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Chapter 6
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Animal Models of Thrombosis
Gemma Vilahur
1
Program ICCC, IR-IIB-HSCiSP, Barcelona, Spain;2CIBERCV-ISCIII, Madrid, Spain;3UAB, Barcelona, Spain
1,2
, Soumaya Ben-Aicha1and Lina Badimon
1,2, 3
BRIEF OVERVIEW OF THE PATHOGENESIS OF THE THROMBOTIC PROCESS
Arterial thrombosis frequently develops on top of a disrupted atherosclerotic plaque (atherothrombosis) because of the
sudden exposure of thrombogenic materialdcollagen and tissue factor (TF)dto the circulation [1]. TF, expressed by either
the vascular subendothelium or the macrophage- and/or vascular smooth muscle-derived foam cells resident within the
atherosclerotic lesions, is crucial in the initiation of thrombosis because of its capability to trigger the coagulation cascade
thereby leading to thrombin generation (a powerful platelet agonist) and subsequent fibrin deposition at sites of plaque
rupture [1]. Platelets adhered at the site of injury also play a key role in thrombus formation. Platelet surface receptors,
mainly glycoproteins (GPs), and particularly GP Ib/IX/V, interact with von Willebrand factor (VWF) and/or vascular
structures (mainly collagens), inducing platelet activation. In addition to plateletevascular interaction, circulating agents
such as serotonin, adenosine diphosphate, and TF-derived thrombin are also capable of inducing platelet activation by
interacting with several platelet surface receptors. Platelet activation, in turn, is accompanied by an increase in the cytosolic
concentration of Ca
the intraplatelet granule content. All these events concur with the activation of the platelet receptor GP IIb/IIIa, which
mediates plateleteplatelet interaction (i.e., platelet aggregation) mainly through its binding with fibrinogen and to a lesser
extent to VWF. Fairly recent reports have suggested that platelet-derived microparticles (submicrometer vesicles released
from the plasma membrane of activated cells) and neutrophil extracellular traps (nuclear chromatin and proteins of nuclear,
granular, and cytosolic origin extruded from neutrophils) also favor thrombus formation and growth [2e4]. In contrast to
this arterial platelet-rich thrombus (so-called “white thrombus”), which develops at sites o f atheroscler ot ic plaque
rupture, venous throm bi are rich in red blood cells (“red thrombus”), since they mostly develop because of the presence
of e ndot he lial injury, flow stasis, and hypercoagulabi lity. In the latter regard, initiation of the coagulation cascade may
occur through the activation of the factor VIIeTF pathway (“exogenous pathway”) or via the contact activati on pathw ay
(“endogenous pathway”), which proceeds through factors XII, XI, and IX to the assem bly of a tenase complex. Both the
exogenous and the endogenous pathways can activate factor X, which induc es the formation of the prothrombinase
complex, consisting of factor Xa, factor Va, and Ca
complex leads to the activation of thrombin, which cleaves fibrinogen to fibrin. The fibrin clot is eventually stabilized by
factor XIIIa [5].
2þ
, cellular shape change with the consequent exposure of a procoagulant surface, and the secretion of
2þ
, on p hosp holi pi d surface s. Asse mbly of the prot hrom bina se
DEVELOPMENT OF ANIMAL MODELS OF THROMBOSIS
Without a doubt in vitro studies have helped to identify molecular and cellular mechanisms involved in thrombus formation. However, they have a major disadvantage, because in vitro approaches are removed from their natural environment, thereby disregarding in vivo interactions with multiple vascular and blood elements, as well as the hemodynamic/
hemorheologic conditions that occur during the generation and propagation of thrombi. In addition, the triggers and
conditions tested in vitro generally differ from those that occur under pathophysiological conditions, resulting in artifacts
that do not reflect real thrombus formation [6].
Cardiovascular Thrombus. https://doi.org/10.1016/B978-0-12-812615-8.00006-5
Copyright © 2018 Elsevier Inc. All rights reserved.
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FIGURE 6.1 Parameters to take into account when choosing the animal model of thrombosis.
In contrast, animal models of thrombosis have b ecome a vital par t of the identi fication of factors and pathways that
drive in vivo thrombosis, and most importantly, have played a crucial role in the discovery and development of new
antithrombotic drugs that are now successfully bein g used for the treatment and prevention of thrombotic diseases
[7,8]. So far, several models of thrombos is have been developed in both small and large animals [9]. Whereas mice,
rats, rabbits, dogs, pigs, and nonhuman primates are well-established animal models of thrombosis, hamsters, cats,
andguineapigshavebeenusedtoalesserextent[10]. Fig. 6.1 highlights the parameters that should be taken into
account when choosing the animal model of thrombosis. Also very important to take into account, although the
majority of animal models of thrombosis are performed in young and healthy animals, thereby not addressing the
underlying pathological cause, the ideal animal model should be as representative as possible of the human thrombotic disease.
SMALL ANIMAL MODELS OF THROMBOSIS: THE MURINE MODELS
Mice are the most common animal species utilized as a research tool in thrombosis for several reasons [11,12]:
1. Mice are a mammalian system.
2. There is a wealth of biological information available for research.
3. Mice are low cost, are readily available, and present reduced ethical concerns compa red with large animals.
4. The mouse genome can be manipulated, providing numerous transgenic, knockout, and knock-in lines for a multitude
of genes, allowing one to determine the impact of multiple platelet receptors, coagulation factors, and blood compo-
nents on thrombus formation.
5. The small size of mice limits the quantities of new agents required for in vivo screening of new antithrombotic
compounds.
In counterpart, however, their utility for extrapolating the findings to human d isease or as preclinical models to prove
the validity of pharmacologic agents is less robust. Furthermore, it has been questioned whether transgenic mouse
models should not be trusted blindly, since compensating mechanisms and redundancies may affect their thrombotic
phenotype.
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FIGURE 6.2 Murine models of carotid (arterial) thrombosis.
So far, multiple murine models of thrombosis have been performed in both the carotid artery and the microvasculature
[13]. Fig. 6.2 depicts the various models of carotid (arterial) thrombosis in which arterial thrombus formation is mainly
assessed by flow monitoring. As per microvascular thrombosis, this has been mainly carried out by intravital microscopy
studies in the cremaster muscle, the connective fascia attached to the mesenteric structures, and/or the hairless ear. Intravital
microscopy uses precise pulses of laser light to induce thermal injury to the exposed vessel, with the subsequent monitoring
of the site (at high magnification) over time using fluorescence microscopy for image acquisition of thrombus-targeting
fluorophores (extensively reviewed in Masedunskas et al. [14]). Quantitation of the relative fluorescence of multiple
labels over time offers the advantage of monitoring the dynamics of thrombus formation in these thin, transparent vessels,
thus providing a great insight into specific cellular and molecular interactions within the developing thrombus [13,14].
Overall, both carotid and microvascular murine thrombosis models have shown strong utility as generic models of
thrombus induction for evaluating how subsequent thrombus generation progresses under the systemic conditions inherent
to the mouse genotype or pharmacologic state [15]. However, the size differential between mice and humans limits the
relevance to clinical thrombosis. In fact, none of these models parallel how we think a clinical thrombus might be
generated. In addition, other species-related differences between rodents and humans should also be considered before
interpreting the data. For instance, platelet counts in mice on average are four times those of humans, and platelets are only
approximately one-half the volume of human platelets. Moreover, rat platelets synthesize little or no thromboxane A
bringing the aspirin data reported in this animal model into question. All these reasons may help to explain why rodents
have not been as widely used as larger animal models to test possible therapeutic usefulness of antithrombotic agents [16].
Another example in which different results have been obtained between small and large animal models of thrombosis
concerns the analysis of relevant TF pools. Studies in groups using the same low-TF-expressing mice reached different
conclusions regarding the relevant source of TF in vascular thrombus formation, one group supporting the contribution of
vessel-related TF [17], whereas the other emphasized the key role of blood-borne TF as a major thrombogeni c stimuli [18].
We and others, by using large animal models of thrombosis, have supported the hypothesis that vessel-wall-derived TF is a
primary contributor to arterial thrombus formation and propagation, yet, blood-borne TF may also contribute, depending
on the triggering lesion and the shear rate [1,19]. On top of all these limitations, an accurate in-depth determination of
the mouse hemostatic system (i.e., coagulation and fibrinolytic systems, platelet structure, and platelet receptor/enzyme
system) is still lacking [20].
,
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LARGE ANIMAL MODELS OF THROMBOSIS
More advanced experimental models, including dogs, pigs, and nonhuman primates, have become essential to our
understanding of the mechanisms that promote throm bogenesis in humans while testing for new antithrombotic therapies
[21]. Because of the animal size, these animal models allow one to perform serial blood collections for coagulation tests,
platelet function assays, pharmacokinetic studies, and molecular analyses. Yet, not all the large experimental animal
models have human resemblance and validity, and species-related differences should be regarded before interpreting the
data Moreover, the disadvantages of large animal models are primarily the reverse of the advantages of small animal
models (high cost, heightened ethical concerns, less precise genetic characterization, difficulties involved in maintaining
the colonies and their handling, and a scarcity of transgenic models and antibodies) [6].
Canine Models of Thrombosis
Folts and colleagues [22] described in 1976 a model of repetitive thrombus formation, assessed by cyclic flow reductions
by an electromagnetic flow probe, in stenosed coronary arteries of open-chest anesthetized dogs. The Folts’ model is based
on the combination of severe , concentric stenosis and focal intimal injury in canine coronaries (circumflex and left anterior
descending) [23]. Yet, this model was lately modified for its use in both femoral and carotid rabbit arteries. The Folts’
model mainly induces platelet-rich thrombus formation and is an attractive choice for initial antithrombotic effectiveness
in vivo, especially for assessing prostaglandin-inhibitor compounds, recombinant factor VII, prostacyclin mimetics, or
platelet GP IIb/IIIa receptor antagonists (e.g., abciximab, tirofiban, etc.) [24e28]. In this regard, the preclinical data
obtained with this animal model has been further corroborated in clinical trials such as the Evaluation of 7E3 for the
Prevention of Ischaemic Complications (EPIC; abciximab) [29] and Platelet Receptor Inhibition in Ischemic Syndrome
Management (PRISM; tirofiban) investigator trials [30].
Canine thrombosis can also be challenged by advancing a thrombogenic coil into a coronary artery in closed-chest
animals [31]. The composition (alloy) and size of the coil determine the time to occlusive thrombus formation
confirmed by a filling defect distal to the coil. This model, in contrast to the Folts model, induces platelet-poor and fibrinrich thrombus and thus enables one to test thrombolytic agents [32]. Important drawbacks, however, are the high incidence
of ventricular fibrillations (which are around 20%) and the requirement of expensive equipment and well-trained
researchers, which limits the efficiency of this approach. To overcome these disadvantages Bush and collaborators [33]
developed a femoral artery version of this model that uses the same thrombogenicity coils but consists of Doppler flow
probes placed proximal to the site of thrombosis and thus is also appropriate to use in rabbits.
Another model of coronary thrombosis in open-chest dogs is based on the local delivery of thrombin in the left
anterior descendin g coronary arte ry . This approa ch induces fibrin-rich clots amenable to lysing upon thrombolytic
administration [34].
Nonhuman Primates, Pigs, and Equines
Pig and nonhuman primate thrombosis models have flow hemodynamics, hematologic parameters, platelet function,
coagulation, fibrinolysis, and, overall, a cardiovascular system that more closely resemble those of humans Fig. 6.4
In addition, nonhuman primates are semiupright animals and present some human immunologic overlap, which allows
utilization of available assays [21].
The most commonly used model of thrombosis in nonhuman primates includes the arteriovenous (AV) shunt models in
which thrombus formation is mainly assessed by platelet radiolabeling (see Arteriovenous Shunt and Perfusion Chambers:
In Vivo/Ex Vivo Models of Thrombosis in Large Animal Models). This model has been demonstrated to be an optimal tool
for preclinical assessment of novel thrombotic agents prior to administration in clinical trials [35]. However, the use of
nonhuman primates has significant disadvantages, including ethical considerations, cost of protocols, necessity for
specialized equipment, viral zoonosis (e.g., Macacine herpesvirus 1 in Macaca genus), and the need for highly trained
personnel to perform research studies. These drawbacks have stressed the use of more accessible and less costly large
animal models, such as the porcine model.
Pigs have a heart that is anatomically similar to that of humans except for the presence of the left azygous (hemiazygous) vein, which drains the intercostal system into the coronary sinus [36], and for the size, which tends to be a bit
smaller. The heart blood supply is mostly right-side dominant, since it originates from the posterior septal artery [37], and
both the anatomy and the function of the pig coronary system, as well as the histological anatomy of the aorta, are
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comparable to those of humans. However, pig blood vessels are more friable and prone to vasospasm during manipulation,
and thus require careful handling during blood withdrawals [38]. In relation to pig hemodynamics, either physiological
cardiac function or mechanically induced myocardial infarction and the subsequent arrhythmogenic activity in reperfusion
are also analogous to those in humans, as well as the wound healing process [39].
As for nonhuman primates, the “ex vivo/in vivo” AV shunt model has been widely implemented.
Finally, while the horse is an extremely appealing model for cartilage-related studies [40], it has not been used in the
field of thrombosis and hemorrhage. Studies with horses not only involve a highly specialized and well-equipped center
with well-trained personnel to carry out equine surgeries, but also require a large specialized habitat, leading, overall, to
substantially increase costs. In addition to these practical/econo mic considerations, horses are also subject to stringent
licensing in some jurisdictions because of their historic status as a companion animal.
ARTERIOVENOUS SHUNT AND PERFUSION CHAMBERS: IN VIVO/EX VIVO MODELS
OF THROMBOSIS IN LARGE ANIMAL MODELS
The ex vivo/in vivo models of thrombosis in large animal models mainly consist of an extracorporeal AV shunt, usually
located between the carotid artery and the jugular vein, into which a perfusion chamber containing a thrombus-promoting
surface (synthetic or vascular derived) is inserted [41,42]. Then, a pump or a clamp positioned distal to the device controls
the flow rate, and thus the shear conditions, allowing blood to circulate from the arterial to the venous vessels. Eventually,
thrombus developing over the exposed thrombogeni c surface may be measured by radiolabeling (platelets and/or fibrinogen) or by section morphometry. This ex vivo/in vivo model of thrombus formation has become essential for testing the
effects of blood elements and rheology, as well as atherosclerotic vessel components in thrombus formation, in a controlled
manner [43e47]. Indeed, it has allowed the evaluation of the thrombogenic effects of different atherosclerotic plaque
constituents (e.g., collagen, fatty streaks, smooth muscle cells, etc.) [43,48], different degrees of shear stress (to mimic
different degrees of stenosis) [44,45], and the antiplatelet effects of new antithrombotic compounds [43,45,49]. In this
regard, this model of thrombosis offers the opportunity to evaluate, in an easy and reproducible manner, the interaction of a
given compound with the blood and vascular compartment and consider any metabolic transformation.
H.R. Baumgartner developed the first popular annular perfusion chamber that helped to advance the knowledge and
understanding of platelet adhesion to the subendothelium under laminar flow [36]. Since then, other ex vivo chamber
systems have been developed for investigating prosthetic and biologic substrate surfaces over a broad range of flow
conditions [50,51]. In fact, although atherosclerosis prefer entially occurs in areas of turbulent blood flow and low fluid
shear stress, thrombosis is induced by high shear stress. Moreover, in atherosclerotic vessels, laminar flow conditions may
not be maintained, since stenotic narrowing induces flow disturbances that modify cellecell and cellevessel interactions as
well as the local concentrations of fluid-phase chemical mediators necessary for cell interaction. In this regard, we have
developed an extracorporeal perfusion system (the Badimon chamber) [52] that allows one to investigate the dynamics of
platelet deposition and thrombus formation:
1. on various surfaces (biological and prosthetic materials) [53,54];
2. under a broad range of pathophysiological flow conditions, including laminar and nonparallel streamline
flows [44,55,56];
3. with varying perfusing blood treatments.
This chamber has helped to improve the understanding of thrombus formation (Fig. 6.3) and of the pathophysiology of
the acute coronary syndromes [52,57], to characterize the thrombogenicity of different degrees of vascular injury [57] and
different atheromatous components [48], and to evaluate the thrombogenicity associated with several synthetic/prosthetic
surfaces [53,54] and several plasma components (cholesterol, glucose levels, etc.) [58,59] (Fig 6.3). It has also become a
useful tool for the study and screening of new antithrombotic and platelet-inhibitory compounds upon exposure to human
atherosclerotic and stented vessels [43,45,48,60e64].
ANIMAL MODELS OF VENOUS THROMBOSIS
Vein thrombosis is mainly attributed to the combination of a hypercoagulable state, blood stasis, and vascular injury [65].
As described for arterial thrombosis, many animal models (mouse [66,67], rat [68], rabbit [69], dog [69,70], pig [71], and
nonhuman primates [72]) have contributed to elucidating the molecular and cellular mechanisms involved in venous
thrombus formation (nicely reviewed in Dorffler-Melly [73] and Levi [74]) and have helped to assess the efficacy and
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FIGURE 6.3 Representative immunophotomicrographs of platelet deposition over a denuded/eroded vessel (mild damage; A) and a platelet-rich
thrombus formed over a disrupted vessel (severely damaged; B). Blood was perfused over the substrates under shear rate conditions of 800/s
(mimicking blood flow in mild stenotic coronary arteries) within the Badimon perfusion chamber. Platelets are labeled with red. White arrowheads
indicate platelet deposits (A) and platelet aggregates (B).
safety of multiple antithrombotic agents [9]. A murine model of inferior vena cava stenosis [75], a model of mechanical or
photochemical injury [76e79] and endothelial stimulation [80e82], should be regarded as the most used macrovascular
and microvascular model of thrombosis.
OTHER MODELS OF THROMBOSIS
Von Willebrand Disease in Large Animal Models
Von Willebrand disease (VWd) is a genetic bleeding disorder that arises from abnormalities in VWF. It is the most
common inherited bleeding disorder in humans, and over the past years several animal species have also been described as
suffering from this disease, whether through spontaneous mutation (pigs and dogs) or through a genetically engineered
mutation (mice) [83]. All these different animal models have been extremely useful in exploring the characteristics of VWd
and in test ing new treatments. Indeed, as described earlier, the interaction of platelets with VWF is crucial in the initiation
and development of any thrombotic process, since VWF enables platelets, via their surface GP receptors, to adhere to
exposed subendothelium [84e86]. The pig is a good model for studying VWd because VWF localization in endothelial
cells and platelets mimics that of humans, as do the clotting and platelet characteristics. In addition, in normal pigs the level
of VWF is close to the human level (Fig. 6.4) [83]. Canine VWd has also been demonstrated to be similar to that in
humans. Over the years, many dog breeds have been identified as suffering from this disease, making VWd the most
common inherited bleeding disorder in dogs. However, under the term “canine VWd,” there seems to be a very heterogeneous group of bleeding disorders with different subtypes and modes of inheritance [87]. Actually, canine VWd can vary
in genetic transmission, clinical severity, and diagnostic laboratory findings. Thus, in contrast to pigs, VWd dogs have not
been used extensively for research purposes [88]. Final ly, it deserves to be mentioned that VWd has also been reported in
other animal species, such as mice [89,90], rabbits [91], cats [92], and nonhuman primates (i.e., baboons) [93]. However,
caution must be taken in extrapolating these observations to the human clinical conditions.
Animal Models of Intracranial Thrombosis
Intracranial venous thrombosis is a relatively rare disease, but constitutes an entity that must be accurately diagnosed in a
timely manner in emergency services, given the need for prompt treatment to avoid serious complications, including
neurological deficits or even death [94]. As stated previously, arterial thrombosis is characterized as being a thrombotic
phenomenon of platelet activation, whereas venous thrombos is is largely a matter of activation of the clotting system. This
important difference implies that the animal models have to be differentiated, as well as the treatments studied, because
arterial and venous thrombosis usually differ in target strategies [95].
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FIGURE 6.4 Differences between large animal models and humans in the thrombotic system and other parameters that may influence antithrombotic
effectiveness.
Several research groups have implemented animal models of cerebral thrombosis in mice, including the induction of
thromboembolic stroke [96] and a model of transient middle cerebral artery occlusion [97]. Thromboembolic stroke is
based on transient cerebral hypoxiceischemic insult, which triggers both thrombosis and infarction [96]. On the other
hand, transient middle cerebral artery occlusion is achieved by inserting and advancing a standardized silicon rubbercoated nylon monofilament via the right internal carotid artery to occlude the origin of the right middle cerebral artery
for 60 min. Mouse thromboembolic stroke models have shown usefulness for optimizing fibrinolytic therapies [96].
However, there is still a lack of information and limited bias reduction so that no intervention has yet been tested in
sufficient range and depth to support translation to clinical trial [98]. Unfortunately, large animal models of cerebral
thrombosis/ischemia are not currently extended so far [99].
CONCLUSIONS AND FUTURE CHALLENGES
In vivo animal models of thrombosis have allowed us to mimic the myriad of hemodynamic and localized cellular and
molecular interactions that occur during the generation, propagation, and lysis of thrombi, improving our understanding
of the etiology and pathogenesis of the disease. In addition, they have supplied valuable information for the investigation of
new antithrombotic drugs, providing pharmacodynamic, pharmacokinetic, and safety data that have helped in the design of
efficient clinical trials. Even though no ideal animal model exists, in this chapter we have tried to highlight the criteria for
the selection and the “pros” and “cons” of the most-used animal models.
The use of transgenic approaches has permitted the uncovering of specific gene functions and has facilitated the
formulation of new strategies for cardiovascular protection and the prevention and treat ment of thrombosis; yet, da ta
obtained in small animal models should not be overinterpreted for their relevance to what occurs in human thrombosis.
In contrast, large a nim al models of thrombosis have a cardiovascular system that closely resembles that of humans,
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making them valuable models to potentially extrapolate the findings to the clinical setting. Nonetheless, efforts need to
be directed toward:
1. standardizing existing models (animal species and mechanisms of induction);
2. implementing new noninvasive imaging techniques to observ e molecular and cellular processes that may underlie the
onset of the disease;
3. generating models with a higher “patient” resemblance (presence of comorbidities and/or cardiovascular risk factors,
atherosclerotic plaques, etc.) [100].
This combination will allow better mimicking of the real pathological scenario with the aim of reducing the global
burden of vascular thrombotic disease in humans.
ACKNOWLEDGMENTS
This work was supported by PNS 2015-71653-R (to G.V.) and PNS SAF2016-76819-R (to L.B.) from the Spanish Ministry of Science and
Innovation and FEDER funds from the Instituto de Salud Carlos III CIBERCV (CN16/11/00411 to L.B.). We are thankful for the continuous
support of the Generalitat of Catalunya (Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat;
2014SGR1303) and the Fundacion Investigación Cardiovascular.
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