Clot Architecture Is Altered in Abdominal Aortic

Aneurysms and Correlates With Aneurysm Size

D. Julian A. Scott, Priya Prasad, Helen Philippou, Sheikh Tawqeer Rashid, Soroush Sohrabi,
Daniel Whalley, Andy Kordowicz, Quen Tang, Robert M. West, Anne Johnson, Janet Woods,
Ramzi A. Ajjan, Robert A.S. Ariëns

Objective—Abdominal aortic aneurysm (AAA) is characterized by widening of the aorta. Once the aneurysm exceeds 5.5
cm, there is a 10% risk of death due to rupture. AAA is also associated with mortality due to other cardiovascular
disease. Our aim was to investigate clot structure in AAA and its relationship to aneurysm size.
Methods and Results—Plasma was obtained from 49 controls, 40 patients with small AAA, and 42 patients with large
AAA. Clot formation was studied by turbidity, fibrin pore structure by permeation, and time to half lysis by turbidity
with tissue plasminogen activator. Plasma clot pore size showed a stepwise reduction from controls to small to large
AAA. Lag phase for plasma clot formation and time to half lysis were prolonged, with smaller AAA samples showing
intermediate response. Clot structure was normal in clots made with fibrinogen purified from patients compared with
controls, suggesting a role for other plasma factors. Endogenous thrombin potential and turbidity using tissue factor
indicated that the effects were independent of changes in thrombin generation.
Conclusion—Patients with AAA form denser, smaller pored plasma clots that are more resistant to fibrinolysis, and these
characteristics correlate with aneurysm size. Clot structure may play a role in AAA development and concomitant
cardiovascular disease. (Arterioscler Thromb Vasc Biol. 2011;31:3004-3010.)
Key Words: aortic diseases 䡲 blood coagulation 䡲 fibrin 䡲 fibrinolysis 䡲 thrombosis

A bdominal aortic aneurysm (AAA) involves progressive

dilatation of the abdominal aorta, which mainly occurs
in men older than 55 years of age. Once the anteroposterior
(inner to inner) aorta diameter exceeds 3.0 cm, it is defined as
aneurysmal.1 Left untreated, the dilated aorta (⬎5.5 cm) will
eventually rupture. Male sex and smoking are the main risk
factors for AAA, whereas aortic diameter is a main risk factor
for rupture.1,2 AAA is currently the 10th most common cause
of death in the Western world.3 This is only partly due to the
risk incurred by rupture, as there is a high risk of mortality
due to other cardiovascular causes, both before and after
surgical intervention.4 The mortality due to other cardiovas-
cular causes in AAA is increasing because current screening
programs lead to a larger number of patients undergoing
surgical intervention, whereas the cardiovascular risk remains
largely untreated.5
Larger aneurysms are often characterized by the presence
of intraluminal thrombus (ILT).6 Although early reports
suggested that ILT has a protective effect on the tensile stress
of the aortic wall,7 recent data show that ILT increases
biochemical stress, leading to accelerated dilatation.8 The
thrombus is metabolically active, and a complex relationship
exists between the luminal surface and the interface with the
aortic wall.8,9 The thrombus is constantly remodeled at the
luminal surface, and it contributes to increased inflamma-
tion.8 –10 ILT has been implicated in recruiting leukocytes,
which release matrix metalloproteinases that cause wall
breakdown.9 In addition, ILT causes local hypoxia, inducing
inflammation and impairing synthesis of structural compo-
nents of the wall.6 Some studies have suggested that aneu-
rysm and thrombus growth are interlinked8 and that ILT
presence is a predictor for AAA rupture.11
The main protein constituent of the thrombus is a mesh-
work of fibrin fibers. The fiber network influences stability of
the clot.12 Networks with small pores and densely packed
fibers incur greater resistance to fibrinolysis.13 Propensity to
form dense fibrin clots may therefore be associated with
increased ILT deposition in AAA because of reduced lysis of
the thrombus. Patients with coronary artery disease have
previously been shown to produce clots with increased
number of shorter fibers, decreased permeability, and in-
creased stiffness.13,14 We have shown altered plasma clot

Received on: June 2, 2010; final version accepted on: August 18, 2011.
From the Division of Cardiovascular and Diabetes Research, Section on Mechanisms of Thrombosis (D.J.A.S., P.P., H.P., S.T.R., S.S., D.W., A.K.,
Q.T., A.J., J.W., R.A.A., R.A.S.A.) Centre of Epidemiology and Biostatistics (R.M.W.), Leeds Institute for Genetics, Health and Therapeutics, Faculty
of Medicine and Health, University of Leeds, Leeds, United Kingdom.
Drs Scott and Prasad contributed equally to this work.
Correspondence to Robert Ariëns, PhD, Division of Cardiovascular and Diabetes Research, Section on Mechanisms of Thrombosis, University of
Leeds, Clarendon Way, Leeds LS2 9JT, United Kingdom. E-mail r.a.s.ariens@leeds.ac.uk
© 2011 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.111.236786

Downloaded from http://atvb.ahajournals.org/ at3004

Universiteitsbibliotheek Utrecht on March 17, 2015

structure in first-degree relatives of patients with coronary
artery disease, suggesting a role of fibrin in predisposition to
thrombosis.15 Based on the common role of thrombosis in
cardiovascular disease and AAA, we tested the hypothesis
that patients with AAA produce clots with altered structure.
The objectives of this study were to investigate (1) clot
structure in patients with AAA compared with controls, and
(2) association of clot structure with aneurysm size. We found
that plasma clots were denser and more resistant to lysis in
patients with AAA and that this was particularly evident in
patients with large aneurysms.
Patients and Methods
Subjects
Eighty-two patients with AAA were enrolled as part of the Leeds
Aneurysm Development Study in the vascular outpatient department,
Leeds Teaching Hospitals. All subjects were white, northern Euro-
pean males over the age of 50. Forty of these patients had small
AAA, with aortic diameter ranging from 3.0 to 5.4 cm. Forty-two
patients had large AAA (ⱖ5.5 cm). Large and small AAA patients
were age matched with each other and with 49 male controls,
recruited at random from cardiology and vascular outpatients.
Control subjects all underwent ultrasound assessment and had aortic
diameters ⱕ2.9 cm. Subjects who were on oral anticoagulation, had
underlying malignancy, had had surgery in the last 3 months, or had
active inflammatory conditions were excluded. Written informed
consent was obtained. Ethical approval was provided by the Leeds
Teaching Hospitals Research Ethics Committee.
Clinical Data
Subjects were fasted and asked to refrain from smoking for 10 hours
overnight. A detailed clinical history was obtained by questionnaire.
Blood pressure, body mass index, and waist:hip ratio measurements
were obtained using standard protocols. A 12-lead ECG was per-
formed to analyze ischemic changes indicative of angina. Maximal
infrarenal anteroposterior aortic diameter (inner to inner) was deter-
mined by a departmental ultrasound scan.
Blood Sampling
Blood was drawn from the antecubital vein with minimal stasis. The
first few milliliters were discarded. Blood was collected on 0.1 mol/L
sodium citrate (9 parts blood per 1 part citrate) and centrifuged at
2400g for 20 minutes within 2 hours to separate platelet-poor
plasma. Samples were stored at ⫺40°C.
Hemostasis Measurements
Fibrinogen was determined by the Clauss method on an Amelung
KC10 coagulometer (Lemgo, Germany).16 Factor VII clotting activ-
ity was measured on an ACL300 Plus (Instrumentation Laboratories,
Warrington, United Kingdom) using factor VII– deficient plasma
(Sigma-Aldrich) and recombinant rabbit thromboplastin (Instrumen-
tation Laboratories). Factor XIII A subunit was measured by in-
house ELISA.16 Tissue plasminogen activator (tPA) (Imulyse,
Biopool International, Umea, Sweden), plasminogen activator inhib-
itor-1 (PAI-1) (Imulyse, Biopool), thrombin-antithrombin complex
(Dade-Behring, Marburg, Germany), and D-dimer (TintElize,
Biopool) were measured by ELISA.
Clot Permeation
Ten ␮L of activation mixture (0.11 mol/L calcium chloride, 11 U/mL
human thrombin [Calbiochem, San Diego, CA] in permeation buffer
[0.05 mol/L Tris, 0.10 mol/L NaCl, pH 7.5]) was mixed with 100 ␮L
of plasma. The mixture was carefully transferred into the tip of a
1-mL pipette and placed in a humidity chamber at room temperature
for 2 hours. Tips were connected to a reservoir containing perme-
ation buffer, with a 4-cm pressure drop. Six drops were allowed to
permeate before measurements. A small container was attached to
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Scott et al Clot Structure in AAA 3005
the tip to collect the percolate. At 30-minute intervals, the container
was changed and weighed for percolate volume. Percolate measure-
ments were performed a minimum of 3 times per clot. The perme-
ation coefficient (Ks), representing the clot surface allowing flow
through the network (pore size), was calculated as described.17
Samples were analyzed in duplicate.
Turbidity Lysis
Plasma (25 ␮L) was loaded into 96-well plates, and 75 ␮L of
permeation buffer was added. Fifty ␮L of activation mix
(22.5 mmol/L calcium chloride and 0.5 U/mL human thrombin in
permeation buffer) was added. The plate was immediately placed in
a Bio-Tek ELx 808 reader. Absorbency was read every 12 seconds
at 350 nm for 60 minutes. For turbidity lysis with purified samples,
0.5 mg/mL fibrinogen was incubated with 0.1 U/mL thrombin and
2.5 mmol/L calcium chloride (final concentrations) in permeation
buffer (150 ␮L final volume).
Lysis was measured in a parallel plate. tPA (Technoclone, Vienna,
Austria) was diluted to 170 ng/mL in permeation buffer, of which 75
␮L was added to the plasma instead of buffer in the above turbidity
assay. Activation mix was added as above. Absorbency at 350 nm
was read every 12 seconds for 1 hour and then every 2 minutes for
9 hours.18 Data were analyzed using custom-written software, which
is available from the authors on request. Samples were analyzed in
duplicate. Turbidity and lysis was also performed in all samples with
platelet-poor plasma tissue factor (TF) (endogenous thrombin poten-
tial [ETP] reagent) at a final concentration of 1.25 pmol/L (correlat-
ing with 5 pmol/L in undiluted plasma) instead of thrombin.
ETP
The ETP was performed according to the manufacturer’s instructions
(Thrombinoscope, Maastricht, the Netherlands), using 5 pmol/L TF.
Fibrinogen Purification
Fibrinogen was purified from 4 controls, 5 patients with small AAA,
and 5 patients with large AAA as previously described,19 selected on
the basis of representative clot permeation characteristics. In brief,
plasma collected in heparin was applied to affinity chromatography
with a calcium-dependent antibody (IF-1, Kamiya Biomedical,
Seattle, WA). Fibrinogen was eluted with EDTA and dialyzed
extensively against permeation buffer. Purity of the samples was
tested by SDS-PAGE.
Confocal Microscopy
Plasma (30 ␮L) was diluted with 25 ␮L of permeation buffer and 5
␮L of Alexa Fluor 488 fibrinogen (5% final concentration, Molec-
ular Probes, Leiden, the Netherlands) and placed in a ␮-slide VI
(Ibidi, Munich, Germany). Six ␮L of activation mix (5 U/mL
thrombin, 100 mmol/L CaCl in permeation buffer) was added. Clots
were left in a humidity chamber overnight. A Zeiss LSM510META
Axioplan2 confocal microscope fitted with a ⫻63 oil immersion
objective was used for imaging. Scan format was 512⫻512 pixels.
Ten scans of 230/230/20 ␮m (x/y/z) were taken to visualize the fibrin
network and to analyze fiber density per cubic area.19,20 The 3D
information was converted into 2D images by projection using the
Zeiss LSM software (version 4.20.0.121).
Statistical Analysis
Based on an average Ks of 4.7, a dispersion of 0.75, and an ␣-value
of 0.05, a minimum of 36 subjects were needed in each group to
detect an average difference of 0.5 with 80% power. Nonparametric
data were described with median and interquartile ranges (IQRs).
Variables with normal distribution were described with mean and
SD. A Kruskal-Wallis test compared Ks and turbidity across the 3
groups, followed by a Mann-Whitney U test for comparison between
2 groups. Spearman’s rank was used to correlate continuous vari-
ables. We used SPSS, version 12.0 (SPSS, Chicago, IL), and
significance was defined as P⬍0.05. Multiple linear regression to
test for independent covariates of Ks and lysis was performed using
R, version 2.13.21
3006 Arterioscler Thromb Vasc Biol December 2011

Table 1. Clinical, Biochemical, and Hemostatic Characteristics

Controls Small AAA Patients Large AAA Patients
(n⫽49) (n⫽40) (n⫽42) P
Age, y* 72.4 (70.1–78.2) 73.7 (69.8 –77.8) 72.6 (69.7–78.3) 0.999
Aortic diameter, cm* 2.0 (1.9–2.3) 4.2 (3.8–4.6) 6.5 (6.0–7.0) 0.0001
Current smokers‡ 7 (14.3) 7 (17.5) 5 (11.9) 0.356
Ever smokers‡ 31 (63) 27 (68) 38 (90) 0.004
Alcohol, U/wk* 4.0 (0.5–6.8) 4.0 (0.0–7.0) 14.0 (9.0–28.0) 0.0001
History of cardiovascular
disease, CVD:
All CVD‡ 21 (42.9) 22 (55) 22 (52.4) 0.475
Angina‡ 9 (18) 12 (30) 10 (24) 0.438
CABG‡ 0 (0) 6 (15) 7 (17) 0.013
CA‡ 2 (4) 2 (5) 1 (2) 0.820
CVA/TIA‡ 4 (8) 9 (23) 11 (26) 0.059
LLA‡ 4 (8) 4 (10) 5 (12) 0.838
MI‡ 3 (6) 12 (30) 13 (31) 0.004
PVD‡ 12 (24) 10 (25) 12 (29) 0.894
Hypertension‡ 17 (34.7) 18 (45) 26 (61.9) 0.046
BMI, kg/m2† 27.1 (4.4) 27.6 (4.4) 26.0 (3.0) 0.159
Waist:hip ratio† 0.97 (0.10) 0.96 (0.06) 0.97 (0.08) 0.652
Systolic BP, mm Hg† 145.4 (19.5) 144.5 (20.0) 146.6 (20.8) 0.890
Diastolic BP, mm Hg† 77.5 (10.9) 82.6 (11.4) 81.6 (10.5) 0.067
Total cholesterol, mmol/L† 4.80 (0.94) 4.75 (0.88) 4.17 (0.94) 0.002
LDL cholesterol, mmol/L* 3.0 (2.0–4.0) 3.0 (2.0–4.0) 2.0 (1.7–2.9) 0.0001
HDL cholesterol, mmol/L* 1.3 (1.1–1.6) 1.2 (1.0–1.8) 1.2 (1.0–1.4) 0.252
Triglyceride, mmol/L* 1.3 (1.0–2.2) 1.4 (1.0–2.1) 1.32 (1.2–1.9) 0.896
Statins‡ 14 (28.6) 14 (35.0) 29 (69.0) 0.0001
Aspirin‡ 17 (34.7) 17 (42.5) 26 (61.9) 0.03
Fibrinogen, g/L† 2.7 (0.71) 2.9 (0.7) 2.8 (0.8) 0.431
ETP, (nmol/L)⫻min* 1461 (1221–1552) 1214 (747–1400) 1344 (1134–1586) 0.002
ETP lag-time, min* 3.67 (3.33–4.33) 4.83 (3.75–7.42) 3.83 (3.50–4.75) 0.0001
ETP peak-height, nmol/L† 232 (60) 177 (84) 216 (70) 0.001
FXIIIA, U/mL† 103.5 (18.2) 102.4 (31.1) 99.1 (23.3) 0.657
TAT, ng/mL* 1.92 (1.59–2.22) 5.12 (2.97–7.21) 4.72 (2.82–8.46) 0.0001
D-dimer, ng/mL* 114 (87.5–166) 355.5 (199–739) 650.1 (341.9–1055.7) 0.0001
P-values refer to tests for nonuniformity between the three groups. Significance is highlighted in bold. All CVD includes angina and
history of CABG, CA, CVA/TIA, LLA, MI, or PVD. AAA indicates abdominal aortic aneurysm; U, units; CVD, cardiovascular disease; CABG,
coronary artery bypass graft; CA, coronary angioplasty; CVA, cerebrovascular event; TIA, transient ischemic attack; LLA, lower limb
angioplasty; MI, myocardial infarction; PVD, peripheral vascular disease; BMI, body mass index; BP, blood pressure; LDL, low-density
lipoprotein; HDL, high-density lipoprotein; ETP, endogenous thrombin potential; FXIIIA, factor XIII A-subunit; TAT, thrombin-
antithrombin.
*Nonparametric data expressed as median (25th, 75th quartiles), analyzed by the Kruskal-Wallis test.
†Parametric data expressed as mean (standard deviation), analyzed by ANOVA.
‡Categorical data expressed as No. (%), analyzed by ␹2 analysis.

Results
Clinical Characteristics
Subjects in the control, small AAA, and large AAA groups
were men who were matched for age (Table 1). Aortic
diameter was 2.0 cm (IQR 1.9 –2.3) in controls, 4.2 cm
(3.8 – 4.6) in small AAA patients, and 6.5 cm (6.0 –7.0) in
large AAA patients. There were no differences in current
smoking, angina, or body mass index. There was a higher
prevalence of ever smoking, hypertension, and alcohol con-
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sumption in AAA, the latter only in the group of large AAA
patients. Use of statins and aspirin was significantly higher in
the patients, particularly with large AAA. A small but
significant decrease in total and low-density lipoprotein
cholesterol was observed in large AAA patients, a likely
reflection of the increased use of statins in this group. There
were no differences in triglyceride levels.
There were no differences in fibrinogen, factor VII
(not shown), or factor XIII level (Table 1). Thrombin-

Figure 1. Clot pore structure in abdominal aortic aneurysm
(AAA). Clot permeation showed a stepwise decrease in average
pore size (permeation coefficient [Ks], 10⫺9 cm2) comparing
controls with small and large AAA patients. Data represent
median (thick line), interquartile range (box), and 95th percen-
tiles (error bars). Individual outliers are shown by circles and
asterisks (*).
antithrombin, a marker of thrombin generation, was increased
in AAA patients. Fibrin degradation product D-dimer was
also increased. ETP, a measure of how much thrombin can be
generated in plasma, was reduced in AAA patients. ETP peak
height showed a similar reduction in AAA patients, and ETP
lag-time was prolonged. PAI-1 and tPA were not different
(not shown).
Clot Structure
Permeation showed a decrease in average plasma clot pore
area, Ks, from 4.7⫻10⫺9 cm2 (IQR 4.1–5.5) in controls, to
4.0⫻10⫺9 cm2 (3.4 – 4.3) in small AAA, and 3.4⫻10⫺9 cm2
(2.5– 4.1) in large AAA (P⬍0.0001; Figure 1). Ks was
3.6⫻10⫺9 cm2 (2.8 – 4.3) in all patients combined (P⬍0.0001
versus controls). There was no correlation between Ks and
aspirin or statins. These results indicate that patients with
AAA show a propensity to form clots with more densely
Figure 2. Clot structure in abdominal aortic aneurysm (AAA) imaged by confocal microscopy. Representative clots from control sub-
jects (A), patients with small AAA (B), and patients with large AAA (C). Images are 2D projections of 10 z-stacks obtained over 230/230/
20 ␮m (x/y/z).
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Scott et al Clot Structure in AAA 3007
packed fibers and smaller pores than controls and that this
correlates with aneurysm size. Differences in Ks were also
significant when comparing subgroups with each other:
control versus small AAA (P⫽0.001), controls versus large
AAA (P⬍0.0001), and small versus large AAA (P⫽0.045).
Turbidity lag phase increased in small (497 seconds [IQR
409 – 687], P⫽0.001) and large AAA (484 seconds [IQR
425–555], P⬍0.0001) compared with controls (399 seconds
[IQR 227– 463]). A similar turbidity lag-phase increase was
observed with TF instead of thrombin as trigger (controls
versus small versus large: 400 seconds [IQR 355– 437] versus
498 seconds [447–588] versus 433 seconds [390 – 479],
P⬍0.0001). There was no difference in lag phase between
patients with small and large AAA. Maximum absorbency
was not different in patients with AAA when compared with
controls, either with thrombin or with TF as trigger (data not
shown).
Confocal microscopy was used to study plasma clot ultra-
structure in large AAA, small AAA, and controls. Clots from
large AAA samples consistently demonstrated a more
densely packed fibrin structure compared with small AAA
and controls, in agreement with the observed differences in
plasma clot pore structure by permeation analysis (Figure 2).
There was an increase in fiber density comparing controls
with small and large AAA (mean⫾SD: 75⫾7.7, 114⫾9.7,
and 159⫾10/105 ␮m3, respectively, P⬍0.0001).
Fibrinolysis
We next investigated whether differences in plasma clot
structure in AAA influenced susceptibility to fibrinolysis.
Lysis rates were analyzed using turbidity with tPA, and time
to 50% lysis was recorded. As with clot pore size, fibrinolysis
rates showed a stepwise change from controls to patients with
small and large AAA (Figure 3). Lysis times were shorter in
clots from controls (1892 seconds [IQR 1758 –2107]), inter-
mediate in patients with small (2101 seconds [IQR 1785–
2285]) and longer in patients with large AAA (2195 seconds
[IQR 2041–2640], P⬍0.0001). The difference in lysis times
between controls and patients with small AAA was not
significant (P⫽0.054), but the differences between controls
and patients with large AAA and between patients with small
3008 Arterioscler Thromb Vasc Biol December 2011

Table 2. Covariates of Permeation Coefficient in a Multiple

Linear Regression Model
Covariate/Factor Coefficient 95% CI P Value
Age ⫺0.022 ⫺0.077, 0.034 0.44
All CVD ⫺0.423 ⫺1.150, 0.304 0.25
Smoking ⫺0.074 ⫺0.895, 0.748 0.86
Statins 0.140 ⫺0.534, 0.813 0.68
Aspirin ⫺0.021 ⫺0.710, 0.667 0.95
Fibrinogen ⫺0.271 ⫺0.689, 0.147 0.20
ETP ⫺0.000 ⫺0.001, 0.000 0.14
Small AAA ⫺0.875 ⫺1.523, ⫺0.227 0.009
Large AAA ⫺2.310 ⫺3.551, ⫺1.069 0.0001
Regression was performed using R, version 2.13. CI: confidence interval. All
CVD was as defined in Table 1. CVD indicates cardiovascular disease; ETP,
endogenous thrombin potential; AAA, abdominal aortic aneurysm.
Figure 3. Fibrinolysis in abdominal aortic aneurysm (AAA). Clots
from patients with large AAA showed longer lysis times than apart from maximum absorbency, which showed a positive
clots from those with small AAA, which in turn were longer than correlation (r⫽0.53, P⫽0.003).
control. Time to 50% lysis was analyzed by turbidity with tissue
plasminogen activator. Data represent median (thick line), inter-
quartile range (box), and 95th percentiles (error bars). Individual Multivariable Analysis
outliers are shown by circles and asterisks (*). We constructed a model to test for independent covariates of
Ks, based on univariate associations and on previously
and large AAA were significant (P⬍0.0001 and P⫽0.007, reported associations with Ks. In this model, only small and
respectively). In all patients combined, lysis was 2192 sec- large AAA showed association with Ks (P⫽0.009 and 0.0001
onds (IQR 1933–2580) (P⬍0.0001 versus controls). PAI-1 respectively), with no associations between Ks and age, all
and tPA were not different between patients and controls (not cardiovascular disease, smoking, statins, aspirin, fibrinogen,
shown). Also, when triggering plasma clot formation with or ETP (Table 2). We constructed a similar model for lysis,
TF, lysis was significantly prolonged in patients with AAA also including tPA and PAI-1 levels. In this model, tPA and
(controls versus small AAA versus large AAA: 1146 seconds PAI-1 levels were the strongest predictors of lysis, but AAA
[IQR 1101–1197] versus 1395 seconds [1293–1686] versus also remained independently associated (P⫽0.014). None of
1302 seconds [1182–1407], P⬍0.0001). the other parameters were associated with lysis (data not
In an attempt to further investigate mechanisms underpin- shown).
ning changes in clot structure and lysis, we purified fibrino-
gen from 4 controls, 5 patients with small AAA, and 5 Discussion
patients with large AAA. There were no differences in fibrin Our study shows significant differences in plasma clot struc-
clot structure by turbidity or in fibrinolysis rates in clots ture in patients with AAA. Patients with large AAA produced
produced from these fibrinogens (data not shown). denser, less porous clots compared with patients with small
AAA who in turn produced denser clots than control subjects.
Clot Structure and Cardiovascular Risk This stepwise decrease in porosity was associated with
In all, 65 subjects had a history of cardiovascular disease. As increased lysis times, showing that clots from patients with
cardiovascular disease has previously been associated with larger aneurysms are more difficult to lyse than those of
dense clot structure,13,14 we analyzed plasma clot structure in patients with small aneurysms and controls. These cross-
the absence of this confounder. When we excluded cases with sectional data suggest that clot structure may be involved in
all cardiovascular disease, Ks also decreased in patients with AAA development and progressive dilation of the aorta,
AAA (controls versus small AAA versus large AAA: however, prospective studies will be required to test this
4.8⫻10⫺9 cm2 [IQR 4.3–5.8⫻10⫺9] versus 3.7⫻10⫺9 [3.4 – hypothesis further.
4.3⫻10⫺9] versus 4.0⫻10⫺9 [3.3– 4.4 ⫻10⫺9], P⫽0.001), Formation of fibrin is the final step in coagulation. Studies
and lysis times increased (1802 seconds [IQR 1725–2018] have shown that clot architecture is dependent on fibrinogen
versus 1943 [1774 –2207] versus 2255 [1967–2640], and thrombin levels.24 Fibrinogen levels are high in patients
P⬍0.0001). Statin use was not associated with changes in Ks, with cardiovascular disease and high fibrinogen associates
lysis, or ETP in this study. with clots of reduced permeability and increased maximum
Because homocysteine levels have previously been asso- absorbency. In our study, the association between plasma clot
ciated with AAA and thrombosis,22,23 we investigated its structure and AAA cannot, however, be explained by changes
relationship to clot structure in AAA. Permeation and turbid- in fibrinogen levels, as these were comparable between the 2
ity (triggered with thrombin or TF) and fibrinolysis measure- AAA groups and the controls. Previous studies have shown
ments did not correlate with homocysteine levels that were that dense clot structures are caused by high levels of
measured in a small subset of samples measured (n⫽36), thrombin generation.24 To elucidate whether thrombin gener-
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ation was responsible for our findings, we performed throm-
bin generation experiments on the plasma samples. However,
the ETP, a direct measure of how much thrombin can be
generated in plasma, was not increased but slightly decreased.
Furthermore, fibrin polymerization studies using TF instead
of thrombin (and therefore incorporating effects of endoge-
nous thrombin generation) showed similar differences be-
tween AAA patients and controls, thereby reflecting that
overall thrombin generation within the plasma samples was
not responsible for our findings.
In an attempt to further elucidate a mechanism for the
observed changes, we purified fibrinogen from a limited
number of controls and AAA patients. Clots made from
purified fibrinogen samples did not show a difference in
structure, unlike those from plasma samples. Together with
the thrombin generation data and the turbidity data using TF,
these results suggest that the alterations in clot structure in
AAA were not caused by changes in fibrinogen levels,
thrombin generation, or modifications of the fibrinogen
molecule itself but that they may be caused by another,
unknown factor. However, although the calcium-dependent
IF-1 antibody, which we used to purify fibrinogen from
plasma, is known to bind a large number of different
fibrinogen variants (including proteins with mutations, dele-
tions, splice variations, glycation, and acetylation), we cannot
exclude that this procedure failed to isolate a subpopulation
of abnormal fibrinogen. There are several candidate factors
that may provide a mechanism responsible for changes in clot
structure in AAA. Factors that change clot structure indepen-
dently of thrombin generation include factor XIIa,25 C3a,26
and lipoprotein(a).27 Future studies are required to determine
whether these or other factors could be responsible for the
observed effects.
Smoking is one of the strongest risk factors for AAA.
Undas et al showed that current smoking in healthy subjects
reduces clot permeability and prolongs lysis times.28 Another
recent study showed that cigarette smoke has direct and
immediate effects on clot structure, leading to thinner fibers,
smaller pores, and increased stiffness.29 Passive exposure to
secondhand smoke has also been shown to influence clot
structure and stiffness.20 In our study, the number of current
smokers did not differ between the groups, which indicates
that smoking was not responsible for the observed differences
in plasma clot structure. We did observe a higher number of
ever smokers in patients with AAA, particularly those with
large AAA, in agreement with previous studies.2
We found a stepwise increase in lysis from the controls to
small and large AAA patients, which agrees with the differ-
ences in plasma clot structure. Statins have previously been
shown to produce beneficial effects on clot structure by
increasing pore size and enhancing fibrinolysis rates.30 How-
ever, we found a decrease in plasma clot porosity in patients
with large AAA, despite the fact that a larger proportion of
these patients were treated with statins. These data suggest
that statins may be insufficient in these patients to counteract
changes in clot structure.
There are several possible consequences of altered clot
structure in AAA. It has been suggested that larger aneurysms
are associated with increased convection flow, which, in the
Downloaded from http://atvb.ahajournals.org/ at Universiteitsbibliotheek Utrecht on March 17, 2015
Scott et al Clot Structure in AAA 3009
context of an aorta with normally very fast-flowing blood,
leads to local pockets of reduced flow and shear stress.31,32
This reduced flow, in combination with the propensity of
patients with large aneurysms to form denser clots that are
slower to lyse, could significantly increase the prothrombotic
burden in the aneurysmal aorta. Furthermore, it is possible
that a more densely packed clot may trap and accumulate a
greater number of polymorphonuclear leukocytes, which in
turn release biologically active proteases, causing AAA dilata-
tion. This is supported by recent studies reporting that ILT leads
to increased release of pro–matrix metalloproteinase-9 and other
proteases, causing degradation of the extracellular matrix.9,33 On
the other hand, a recent report has suggested that ILT composed
of dense fibrin may deprive the thrombus of cells, thereby
reducing proteolysis.34 Finally, altered clot structure may influ-
ence the elastic properties of the thrombus, with potential
implications for AAA extension and growth. Additional studies
will be required to investigate the effects of a densely packed
thrombus and clot elasticity on the vascular wall.
Fibrin polymerization showed no differences in maximum
absorbency in AAA. It has been reported that maximum
absorbency reflects average fiber thickness.35 The reason for
a lack of significance in maximum absorbency is not clear,
but it may reflect the small study size. Overall, our data
suggest that maximum absorbency may not be a strong
determinant of clot structure. This is supported by Collet et al,
who reported that tighter fiber arrangement is more important
than fiber diameter in determining fibrinolysis speed.36 We
observed a prolongation of the lag phase by turbidity in AAA.
Lag phase represents rate of lateral aggregation of fibrin
protofibrils.35 The prolonged lag phase in AAA therefore
suggests a decrease in lateral fiber aggregation, which is in
agreement with reduced porosity for the fibrin network.
The abnormal plasma clot structure in AAA appears
similar to those previously described for other cardiovascular
disease, both in terms of type and magnitude of the changes.
Studies in patients with coronary artery disease have found
denser clots, with increased stiffness, decreased permeability,
and decreased susceptibility to lysis.13,14 Changes in clot
structure in patients with acute coronary disease are associ-
ated with markers of inflammation and oxidative stress.37
Clot structure also show similar changes in patients with
venous thrombosis and their first-degree relatives, suggesting
that clot structure provides a common mechanism for venous
and arterial thrombosis.38 Our current data suggest that clot
structure may represent an important common link between
other cardiovascular disease and AAA. These findings further
suggest that drugs that counteract abnormal clot structure
may be beneficial to reduce the risk for other cardiovascular
events in patients with AAA. Aspirin and statins have been
shown to change clot structure beneficially30,39; however,
new drugs may be required to address this issue without
incurring a bleeding risk.
In conclusion, large AAA patients produce clots with lower
permeability than small AAA and controls. Lysis time was
increased in large AAA patients compared with small AAA
and controls. These results support the following conclusions:
(1) plasma samples from large AAA produce denser clots
with smaller pores that are more resistant to fibrinolysis than
3010 Arterioscler Thromb Vasc Biol December 2011
small AAA; (2) the propensity to form dense clots resistant to
fibrinolysis is associated with aortic diameter; and (3) altered
clot structure may represent a common mechanism that links
AAA and cardiovascular disease.
Sources of Funding
This study was supported by the Garfield Weston Trust for Research
into Heart Surgery and the Circulation Foundation.
Disclosures
None.
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 Clot Architecture Is Altered in Abdominal Aortic Aneurysms and Correlates With
Aneurysm Size
D. Julian A. Scott, Priya Prasad, Helen Philippou, Sheikh Tawqeer Rashid, Soroush Sohrabi,
Daniel Whalley, Andy Kordowicz, Quen Tang, Robert M. West, Anne Johnson, Janet Woods,
Ramzi A. Ajjan and Robert A.S. Ariëns

Arterioscler Thromb Vasc Biol. 2011;31:3004-3010; originally published online September 15,
2011;
doi: 10.1161/ATVBAHA.111.236786
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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Copyright © 2011 American Heart Association, Inc. All rights reserved.
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