated innate and adaptive immune mise the arterial lumen leading to the presentation
responses, proliferation of vascular smooth mus- of acute ischaemic syndromes. In this review, we
cle cells and remodelling of the extracellular will describe the progression of the atherosclerotic
matrix, resulting in the formation of an athero- lesion along with the main morphological charac-
sclerotic plaque. High-risk plaques have a large teristics that predispose to plaque rupture, and
acellular lipid-rich necrotic core with an overlying discuss the multifaceted mechanisms that drive
thin fibrous cap infiltrated by inflammatory cells platelet activation and subsequent thrombus
and diffuse calcification. The formation of new formation. Finally, we will consider the current
fragile and leaky vessels that invade the expanding scientific challenges and future research
intima contributes to enlarge the necrotic core directions.
increasing the vulnerability of the plaque. In
addition, biomechanical, haemodynamic and Keywords: atherosclerosis, atherosclerotic plaque,
physical factors contribute to plaque destabiliza- coagulation, lipids, platelets, thrombosis.
tion. Upon erosion or rupture, these high-risk

excess lipid (LDLs) with concomitant endothelial

Molecular and cellular basis of atherosclerotic plaque formation
activation/dysfunction and the internalization and
Atherosclerosis and its cardiovascular ischaemic deposition of lipids in the intima (Fig. 1). The
complications are the most common causes of continuous exposure to other pathogenic factors,
death and disability worldwide [1]. Indeed, the such as infectious disease, chronic subclinical
World Health Organization (WHO) reported in 2010 inflammation, hypertension, diabetes, stress and
that cardiovascular disease represents around smoking, contributes to endothelial injury. The
30% of global deaths and estimated that by 2030 dysfunctional and permeable endothelium allows
more than 23.3 million persons will die annually LDL particles to further infiltrate and accumulate in
from cardiovascular disease [24]. the extracellular matrix (ECM) where they become
targets for oxidative and enzymatic modifications
Atherosclerosis is a chronic lipid-driven inflamma- (Fig. 1). Modified LDLs enhance a series of pro-
tory disease of the arterial wall characterized by the inflammatory reactions perpetuating the activa-
involvement of the innate and adaptive immune tion, recruitment and transmigration of different
systems [5, 6]. The first step in the development of innate immune cells (monocytes, mast cells, neu-
atherosclerosis is the exposure of vascular cells to trophils, natural killer cells and dendritic cells)

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

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

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

promoting thrombus formation upon plaque rup- stress, further expanding the lipid core and,
ture [35]. We have demonstrated that the athero- thereby, increasing plaque vulnerability. In fact, it
matous TF-rich core is the most thrombogenic has been shown that the degree of reactivity of
component of human atherosclerotic plaques (six- glycophoryn A (a membrane sialoglycoprotein of
fold greater than collagen) [36]. Interestingly, red blood cells) and iron deposits in the plaque
recent data show that TF signalling is also involved correlates with the increasing size of the necrotic
in VSMC migration, thus contributing to plaque core and macrophage density, indicating that
remodelling [37, 38]. haemorrhage per se elicits an inflammatory
response [49]. Microvessel density is much higher
in rupture-prone and ruptured plaques than in
Thin fibrous cap
stable plaques (2- to 4-fold increase) [23]. In
The fibrous cap is located between the vascular human coronary atherosclerotic lesions from
lumen and the necrotic core. Autopsy studies have excised hearts at transplantation, we have demon-
determined that ruptured plaques are extremely strated that the most advanced-stage plaques,
thin (<65 lm thick), have a macrophage density of which contain the highest neovessel content, are
around 26% and have a low collagen content [19]. associated with the highest rate of thrombotic
Apoptosis of macrophages in the thin fibrous cap events [50]. Furthermore, using laser dissection
derive in the continuous release of MMPs which in microscopy approaches, we found that new angio-
turn degrade collagen in the fibrous cap [39, 40]. In genic factors appear to contribute to plaque vas-
addition, loss of VSMCs (the main source of colla- cularization/vulnerability [51, 52] and observed
gen synthesis) also affects the thinning of the modulation of angiogenesis through signalling of
fibrous cap, thus contributing to plaque vulnera- the TF cytoplasmic domain [5355].
bility to rupture [41].
Culprit plaques that account for acute coronary
There is substantial evidence confirming a close events were found to be less calcified than those
relationship between intralesion angiogenesis and associated with stable angina, indicating that the
culprit atherosclerotic plaques [42]. Moreover, in- presence of calcium provides stability [5658]. Of
traplaque neovascularization has been associated note, Virmani et al. reported that calcified nodules
with the occurrence of prior, and shown to predict (calcifications with a protruding, irregular and
future cardiovascular events [43, 44]. It has been convex luminal surface), although rare (27%),
suggested that the oxygen supply is restricted once are a cause of thrombotic events. The findings of
the wall thickness of the vessel exceeds 100 lm, the recent PROSPECT trial, [59] however, have
triggering hypoxia-inducible transcription factor- demonstrated that the incidence of such calcified
1a accumulation and the subsequent expression of nodules is linked to greater plaque burden, an
vascular endothelial growth factor (VEGF) and increased number of thick-cap fibroatheromas and
other angiogenic modulators. Together these a lower rate of events. Intravascular ultrasound
angiogenic factors promote vessel formation from imaging studies have provided evidence that the
the adventitial vasa vasorum towards the intima in coronary calcification pattern determines plaque
order to provide oxygen and nutrients, thus allow- vulnerability. As such, spotty calcifications
ing plaque growth. Yet, in more advanced plaques, (defined by lesions 14 mm in length with an arc
inflammatory cell infiltration and the simultaneous of calcium < 90), in contrast to extensive calcified
release of several pro-angiogenic cytokines result atherosclerotic lesions, are associated with ischae-
in the induction of uncontrolled neointimal micro- mic events [18]. Finally, recent work has also
vessel formation without supporting VSMCs. These suggested that the presence of microcalcifications
new microvessels are weak and permeable, and in the fibrous cap may increase the risk of rupture
express cellular adhesion molecules favouring [60].
local extravasation of red blood cells, plasma
proteins and inflammatory cells which conse-
Nonplaque determinants of rupture
quently may lead to recurrent intraplaque haem-
orrhage [13, 4548]. Moreover, released lipid-rich Local dynamic forces, including circumferential,
erythrocyte cell membranes and free haemoglobin flexion and shear stresses, may participate in
enhance the inflammatory response and oxidative vulnerable plaque disruption [61]. On the one

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

hand, atherosclerosis usually develops at bifurca- and mediate the passage of water, ions and other
tions or arterial curvatures where blood flow is slow small molecules. Integrins act as receptors for
and nonlinear [62]. Low shear stress has recently vitronectin and fibronectin and, therefore, are
been related to the progression of vulnerable responsible for adhesion of the endothelial mono-
plaques [63]. Moreover, it has also been shown layer to the extracellular matrix (Fig. 2) [69]. The
that low shear stress upregulates inflammatory healthy endothelium, devoid of atherosclerotic
signalling in endothelial cells and leucocytes, con- lesions, is also a highly thromboresistant surface
tributing to vulnerable plaque development [62]. that limits thrombus formation and the occurrence
On the other hand, according to Laplaces law, of ischaemic events. Indeed, the endothelial layer
circumferential stress is directly related to the expresses a vast array of molecules with antiplat-
luminal diameter and intraluminal pressure of a elet, anticoagulant and fibrinolytic properties
vessel and inversely related to wall thickness [64]. It (Fig. 2).
is also generally considered that fibrous cap rupture
takes place near the shoulder region where there is a
Platelets: the key triggers of haemostatic plug and thrombus
high density of inflammatory cells. However, in an
formation on damaged endothelium
interesting study it was revealed that rupture of the
plaque occurred in the mid-portion in those patients Platelets are anucleated cells (2 lm in diameter)
who were performing intense physical exercise dur- that are devoid of genomic DNA but contain mes-
ing sudden coronary death whereas ruptured senger RNA and the capability for synthesizing
occurred in the shoulder region in those who died proteins. They are released into the circulation as
at rest [65]. These observations support the contri- cytoplasmic fragments of bone marrow-derived
bution to plaque rupture of biomechanical forces megakaryocytes and circulate in the blood stream
rather than an inflammatory component. It has been for 710 days without interacting with other blood
shown that physical, environmental or emotional elements and/or the vascular wall [71]. Upon
stressors, although insufficient to cause coronary endothelial disruption or rupture of an atheroscle-
instability per se, activate the sympathetic nervous rotic plaque, a sequence of events leads to the
system and induce the release of catecholamines development of a platelet-rich thrombus [72].
leading to local vasoconstriction in addition to a rise Platelets avidly adhere to dysfunctional, damaged
in cardiac rate and blood pressure. In fact, coronary or disrupted endothelium.
vasospasm is believed to contribute to plaque ero-
sion. Together, these events may precipitate plaque Haemostatic plug formation, which usually occurs
rupture and consequent acute coronary syndrome at very high blood shear rates, and thrombosis on
[66]. In addition, activation of the sympathetic endothelial injured vessels lead to rapid platelet
nervous system also affects haemostasis and hae- recruitment. The initial tethering is mainly mediated
morheology (higher platelet reactivity, coagulability by the interaction between platelet glycoprotein (GP)
and microvascular constriction), further contribut- Iba receptor (the main binding region of the platelet
ing to plaque rupture and consequently to the acute GPIbIXV complex) and the A1 domain of von
clinical ischaemic event [67]. Willebrand factor (vWF). vWF is already anchored
to collagen through its A3 domain. GPIba also
Atherothrombosis: a complication of the atherosclerotic plaque contains binding sites for Leukocyte integrin mac-
rophage antigen and P-selectin which favours fur-
Healthy endothelium: a critical feature of thromboresistance
ther recruitment of GPIbavWF interaction,
The endothelial layer is a semi-permeable barrier however, triggers a weak intracellular signal and
that controls the diffusion of plasma molecules, maintains platelets in contact with the endothelial
regulates vascular tone and inflammation, and layer but does not mediate irreversible adhesion
prevents thrombus formation [68, 69]. The integrity [34]. Stable and strong platelet binding is brought
of the endothelial barrier is determined by the about by two well-established platelet collagen
presence of intercellular junction complexes (i.e. receptors [GPIa/IIa (integrin a2b1) and GPVI (immu-
occludin, claudin, junctional adhesion molecules noglobulin)] which also induce platelet activation/
[70], cadherin and gap junctions) and integrin aggregation. GPVI signals through tyrosine kinases
receptors (Fig. 2). Tight and adherent junctions and activates phospholipase (PL) C which mobilizes
maintain intercellular binding and regulate endo- calcium, activates the GPIIb/IIIa (integrin aIIbb3)
thelial cell growth and survival whereas gap junc- receptor and induces granule secretion, further
tions are mainly involved in intercellular binding promoting platelet activation and aggregation [34,

6 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


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

AMP adenosin X Protein S Protein C

Anthrombin III



u lsi


Protacyclin Platelet NO Plasmin Fibrin

Tissue plasminogen Endothelial cell
AA acvator
L-Arginine GAP
L-citruline juncons
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- ing the surface of the exposed vascular damage
acting with several collagen-related binding sites. (Fig. 3). Later thrombosis evolves with a predomi-
Laminin, fibronectin, thrombospondin and vitro- nance of platelets that are rapidly activated and
nectin also contribute to platelet adhesion by bind- recruited to the growing thrombus. Exposure of the
ing to GPIc-IIa, GPIc-IIa, GPIV and vitronectin extracellular domain of TF to flowing blood initiates
receptors, respectively, although to a lesser extent. 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
TF plays a key role in atherothrombosis
activates both FIX and FX, and in turn, activated
In atherosclerotic plaques, the initial triggering is FX (FXa), and its cofactor FVa, form the prothom-
the exposed TF that induces thrombin and the binase complex which transforms prothrombin to
subsequent formation of a fibrin monolayer cover- 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

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

(a) (b)

L Platelets
Platelets L


Platelets Platelets
(c) L (d)

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

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

2014 The Association for the Publication of the Journal of Internal Medicine 9
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L. Badimon & G. Vilahur Review: Thrombosis formation on atherosclerotic lesions and plaque rupture

Platelet aggregaon

Fibrinogen binding Thr


PAR-1 Thrombin proteolyc

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





AC AMPc Ca2+ Ca2+

Adenylyl cyclase (AC) Ca2+

AMPc decrease TP

Calcium cytosol TXA2
TXA2 generaon
acvaon PLA2 acvaon

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

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 contributors to plaque development, and the loss of
fibrinolysis. Thrombin may activate TAFI but the VSMCs and increased intraplaque haemorrhage
rate of activation is increased by the thrombin are critical steps in necrotic core destabilization
thrombomodulin complex. Once activated, TAFI and enlargement. In fact, reduction of apoptosis
downregulates fibrinolysis by the removal of C- execution has been detected in therapies
terminal lysines from fibrin. As a consequence the addressed towards atherosclerosis stabilization
binding of plasminogen and t-PA to the fibrin clot is [108] highlighting the need to assess new therapies
inhibited [101]. directed towards apoptosis modulation. Similarly,
selectively targeting new vessel formation appears
to be a promising strategy to promote plaque
Regulation of thrombus growth by local and systemic factors
stabilization. During recent years, although incon-
Cardiovascular disease risk factors (such as dy- sistent and conflicting results have been reported,
slipidaemia, diabetes and hypertension) have been the advent of state-of-the-art technologies has
shown to affect blood thrombogenic potential by created considerable enthusiasm for the use of
increasing coagulability, lowering the fibrinolytic stem cell-based therapies in combination with gene
potential and enhancing platelet reactivity [102]. therapies (i.e. stem cell preconditioning) to prevent
Conversely, interventions to modify cardiovascular and/or cause the regression of atherosclerotic
disease risk factors have been shown to lower the lesions [109]. In line with these approaches, the
prothrombotic risk [103]. There has been increas- concomitant use of molecular and conventional
ing interest in the contribution of the cross-talk imaging modalities may enable the detection of
between MPs and cells involved in atherosclerosis plaque composition, activity and progression,
progression and thrombus formation. Although MP allowing the success of different new therapeutic
formation is a physiological process, it has been strategies for plaque stabilization to be monitored.
demonstrated in several studies that the levels of Conversely, taking into consideration that super-
circulating MPs are raised in a wide range of imposition of a thrombus at the site of plaque
cardiovascular diseases, including atherosclerotic rupture with the consequent luminal obstruction is
lesion progression, thrombosis, heart failure, ar- the main cause of cardiovascular ischaemic events,
rhythmias and inflammation-related vascular dis- an in-depth understanding of platelet biology using
ease, as well as in conditions in which omic approaches as well a better knowledge of the
cardiovascular disease risk factors are not con- molecular/cellular interactions that govern plate-
trolled [104]. MPs have also been also detected in let aggregation and clot formation may guide the
atherosclerotic lesions, mainly originating from proper use of existing treatments and the develop-
macrophages, red blood cells and VSMCs, whereas ment of new anti-thrombotic therapies, eventually
circulating MPs mainly originate from platelets. Of improving patient outcomes and helping to reduce
note, we have recently demonstrated that statin the economic burden of cardiovascular disease.
treatment reduces the number of circulating and Finally, new paradigms are emerging that
platelet-derived MPs carrying markers of activated strengthen the critical role of inflammation in the
cells, suggesting a new mechanism of statin- pathogenesis of atherothrombosis. As such, better
dependent protection against vascular disease characterization of the immune-modulating role of
[105]. In addition, we have recently reported that platelets as well as of microRNAs (endogenous
both circulating and platelet-derived MPs enhance small noncoding RNAs with the ability to regulate
thrombosis on atherosclerotic plaques [106]. In gene expression at transcriptional and post-
fact, it has been shown that MPs are captured by transcriptional levels) may give rise to novel
thrombus-associated platelets through the inter- experimental strategies for the prevention and
action between MP-exposed P-selectin glycoprotein treatment of atherosclerosis-related diseases.
ligand 1 and P-selectin from platelets [107].
Conflict of interest statement
Conclusions and future scientific challenges
No conflicts of interest to declare.
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 The work in the authors laboratory discussed in
(cellular and humoral) are considered the major 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 American Heart Association. Circulation 1995; 92: 1355
Ministry of Science and Innovation; Spanish Car- 74.
16 Virmani R, Kolodgie FD, Burke AP, Farb A, Schwartz SM.
diovascular Network (Red Cardiovascular) and the
Lessons from sudden coronary death: a comprehensive
Spanish Cell Therapy Network (Red de Terapia morphological classification scheme for atherosclerotic
Celular - TERCEL) from Instituto Salud Carlos III (to lesions. Arterioscler Thromb Vasc Biol 2000; 20: 1262
LB) and a grant from lOreal-UNESCO (for Women 75.
in Science to GV). We thank Fundacion Jesus Serra 17 van der Wal AC, Becker AE, van der Loos CM, Das PK. Site of
for their continuous support. GV is a Ramon y intimal rupture or erosion of thrombosed coronary athero-
sclerotic plaques is characterized by an inflammatory pro-
Cajal scientist funded by the Spanish Ministry of
cess irrespective of the dominant plaque morphology.
Science and Innovation (MICINN). Circulation 1994; 89: 3644.
18 Sakakura K, Nakano M, Otsuka F, Ladich E, Kolodgie FD,
Virmani R. Pathophysiology of atherosclerosis plaque pro-
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Journal of Internal Medicine