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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 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
Obesity
Hyperhomocysteinaemia
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

2014 The Association for the Publication of the Journal of Internal Medicine 3
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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 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

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- 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].
Calcification
Neovascularization
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

2014 The Association for the Publication of the Journal of Internal Medicine 5
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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

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- 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

2014 The Association for the Publication of the Journal of Internal Medicine 7
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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
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
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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
Coagulation
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


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 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
Acknowledgements
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-
gression. Heart Lung Circ 2013; 22: 399411.
References 19 Falk E, Nakano M, Bentzon JF, Finn AV, Virmani R. Update
1 Vilahur G, Badimon J, Bugiardini R, Badimon L. Perspec- on acute coronary syndromes: the pathologists view. Eur
tives: the burden of cardiovascular risk factors and coronary Heart J 2013; 34: 71928.
heart disease in Europe and worldwide. Eur Heart J 2014; 20 Tomey MI, Narula J, Kovacic JC. Year in review: advances in
16: A711. understanding of plaque composition and treatment
2 Lozano R, Naghavi M, Foreman K et al. Global and regional options. J Am Coll Cardiol 2014; 63: 160416.
mortality from 235 causes of death for 20 age groups in 1990 21 Brugaletta S, Cola C, Martin-Yuste V et al. Qualitative and
and 2010: a systematic analysis for the Global Burden of quantitative accuracy of ultrasound-based virtual histology
Disease Study 2010. Lancet 2012; 380: 2095128. for detection of necrotic core in human coronary arteries. Int
3 Falk E, Shah PK, Fuster V. Coronary plaque disruption. J Cardiovasc Imaging 2014; 30: 46976.
Circulation 1995; 92: 65771. 22 Joshi F, Rosenbaum D, Bordes S, Rudd JH. Vascular
4 Mathers CD, Loncar D. Projections of global mortality and imaging with positron emission tomography. J Intern Med
burden of disease from 2002 to 2030. PLoS Med 2006; 3: 2011; 270: 99109.
e442. 23 von Birgelen C, Klinkhart W, Mintz GS et al. Plaque distri-
5 Libby P. Inflammation in atherosclerosis. Arterioscler bution and vascular remodeling of ruptured and nonrup-
Thromb Vasc Biol 2012; 32: 204551. tured coronary plaques in the same vessel: an intravascular
6 Badimon L, Storey RF, Vilahur G. Update on lipids, inflam- ultrasound study in vivo. J Am Coll Cardiol 2001; 37: 1864
mation and atherothrombosis. Thromb Haemost 2012; 105 70.
(Suppl 1): S3442. 24 Schoenhagen P, Ziada KM, Kapadia SR, Crowe TD, Nissen
7 Hansson GK, Libby P, Schonbeck U, Yan ZQ. Innate and SE, Tuzcu EM. Extent and direction of arterial remodeling in
adaptive immunity in the pathogenesis of atherosclerosis. stable versus unstable coronary syndromes: an intravascu-
Circ Res 2002; 91: 28191. lar ultrasound study. Circulation 2000; 101: 598603.
8 McLeod O, Silveira A, Fredrikson GN et al. Plasma autoan- 25 Kojima S, Nonogi H, Miyao Y et al. Is preinfarction angina
tibodies against apolipoprotein B-100 peptide 210 in sub- related to the presence or absence of coronary plaque
clinical atherosclerosis. Atherosclerosis 2014; 232: 2428. rupture? Heart 2000; 83: 648.
9 Badimon L. Interleukin-18: a potent pro-inflammatory cyto- 26 Felton CV, Crook D, Davies MJ, Oliver MF. Relation of
kine in atherosclerosis. Cardiovasc Res 2012; 96: 1725; plaque lipid composition and morphology to the stability of
discussion 6-80. human aortic plaques. Arterioscler Thromb Vasc Biol 1997;
10 Libby P, Nahrendorf M, Swirski FK. Monocyte heterogeneity 17: 133745.
in cardiovascular disease. Semin Immunopathol 2013; 35: 27 Loree HM, Kamm RD, Stringfellow RG, Lee RT. Effects of
55362. fibrous cap thickness on peak circumferential stress in
11 Koga J, Aikawa M. Crosstalk between macrophages and model atherosclerotic vessels. Circ Res 1992; 71: 8508.
smooth muscle cells in atherosclerotic vascular diseases. 28 Richardson PD, Davies MJ, Born GV. Influence of plaque
Vascul Pharmacol 2013; 57: 248. configuration and stress distribution on fissuring of coro-
12 Tabas I. Macrophage death and defective inflammation nary atherosclerotic plaques. Lancet 1989; 2: 9414.
resolution in atherosclerosis. Nat Rev Immunol 2010; 10: 29 Padro T, Pena E, Garcia-Arguinzonis M, Llorente-Cortes V,
3646. Badimon L. Low-density lipoproteins impair migration of
13 Virmani R, Kolodgie FD, Burke AP et al. Atherosclerotic human coronary vascular smooth muscle cells and induce
plaque progression and vulnerability to rupture: angiogen- changes in the proteomic profile of myosin light chain.
esis as a source of intraplaque hemorrhage. Arterioscler Cardiovasc Res 2008; 77: 21120.
Thromb Vasc Biol 2005; 25: 205461. 30 Garcia-Arguinzonis M, Padro T, Lugano R, Llorente-Cortes
14 Study Group on the Classification of Atherosclerotic Lesions. V, Badimon L. Low-density lipoproteins induce heat shock
World Health Organ Tech Rep Ser 1958; 57: 120. protein 27 dephosphorylation, oligomerization, and subcel-
15 Stary HC, Chandler AB, Dinsmore RE et al. A definition of lular relocalization in human vascular smooth muscle cells.
advanced types of atherosclerotic lesions and a histological Arterioscler Thromb Vasc Biol 2010; 30: 12129.
classification of atherosclerosis. A report from the Commit- 31 Bauriedel G, Hutter R, Welsch U, Bach R, Sievert H, Luderitz
tee on Vascular Lesions of the Council on Arteriosclerosis, B. Role of smooth muscle cell death in advanced coronary

12 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

primary lesions: implications for plaque instability. Cardio- 49 Kolodgie FD, Gold HK, Burke AP et al. Intraplaque hemor-
vasc Res 1999; 41: 4808. rhage and progression of coronary atheroma. N Engl J Med
32 Stoneman VE, Bennett MR. Role of apoptosis in atheroscle- 2003; 349: 231625.
rosis and its therapeutic implications. Clin Sci (Lond) 2004; 50 Juan-Babot JO, Martinez-Gonzalez J, Berrozpe M, Badimon
107: 34354. L. [Neovascularization in human coronary arteries with
33 Boyle JJ, Weissberg PL, Bennett MR. Tumor necrosis lesions of different severity]. Rev Esp Cardiol 2003; 56:
factor-alpha promotes macrophage-induced vascular 97886.
smooth muscle cell apoptosis by direct and autocrine 51 Slevin M, Krupinski J, Badimon L. Controlling the angio-
mechanisms. Arterioscler Thromb Vasc Biol 2003; 23: genic switch in developing atherosclerotic plaques: possible
15538. targets for therapeutic intervention. J Angiogenes Res 2009;
34 Freynhofer MK, Bruno V, Wojta J, Huber K. The role of 1: 4.
platelets in athero-thrombotic events. Curr Pharm Des 2012; 52 Slevin M, Turu MM, Rovira N et al. Identification of a
18: 5197214. snapshot of co-expressed angiogenic markers in laser-dis-
35 Tedgui A, Mallat Z. Apoptosis as a determinant of athero- sected vessels from unstable carotid plaques with targeted
thrombosis. Thromb Haemost 2001; 86: 4206. arrays. J Vasc Res 2010; 47: 32335.
36 Fernandez-Ortiz A, Badimon JJ, Falk E et al. Characteriza- 53 Arderiu G, Pena E, Aledo R, Espinosa S, Badimon L. Ets-1
tion of the relative thrombogenicity of atherosclerotic plaque transcription is required in tissue factor driven microvessel
components: implications for consequences of plaque rup- formation and stabilization. Angiogenesis 2012; 15: 657
ture. J Am Coll Cardiol 1994; 23: 15629. 69.
37 Pena E, Arderiu G, Badimon L. Tissue factor induces human 54 Arderiu G, Pena E, Aledo R, Badimon L. Tissue factor-Akt
coronary artery smooth muscle cell motility through signaling triggers microvessel formation. J Thromb Haemost
Wnt-signalling. J Thromb Haemost 2013; 11: 188091. 2012; 10: 1895905.
38 Pena E, Arderiu G, Badimon L. Subcellular localization of 55 Arderiu G, Pena E, Aledo R, Juan-Babot O, Badimon L.
tissue factor and human coronary artery smooth muscle cell Tissue factor regulates microvessel formation and stabiliza-
migration. J Thromb Haemost 2012; 10: 237382. tion by induction of chemokine (C-C motif) ligand 2 expres-
39 Shah PK, Falk E, Badimon JJ et al. Human mono- sion. Arterioscler Thromb Vasc Biol 2011; 31: 260715.
cyte-derived macrophages induce collagen breakdown in 56 Sangiorgi G, Rumberger JA, Severson A et al. Arterial
fibrous caps of atherosclerotic plaques. Potential role of calcification and not lumen stenosis is highly correlated
matrix-degrading metalloproteinases and implications for with atherosclerotic plaque burden in humans: a histologic
plaque rupture. Circulation 1995; 92: 15659. study of 723 coronary artery segments using nondecalcify-
40 Galis ZS, Khatri JJ. Matrix metalloproteinases in vascular ing methodology. J Am Coll Cardiol 1998; 31: 12633.
remodeling and atherogenesis: the good, the bad, and the 57 Ehara S, Kobayashi Y, Yoshiyama M et al. Spotty calcifica-
ugly. Circ Res 2002; 90: 25162. tion typifies the culprit plaque in patients with acute
41 Geng YJ, Libby P. Evidence for apoptosis in advanced myocardial infarction: an intravascular ultrasound study.
human atheroma. Colocalization with interleukin-1 Circulation 2004; 110: 34249.
beta-converting enzyme. Am J Pathol 1995; 147: 25166. 58 Beckman JA, Ganz J, Creager MA, Ganz P, Kinlay S.
42 Tenaglia AN, Peters KG, Sketch MH Jr, Annex BH. Neovas- Relationship of clinical presentation and calcification of
cularization in atherectomy specimens from patients with culprit coronary artery stenoses. Arterioscler Thromb Vasc
unstable angina: implications for pathogenesis of unstable Biol 2001; 21: 161822.
angina. Am Heart J 1998; 135: 104. 59 Xu Y, Mintz GS, Tam A et al. Prevalence, distribution,
43 Koole D, Heyligers J, Moll FL, Pasterkamp G. Intraplaque predictors, and outcomes of patients with calcified nodules
neovascularization and hemorrhage: markers for cardiovas- in native coronary arteries: a 3-vessel intravascular ultra-
cular risk stratification and therapeutic monitoring. J Car- sound analysis from Providing Regional Observations to
diovasc Med (Hagerstown) 2012; 13: 6359. Study Predictors of Events in the Coronary Tree (PROS-
44 Hellings WE, Peeters W, Moll FL et al. Composition of carotid PECT). Circulation 2012; 126: 53745.
atherosclerotic plaque is associated with cardiovascular 60 Maldonado N, Kelly-Arnold A, Vengrenyuk Y et al. A mecha-
outcome: a prognostic study. Circulation 2010; 121: 1941 nistic analysis of the role of microcalcifications in atheroscle-
50. rotic plaque stability: potential implications for plaque
45 Barger AC, Beeuwkes R 3rd, Lainey LL, Silverman KJ. rupture. Am J Physiol Heart Circ Physiol 2012; 303: H61928.
Hypothesis: vasa vasorum and neovascularization of human 61 Yuan C, Mitsumori LM, Ferguson MS et al. In vivo accuracy
coronary arteries. A possible role in the pathophysiology of of multispectral magnetic resonance imaging for identifying
atherosclerosis. N Engl J Med 1984; 310: 1757. lipid-rich necrotic cores and intraplaque hemorrhage in
46 McCarthy MJ, Loftus IM, Thompson MM et al. Angiogenesis advanced human carotid plaques. Circulation 2001; 104:
and the atherosclerotic carotid plaque: an association 20516.
between symptomatology and plaque morphology. J Vasc 62 Seneviratne A, Hulsmans M, Holvoet P, Monaco C. Biome-
Surg 1999; 30: 2618. chanical factors and macrophages in plaque stability. Car-
47 Polverini PJ, Cotran PS, Gimbrone MA Jr, Unanue ER. diovasc Res 2013; 99: 28493.
Activated macrophages induce vascular proliferation. Nature 63 Giannoglou GD, Antoniadis AP, Koskinas KC, Chatzizisis
1977; 269: 8046. YS. Flow and atherosclerosis in coronary bifurcations.
48 Moreno PR, Purushothaman M, Purushothaman KR. Plaque EuroIntervention 2010; 6(Suppl J): J1623.
neovascularization: defense mechanisms, betrayal, or a war 64 Lee RT, Kamm RD. Vascular mechanics for the cardiologist.
in progress. Ann N Y Acad Sci 2012; 1254: 717. J Am Coll Cardiol 1994; 23: 128995.

2014 The Association for the Publication of the Journal of Internal Medicine 13
Journal of Internal Medicine
L. Badimon & G. Vilahur Review: Thrombosis formation on atherosclerotic lesions and plaque rupture

65 Burke AP, Farb A, Malcom GT, Liang Y, Smialek JE, Virmani 85 Fuster V, Badimon L, Badimon JJ, Chesebro JH. The
R. Plaque rupture and sudden death related to exertion in pathogenesis of coronary artery disease and the acute
men with coronary artery disease. JAMA 1999; 281: 9216. coronary syndromes (1). N Engl J Med 1992; 326: 24250.
66 Crea F, Liuzzo G. Pathogenesis of acute coronary syndromes. 86 Fuster V, Badimon L, Badimon JJ, Chesebro JH. The
J Am Coll Cardiol 2013; 61: 111. pathogenesis of coronary artery disease and the acute
67 Thrall G, Lane D, Carroll D, Lip GY. A systematic review of coronary syndromes (2). N Engl J Med 1992; 326: 3108.
the effects of acute psychological stress and physical activity 87 Badimon L, Vilahur G, Padro T. Lipoproteins, platelets and
on haemorheology, coagulation, fibrinolysis and platelet atherothrombosis. Rev Esp Cardiol 2009; 62: 116178.
reactivity: implications for the pathogenesis of acute coro- 88 Celi A, Merrill-Skoloff G, Gross P et al. Thrombus formation:
nary syndromes. Thromb Res 2007; 120: 81947. direct real-time observation and digital analysis of thrombus
68 Mehta D, Malik AB. Signaling mechanisms regulating endo- assembly in a living mouse by confocal and widefield
thelial permeability. Physiol Rev 2006; 86: 279367. intravital microscopy. J Thromb Haemost 2003; 1: 608.
69 Otsuka F, Finn AV, Yazdani SK, Nakano M, Kolodgie FD, 89 Mailhac A, Badimon JJ, Fallon JT et al. Effect of an
Virmani R. The importance of the endothelium in athero- eccentric severe stenosis on fibrin(ogen) deposition on
thrombosis and coronary stenting. Nat Rev Cardiol 2012; 9: severely damaged vessel wall in arterial thrombosis. Rela-
43953. tive contribution of fibrin(ogen) and platelets. Circulation
70 Majamaa H, Isolauri E, Saxelin M, Vesikari T. Lactic acid 1994; 90: 98896.
bacteria in the treatment of acute rotavirus gastroenteritis. J 90 Meyer BJ, Badimon JJ, Mailhac A et al. Inhibition of growth
Pediatr Gastroenterol Nutr 1995; 20: 3338. of thrombus on fresh mural thrombus. Targeting optimal
71 Kaushansky K. The molecular mechanisms that control therapy. Circulation 1994; 90: 24328.
thrombopoiesis. J Clin Invest 2005; 115: 333947. 91 Toschi V, Gallo R, Lettino M et al. Tissue factor modulates
72 Badimon L, Vilahur G. Platelets, arterial thrombosis and the thrombogenicity of human atherosclerotic plaques. Cir-
cerebral ischemia. Cerebrovasc Dis 2007; 24(Suppl 1): 309. culation 1997; 95: 5949.
73 Penz S, Reininger AJ, Brandl R et al. Human atheromatous 92 Badimon JJ, Lettino M, Toschi V et al. Local inhibition of
plaques stimulate thrombus formation by activating platelet tissue factor reduces the thrombogenicity of disrupted
glycoprotein VI. FASEB J 2005; 19: 898909. human atherosclerotic plaques: effects of tissue factor
74 Owens AP 3rd, Mackman N. Tissue factor and thrombosis: pathway inhibitor on plaque thrombogenicity under flow
the clot starts here. Thromb Haemost 2010; 104: 4329. conditions. Circulation 1999; 99: 17807.
75 Grad E, Danenberg HD. C-reactive protein and atherothrom- 93 Badimon JJ, Ortiz AF, Meyer B et al. Different response to
bosis: cause or effect? Blood Rev 2013; 27: 239. balloon angioplasty of carotid and coronary arteries: effects
76 Molins B, Pena E, Vilahur G, Mendieta C, Slevin M, Badimon on acute platelet deposition and intimal thickening. Athero-
L. C-reactive protein isoforms differ in their effects on sclerosis 1998; 140: 30714.
thrombus growth. Arterioscler Thromb Vasc Biol 2008; 28: 94 Egorina EM, Sovershaev MA, Bjorkoy G et al. Intracellular
223946. and surface distribution of monocyte tissue factor: applica-
77 Molins B, Pena E, de la Torre R, Badimon L. Monomeric tion to intersubject variability. Arterioscler Thromb Vasc Biol
C-reactive protein is prothrombotic and dissociates from 2005; 25: 14938.
circulating pentameric C-reactive protein on adhered acti- 95 Camino-Lopez S, Llorente-Cortes V, Sendra J, Badimon L.
vated platelets under flow. Cardiovasc Res 2011; 92: 32837. Tissue factor induction by aggregated LDL depends on LDL
78 de la Torre R, Pena E, Vilahur G, Slevin M, Badimon L. receptor-related protein expression (LRP1) and Rho A trans-
Monomerization of C-reactive protein requires glycoprotein location in human vascular smooth muscle cells. Cardiovasc
IIb-IIIa activation: pentraxins and platelet deposition. Res 2007; 73: 20816.
J Thromb Haemost 2013; 11: 204858. 96 Llorente-Cortes V, Otero-Vinas M, Camino-Lopez S, Llam-
79 Habersberger J, Strang F, Scheichl A et al. Circulating payas O, Badimon L. Aggregated low-density lipoprotein
microparticles generate and transport monomeric C-reactive uptake induces membrane tissue factor procoagulant activ-
protein in patients with myocardial infarction. Cardiovasc ity and microparticle release in human vascular smooth
Res 2012; 96: 6472. muscle cells. Circulation 2004; 110: 4529.
80 Cimmino G, Conte S, Morello A, DElia S, Marchese V, Golino 97 Day SM, Reeve JL, Pedersen B et al. Macrovascular throm-
P. The complex puzzle underlying the pathophysiology of bosis is driven by tissue factor derived primarily from the
acute coronary syndromes: from molecular basis to clinical blood vessel wall. Blood 2005; 105: 1928.
manifestations. Expert Rev Cardiovasc Ther 2012; 10: 98 Ramaiola I, Padro T, Juan-Babot O et al. Relationship
153343. between time of ischemia and thrombus composition in
81 http://www.acc.org ACoCaAHAAAEpgmopwafoAfU. STEMI. ESC congress 2012.Eur Heart J 2012; 33(Suppl 1):
82 Andrews RK, Gardiner EE, Shen Y, Berndt MC. Platelet 473 (P2730).
interactions in thrombosis. IUBMB Life 2004; 56: 138. 99 Silvain J, Collet JP, Nagaswami C et al. Composition of
83 Peterson ED, Pollack CV Jr, Roe MT et al. Early use of coronary thrombus in acute myocardial infarction. J Am Coll
glycoprotein IIb/IIIa inhibitors in non-ST-elevation acute Cardiol 2011; 57: 135967.
myocardial infarction: observations from the National Reg- 100 Abbate R, Cioni G, Ricci I, Miranda M, Gori AM. Thrombosis
istry of Myocardial Infarction 4. J Am Coll Cardiol 2003; 42: and acute coronary syndrome. Thromb Res 2012; 129: 235
4553. 40.
84 Hemker HC, van Rijn JL, Rosing J, van Dieijen G, Bevers 101 Foley JH, Kim PY, Mutch NJ, Gils A. Insights into thrombin
EM, Zwaal RF. Platelet membrane involvement in blood activatable fibrinolysis inhibitor function and regulation. J
coagulation. Blood Cells 1983; 9: 30317. Thromb Haemost 2013; 11(Suppl 1): 30615.

14 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

102 Woodward M, Lowe GD, Rumley A et al. Epidemiology of 106 Suades R, Padro T, Vilahur G, Badimon L. Circulating and
coagulation factors, inhibitors and activation markers: the platelet-derived microparticles in human blood enhance
Third Glasgow MONICA Survey. II. Relationships to cardio- thrombosis on atherosclerotic plaques. Thromb Haemost
vascular risk factors and prevalent cardiovascular disease. 2012; 108: 120819.
Br J Haematol 1997; 97: 78597. 107 Celi A, Lorenzet R, Furie BC, Furie B. Microparticles and a
103 Osende JI, Badimon JJ, Fuster V et al. Blood thromboge- P-selectin-mediated pathway of blood coagulation. Dis
nicity in type 2 diabetes mellitus patients is associated Markers 2004; 20: 34752.
with glycemic control. J Am Coll Cardiol 2001; 38: 1307 108 Beohar N, Flaherty JD, Davidson CJ et al. Antirestenotic
12. effects of a locally delivered caspase inhibitor in a balloon
104 Amabile N, Rautou PE, Tedgui A, Boulanger CM. Micropar- injury model. Circulation 2004; 109: 10813.
ticles: key protagonists in cardiovascular disorders. Semin 109 Zhang N, Xie X, Chen H, Yu H, Wang JA. Stem cell-based
Thromb Hemost 2010; 36: 90716. therapies for atherosclerosis: perspectives and ongoing
105 Suades R, Padro T, Alonso R, Mata P, Badimon L. Lipid-low- controversies. Stem Cells Dev 2014; 23: 173140.
ering therapy with statins reduces microparticle shedding
from endothelium, platelets and inflammatory cells indicat- Correspondence: Lina Badimon, Cardiovascular Research Center,
ing protection against cell activation. Thromb Haemost c/Sant Antoni MClaret 167, 08025 Barcelona, Spain.
2013; 110: 36677. (fax: (34)-935565559; e-mail: lbadimon@csic-iccc.org).

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