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Fibrin Clot Structure and Function: A Novel Risk Factor for Arterial and Venous Thrombosis Chapter | 3 35
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FXIIIa. As a result, studies have shown a direct relationship between final clot structure and FXIIIa in a manner that is
dependent on fibrinogen concentration [72]. FXIII deficiency, resulting in clots that are weaker and more susceptible to
lysis, is a risk factor for severe bleeding and hemostatic-related diseases [73]. Little is known about the effect of excess
FXIII, likely because of the complex regulation of FXIII activation and fibrinogen concentration.
FIBRINOLYSIS
Fibrin clots are meant to be temporary structures formed to stop bleeding. After the process of wound healing has started,
the ridged clot must be broken down to reduce the risk of thrombosis. The process of clot breakdown or fibrinolysis is
orchestrated by the plasmin system. Physiologically, plasmin’s precursor, plasminogen, circulates in the blood and binds to
fibrin at specific binding sites, mainly lysine side chains [14,74,75]. Once bound to fibrin, this 92-kDa glycoprotein is
converted to plasmin by one of two different activators: tissue plasminogen activator (tPA) or urokinase plasminogen
activator (uPA). Plasminogen and tPA binding sites as wel l as initial plasmin cleavage sites are shown in Fig. 3.3 (left).
tPA and uPA act in two different ways to produce plasmin. tPA, which is released by endothelial cells found on the lumen
of blood vessels, can bind directly to fibrin [27]. As plasmin begins to degrade the clot, newly created lysine binding sites
allow for further degradation of the clot [76].Iffibrin is absent, tPA is very weakly bound to plasminogen, resulting in a
weak activation, but in the presence of fibrin, tPA can activate plasminogen in a highly accelerated manner [77]. This
acceleration is a result of plasminogen and tPA binding sites on fibrin being close together. Conversely, uPA does not bind
to fibrin under normal physiological conditions and therefore does not require fibrin to convert plasminogen into plasmin
[78].
Due to the nature of normal clot structure and lateral aggregation of fibrin protofibrils during clot formation, plasmin
degradation of aC-regions occurs early in the fibrinolysis process, resulting in aC fragments [79,80]. This is followed by
degradation of the coiled-coil region [81]. The resulting by-product of this degradation, found only in the presence of
cross-linked fibrin, is the geg cross-linked domain termed D-dimer [82,83]. In certain cases, although Aea and Beb
interactions are not covalently cross-linked like geg chains are, the central E-region is still attached to the D-dimer [79].
This and other common proteolysis products are shown in Fig. 3.3 (right). The efficiency of clot lysis is determined by clot
structure and fiber diameter. Clots made from thicker fibers are more porous and less densely packed than those made from
thinner fibers. The resulting architecture allows for more plasminogen and tPA integration into clots with larger pores and
accelerates fibrinolysis rates [84]. Due to the fragile balance of lysis inhibitors, the speed of clot lysis is a complex
combination of many different factors.
FIGURE 3.3 Plasminogen and tissue plasminogen activator (tPA) binding sites, initial plasmin cleavage sites, and common fibrinolysis products.
Schematic showing the tPA and plasminogen binding sites on a fibrin molecule as well as the initial plasmin cleavage sites (left). Binding sites for tPA and
plasminogen (green arrows) are located at residues a148e160 (low affinity) and in the aC domain (392e610, high affinity), likely attaching to lysine
residues in the regions [184,185]. A lysine-independent tPA binding site (blue arrows) is located at residues g312e324 [186]. Plasmin first cleaves fibrin
(red scissors) at residues aK583, aK206, and aK230, which removes the entire aC-region, leaving fragment X (shown on the right). Subsequent cleavage
sites are located on each chain at residues a (K78, K81, R95, R104, R110), b (K122, K133), and g (K53, K58, K62, K85, K88), all located in the coiledcoil region of fibrin [187]. Other common fibrinolysis products are shown on the right, including fragment Y (one coiled-coil is cleaved), D-D (commonly
referred to as the D-dimer, in which two cross-linked D domains are left following fibrinolysis), and D-E-D (a D-dimer with an un-cross-linked E-region
still attached).
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Various factors in the blood are counteracting the conversion of plasminogen to plasmin while also competing with
plasminogen to bind to fibrin. These regulators include a
plasminogen activator inhibitor-1 and -2 (PAI-1 and PAI-2) [27]. The most potent of these inhibitors, a
described earlier, is incorporated into the clot by FXIIIa. Through this incorporation into the clot, a
plasminogen binding to fibrin [74]. In addition, a
a
-AP-deficient plasma have been shown to lyse very rapidly [85]. Lysis time can be increased by the addition of a2-AP,
2
-AP rapidly inactivates plasmin , regulating fibrinolysis. Clots made with
2
but this recovery is not as efficient a regulator as FXIIIa incorporation of a
TAFI is activated by thrombin with the help of thrombomodulin [87]. Similar to a
-AP, tissue activatable fibrinolysis inhibitor (TAFI), and
2
-AP, which was
2
-AP interferes with
2
-AP into the clot directly [86].
2
-AP, FXIIIa cross-links TAFI to
2
fibrin [66]. TAFI, in its activated form, cleaves C-terminal lysine residues from fibrin, thereby inhibiting plasminogen-toplasmin conversion by tPA. As a result, the presence of TAFIa increases the lysis time and reduces the amount of plasmin
generated.
PAI-1 and PAI-2 act similarly to prevent lysis by inhibit ing tPA and/or uPA. Both PAI-1 and PAI-2 actively bind fibrin
and inhibit the ability of tPA or uPA to activate plasminogen into plasmin [88]. tPA is released by platelets and endothelial
cells, and is therefore most important in the regulation of lysis in platelet-rich thrombi and in areas near the blood vessel
wall where endothelial cells are present. PAI-2 is typically found at high volumes in the blood only during pregnancy [89].
CLOT MECHANICS
The mechanical properties of a clot are pivotal in supporting hemostasis. A clot must be stiff enough to stem the flow of
blood, yet elastic enough to promote cell migration to the site of injury. There are two main mechanisms for deformation,
elastic and plastic (inelastic), and fibrin clots display properties of both [90]. Elastic deformation is seen in a wide variety of
biopolymers, including collagen and actin [91]. Similar to what is found in rubber, elastic deformation is a reversible
process. In biopolymers, this property allows for cells and substrates to resist mechanical changes from an applied stress.
Plastic or inelastic deformation is most commonly found in metals, in which local cracking due to stress can stop the
overall failure of the substrate [92,93]. Physiologically, tissues can display plastic deformation as a defense mechanism
against outside stimuli. If a polymer is able to display both elastic and inelastic proper ties it is known as viscoelastic.
Fibrin’s remarkable viscoelastic properties make it unique among biological and synthetic polymers [94].
To truly understand the unique mechanical properties of fibrin clots, it is important to discuss each hierarchical level of
a fibrin clot, starting at the molecular level and working our way up in scale from microscopic single fibers to the
macroscopic fiber network. Fibrin molecules, when extended, first unfold in the coiled-coil region, which contributes to
much of the individual molecule’s elasticity [95]. While this is occurring, aC-regions are lining up and beginning to
stretch. This is followed by unfolding of the D-region and E-region, which contains elastic and plastic or irreversible
deformation [94].
On the protofibril level, it is believed that slipping of protofibrils past one another and possible unbinding of noncovalent bonds contribute to the viscous or irreversible deformation of the clot. Single fibrin fibers have been shown to
have extreme elasticity and extensibility, withstanding stretching up to 3.3 times their original length before breaking [95].
In the presence of FXIII, this extension occurs to a lesser degree, occurring at near 2.5 times their original length before
rupturing. As might be expected, the presence of FXIII also stiffens individual fibers up to twofold higher than those that
are un-cross-linked, maintaining a stiffness in the megapascal range. Here it should be noted that these elastic, mechanical
properties behave in a nonlinear way [93,96]. Indicative of nonlinear response is the presence of strain hardening, an
increase in stiffness while strain increases, during individual fiber mani pulations [97]. In the presence of FXIII, fibrin
clots still display strain hardening, though to a lesser degree than in un-cross-linked fibers [95]. This is an important
phenomenon in vivo, allowing clots to be more easily deformed at low strains while becoming stiffer at higher strains,
preventing damage to the clot.
At the macroscopic level, individual fibers are branched and fiberefiber interactions can occur. The elasticity of fibrin
can be further understood by looking at the effect FXIII has on the stiffness of these branch points. Carlisle et al. used an
atomic force microscope to study how the branch points ruptured under strain [98]. This rupture could occur either at the
node of the branch point or along the fibers connected to the branch point. In the absence of FXIII, ruptures occurred
68.5% of the time at the node and 31.5% of the time along the adjoining fiber. When fibers were cross-linked, these
findings were nearly opposite, with ruptures occurring 39.5% of the time at the node and 60.5% of the time along the fiber.
While this shows that the presence of covalent cross-links increases the stiffness at the node, it also suggests that crosslinked fibers are weaker along the fiber than along the nodes. When we look at the clot as a whole, individual fibers begin
to align when they are under a mechanical strain, resulting in a reduced number of pores as the strain is further increased
[99,100].
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The mechanical properties and structure of the clot are at a delicate balance with various other factors found in blood
plasma. Thrombin concentration can greatly affect clot structure and mechanics. Domingues et al. showed that increased
thrombin concentration leads to thinner fibers made from less tightly packed protofibrils [101]. Fibrin clots were also found
to be less stiff with increasing concentrations of thrombin. In whole blood, incorpo ration of red blood cells (RBCs) into the
clot has been shown to increase the clot viscosity while also decreasing its stiffness. DesA-fibrin, which can be produced in
the presence of a snake venomederived enzyme, batroxobin, cleaves only FpA and not FpB. Clots made with desA-fibrin
are two to four times less stiff and up to eight times less elastic than clots formed with thrombin, cleaving both fibrinopeptides [102,103]. This suggests that Beb interactions play an important role in clot stiffness and elasticity. This delicate
balance can also be greatly affected by changes in pH, calcium concentration, and concentrations of other plasma proteins
that contribute to the coagulation cascade.
ALTERNATIVE mRNA FIBRINOGEN SPLICING
Two common fibrinogen variants occur due to alternative splicing in the mRNA transcription, which results in mutations on the a chain (fibrinogen-420) or the g chain (fibrinogen g
0
). Fibrinogen-420 is found at normal conc entratio ns
of 1%e2% of the total fibrinogen concentration in b lood plasma [104]. Fibrinogen-420 gets its name from the high er
molecular mass, 420 kDa, compared with the more abundant form of fibrinogen, which has a mo lecula r mass of
340 kDa [105]. The additional molecular mass comes from each of the normal a chains being replaced by an extended a
chain (a
). This extended a chain leads to two additional globular domains now found at the C terminus named aEC
E
(Fig. 3.4, left). Although this additional globular domain does not contain a fibrin binding pocket similar to those found
in the C terminus of the g and b chains, it does contain a binding site for b
-integrins, suggesting that it plays a role in
2
cellular adh esion [106]. Structurally, clots f ormed with purified fibrinogen-420 show less lateral aggregation than those
formed with fibrinogen-340, which leads to thinner fibers that ar e more h ighl y branc he d.
Fibrinogen g
chain found in normal fibrinogen (gA) contains 411 amino acid residues, whereas the g
During transcription of the g
additional amino acids (g
heterodimer, gA/g
at very low concentrations in normal blood plasma, mainly <1%. The g
0
is a much more abundant variant, found at normal concentrations of 8%e15% in blood plasma. The g
0
chain, the four terminal ends of the g chain (g408e411) are lost and replaced with 20
0
408e427), which are more negatively charged [30,107]. The majority of this variant occurs in a
0
, in which only one of the two g chains contains the g0sequence. The homodimer, g0/g0, is found only
0
mutation is found in the D-region and may
0
chain contains 427 residues.
interfere with DeD binding during polymerization [108]. These variants are shown in Fig. 3.4 (right).
0
The g
chain has also been shown to have a high binding affinity for thrombin [108]. It has been reported that this high-
affinity binding leads to decreased thrombin activity [109]. It should be noted, however, that g
0
does not inhibit FpA
cleavage from fibrinogen or FXIII activation, possibly even enhanci ng its activation. It has been proposed that fibrinogen
0
g
acts as a modulator of thrombin activity, specifically inhibiting acti vity driven through thrombin exosite II [110].Asa
result, g
0
does not have a direct inhibitory effect on fibrin clot formation due to its thrombin binding affinity and is believed
to be largely directed toward the intrinsic pathway through inhibition of FVIIIa and FVa [111,112].
FIGURE 3.4 Alternative fibrinogen mRNA splice variants. Schematic of fibrinogen-420 showing the extended aC-region (aEC, left). On the right are
shown the g splice variants, with one side of the fibrinogen molecule having the extended g chain (gA/g
molecule having the extended g chain (g
0/g0
fibrinogen).
0
fibrinogen) and both sides of the fibrinogen
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Structurally, clots made from purified gA/g0have been shown to undergo altered polymerization, with resulting clots
containing thinner fibers, a less porous architecture, and increased branching compared with clots made from gA/gA
[113e115]. Furthermore, it has been shown that protofibril packing of clots made from gA/g
0
contributes to the decrease in
fiber radius [101]. This is contradictory to what would be expected from decreased thrombin activity since decreased
thrombin activity would lead to larger fibers. Some of these results may be explained by studies looking into early fibrin
oligomer and protofibril formation. Allan et al. showed that g
0
directly interferes with early fibrin polymerization, resulting
in fibers that are thinner and mechanically weaker, but are more resistant to lysis [115]. The reduced lysis is due to reduced
plasminogen and tPA binding leading to reduced plasmin generation [116].
FIBRIN CLOT STRUCTURE AND CORONARY ARTERY DISEASE
One of the major new developments in hemostasis and thrombosis research has been the recognition that the structure of
the fibrin clot is an important player in determining risk of thrombotic vascular occlusion. The reasons for this are that the
structure of the fibrin network determines the mechanical properties of the clot and its resistance to deformation and
embolization. Furthermore, fibrin structure also regulates clot contractibility, its resistance to fibrinolysis by the plasmin
generation system, interactions with blood cells during clotting, and interactions with other cells during processes involved
in tissue repair. Fibrin clot structure has been implicated in thrombosis of both the arterial and the venous circulation, each
of which is discussed in further detail later. Confocal micrographs of a loose and a dense fibrin clot are shown in Fig. 3.5.
The first studies showing a link between fibrin clot structure and risk of thrombosis were of fibrin clot structure in
patients with CAD and MI. It was found that patients with MI produced clots that were denser, with smaller pores and
increased resistance to fibrinolysis [117,118]. These early studies indicated that in patients with thrombosis and cardiovascular occlusion, the structure of the fibrin clot was abnormal and contributed to the pathophysiology of cardiovascular
disease.
Studies from our laboratory further showed that clot structure abnormalities also occur in the first-degree relatives of
patients with CAD [119]. First-degree relatives of patients with CAD produced clots with reduced pore size and increased
fiber branching compared with healthy age- and sex-matched controls. These studies suggest that patients with CAD may
be predisposed to clot abnormalities that contribute to the increased risk of thrombosis. In support of this, studies of the
heritability of fibrin clot structure in monozygous compared with heterozygous twins showed that as much as around 40%
of the variation in clot structure is determined by genes, while the remaining 60% of the variation is determined by the
environment [120]. Which genes contribute to this regulation of clot structure remains to be established.
It is of interest to note that such relatively high degree of heritability exists for a complex molecular phenotype like clot
structure, which is the product of many factors from the coagulation system acting together to form the three-dimensional clot
structure. In agreement with the significant heritability of complex biological and pathological pathways, high contributions
of heritability have also been found in activation products of the coagulation cascade, including thrombineantithrombin
complex, prothrombin fragment 1þ2, and cross-linked fibrin degradation product D-dimer [121].
While there is considerable genetic control of fibrin clot structure, which may help to explain the predisposition of
patients with cardiovascular disease to thrombosis, this naturally does not preclude the beneficial effects of treatment
FIGURE 3.5 Confocal images of loose
and densely packed clots. Representative
confocal micrographs of purified fibrin clots
with loosely (left) and densely (right) packed
fibers. Densely packed fibrin clots are associated with an increased risk of thrombosis in
both the venous and arterial circulation. Both
clots are imaged at the same magnification.
Original scale of each micrograph is
160 160 mm. Images courtesy of Fraser
Macrae.
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directed toward clot structure. For example, statins, aspirin, and clopidogrel have all been reported to have a beneficial impact
on clot structure in patients with cardiovascular disease; more about this later. Further studies are required to determine the
mechanisms by which clot structure is regulated by genes. While genetic polymorphisms in fibrinogen and FXIII contribute
to changes in clot structure [71,122,123], it is unlikely that they fully explain the genetic regulation of clot structure [124].
Studies have shown that a number of coagulation proteins are present in the atherosclerotic plaque [125]. Significant
colocalization of FXII and fibrinogen has been shown in atherosclerotic plaque specimens obtained by atherectomy [126].
These findings suggest that coagulation factors and fibrin may contribute to plaque morphology and development. It also
opens up the possibility that fibrin or other coagulation or fibrinolytic factors may affect plaque stability and possible
rupture [127,128]. The mechanism(s) by which fibrin and coagulation factors recruit to the atherosclerotic plaque is not
entirely clear. It may be that fibrin binds to monocytes and macrophages that infiltrate the plaque. Alternative ly, or in
addition, subclinical plaque lesions may trigger clot formation without complete occlusion of the coronary artery, after
which the formed fibrin clot is incorporated into the growing plaque. Furthermore, platelets incorporated into the clot and
atherosclerotic plaques contain fibrinogen stored in the alpha granules [129]. Further studies into the role of fibrin and
coagulation proteins in atherosclerosis and plaque formation are required to investigate mechanisms involved in some of
these processes.
FIBRIN CLOT STRUCTURE AND VENOUS THROMBOSIS
Studies have indicated that fibrin clot structure is altered not only in arterial thrombotic diseases, but also in venous
thromboembolic diseases. Patients with DVT produced clots with smaller pores that were more resistant to fibrinolysis
compared with control clots [130]. Furthermore, the first-degree relatives of these patients also showed clot abnormalities
compared with controls, in accordance with the reported heritability of fibrin clot structure and function [120]. Interestingly, patients with DVT who also developed pulmonary embol ism showed clots that were less compact and lysed more
readily compared with those from patients with DVT alone [130]. These findings suggest that clot structure may play a role
in the risk of clot embolization that occurs following DVT. In addition, studies show that clots from patients with pulmonary embolism differed from those with DVT alone by the accelerated establishment of viscoelastic properties [131].
How exactly the early establishment of clot viscoelastic properties increases the risk for clot embolization remains to be
elucidated.
Venous thrombi are typically rich in fibrin and RBCs. Fibrin cross-linking by FXIIIa and clot elastic properties have
been shown to play an important role in the retention of RBCs by the clot. In normal cross-linked clots, RBCs are retained
inside the clots when they retract. Clot retraction is a physiological mechanism that follows clot formation. Activated
platelets pull on the fibrin fibers and reduce the size of the clot, making it more compact [132]. Aleman et al. showed that
clots made from mice that do not support fibrin cross-linking by FXIIIa (FXIII-deficient mice and fibrinogen g390e396A
mice in which the FXIII binding site is mutated) retain many fewer RBCs during clot retraction [133]. Thrombus size was
reduced due to the smaller number of RBCs retained, and human clots deficient in FXIII also retained fewer RBCs [133].
In a follow-up study, Byrnes et al. investigated the mechanism by which FXIII regulates RBC retention in the clot. FXIIIa
does not appear to directly cross-link the RBC with fibrin, but cross-linking retains RBCs by strengthening the mechanical
properties of fibrin, particularly through cross-linking of the fibrin a chains [134].
Around 20%e40% of patients with a DVT develop postthrombotic syndrome. Postthrombotic syndrome is a long-term
complication due to residual vascular occlusion and may lead to limb ulceration. Studies showed augmented clot structure
abnormalities in patients with postthrombotic syndrome, which may contribute to long-term complications [135]. Further
studies are required to investigate the role of fibrin clot structure in pathological processes that underpin the postthrombotic
syndrome and ulceration.
Air pollution has been reported to increase the risk of venous as well as atherothrombotic disease. Some of the
increased risk of thrombosis by air pollution could be mediated through abnorm al clot structure. Studies by Metassan et al.
have shown that particulate matter obtained from air pollution and diesel exhaust is capable of inducing changes in fibrin
clot structure, leading to clots that are more tightly packed, with smaller pores and increased resistance to fibrinolysis [136].
However, in young healthy individuals who were transiently exposed to a controlled amount of diesel exhaust, while
exercising moderately in an exposure chamber for 2 h, postexposure clot structure did not change compared with preexposure samples [137]. These data are in some way reassuring, as they suggest that healthy individuals may not be at
increased risk for abnormal clots, if exposed to air pollution for a short period of time. In a study in patients with venous
thrombosis, Pan et al. showed that patients with previous DVT living close to busy roads showed denser and compact clot
structures compared with patients living farther away, while no changes were found for control subjects with high exposur e
compared with low exposure. Together, these data indi cate that high levels of air poll ution provoke prothrombotic changes
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in clot structure in patients who are at risk for thrombosis, whereas healthy subjects remain unaffected [138]. Changes in
clot structure represent a potential mechanism by which air pollution contributes to the risk of thrombosis in patients with
previous thrombosis.
ROLE OF SMOKING, DIABETES, AND GLYCATION
In addition to the aforementioned effects of air pollution on clot structure, a number of other risk factors have been
associated with prothrombotic clot alterations. Smoking is one of the strongest risk factors for atherothrombotic disease and
has been associated with clear alterations in clot structure. In a group of healthy volunteers, smoking had acute effects on
clots, producing thinner fibers, increased number of fibers per area in the clot, and increased maximum turbidity compared
with nonsmokers [139]. In addition, clots were significantly stiffer after smoking as measured by thromboelastography
[139]. In a large group of patients with type 2 diabetes mellitus, smoking was significantly associated with prothrombotic
clot structural changes, independent of the patients’ gender [140]. Interestingly, Kovács et al. found that thrombi obtained
by thromboaspiration in patients with acute MI showed a higher fibrin content in smokers compared with nonsmokers
[141]. In a separate study, Silvain et al. showed that thrombi obtained by thromboaspiration from patients with ST-segment
elevation MI were composed largely of fibrin (56%), followed by a minor contribution by platelets (17%), RBCs (12%),
cholesterol (5%), and white blood cells (1.5%). In addition, the relative fibrin content of the thrombus was positively
correlated with ischemic time [142]. These studies clearly show the importance of fibrin in coronary artery thrombosis, and
warrant further studies into fibrin clot structure in patients with CAD. Furthermore, these studies indicate that some of the
prothrombotic effects of smoking are mediated through fibrin and clot structure.
Studies have shown clear links between both type 1 and type 2 diabetes and abnormal, prothrombotic clot structures.
Several mechanisms that drive these prothrombotic clot changes have also been identified. Jörneskog et al. was the first to
show that clot porosity was reduced, indicative of a more compact clot structure, in a small study of 20 patients with type 1
diabetes compared with healthy control subjects [143]. Carr et al. showed that the clot elastic modulus was significantly
increased in patients with chest pain and diabetes, compared with those without diabetes or normal controls [144]. In a larger
study of 150 patients with type 2 diabetes compared with 50 controls, Dunn et al. showed that fibrinogen purified from
patients with diabetes produced clots with reduced pore size, increased number of fiber branch points, and increased fiber
density compared with those produced from fibrinogen from healthy controls. Furthermore, the prothrombotic changes
correlated significantly with HbA1C (glycated hemoglobin) in the patients, a sensitive indicator of the glycemic index [145].
In a second study, Dunn et al. showed that the fibrinogen protein in patients with diabetes showed signs of glycation,
particularly of the Bb and g chains, as determined by mass spectrometry [146]. It was also found that clots produced with
fibrinogen from patients with type 2 diabetes were slower to lyse, bound tPA and plasminogen with lower affinity,
supported less plasmin generation, and incorporated more a
comprehensive studies of the effects of glycation in diabetes on fibrin clot structure and function as of this writing, and they
clearly point to a mechanism by which increased glycation of the fibrinogen molecule in diabetes incurs prothrombotic
changes at the level of the fibrin network.
The glycation of fibrinogen in diabetes can be reversed by improved glycemic control as shown in patients with
uncontrolled diabetes treated with insulin by Pieters et al. [147]. However, no differences were observed in the clot
structure characteristics between patients with diabetes and controls in this study. The reasons for this are unclear, but may
be due to the relatively high fibrinogen concentrations in both patients and controls in this study, by itself a strong regulator
of clot structure, which may have outweighed any effects of glycation on clot structure.
Diabetic retinopathy, a microvascular complication of diabetes, has been associated with denser clots that are slower to
lyse [148]. Patients with diabetes are also at increased risk for macrovascular disease. Indeed, in patients with CAD, type 2
diabetes has been shown to exacerbate prothrombotic clot alterations [149,150]. In a large study of 875 patients, clot
structure was further impaired in women compared with men when both had type 2 diabetes [140]. These studies indicate
that abnormal clot structure is one of the mechanisms by which risk of thrombosis is increased in patients with diabetes.
Furthermore, clot structure is a potential mechanism by which diabetes increases the risk for CAD and its atherothrombotic
complications. Future treatments targeting fibrin clot structure may prove beneficial to treat risk of thrombosis in patients
with CAD and diabetes.
-AP than control clots [146]. These are some of the most
2
STROKE, PERIPHERAL DISEASE, AND ANEURYSM
Several other atherothrombotic diseases have been associated with abnormal clot structure. Ischemic stroke has been
associated with prothrombotic clots that were slower to lyse [151]. Furthermore, the presence of compact clots was
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associated with neurological deficit, at both admission and discharge of patients with acute ischemic stroke [152]. Also
fractal dimension, a measure of clot architecture, is increased in patie nts with ischemic stroke, along with an increase in the
elastic modulus at gel point [153]. Not much is known about the role of clot structure in hemorrhagic stroke, or in the
resolution of ischemic stroke by thrombolysis with tPA or other thrombolytics.
In patients with peripheral arterial disease (PAD), intermittent claudication was associated with less permeable clots,
with thicker fibers and increased resistance to fibrinolysis [154]. Moreover, the first-degree relatives of patients with PAD
showed clot structure perturbations, indicative of a genetic basis for the observed changes [155]. Interestingly, thrombi
obtained from patients with PAD by thrombendarterectomy showed thicker fibrin fibers overall than those obtained by
thromboaspiration in patients with MI [141]. This may be related to the local vascular differences in the concentrations of
thrombin and fibrinogen, as well as differences in flow rates and shear stress between the different arteries.
Abnormal clot structure that increases the risk of thrombosis has also been found in patients with abdominal aortic
aneurysm (AAA) [156]. AAA is a progressive dilatation of the aorta, situated in the abdomen between the renal arteries and
the femoral bifurcation. AAA may develop over many months or years, and once it exceeds 5.5 cm (2.75-fold the normal
size of the aorta), the risk of rupture and life-threatening bleeding is very high. In approximately half of patients with a
larger AAA, the aneurysm contains an intraluminal thrombus. The thrombus is composed of fibrin, RBCs, platelets, and
leukocytes, and it increases the hypoxic and proinflammatory stress on the underlying aneurysmal aortic wall [157].
Furthermore, patients with AAA have a significantly increased risk of other cardiovascular diseases, which persists even
after surgical repair of the AAA [158]. Patients with AAA have high levels of thrombineantithrombin complex and
prothrombin fragment 1þ2, indicative of elevated coagulation activation, and also D-dimer, demonstrating fibrin deposition and turnover [159]. The only current therapeutic option for patients with large AAA is elective surgical repair. The
complexity with treatment of coagulation abnormalities in AAA is that there is a need to reduce thrombotic burden in these
patients, while avoiding exacerbation of the risk of fatal bleeding in the event of an AAA rupture.
Patients with AAA show a prothrombotic clot phenotype, characterized by densely packed fibrin fibers, reduced clot
porosity, and increased resistance of the clot to fibrinolysis. Evidence was provided that the changes in clot structure
depend on factors other than fibrinogen and thrombin generation in these patients [156]. Macrae et al. showed that
His95Arg, a common genetic polymorphism in the FXIII B-subunit, is associated with risk of AAA, indicating a possible
role for FXIII in the disease [160]. Furthermore, complement has been implicated in AAA pathogenesis, with evidence for
activation of all three complement pathways, the lectin [161], alternative [162], and classical [163]. The complement and
coagulation pathways are very closely linked, with several key players in coagulation playing a role in complement and
vice versa [164]. There is strong evidence for at least three key compleme nt factors binding to fibrinogen and/or playing a
role in clot structure, i.e., C1q inhibitor [165],C3[166e168], and MASP-1
complement interactions with fibrin and clot structure that elicit or promote AAA, deserving of further attention in future
studies (Table 3.1).
[169]. These data suggest a possible role for
MEDICATION AND CLOT STRUCTURE
In view of the clear associations between clot structure and thrombosis risk in both the arterial and the venous circulation,
fibrin seems an attractive target for the development of novel antithrombotic drugs. However, as of this writing there are no
agents that selectively target clot structure. Future studies into the mechanisms that regulate the fibrin network, its
mechanical properties, and resistance to lysis are required to identify possible targets for therapeutic intervention.
Notwithstanding eagerly awaited future studies into new targets, a number of current medications used in cardiovascular
disease have been shown to modify clot structure and function. The effects of these drugs on clot structure are important for
our understanding of the drug’s antithrombotic action and also may help to illuminate novel mechanisms of disease and its
treatment.
There are few studies on the effects of oral anticoagulation with vitamin K antagonists on clot structure, as patients on
oral anticoagulation are normally excluded from studies because of the effects on thrombin generation. Thrombin generation and prothrombin levels have an impact on clot structure, whereby reduced thrombin concentration produces clots
with fibers that are thicker and more loosely packed [55,170]. Furthermore, a 2016 study showed that low concentrations of
thrombin enhance fibrin intrafibrillar structure by increasing protofibril packing and thereby enhancing the elastic properties of the fibers [101]. Since vitamin K antagonists inhibit g carboxylation of FVII, FIX, FX, and prothrombin, and
thereby reduce their ability to bind to negatively charged phospholipid surfaces presented by the activated platelet, the
catalytic activity of these enzymes and thrombin generation are impaired. Therefore, oral anticoagulants are expected to
affect clot structure by producing thicker fibers that are more loosely packed. This is indeed the case, as demonstrated by
Ząbczyk et al., who showed that fibrin clot porosity increased with the INR (international normalized ratio, a standardized
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TABLE 3.1 Cardiovascular Diseases, Risk Factors and Clot Structure
Cardiovascular Diseases and Risk Factors Effect on Clot Structure Reference
Myocardial infarction
First-degree relatives of CAD
DVT
First-degree relatives of DVT
DVT and pulmonary embolism
Air pollution
Smoking
Type 1 and type 2 diabetes
Diabetic retinopathy
Ischemic stroke
PAD
First-degree relatives of PAD Clot structure perturbations [155]
Abdominal aortic aneurysm
Green arrows indicate increased or decreased pore size, blue arrows indicate increased or decreased fiber size, and red arrows indicate increased or
decreased fibrinolysis. CAD, coronary artery disease; DVT, deep vein thrombosis; PAD, peripheral arterial disease.
Increased branching [119]
(Compared with patients with DVT alone) [130]
Increased number of fibers and increased maximum turbidity [139]
Increased branching, increased number of fibers [143,145,146]
[117,118]
[130]
[120]
[136,138]
[148]
[151,152]
[154]
[156]
measure of oral anticoagulation), if clotting was triggered with low-dose tissue factor (10 pM) [171], a situation that relies
on the intrinsic thrombin-generating capacity of the plasma sample. Interestingly, if 1 IU/mL thrombin was used to trigger
clotting, clot pore size did not correlate with INR, which is in agreement with the fact that the intrinsic coagulation pathway
in this situation is largely bypassed by the excess thrombin added to the sample [171].
Anticoagulation with heparin has clear effects on clot structure due to the inhibition of thrombin. Clots made in the
presence of therapeutic doses of heparin show larger pores and thicker fibers and are less resistant to fibrinolysis [172].
Addition of low-molecular-weight heparin had similar effects, although perhaps somewhat less pronounced than those of
unfractionated heparin [173]. Protamine has been shown to reverse these effects of heparin on clot structure, but excess
protamine compromises clot structure in its own right [174].
Direct thrombin inhibitors such as dabigatran or argatroban affect clot structure by producing thicker fibers and larger pores,
and increase fibrinolysis through their effects on clot structure, but also through effects on TAFI [175e177]. Also rivaroxaban,
a direct inhibitor of FXa, showed beneficial effects on clot structure, with larger pores and reduced resistance to fibrinolysis
[178]. Further studies are needed to investigate the effects of other direct oral anticoagulants on clot structure and function.
Aspirin has been shown to directly modify fibrin clot structure, independent of its effects on platelet activation.
Acetylsalicylic acid beneficially changed clot structure by increasing clot porosity [175,179]. Interestingly, low-dose aspirin
(75 mg/day) showed increased beneficial effects on clot structure compared with a high dose (320 mg/day) [180] .
Aspirin also reduced resistance of the clot to fibrinolysis [181]. Studies in cells expressing human fibrinogen showed that
aspirin directly modifies fibrinogen, through acetylation of lysine residues, which leads to clots that are more permeable,
less stiff, and easier to lyse [182]. Altogether, these studies indicate a mechanism by which aspirin acetylates lysine residues
in fibrinogen, which then have an impact on clot structure and its stability. These actions of aspirin contribute to the
antithrombotic effect of this commonly used prophylactic drug for the treatment of atherothrombotic diseases.
Other commonly prescribed drugs in atherothrombotic diseases such as statins have also been associated with beneficial
effects on clot structure. Undas et al. showed that simvastatin and atorvastatin were associated with increased clot pore size,
increased fiber thickness, and reduced resistance to fibrinolysis in patients with CAD [183]. Effects of simvastatin on clot
structure were found even if C-reactive protein remained unaltered, and were independent of the effects of the drug on
cholesterol levels. Fenofibrate, which controls lipids via a different mechanism, also had beneficial effects on clot structure.
Furthermore, the angiotensin-converting enzyme inhibitor quinapril also showed modest beneficial effects on clot structure
[183]. These findings suggest important pleiotropic effects of statins and other common cardiovascular drugs, which may
contribute to the overall antithrombotic effects of these drugs and may indicate novel mechanisms that regulate clot
structure (Table 3.2).
Fibrin Clot Structure and Function: A Novel Risk Factor for Arterial and Venous Thrombosis Chapter | 3 43
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TABLE 3.2 Common Cardiovascular Medications and Clot Structure
Medication Effect on Clot Structure Reference
Heparin (therapeutic doses)
Heparin (low molecular weight)
Protamine
Dabigatran (direct thrombin inhibitor)
Argatroban (direct thrombin inhibitor)
Rivaroxaban (direct factor Xa inhibitor)
Aspirin (low dose)
Simvastatin
Atorvastatin
Fenofibrate
Green arrows indicate increased or decreased pore size, blue arrows indicate increased or decreased fiber size, and red arrows indicate increased or
decreased fibrinolysis. TAFI, thrombin-activatable fibrinolysis inhibitor.
(Less pronounced than unfractionated heparin) [173]
(Reverses the effects of heparin) [174]
Reduced TAFI activation [176]
Reduced TAFI activation [175,177]
Decreased fiber stiffness [175,179e182]
[172]
[178]
[183]
[183]
[183]
CONCLUSIONS
There is compelling evidence for a role of abnormal fibrin clot structure in thrombosis and cardiovascular disease. When
produced by the coagulation system, fibrin polymerizes through a remarkable mechanism into a three-dimensional scaffold
that supports the blood clot. Fibrin is stabilized by FXIIIa and orchestrates its own breakdown by the fibrinolytic system.
Furthermore, fibrin has remarkable mechanical properties and is one of the most elastic biological polymers. Studies
demonstrate that the structure of the fibrin network is a major determinant of its stability, resistance to fibrinolysis, and
elastic characteristics under flow and shear stress. Consistent associations between clot structure and thrombosis in
different vascular systems have been found. Compact clots, with increased numbers of fibers and small pores associate with
increased risk for CAD, MI, stroke, venous thrombosis, PAD, diabetes, and abdominal aneurysms. Cardiovascular risk
factors have detrimental effects on clot structure, while anticoagulation and cardiovascular medication can help to reverse
these detrimental effects. Future developments of specific medicines targeting clot structure are eagerly awaited to assess
their potential benefit in the treatment of thrombotic diseases.
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