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REVIEWS

Arterial thrombus formation in cardiovascular


disease
Giuseppe Lippi, Massimo Franchini and Giovanni Targher
Abstract | The pathogenesis of arterial thrombosis is complex and dynamic. Unlike venous thrombi, arterial
thrombi typically form under conditions of high blood flow and are mainly composed of platelet aggregates,
giving them the appearance of white clots. Strong evidence suggests that arterial thrombi originate as a
consequence of an injured atherosclerotic plaque, and that their formation involves the release of prothrombotic
material (such as tissue factor), platelet aggregation, and platelet adhesion to the vascular wall. The initially
labile platelet plaque is then stabilized by insoluble fibrin produced upon activation of the coagulation
cascade. Inherited genetic factors (gene polymorphisms) and acquired predisposing conditions (such as the
concentration and activity of clotting factors) can influence both the composition and the size of an arterial
thrombus. Further research is needed to elucidate the functions of blood coagulation proteins and cellular
elements that are critical to the pathogenesis of arterial thrombosis. This Review explains mechanisms of
pathological arterial thrombus formation and discusses genetic and acquired risk factors of atherothrombosis.
Lippi, G. etal. Nat. Rev. Cardiol. 8, 502512 (2011); published online 5 July 2011; doi:10.1038/nrcardio.2011.91

Introduction
Hemostasis is the host defense system aimed at preserv Notably, arterial and venous thrombi differ in their com
ing the integrity of the circulatory system in mammals. position. Arterial thrombi are typically composed mainly
By necessity, hemostasis is a highly complex and tightly of platelet aggregates, giving the appearance of white clots.
regulated process that involves blood cells (erythrocytes, Venous thrombi largely consist of fibrin and red blood cells
platelets, and leukocytes), soluble plasma proteins, and and are thereby identified as red clots. Although platelets
the vessel wall. The endothelium is an inert surface that accumulate at the head of the venous thrombus, their
separates blood cells and soluble plasma proteins involved incorporation gradually decreases and the clots become
in blood coagulation and fibrinolysis from subendothelial more red, because they are predominantly composed of
vessel components. Physiologically, hemostasis is activated fibrin and erythrocytes.2,3
after injury of the vessel wall. Pathological thrombosis occurs when the hemostatic
Exposure of the subendothelial matrix and activation pathway is so strongly activated that it exceeds the normal
of endothelial cells are the two most important events regulatory counterbalance by anticoagulant factors, which
following vessel injury. Two consequences of endothelial are supposed to limit and localize thrombus formation
activation are the expression of adhesion receptors, to the injured area (Figure1).47 Such strong activation
Clinical Chemistry
including intercellular adhesion molecule (ICAM)1, of the hemostatic pathway in arteries can result from
Laboratory (G. Lippi), ICAM2, ICAM3, vascular adhesion molecule, and plate atherosclerotic plaque disruption. This Review details
Immunohematology let endothelial cell adhesion molecule on the endothelial our current knowledge of the mechanisms of pathological
and Transfusional
Medicine Service cell membrane, and the release of WeibelPalade bodies arterial thrombus formation, and we also discuss genetic
(M.Franchini), via exocytosis.1 Alterations of the plasma membrane lipid and acquired risk factors of atherothrombosis.
Department of Clinical
Pathology and
bilayer result in the exposure of an endothelial surface to
Laboratory Medicine, the blood flow that is rich in negatively charged phospho Pathological arterial thrombosis
Academic Hospital,
lipids, which can bind coagulation factors and other Pathological arterial thrombosis is preceded by a complex
University of Parma,
Via Gramsci 14, hemostatic molecules (such as tissue factor), thereby pro interplay between environmental and genetic factors, and
43126Parma, Italy. moting prothrombotic signaling. After release of tissue represents the most-frequent cause of death worldwide.8
Section of
Endocrinology and factor from injured endothelial cells, inactive blood coagu Our understanding of the molecular and cellular basis of
Metabolism, lation proteins (zymogens) are sequentially converted into arterial thrombus formation has advanced greatly through
Department of
Medicine, University
the corresponding active enzymes through a cascade of the use of novel mouse models of thrombosis.
of Verona, Piazzale sequential, calcium-dependent enzymatic reactions. The
Aristide Stefani 1, rapid production of thrombin marks the initiation of Atherosclerotic plaque disruption
37126Verona, Italy
(G.Targher). thrombus formation. Atherosclerosis is widely recognized as a chronic disease
that is characterized by lipid retention and chronic inflam
Correspondence to:
G. Lippi Competing interests mation in the arterial intima.4,5 The prevailing hypothesis
glippi@ao.pr.it The authors declare no competing interests. is that rupture of the fibrous cap of atherosclerotic plaques

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is responsible for 6080% of acute myocardial infarc Key points


tions, whereas the remaining 2040% might result from
Arterial thrombus formation is a complex and dynamic pathological process that
superficial erosion of the intima.47
is initiated at an injured atherosclerotic plaque
The atherosclerotic process initiates with the accumula
Several factors are involved in arterial thrombus formation, including the platelet
tion of cholesterol-containing LDL in the intima, which
glycoprotein VI, glycoprotein receptor Ia/IIa, collagenous plaque components, and
triggers the activation of endothelial cells. As a result, blood coagulation protein
endothelial cells express leukocyte adhesion molecules
Coronary artery disease (CAD) in young individuals often has a genetic basis, but
and chemokines that promote the recruitment of mono whether genetic variants or disorders predispose to an increased risk of CAD in
cytes and Tcells to the site. Monocytes differentiate into adults remains unclear
macrophages and upregulate pattern-recognition receptors, Reliable associations have been demonstrated between CAD and acquired
such as scavenger and toll-like receptors (TLRs). Pattern- risk factors such as antiphospholipid antibodies, hyperhomocysteinemia, and
recognition receptors mediate the uptake of lipoproteins, elevated levels of fibrinogen, C-reactive protein, and lipoprotein(a)
which results in the formation of foam cells. TLRs can be Future research should aim to further elucidate the functions of blood
activated by microbial agents as well as by endogenous coagulation proteins and cellular elements that are critical to thrombus formation
proteins, including degradation products of extracellu A better knowledge of the pathophysiological mechanisms of arterial thrombosis
lar matrix macromolecules, oxidized LDL or minimally- might open up new therapeutic options in the treatment of atherothrombosis
modified LDL, and serum amyloidA. Ligand binding to
TLRs transmits macrophage-activating signals9 and leads
to the release of proteases, cytokines, and other vasoactive in detail and shown to occur in two discrete stages.
molecules. Within the atherosclerotic lesion, Tlymphocytes Reininger and colleagues demonstrated that glycoprotein
interact with local antigens and support Thelper (TH)1 cell (GP)VI-mediated and GPreceptor-Ia/IIa (integrin21)-
responses by producing proinflammatory cytokines that mediated platelet adhesion and platelet aggregation on
further enhance local inflammation and promote plaque collagenous plaque components (such as fibronectin, colla
growth. Sustained local inflammation can lead to prote gen, and VWF) occur within 1min after rupture of athero
olysis and the disruption of the atheroscleroticplaque.4,5 sclerotic plaques (Figure2).12 After 3min, the thrombus
Indeed, the composition of the atherosclerotic plaque, is characterized by the formation of thrombin and fibrin,
rather than the severity of arterial stenosis, is the major and by the activation of coagulation, which is entirely
determinant of plaque rupture and thrombosis.10 triggered by tissue factor (Figure3). Importantly, under
Plaque erosion and rupture are promoted by the rela these circumstances, tissue factor is mainly derived from
tive abundance of the various subgroups of inflammatory atherosclerotic plaques and not from the circulation.
leukocytes (such as TH1, TH2, or TH17), a paucity of col In their study, Reininger etal. have shown that the
lagen, high tissue-factor expression, matrix-degrading inhibition of GPVI, but not that of plaque tissue factor,
proteinases, and oxidative-stress-mediated apoptosis of suppresses plaque-induced thrombus formation exvivo.12
cells contained in the atherosclerotic plaque (especially In addition, exposure of plaques to tissue factor does not
if mediated by myeloperoxidase).6,7 Ulceration, fissure, allow the development of clotting under physiological flow
or rupture of a pre-existing plaque exposes the subendo conditions.12 These findings could be of clinical relevance
thelial matrix and a variety of thrombogenic material because inhibition of the first step of platelet adhesion
located in the core of the plaque to the arterial circulation. and aggregation, rather than that of later stages, might be
In uninjured vessels, the endothelium releases three impor the most-effective way to inhibit atherothrombus forma
tant thromboregulators (nitric oxide, prostacyclin, and the tion.12 This hypothesis is supported by the disappointing
ectonucleotidase CD39), as well as thrombomodulin, tissue results of a large, randomized clinical trial of adjunctive
factor pathway inhibitor, and cerebral anticoagulants (such therapy with rivaroxaban (an oral direct factorXa inhibi
as amyloid protein precursor and amyloid-precursor-like tor) in patients that were stabilized after acute coronary
protein2), all of which efficiently inhibit arterial throm syndromes. Rivaroxaban was associated with an increased
bus formation.6,7 In addition to lipid droplets, matrix risk of bleeding in a dose-dependent manner, but did not
constituents, foam cells, necrotic cell debris, and tissue significantly affect the rates of the primary efficacy end
factor, atherosclerotic plaques also contain other platelet- point, which included myocardial infarction, stroke, severe
activating molecules, such as von Willebrand factor (VWF), recurrent ischemia requiring coronary revasculariza
fibrin, fibrinogen, thrombospondin, vitronectin, fibro tion, or death during a 6month follow-up period (5.6%
nectin, various types of collagens, stromal-derived factor1, with rivaroxaban versus 7.0% with placebo; hazard ratio
oxidized LDL, and LDL-cholesterol sulfate. When released [HR]0.79; 95%CI 0.601.05; P=0.10).13
into the arterial circulation, these molecules actively stimu
late adhesion and aggregation of platelets, as well as secre Platelet activation and aggregation
tion of their dense granules. Moreover, these factors elicit The process of platelet activation and aggregation is
plateletmonocyte aggregation and platelet-dependent depicted in Figure2.
activation of blood coagulation proteins.7,11
Role of glycoprotein receptors
Two discrete stages of arterial thrombosis The primary role of VWF and the extracellular matrix,
The formation of arterial thrombi induced by vulner particularly that of collagen, in initiating the arte
able human atherosclerotic plaques has been studied rial clot is widely recognized.6,7,14 The formation of the

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Platelet-activating factor Prostac


Prostacyclin
Endothelin 1 oxide
Nitric o
Thromboxane A2 Carbon monoxide
Tissue factor Anticoagulant Antithrombin
Antithr
s
Tissue-factor-bearing microparticles forces Protein C/protein S/thrombomodulin system
Clotting factors Tissue factor pathway inhibitor
Von Willebrand factor Tissue-type plasminogen activator
Tissue-
Plasminogen activator inhibitor 1 Procoagulant Urokinase-type plasminogen activator
Urokina
2-plasmin inhibitor forces Others
Others
Carboxypeptidase B2
Others

Thrombus formation

Figure 1 | An imbalance between prothrombotic and antithrombotic forces in the blood promotes the development
of arterial thrombi.

platelet aggregate begins with the interaction of the Role of CD36


platelet surface receptors GPIbIXV and GPVI with The scavenger receptor CD36 is expressed on platelets
VWF and collagen, respectively.15,16 GPIbIXV func and macrophages where it mediates atherogenesis and
tions irrespectively of platelet activation and thereby thrombosis.19 The interaction between platelet CD36
facilitates rapid interactions between platelets and VWF and oxidized LDL enhances platelet activation and thus
in the plaque that can resist arterial shear forces acting provides a mechanistic link between dyslipidemia, oxida
on the cells.16 tive stress, and a prothrombotic state. Platelet CD36 also
The pivotal role of GPVI in arterial thrombus forma binds microparticles generated from endothelial cells,
tion is supported by a study of Massberg and colleagues, monocytes, or platelets in response to vascular injury,
who showed that the genetic knockout of GPVI, or the especially in patients with prothrombotic diseases. 20
inhibition of GPVI with a specific monoclonal antibody, Activation of CD36 promotes thrombus formation even
virtually abolished plateletvessel-wall interactions in the absence of dyslipidemia, as supported by experi
in mice after endothelial denudation.17 Accordingly, mental data showing that thrombus formation and micro
Cheli etal. showed that a subset of Gpvi-knockout mice particle accumulation in thrombi are markedly reduced
bearing a single-nucleotide polymorphism on chromo in mice with a deficiency in CD36.21 Inhibition of CD36
some4which was designated as modifier of hemo might, therefore, be a promising strategy for the treatment
stasiswere characterized by extremely prolonged ofatherothrombosis.
bleeding times and impaired thrombus formation when
the carotid artery was injured with ferric chloride. Mice Potential role of thrombin
that did not have this polymorphism, however, had Protease-activated receptor (PAR)4 is the main thrombin
normal bleeding times despite their genetic deficiency receptor in platelets.22 In an experimental setting, mice
in GPVI.18 lacking PAR4 are protected from arterial thrombosis,23
indicating that the activation of platelets by thrombin
Role of integrins is essential for the initiation of the hemostatic process.
The initial phase of platelet activation, which might be Accordingly, both PAR-4 and PAR-1, which is a high-
reinforced by soluble mediators released from platelets, is affinity thrombin receptor, represent promising antithrom
followed by a shift in the conformation of platelet integrins. botic targets that might provide effective platelet inhibition
In particular, integrinIIb3 (also known as GPII/III, without the liability of increased bleeding associated with
integrin32, or GPIIb/IIIa), which binds VWF and dual antiplatelet therapy.24,25 However, thrombin is not at
fibrinogen, changes into a high-affinity state upon plate all, or is only marginally, generated in the early phase of
let activation. The release from platelets of secondary arterial thrombus formation26 and, therefore, an impor
mediators such as ADP and thromboxaneA2 mediates tant role for thrombin in initial platelet activation seems
firm adhesion, spreading, coagulant activity, and aggre unlikely. Instead, tissue-factor-induced thrombin accu
gation of platelets.16 Platelet integrin21 is also essential to mulation might be important in triggering secretion of
this process because it mediates collagenplatelet binding platelet granules and the exposure of phosphatidylserine
under conditions of low shear rates (for example near on the platelet membrane, which would provide an ideal
an atherosclerotic plaque) and promotes the association procoagulant (platelet) surface for supporting the assembly
between collagen and GPVI. and activation of coagulation factors.27

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Activation of blood coagulation Blood vessel lumen


Blood coagulation generally develops after the activa
tion and aggregation of platelets. Blood coagulation ADP TXA2
involves several sequential, calcium-dependent enzyma
Platelet
tic reactions encompassing the conversion of inactive activation
proenzymes into the respective serine proteases, and
culminates in the generat ion of thrombin (Figure3
andFigure4). The activation of the coagulation cascade
and the subsequent stabilization of the blood clot are the
result of a delicate balance between the activity of spe Platelet
cific procoagulant proteases and respective anticoagulant aggregation
enzymeinhibitors.

Intrinsic and extrinsic pathways Endothelial


cells
Since 1964,28,29 the activation of blood coagulation has
been schematically subdivided into two main pathways Atherosclerotic plaque Intima
Collagen
the intrinsic and the extrinsic pathways (Figure4). Media
This model has gained much success in both clinical and
laboratory practice, especially owing to its ideal reflec
tion in first-line coagulation tests such as prothrombin
time and activated partial thromboplastin time. The GPVI GPIIb/IIIa VWF Necrotic cells,
cell-derived microparticles,
modern concept of the activation of blood coagulation fat droplets,
invivo, however, is centered on the extrinsic pathway, GPIb-IX-V GPIa/IIa Fibrinogen oxidized LDL etc.
because the activation of the intrinsic pathway does not Figure 2 | Platelet activation and aggregation in the context of arterial thrombus
seem to contribute substantially to the initiation of the formation. The formation of a platelet aggregate is initiated by the interaction
physiological hemostatic process (but is involved in the of platelet surface receptors GPIbIXV and GPVI with VWF and collagen,
boost phase).27 This idea is supported by clinical evi respectively. The initial phase of platelet activation is followed by a shift in the
dence that patients with severe deficiencies in the coagu conformation integrins on platelets. GPIIb/IIIa (also known as GPII/III,
lation factors upstream of the intrinsic pathway (such integrinIIb3, or integrin32) binds VWF and fibrinogen, mediating firm adhesion,
spreading, coagulant activity, and aggregation of platelets. Platelet GPIa/IIa (also
as clotting factorXIIa [FXIIa], prekallikrein, and high-
know as integrin 21) is also essential for platelet activation, since it mediates
molecular-weight kininogen) do not have a substantial collagenplatelet binding under low-shear-rate conditions (such as those near an
risk of bleeding.30 However, the importance of FXIIa in atherosclerotic plaque) and promotes the association between collagen and
arterial thrombosis remains controversial, as evidence GPVI. ADP further promotes platelets to undergo a shape change, to release
has been provided that FXII (F12) knockout mice are granule contents, and to aggregate, whereas TXA2 exerts a positive feedback in
characterized by defective arterial thrombus formation terms of activation and recruitment of additional platelets to the primary
invivo.31 These experimental findings raise the possibil hemostatic plug. Abbreviations: GP, glycoprotein; TXA2, thromboxaneA2;
ity that FXIIa could influence the extent of a develop VWF, von Willebrand factor.
ing thrombus in the intravascular compartment and
might thus contribute to pathological arterial thrombus thrombus.26 Biologically active tissue factor then associ
formation without influencing hemostasis.32,33 ates within the thrombus, which triggers the generation of
fibrin. Leukocytes or leukocyte-derived circulating micro
Sources of tissue factor particles, which accumulate at the site of the lesion shortly
Evidence is growing in support of the hypothesis that after plaque rupture through interaction of Pselectin
tissue factor might act as a major procoagulant factor for with Pselectin counter-receptor1, might represent an
pathological arterial thrombosis and is essential for coagu additional source of tissue factor that supports thrombus
lation to occur. Nevertheless, the major source of tissue propagation at the site of vascular injury.35 In fact, anti
factor involved in arterial thrombosis formation is still bodies that block Pselectin inhibit the accumulation
being debated.2 Plaque tissue factor, which is expressed of leukocytes or leukocyte-derived circulating micro
on apoptosis-derived microparticles from endothelial cells particlesin the developing thrombus, and reduce fibrin
and macrophages and found in the lipid-rich core together formation inanimal models ofthrombosis.36
with macrophages and smooth muscle cells, seems to play Patients with coronary artery disease (CAD), hyper
a pivotal role in this process.12 lipidemia, disorders associated with enhanced intra
Confocal and intravital videomicroscopy are important vascular coagulation (such as sickle cell anemia and
techniques to study thrombus formation in real time in disseminated intravascular coagulation), or those with
genetically altered animals.33 Falati and colleagues used certain tissue factor genotypes (such as I/I)37,38 exhibit
this technique to study thrombus formation after laser increased circulating levels of tissue factor. One cannot
injury to the cremaster arteriole of a live mouse, and definitely exclude that these patients might feature higher
showed that the activation of blood coagulation devel blood thrombogenicity than patients with normal tissue-
ops through initial accumulation of tissue factor on the factor levels, and that the increased thrombogenicity
upstream and thrombusvessel-wall interface of the initial promotes the development of acute thrombotic events.

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FXaFVa FX Role of factor XIII


Blood vessel lumen FXIII has a tetrameric structure (also abbreviated as
FXIIIA2B2), consisting of two potentially active A sub
Fibrin Fibrinogen units (FXIIIA) and two inhibitory or carrier B subunits
(FXIIIB). FXIII inhibits plasma fibrinolysis via two main
mechanismsthe cross-linking of fibrinchains into
Prothrombin Thrombin FIXaFVIII
high-molecular-weight polymers, which renders the
clot more resistant to fibrinolysis,39 and the binding to
FXIIIa TAFI FX FXaFVa FXIa 2-plasmin inhibitor (2PI) and other components of the
FIX
fibrinolytic system, including plasminogen activator inhib
itor1 (PAI1), TAFI, plasminogen, and lipoprotein(a),
FXI which protects the fibrin from degradation by plasmin.41
TFFVIIa Interestingly, increased plasma levels of FXIII might
increase the extent of fibrin cross-linking and inhibit the
degradation of fibrin by exogenous plasmin.42

Endothelial Role of fibrinogen


cells
Fibrinogen is a key component of the hemostatic system.
Atherosclerotic plaque Intima Thrombin-catalyzed cleavage of fibrinopeptideA and
Collagen
fibrinopeptideB converts fibrinogen into fibrin, which
Media
spontaneously polymerizes and forms double-stranded
protofibrils; these protofibrils then assemble into branched
fibrin fibers that form the fibrin clot. Fibrinogen is com
VWF TF Necrotic cells,
cell-derived microparticles,
posed of pairs of three polypeptide chains (chain, chain,
Fibrinogen Fibrin fat droplets, and chain). A causal role of fibrinogen in the pathogenesis
oxidized LDL etc.
of CAD is suggested by at least two important pieces of evi
Figure 3 | The activation of blood coagulation in arterial thrombus formation. Blood dence. Firstly, fibrin is a prominent part of atheromatous
coagulation is initiated by the binding of tissue factor to FVIIa and supported by the plaques and, secondly, fibrin is a major component of final
thrombin burst, which encompasses a series of sequential conversions of inactive occlusive thrombi. As such, both the concentration and the
proenzymes into the respective serine proteases, culminating in the generation of
composition of the fibrin clot might determine the compo
thrombin. FXI, FX, and FIX are essential for the formation of the thrombin burst,
which acts as a physiological amplificatory mechanism that sustains and
sition and size of the thrombus, as well as the severity of the
propagates thrombin generation. Fibrin is then generated by the enzymatic action clinical outcome of an arterial thrombosis.43
of FX on fibrinogen. TAFI contributes to the downregulation of plasma fibrinolysis
by attenuating plasmin-mediated fibrin degradation. Abbreviations: F, factor; Potential role of Creactive protein
TAFI, carboxypeptidase B2 (also known as thrombin-activatable fibrinolysis Chronic inflammation plays a key role in the pathogenesis
inhibitor); TF, tissue factor; VWF, von Willebrand factor. of atherosclerosis and CAD.46 Inflammatory cytokines
can stimulate the generation of endothelial adhesion mole
Role of the thrombin burst cules, proteases, and other mediators, all of which enter the
Although tissue factor has a pivotal role in initiating blood blood flow in soluble form.47 Inflammatory cytokines can
coagulation, further activation of coagulation factorX also trigger the production of interleukin6 from a variety
through the formation of a complex between tissue factor of cells (especially lymphocytes and macrophages), which,
and factorVIIa (FVIIa) seems insufficient to support and in turn, stimulates the hepatic synthesis of acute-phase
maintain the next step of blood coagulation. This step reactants such as Creactive protein (CRP). Platelets44 and
coincides with the so-called thrombin burst and involves visceral adipose tissue45 are additional sources of other
all coagulation factors of the intrinsic pathway.27 The low proinflammatory mediators and potentially involved in
amount of thrombin generated by the extrinsic pathway the pathogenesis of atherothrombosis.4,46
(<2%) is, in fact, efficiently balanced by the much higher The close correlations between inflammation and
concentration of thrombin generated in the thrombin atherosclerosis have encouraged the use of inflamma
burst. This mechanism also exerts positive feedback on the tory biomarkers in the plasma for the prediction of major
intrinsic pathway, resulting in the generation of numerous cardiovascular events and death.4,5 The best-validated
procoagulant enzymatic complexes. The thrombin burst inflammatory biomarker to date is the plasma CRP level.
is also necessary for the activation of carboxypeptidase B2 A growing body of evidence suggests that CRP might
(also known as thrombin-activatable fibrinolysis inhibi also exert a thrombogenic effect. However, whether
tor [TAFI]), which contributes to the downregulation CRP actively participates in the pathogenesis of arterial
of plasma fibrinolysis by attenuating plasmin-mediated thrombosis, or whether CRP plasma concentrations
fibrin degradation.27 Blood coagulation culminates in merely correlate with the course of the disease, remains
the conversion of fibrinogen to fibrin, the polymeriza to be elucidated. Matsuda etal. have assessed whether
tion of fibrin, and in the stabilization of the fibrin network CRP influences arterial thrombus formation after balloon
through the enzymatic activity of coagulation factorXIII injury of smooth-muscle-cell-rich or macrophage-rich
(FXIII) and TAFI (Figure3 and Figure4).39,40 neointima in rabbits.47 The investigators found that

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thrombus size was remarkably increased in transgenic Extrinsic pathway


rabbits overexpressing human CRP and correlated with an Endothelial cell
elevated fibrin content compared with wild-type animals. TFPI

Moreover, mRNA expression and the activity of tissue


factor in neointimal lesions were increased in the trans TF + FVIIa FVIIIa + FIXa
genic rabbits. Notably,the degree of CRP deposition in
plaques correlated with the elevated expression of tissue FX FXa + FVa FX APCPS
factor mRNA and enzymatic activity, as well as with the FXIa
APCPS
size of the thrombus on the injured neointima.47 These FIX
findings are supportive of the hypothesis that CRP might, PC
at least partially, contribute to arterial thrombus formation Prothrombin Thrombin
by enhancing the expression of tissue factor. FXI Intrinsic pathway
However, other mechanistic and animal studies have
TAFI TAFIa
provided evidence against a causal role of CRP in the 2-plasmin
tPA
development of atherothrombosis.4,5 Teupser etal. studied inhibitor
Lipoprotein(a) PAI-1
atherosclerosis-susceptible mice (deficient in apolipo
proteinE [Apoe/] or LDL receptor [Ldlr/]) with addi FXIII FXIIIa Plasmin Plasminogen
tional genetic deletion of Crp.48 The investigators observed
that atherosclerotic lesions were similar, or even enlarged, Fibrinogen Soluble Stabilized FDP
fibrin fibrin Fibrinolysis
in Crp-deficient mice compared with atherosclerosis-prone
control animals.48 Similarly, Koike and colleagues failed Figure 4 | Coagulatory and fibrinolytic systems. Blood coagulation is initiated by
to observe a difference in atherosclerotic lesion formation endothelial-derived tissue factor and then supported by the thrombin burst after
in the aorta or coronary arteries of transgenic rabbits that a sequence of calcium-dependent conversion of inactive proenzymes into the
express human CRP.49 The disparities observed among respective serine protease that culminates in the generation of thrombin. Thefactors
these studies have been attributed to the fact that these of the intrinsic pathway (FXI and FIX) are mainly responsible for the thrombin burst,
which acts as a physiological amplificatory mechanism that sustains and propagates
mouse models might inappropriately reproduce human
thrombin generation. Fibrin is then generated by thrombin from fibrinogen. Blood
physiology, as the concentration of CRP in mice is much coagulation is inhibited by aPCPS through enzymatic degradation of activated FV and
lower that that in humans.50 Moreover, CRP cannot activate FVIII. TAFI attenuates plasmin-mediated fibrin degradation and thereby contributes
the complement system in mice andrabbits.51 to the inhibition of fibrinolysis. The subsequent activation of fibrinolysis involves the
degradation of fibrin polymers during the removal of blood clots and thereby
Fibrinolysis contributes to physiological wound healing and hemostasis. The main component of
Fibrinolysis is the third important hemostatic pathway the fibrinolytic pathway is plasminogen, which is converted into the serine proteinase
besides primary and secondary hemostasis and is acti plasmin by plasminogen activators. Abbreviations: APCPS, activated PC; F, factor;
FDP,fibrin/fibrinogen degradation products; PAI1, plasminogen activator inhibitor1;
vated once the coagulation cascade has stabilized the clot
PC,proteinC; PG, plasminogen; PS, proteinS; TAFI, carboxypeptidase B2 (also
(Figure4). Fibrinolysis involves the degradation of fibrin known as thrombin-activatable fibrinolysis inhibitor); TF, tissue factor; TFPI, tissue
polymers during the removal of blood clots and thereby factor pathway inhibitor; tPA, tissue-plasminogen activator.
contributes to physiological wound healing and hemo
stasis. The main component of the fibrinolytic pathway is
plasminogen, which is converted into the serine proteinase disorders or variants predispose to an increased risk for
plasmin by plasminogen activators (PA), including tissue- CAD in older individuals has not been fully elucidated.
type PA (tPA) and urokinase-type PA (uPA). Plasmin then Thus, genetic studies aimed at examining the inherited
degrades fibrin and extracellular matrix proteins such as basis of CAD are warranted.62 Although the identification
fibronectin,52 laminin,52 proteoglycan,53 and typeIV col and management of established cardiovascular risk factors,
lagen.54 The fibrinolytic system is tightly regulated by such as dyslipidemia, hypertension, smoking, diabetes
the naturally occurring inhibitors PAI1 and 2-plasmin mellitus, and obesity remain the cornerstones for reduc
inhibitor.55 The size and composition of an arterial throm ing the global burden of CAD, investigating the genetic
bus is the result of a fine and complex balance between basis of CAD might improve our understanding of the
continuous fibrin formation and fibrinolysis. The activ etiologyof this pathology.62
ity of the plasma fibrinolytic system is markedly down Unfortunately, the majority of studies searching for
regulated in patients with CAD and other manifestations genes that promote CAD have produced inconclu
of atherosclerosis.5659 sive findings and are plagued by drawbacks. Problems
include an inadequate sample size that does not enable
Risk factors of arterial thrombosis true positive signals to be distinguished from casual
Genetic risk factors associations, or the uncertainty of the basic assump
Genetic polymorphisms associated with CAD tion that the proteins encoded by the chosen candidate
Complex diseases such as CAD are caused by multiple genes are mechanistically relevant.6371 However, two
interactions between genes, or between genes and the major advances over the past decade have renewed the
environment. Although the development of CAD in young enthusiasm in this field of researchthe completion
individuals (typically under the age of 40years) is often of the reference sequence of the human genome through
related to genetic factors,60,61 whether or not certain genetic the Human Genome and the International HapMap

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Table1 | Genes and chromosomal loci associated with CAD or myocardial infarction
Study n Chromosome/SNP Genes Odds ratio
(95% CI)
Cases Controls
Helgadottir etal.118 7,790 624 13q1213/HapA* ALOX5AP 1.80
Zwicker etal. 119
1,425 1,425 N700S
TSP1 1.40 (1.11.8)
Helgadottir etal.120 1,553 863 12q22/HapK* LTA4H 1.45||
Helgadottir etal. 84
4,587 12,767 9p21.3/rs2383207 ALOX5AP 1.25 (1.181.31)
9p21.3/rs10757278 CDKN2A, CDKN2B 1.28 (1.221.35)
McPherson etal.86 3,989 18,808 9p21.3/rs10757274 CDKN2A, CDKN2B 1.38 (1.191.60)
9p21.3/rs1333049 CDKN2A, CDKN2B 1.37 (1.261.48)
Samani etal.87 875 1,644 9p21.3/rs4977574 CDKN2A, CDKN2B 1.36 (1.201.53)
9p21.3/rs1333049 CDKN2A, CDKN 2B 1.33 (1.181.51)
1p13.3/rs599839 CELSR2, PSRC1, SORT1 1.20 (1.101.31)
2q36.3/rs2943634 Pseudogene 1.21 (1.131.30)
10q11.2/rs501120 Unknown 1.33 (1.201.48)
6q25.1/rs6922269 MTHFD1L 1.23 (1.151.33)
WTCCC88 1,926 3,000 9p21.3/rs4977574 CDKN2A, CDKN2B 1.35 (1.241.46)
9p21.3/rs1333049 CDKN2A, CDKN2B 1.37 (1.261.48)
1p13.3/rs599839 CELSR2, PSRC1, SORT1 1.29 (1.181.40)
1q41/rs17465637 MIA3 1.20 (1.121.30)
6q25.1/rs6922269 MTHFD1L 1.23 (1.011.50)
MIGen92 12,713 12,821 21q22/rs9982601 SLC5A3, MRPS6, KCNE2 1.20 (1.141.27)
6p24/rs12526453 PHACTR1 1.12 (1.081.17)
2q33/rs6725887 WDR12 1.17 (1.111.23)
10q11/rs1746048 CXCL12 1.17 (1.111.24)
1q41/rs17465637 MIA3 1.14 (1.101.19)
19p13/rs1122608 LDLR 1.15 (1.101.20)
1p13.3/rs646776 CELSR2, PSRC1, SORT1 1.19 (1.131.26)
9p21.3/rs4977574 CDKN2A, CDKN2B 1.29 (1.251.34)
1p32/rs11206510 PCSK9 1.15 (1.101.21)
Erdmann etal.93 19,407 21,366 3q22.3/rs9818870 MRAS 1.15 (1.101.19)
12q24/rs2259816 HNF1A 1.08 (1.051.11)
Gudbjartsson etal. 121 6,650 40,621 12q24/rs3184504 SH2B3 1.13 (1.081.18)
Tregouet etal.122 8,999 10,263 6q2627/CTTG* SLC22A3-LPAL2-LPA 1.20 (1.131.28)
6q2627/CTTC* SLC22A3-LPAL2-LPA 1.82 (1.572.12)
He etal.123 1,003 1,003 +190CC HSPA1A 1.56 (1.102.20)
110C/+190C* HSPA1A 1.17 (1.011.34)
Clarke etal.124 3,145 3,352 6q2627/rs10455872 LPA 1.70 (1.491.95)
6q2627/rs3798220 LPA 1.92 (1.482.49)
*Haplotype; genotype; polymorphism; ||relative risk. Abbreviations: CAD, coronary artery disease; SNP, single-nucleotide polymorphisms.

projects,7274 and the availability of high-throughput Polymorphisms of blood coagulation proteins


genotyping platforms,72,75 which made possible a new In the context of arterial thrombus formation, five frequent
type of genetic study known as genome-wide association polymorphisms resulting in an amino acid exchange in
studies (GWAS). GWAS are large-scale population-based the A subunit of FXIII (Val34Leu, Tyr204Phe, Leu564Pro,
studies based on chip technologies in which identification Val650Ile, and Glu651Gln) have been described.41 The
of associations between genetic markers and particular Val34Leu polymorphism has attracted considerable atten
clinical phenotypes, including CAD, are attempted.7678 tion owing to a suspected thromboprotective effect. From
Large collaborative efforts have led to the identification a biochemical perspective, the Val34Leu FXIII variant
of some genetic variants that are consistently associated displays an approximately 2.5fold higher rate of throm
with CAD, but which globally confer a relatively small bin activation, which is associated with enhanced fibrin
increased risk of disease (with odds ratios ranging from cross-linking and an increased rate of incorporation of
1.21.6) owing to their relatively high frequency (>5%) 2plasmin inhibitor into fibrin. Another mechanism
in the general adult population.7995 Table1 summarizes linking this polymorphism with thrombosis is related
the most important chromosomal loci associated with an to the final structure of fibrin clots inasmuch as blood
increased risk of CAD that have been identified by GWAS. clots generated in the plasma of Val34Leu homozygotes
However, more research is needed to define the culprit are characterized by a loose structure with increased
genes and causal variants as well as to better elucidate the clot permeability and thick fibers and, therefore, display
pathophysiological mechanisms through which they higher susceptibility to fibrinolysis.41,96 The odds ratios for
might influence the protein/peptide risk factors that play CAD for carriers of the FXIIIVal34Leu variant are 0.82
a role in the pathogenesis of arterialthrombosis. forheterozygotes and 0.89 for homozygotes.96

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Findings from several studies have convincingly the association between plasma homocysteine levels and
demonstrated an influence of fibrinogen polymorphisms CAD were assessed.109 The investigators concluded that
on fibrinogen plasma concentration. The most striking hyperhomocysteinemia is a weak predictor of CAD in
association has been demonstrated for the 455A allele, low-risk, healthy people.109 Conversely, clinical and experi
which can increase the mean concentration of plasma mental evidence attests that increased levels of homo
fibrinogen by ~0.15g/l (the normal plasma concentration cysteine might play a synergic role in the pathogenesis of
of fibrinogen is 1.54.0g/l).97 CAD in combination with other risk factors, such as raised
Significant associations between the genetic variants of concentrations of LDL, lipoprotein(a), orboth.110
fibrinolytic markers, such as the 4G/5G PAI1 gene poly Using data from the Atherosclerosis Risk in Com
morphism, or the 325Thr/Ile TAFI gene polymorphism, munities (ARIC) cohort, 111 Kucharska-Newton and
and the risk of CAD have also been reported, but data are colleagues assessed the risk of incident CAD among indivi
conflicting.98 These inconclusive findings have produced duals with elevated levels of markers of inflammation and
a general skepticism among investigators about the feasi hemostasis (including fibrinogen, VWF, and FVIIIc)
bility of linking polymorphisms of blood coagulation pro compared with those with normal levels. Third versus first
teins with the prevalence of CAD. Even more importantly tertile hazard ratios for the association between nonfatal
the costbenefit ratio of routine genetic screening of myocardial infarction and fibrinogen were 1.91 (95%CI
patients has not been investigated in any of these studies 1.552.36), for VWF were 1.37 (95%CI 1.131.66), and
and therefore represents a hot topic for futureresearch. for FVIIIc were 1.31 (95%CI 1.081.60).111
The antiphospholipid antibody syndrome (APS)
Acquired risk factors usually describes the association between the presence of
A number of epidemiological studies have identified antiphospholipid (aPL) antibodies (comprising anticardio
several acquired states that might predispose patients to lipin antibodies, anti-2GP1 antibodies and lupus anti
arterial thrombosis.99 These include elevated blood levels coagulants) and the risk of thrombosis. The prevalence of
of homocystein, CRP, fibrinogen, factorVIIc (FVIIc), aPL antibodies in the general population is relatively low
factorVIIIc, or PAI1, the presence of antiphospholipid (15%), but increases substantially inpatients with venous
antibodies in the blood, hereditary thrombophilias, and thrombosis (up to 10%) and in those with systemic lupus
platelet hyper-reactivity.99 At present, clinical evidence for erythematosus (up to 50%).112 Ben-Ami etal. investigated
elevated fibrinogen, CRP (high-sensitivity test; hs-CRP), associations between APS and arterial stenosis.113 They
hyperhomocysteinemia, and antiphospholipid anti found that APS involves arteries of varying sizes, occurs
bodies as potential risk factors for arterial thrombosis at several different locations (with higher prevalence in
is convincing. Data on FVIIc and platelet receptor poly renal, gastrointestinal, and intracranial arteries), and
morphisms are contradictory or lacking.99 Two indepen encompasses pathogenetic mechanisms such as acceler
dent studies failed to observe any significant association ated atherosclerosis, proliferation of smooth muscle cells,
between FVIIc and FVIIa with prevalent cardiovascular and superimposed thrombosis. Notably, acute myocardial
disease.100,101 Similarly, large prospective studies have infarction and stroke accounted for up to 30% of deaths
failed to demonstrate a significant association between in patients with APS.114
any platelet receptor polymorphism investigated and Plasma levels of PAI1 have been studied in various
thromboticoutcomes.102106 clinical settings characterized by acute thrombotic
Findings of a meta-analysis involving 31 prospective complications, such as CAD, ischemic stroke, vascu
studies and a total of 154,211 individuals indicated a signifi lar death, and the metabolic syndrome.46,5759 However,
cant positive association between plasma fibrinogen levels findings are conflicting, possibly owing to concerns
and cardiovascular risk.107 After adjustment for age and sex, about study design, the presence of confounders (such
the hazard ratio for an increase in plasma fibrinogen (per as traditional risk factors), as well as methodological
gram of fibrinogen per liter) was 2.42 (95%CI 2.242.60) and biological issues. For example, plasma PAI1 levels
for CAD, 2.06 (95%CI 1.832.33) for ischemic stroke, do not mirror the local PAI1 release from activated
and for vascular mortality 2.76 (95%CI 2.283.35). platelets during thrombogenesis, the periprocedural
Although the question of whether CRP is causative, platelet activation might spuriously increase PAI1,
or represents an epiphenomenon of underlying chronic and uncertainty still exists as to how to measure PAI1
inflammation, remained unanswered, a meta-analysis of antigen or PAI1 activity. Moreover the number of nega
54 long-term prospective studies involving 160,309 indivi tive studies, in which no association or predictive power
duals concluded that blood hsCRP levels are linearly cor was found, is equal to those reporting a positive associa
related with the risk of cardiovascular disease.108 When tion; therefore, well-conducted prospective studies are
adjusted for age, sex, traditional risk factors, and fibrino needed to clarify whether PAI1 plasma levels influence
gen, the relative risks per threefold increase in hs-CRP the risk of atherothrombosis.98,115116 Nevertheless, the
concentration were 1.23 (95%CI 1.071.42) for CAD, 1.32 potential involvement of PAI1 and other fibrinolytic
(95%CI 1.181.49) for ischemic stroke, and 1.34 (95%CI markers in the plasma in adverse thrombotic events
1.181.52) for vascular mortality. related to modern, drug-eluting coronary stents has
Lhmann etal. performed a systematic overview of renewed the scientific interest in unraveling the molecu
11 reviews and 33 epidemiological studies (prospective lar processes involved in fibrinolysis and in modulating
cohort studies and nested casecontrol studies) in which fibrinolysispharmaceutically.5759

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REVIEWS

With regards to lipoprotein(a), the important role of this an injured atherosclerotic plaque, the formation of an
unique lipoprotein in atherothrombosis is supported by arterial thrombus involves platelet adhesion and aggre
several lines of evidence, attesting that lipoprotein(a) might gation. This process initially leads to the formation of a
accelerate atherosclerosis (by delivering large amount labile platelet plaque, which is then stabilized by insoluble
ofcholesterol within the plaque, triggering production of fibrin, mainly produced as a result of the activation of the
proinflammatory cytokines, and promoting the recruit coagulation cascade. Factors involved in the formation of
ment of monocytes and the migration of smooth muscle arterial thrombi include GPIb-IXV, GPVI, integrin21,
cells), and that lipoprotein(a) also impairs fibrinolysis GPIIb/IIIa, collagenous plaque components (fibronectin,
(by inhibiting the generation of the tPAfibrin complex collagens, and VWF), and blood coagulation proteins.
through direct interaction with tPA and by enhancing Accumulating evidence also suggests that some inherited
expression of PAI1).95,110 factors related gene polymorphisms and acquired pre
Although plasma fibrinolysis has a central role in art disposing conditions (for example the concentration and
erial thrombosis, we are disappointed by the results of the activity of clotting factors) can contribute to the compo
published studies evaluating the predictive value of plasma sition and size of arterial thrombus. Therefore, although
fibrinolytic parameters for CAD. One reason is the uncer we already have a good basic knowledge of pathological
tainty about what to measure. Proposed markers include arterial thrombus formation, more research is required to
antigens, enzymatic activity, or genetic polymorphisms further elucidate the functions of the many proteins and
of fibrinolytic proteins. Another concern is the inherent cellular elements that have important roles in this process.
limitation of using a simple factorial assessment to quantify A better knowledge of the pathophysiological mechanisms
the complex and dynamic process of plasma fibrinolysis.98 of arterial thrombus formation should enable the develop
So far, no parameter provides a quick and meaningful, ment of new therapeutic approaches to the treatment
individualized assessment of a patients fibrinolytic status as ofatherothrombosis.117
it would be needed in the clinic. Large, prospective studies
are required to examine the predictive value of plasma Review criteria
fibrinolytic markers for CAD, ideally using a simple, rapid,
Relevant articles published between 2001 and 2011
comprehensive, point-of-care test that measures overall were selected from electronic databases (MEDLINE,
plasma fibrinolytic capacity. EMBASE, and Google scholar) using the key words
arterial thrombus formation, mechanisms of
Conclusions thrombosis, pathogenesis of atherosclerosis,
The development of arterial thrombosis is a highly complex pathogenesis of thrombosis, and review. Articles
and dynamic pathological process that differs from that published in languages other than English were excluded
from the analysis.
of venous thrombosis. Originating as a consequence of

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