Regina M. Herges, Thomas G. McLeod, Paul A. Friedman and Waldemar E.

Wysokinski
Naser Ammash, Ewa A. Konik, Robert D. McBane, Dong Chen, Julie I. Tange, Diane E. Grill,
Fibrillation
ADAMTS13 Homeostasis in Atrial Left Atrial Blood Stasis and Von Willebrand Factor
Print ISSN: 1079-5642. Online ISSN: 1524-4636
Copyright 2011 American Heart Association, Inc. All rights reserved.
Greenville Avenue, Dallas, TX 75231
is published by the American Heart Association, 7272 Arteriosclerosis, Thrombosis, and Vascular Biology
doi: 10.1161/ATVBAHA.111.232991
2011;
2011;31:2760-2766; originally published online August 18, Arterioscler Thromb Vasc Biol.
http://atvb.ahajournals.org/content/31/11/2760
World Wide Web at:
The online version of this article, along with updated information and services, is located on the

http://atvb.ahajournals.org//subscriptions/
at:
is online Arteriosclerosis, Thrombosis, and Vascular Biology Information about subscribing to Subscriptions:

http://www.lww.com/reprints
Information about reprints can be found online at: Reprints:

document. Question and Answer
Permissions and Rights page under Services. Further information about this process is available in the
which permission is being requested is located, click Request Permissions in the middle column of the Web
Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for
can be obtained via RightsLink, a service of the Arteriosclerosis, Thrombosis, and Vascular Biology in
Requests for permissions to reproduce figures, tables, or portions of articles originally published Permissions:
by guest on April 25, 2014 http://atvb.ahajournals.org/ Downloaded from by guest on April 25, 2014 http://atvb.ahajournals.org/ Downloaded from
Left Atrial Blood Stasis and Von Willebrand
FactorADAMTS13 Homeostasis in Atrial Fibrillation
Naser Ammash, Ewa A. Konik, Robert D. McBane, Dong Chen, Julie I. Tange, Diane E. Grill,
Regina M. Herges, Thomas G. McLeod, Paul A. Friedman, Waldemar E. Wysokinski
ObjectiveLeft atrial blood stasis is associated with increased risk for left atrial appendage thrombus (LAAT) and stroke
in atrial fibrillation (AF). Von Willebrand factor (VWF) is associated with thromboembolism in AF. VWF
thrombogenic activity is proportional to multimer size, which is regulated by VWF-cleaving protease (ADAMTS13).
Methods and ResultsTo assess the association between left atrial blood stasis and VWF-ADAMTS13 system, plasma VWF
antigen (VWF:Ag), VWF activity (VWF:Act), and ADAMTS13 activity were measured in 414 consecutive patients with
nonvalvular AF (age 6313 years; 25% women) and in 100 patients (age 6414 years; 39% women) with normal sinus
rhythm. Spontaneous echocardiographic contrast (SEC), left atrial appendage emptying velocity, and LAAT were assessed by
transesophageal echocardiography. Presence and intensity of SEC varied directly with VWF:Ag and VWF:Act but not with
ADAMTS13 activity. AF patients with LAAT had higher VWF:Ag (20061 versus 15552, P0.0006) and VWF:Act
(17957 versus 14151 P0.0026) compared with those without LAAT. VWF:Ag and VWF:Act were independent
predictors of LAAT after adjustment for CHADS
2
score (P0.0179 and P0.0497, respectively).
ConclusionThe association between VWF and SEC may explain the thrombotic propensity in AF. Elevated VWF:Ag
may help identify AF patients at risk for LAAT. (Arterioscler Thromb Vasc Biol. 2011;31:2760-2766.)
Key Words: stroke

thrombosis

von Willebrand factor

atrial fibrillation
C
ardioembolic stroke in the setting of atrial fibrillation
(AF) begins with the left atrial appendage thrombus
(LAAT) formation.
13
The risk for LAAT development and
stroke increases with left atrium mechanical function deteri-
oration that is reflected by reduction in left atrial appendage
emptying velocity (LAAEV), development of spontaneous
echocardiographic contrast (SEC), and progression of left
atrial distension.
47
However, the underlying mechanism
behind the relationship between AF, atrial stasis, and throm-
botic propensity is complex and poorly understood.
Von Willebrand factor (VWF) a large plasma glycoprotein
that mediates platelet adhesion and aggregation,
8
has been
associated with stroke risk in AF.
913
Prior studies comparing
VWF levels between AF patients and subjects in normal sinus
rhythm (NSR) were limited by small sample size.
1416
As-
sessment of archived plasma samples from Stroke Prevention
in Atrial Fibrillation III participants lacked a comparator.
911
Moreover, a prior association between elevated VWF and
stroke became nonsignificant after adjustment for other clin-
ical predictors.
11
The association between VWF and AF
therefore remains largely unexplored.
The thrombogenic potential of VWF is directly propor-
tional to VWF activity (VWF:Act) determined by both
plasma concentration and multimer size,
17
which is regulated
by VWF-specific protease ADAMTS13.
18
Two patients may
have similar plasma VWF content, yet thrombotic propensity
will vary considerably depending on the proportion of high-
molecular-weight multimer content. This nuance would be
missed if mere antigen content was assessed.
Atrial distension is associated with overexpression of VWF
multimers,
19
and blood stasis has been shown to downregu-
late ADAMTS13 activity.
20
Previous studies, however, have
not systematically accounted for degrees of atrial stasis or
measured complete variables within the VWF system.
We hypothesized that atrial distension and stasis in AF are
associated with the higher VWF and increased proportion of
large multimers that lead to the development of LAAT. To
address this hypothesis, transesophageal echocardiography (TEE)
measures of stasis and LAAT and measures of VWF antigen
(VWF:Ag), VWF:Act, and ADAMTS13 activity were compared in
consecutive patients with nonvalvular AF. To our knowledge, this
concept has not previously been tested in these patients.
Methods
Patient Recruitment
All patients with nonvalvular AF who had TEE (October 4, 2007, to
April 27, 2009) were approached for study participation. Exclusion
Received on: February 16, 2011; final version accepted on: July 26, 2011.
From the Department of Internal Medicine (N.A., E.A.K., R.D.M., D.C., J.I.T., D.E.G., R.M.H., T.G.M., P.A.F., W.E.W.), Division of Cardiovascular
Medicine (N.A., E.A.K., R.D.M., P.A.F., W.E.W.), Primary Care Internal Medicine (T.G.M.), Department of Laboratory Medicine and Pathology (D.C.,
J.I.T.), Division of Biomedical Statistics and Informatics (D.E.G., R.M.H.), Mayo Clinic and Foundation for Education and Research, Rochester, MN.
Correspondence to Waldemar E. Wysokinski, MD, Division of Cardiovascular Medicine, Mayo Clinic and Foundation for Education and Research,
200 SW First St, Rochester, MN 55905. E-mail wysokinski.waldemar@mayo.edu
2011 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.111.232991
2760 by guest on April 25, 2014 http://atvb.ahajournals.org/ Downloaded from
criteria included (1) acute illness, stroke, myocardial infarction, or
surgery within 30 days; (2) more than moderate heart valvular
disease; (3) artificial heart valves; (4) prior unprovoked venous or
arterial thrombosis; (5) prior major bleeding unrelated to warfarin
therapy; (6) liver disease; (7) active malignancy; or (8) hormonal
stimulation (estrogen/progesterone therapy or pregnancy). The de-
tails related to AF onset, timing, chronicity, underlying etiologies,
cardiac anatomy and physiology, relevant comorbidities, and medi-
cal and interventional treatment of AF, particularly anticoagulation,
were collected prospectively and entered into SAS GEN database,
which is an SAS-based system for managing clinical data. Control
subjects consisted of patients in NSR with no prior history of AF.
These subjects were recruited from the Primary Care Internal
Medicine clinic during their annual medical examination.
Evaluation With TEE
TEE was performed as previously described using commercially
available ultrasound instruments and a multiplane probe.
21,22
LAAT
was defined as an echogenic mass in the appendage or body of the
atrium, distinct from the underlying endocardium and pectinate
muscles and detected in more than 1 imaging plane.
21,22
SEC was
defined as a pattern of dynamic smokelike, slowly swirling,
intracavitary echo-densities imaged with gain settings adjusted to
eliminate background noise. SEC was graded as absent, mild,
moderate, or severe according to the published echocardiographic
criteria.
23
The LAAEV profiles were measured over 5 consecutive
cardiac cycles using pulsed wave Doppler interrogation with the
sample volume positioned 1 cm within the orifice of the LAA.
19
The
left ventricular ejection fraction was visually estimated. Aortic
atherosclerosis severity was defined as simple when atheroma
thickness was 4 mm and immobile. Severe atheroma exceeded
4 mm or contained mobile components.
2124
Given the known
difficulties in measuring left atrial volume by TEE, left atrium
volume index (LAVI) was assessed by transthoracic echo performed
within 1 month of the TEE study and calculated by the biplane
area-length method.
24
All echocardiographic images were analyzed
by the study cardiologist (N.A.), who was blinded to clinical and
laboratory data.
Study Definitions and Event Adjudication
The CHADS
2
score was assigned for each patient.
25
The presence of
AF was confirmed by either ECG or Holter monitoring. Congestive
heart failure was defined as the presence of clinical symptoms and
signs of heart failure within the last 3 months with or without
evidence of left ventricular systolic dysfunction by echocardiogra-
phy.
25
Diabetes mellitus was diagnosed based on the criteria recom-
mended by the American Diabetes Association.
26
Stroke and tran-
sient ischemic attack were defined by criteria proposed by the
American Heart Association.
27
AF was classified as paroxysmal,
persistent, or permanent in accordance with current guidelines.
28
Sample Collection
For each patient, 20 mL of citrate blood was collected by antecubital
venipuncture using a 19-gauge thin-wall butterfly needle with a short
plastic tube extension. For AF patient scheduled for electric cardio-
version or radiofrequency ablation, phlebotomy was uniformly
performed before this procedure.
Assays of Plasma VWF:Ag, VWF:Act, and
ADAMTS13 Activity
VWF:Ag in plasma was measured using HemosIL von Willebrand
Factor Antigen latex immunoassay kits (Instrumentation Laboratory,
Lexington, MA) with 2 ACL TOP coagulation system analyzers
(Beckman Coulter, Brea, CA), following the manufacturers instruc-
tions. VWF:Act in plasma was measured using HemosIL von
Willebrand Factor Activity latex immunoassay kits (Instrumentation
Laboratory) on 2 ACL TOP coagulation analyzers, following the
manufacturers instructions. Briefly, prediluted patient samples (1:1
ratio of plasma to diluent) and latex particleenhanced immunotur-
bidity reagents were added to testing cuvettes. Optical densities were
measured at 405 nm, and VWF:Act was extrapolated from standard
curves.
29
The lower limit of normal for VWF:Ag is 55 IU/dL, and
that of VWF:Act is 55 U/dL. The standard relationship between
activity and antigen is greater than 0.7 (ratio of VWF:Act to
VWF:Ag). The validated normal range of the VWF:Act/VWF:Ag
ratio (n452 normal donors) is 0.7 to 1.4, with mean at 1.0.
ADAMTS13 protease activity (expressed as percentage of normal)
was measured by a fluorescence resonance energy transfer based
assay using a VWF 73 amino-acid peptide substrate
30
(commercial
kit, GTI Diagnostics, Waukesha, WI).
Plasma VWF Multimer Analysis
Plasma VWF multimers were analyzed using an intermediate reso-
lution discontinuous electrophoretic agarose gel system, with in-gel
immunostaining, and near-infrared fluorescent images were acquired
and processed using an Odyssey imaging system (LI-COR Biosci-
ences, Lincoln, NE) as previously described.
31
Statistical Methods
Demographic, clinical, and echocardiographic patients characteris-
tics were tested for statistical significance using the non parametric
Kruskal-Wallis analysis of variance for continuous variables or
Pearsons
2
test for categorical variables. Wilcoxon rank-sum tests
were used for 2-level group comparisons between continuous vari-
ables. The relationship between LAAEV, LAVI, the intensity of SEC
and the presence of LAAT was tested using Kruskal-Wallis
ANOVA. Pearsons correlation was calculated to assess the relation-
ship between LAAEV, LAVI, and both VWF:Ag and VWF:Act.
Logistic regression models were fit to assess the relationship of the
VWF and ADAMTS13 variables with LAAT after adjusting for
comorbidities using the CHADS
2
score. Results are presented as
c-statistics, which are equivalent to the area under the receiver
operating characteristic curve. To provide consistency and allow for
comparisons between the VWF ADMATS 13 system variables, the
odds ratio for developing LAAT is presented as the odds of a value
at the 75th percentile of the distribution relative to a value at the 25th
percentile was also calculated. The effect of medications on the
VWF ADAMTS13 system was tested using linear regression after
adjusted for CHADS
2
and the type of AF.
Results
Demographic, Clinical and
Echocardiographic Data
Of 1423 AF patients screened, 520 had nonvalvular AF, met
inclusion and exclusion criteria, and agreed to participate.
Ninety-five patients were excluded because the TEE was
canceled, the phlebotomy was not performed, or the phlebot-
omy was performed postprocedure. Eleven patients were
excluded because the only documented rhythm was atrial
flutter. The remaining 414 patients with documented AF and
TEE assessment constituted the study group. TEE studies
were requested to exclude intracardiac thrombi before elec-
trophysiological procedures (69%), cardioversion (17%), or
for cardioembolic risk assessment (14%).
There were fewer women in the AF group compared with
NSR controls (Table 1). Antiplatelet and statin therapy was
similar between groups. Significantly more AF patients were
on warfarin therapy compared with controls. Three of 414
patients had no SEC assessment by TEE because of subop-
timal left atrium appendage image. A varying degree of SEC
was noted in 234 patients (57%), including 20 with LAAT
(5%). Gradual intensification of SEC, as noted by TEE, up to
the development of LAAT was associated with older age,
permanent type of AF, lower left ventricular ejection fraction,
and the presence of atherosclerotic plaques in aorta. With the
Ammash et al VWF and Stasis in Fibrillating Atrium 2761
by guest on April 25, 2014 http://atvb.ahajournals.org/ Downloaded from
exception of AF patients without SEC, warfarin use was very
high in all other AF subgroups.
Although the mean CHADS
2
scores were similar in NSR
and AF groups, a history of prior stroke/transient ischemic
attack was more prevalent in AF patients (Table 2). The
prevalence of congestive heart failure, hypertension, diabetes
mellitus, and prior stroke/transient ischemic attack was asso-
ciated with greater intensity of SEC and development of
LAAT. The overall distribution of CHADS
2
scores was
similar between NSR subjects and AF patients. In the
aggregate, a direct relationship of CHADS
2
score with the
development and intensity of SEC was apparent. Although
significantly more AF patients with SEC or LAAT had high
CHADS
2
scores (46), the majority of AF patients with
LAAT (75%) had relatively low CHADS
2
scores (13).
LAAEV measurement was obtained in 343 (83%) of AF
patients. A strong inverse relationship between LAAEV and
both the intensity of SEC and LAAT was evident (Figure 1A,
P0.0001). Transthoracic echocardiogram with LAVI calcu-
lation was available in 346 AF patients (84%). A strong direct
association was observed between LAVI and degree of SEC
intensity (Figure 1B, P0.0001). LAVI did not differ in AF
patients with LAAT compared with those with severe SEC
(P0. 99).
VWF-ADAMTS13 System Evaluation
Overall, the VWF:Ag concentration did not differ signifi-
cantly between AF patients and NSR controls (15753
IU/dL versus 14448 IU/dL, P0.08). AF patients without
SEC (n177) had VWF:Ag essentially identical to that of
NSR controls (14351 IU/dL versus 14448 IU/dL). AF
patients with SEC, however (n214), had significantly
higher VWF:Ag compared with NSR controls (16452
IU/dL versus 14448 IU/dL, P0.006). AF patients with
LAAT had significantly higher VWF:Ag compared with
those without LAAT (20061 IU/dL versus 15552 IU/dL,
P0.001).
VWF:Act was proportional to antigen concentration, with a
VWF:Ag/VWF:Act ratio of 0.9 in AF patients across all SEC
subgroups and in NSR controls (Figure 2). Six patients had
abnormal VWF:Act/VWF:Ag ratios (0.7), and 1 patient had
an inverted ratio (1.46). To further evaluate these 7 patients,
VWF multimer analyses were performed. Although grossly
normal, subtle differences in VWF multimer distribution can
Table 1. Demographic and Clinical Characteristics of Individuals With Normal Sinus Rhythm and Patients With Atrial Fibrillation in
Relation to SEC Intensity and LAAT
Variable (%)
NSR Controls
(n100)
AF Cases
(n414)* P
No SEC
(n177)
SEC
LAAT
(n20) P Mild (n104) Moderate (n73) Severe (n37)
Age (meanSD) 6414 6313 0.44 5813 6512 6712 6812 7112 0.001
Women 39 25 0.005 21 24 33 30 30 0.31
AF
Type 0.001
Uncertain 7 2 11 10 11 10
Paroxysmal 49 72 39 30 16 10
Persistent 28 18 35 40 32 30
Permanent 17 7 15 21 41 50
Duration 0.17
Uncertain 1 2 0 1 0 0
48 h 5 9 4 4 0 0
27 d 5 7 5 1 8 5
14 wk 6 3 11 8 5 5
112 mo 22 19 27 22 22 20
1 y 61 63 54 64 65 70
CAD 18 20 0.72 7 25 30 27 50 0.001
Aspirin 51 47 0.51 49 44 49 43 45 0.92
Plavix 0 4 0.05 3 4 4 3 10 0.59
Warfarin 2 77 0.001 64 82 93 97 85 0.001
Statin 35 38 0.59 29 39 48 46 60 0.008
LVEF 5511 588 5610 5313 4914 4616 0.001
Aortic arch atheroma 0.001
4 mm 55 45 64 61 63 70
4 mm 7 2 8 13 11 20
SEC indicates spontaneous echo contrast; LAAT, left atrial appendage thrombus; NSR, normal sinus rhythm; AF, atrial fibrillation; CAD, coronary artery disease; LVEF,
left ventricular ejection fraction.
*Total no. of AF patients also includes 3 cases that had no SEC assessment, only left atrial appendage emptying velocity measurement, because of technical
difficulties.
2762 Arterioscler Thromb Vasc Biol November 2011
by guest on April 25, 2014 http://atvb.ahajournals.org/ Downloaded from
be appreciated (Figure 3). Individuals with reduced activity/
antigen ratios are associated with a reduction in the highest
molecular weight multimers compared with normal pooled
plasma. In contrast, the individual with an inverted ratio
displayed a relative abundance of the highest molecular
weight multimers. Of interest, this particular patient had
severe SEC by TEE.
AF patients with LAAT had significantly higher VWF:Act
compared with those without LAAT (17957 U/dL versus
14151 U/dL, P0.0026). When stratified by SEC intensity
and LAAT presence, there was a direct and significant
(P0.001) relationship between VWF:Ag and VWF:Act (Fig-
ure 2). This relationship was not evident for ADAMTS13, which
was similar across all AF subsets and NSR controls.
An inverse, albeit weak, correlation between LAAEV and
both VWF:Ag (Pearsons correlation r0.152, P0.005) and
VWF:Act (Pearsons correlation r0.148, P0.006) was
observed. Similarly, LAVI correlated directly, though weakly
with VWF:Ag (Pearsons correlation r0.170, P0.001) and
VWF:Act (Pearsons correlation r0.165, P0.002).
Logistic regression analysis revealed that both VWF:Ag and
VWF:Act were independent predictors of LAAT after adjust-
ment for CHADS
2
score (P0.0179 and P0.0497, respec-
tively) and every element of CHADS
2
system separately.
LAAEV (c-stat0.958, P0.005), congestive heart failure
(c-stat0.747, P0.0001), VWF:Ag (c-stat0.730, P0.0005)
and VWF:Act (c-stat0.695, P0.0025) had the best discrim-
ination in AF patients with LAAT compared with those without
thrombus. LAAEV analysis was performed based on data
available from 343 patients; however, only 9 of the 20 patients
with LAAT had LAAEV measurements performed.
The odds of developing LAAT was 1.5 times greater if AF
patients VWF:Ag was at the 75th percentile relative to being
at the 25th percentile (VWF:Ag values of 184% versus 120%;
P0.001). For VWF:Act values, this odds ratio relative to AF
was almost identical (1.47).
Because gradual intensification of SEC was associated
with permanent type of AF, both VWF:Ag and VWF:Act
were higher in patients with permanent AF (17457 IU/dL
and 16055 U/dL) compared with paroxysmal AF (14853
IU/dL and 13348 U/dL, P0.001 and P0.001, respec-
tively) but not significantly different when compared with
patients with persistent AF (16055 IU/dL and 14853
U/dL, P0.11 and P0.18, respectively).
Measures of VWF system (VWF:Ag, VWF:Act, ADAMTS13)
were not affected by warfarin, aspirin, or statin therapy (data
not shown). There was no significant association between
VWF measures and gender.
Discussion
The main finding of this study is that AF patients with SEC
and particularly with LAAT have significantly higher VWF
levels compared with AF patients without SEC or control
subjects in NSR. A direct relationship was observed between
the degree of left atrial stasis (SEC intensity and LAAT) and
elevated VWF. The other 2 TEE measures of left atrium
blood stasis, LAAEV and LAVI, were also associated with
VWF, although the relationship was less pronounced. SEC
generation is complex and influenced not only by blood stasis
but also age, left ventricular function, aortic atherosclerosis,
plasma fibrinogen, and erythrocytes.
5,32
In our patients with
AF, VWF levels remained normal until SEC developed.
Others have reported conflicting results of VWF levels in AF
patients compared with NSR controls.
1216
These findings
may reflect differences in accrual rates of AF patients with
SEC which was not accounted for in those studies.
1216
Furthermore, VWF:Ag and VWF:Act are independent
predictors for LAAT after adjustment for CHADS
2
score
Table 2. CHADS
2
Score in Subjects With NSR and Patients With AF in Relation to SEC Intensity and LAAT
Variables (%)
NSR Controls
(n100)
AF Cases
(n414)* P
No SEC
(n177)
SEC
LAAT
(n20) P Mild (n104) Moderate (n73) Severe (n37)
CHF 15 23 0.08 8 24 34 46 70 0.001
Hypertension 50 60 0.07 44 68 71 78 85 0.001
Age75 y 25 18 0.09 7 21 23 32 45 0.001
Diabetes 21 14 0.06 6 14 22 24 30 0.001
Stroke/TIA prior 6 14 0.03 9 11 18 30 35 0.001
CHADS
2
(meanSD) 1.21.3 1.41.4 0.18 0.81.1 1.51.2 1.91.3 2.41.7 3.01.4 0.001
CHADS
2
score 0.85 0.001
0 37 31 49 20 16 16 0
1 31 31 32 40 27 14 10
2 13 17 11 19 21 24 30
3 12 14 5 12 27 22 35
4 5 5 3 6 7 11 5
5 2 3 0.6 2 0 11 15
6 0 1 0 0 1 3 5
NSR indicates normal sinus rhythm; AF, atrial fibrillation; SEC, spontaneous echo contrast; LAAT, left atrial appendage thrombus; CHF, congestive heart failure; TIA,
transient ischemic attack.
*Total no. of AF patients also includes 3 cases that had no SEC assessment, only left atrial appendage emptying velocity measurement, because of technical
difficulties.
Ammash et al VWF and Stasis in Fibrillating Atrium 2763
by guest on April 25, 2014 http://atvb.ahajournals.org/ Downloaded from
effectively separating those with LAAT. In a large cohort
from the Stroke Prevention in Atrial Fibrillation III trial, after
adjustment for other clinical predictors, the relationship
between VWF and stroke became nonsignificant.
11
However,
nearly half of strokes occurring in AF patients are due to
noncardioembolic mechanisms,
33
and in this trial, stroke
subtypes were not analyzed. Whereas VWF is integral in both
the initiation and propagation of thrombus formation, these
findings may help explain the association between atrial
stasis and thrombotic propensity in AF. Atrial distension
both injures local endothelium and stimulates endothelial
release of ultralarge VWF multimers.
19
Under laminar
blood flow conditions, ultralarge VWF is rapidly degraded by
ADAMTS13.
34
Under static conditions, proteolysis is re-
tarded 1000-fold.
20,29
Therefore, within the fibrillating left
atrial appendage, excess VWF secretion and inadequate
ADAMTS13 cleavage result in elevated local concentrations
of ultralarge VWF multimers, thus favoring thrombus forma-
tion. Within the flow conditions of the fibrillating atrium,
laminar flow is abolished, velocity (implying both direction
and speed) is chaotic, and one might envision undulating
unfolding, refolding, and chaotic bending and twisting of the
released ultralarge multimer, in turn activating the protein while
simultaneously inducing and then inhibiting ADAMTS13 cleav-
age. Combining variable degrees of atrial endothelial injury
further complicates the localized propensity for thrombus
formation.
As demonstrated by others, VWF measures may improve
thrombosis risk stratification in AF patients.
35
We now
provide evidence that that measures of atrial stasis are
strongly associated with VWF levels. This benefit of adding
VWF measures to the risk assessment was illustrated in 20
AF patients with LAAT in our analysis. Among these
patients, 40% had low CHADS
2
scores, and 10% of patients
had scores of 1. This latter group might not have received
warfarin anticoagulation if the risk assessment was limited to
the CHADS
2
system.
Left atrial appendage velocity and VWF concentrations
were independently associated with LAAT in a previous
study of 109 patients with AF.
36
Moreover, a significant
correlation has been observed between the endocardial VWF
expression, platelet adhesion, and thrombus formation in the
left atrial appendage.
19,37
These combined data corroborate
the relationships between AF, atrial whole blood stasis, VWF
levels, and thrombotic propensity found in our study.
There was no evidence of excessive release of large VWF
multimers into the general circulation. Only one patient with
severe SEC had an inverted ratio of VWF:Act to VWF:Ag
ratio (ratio1.4). In this individual, a relative abundance of
the highest molecular weight multimers was evident (Figure
3). In contrast to others,
13
we found no relationship between
VWF and ADAMTS13 activity, which was similar for AF
patients and control subjects in NSR.
VWF levels rise with inflammation and represent acute
phase reactivity. This raises concerns of reproducibility. A
prospective cohort study of nearly 19 000 individuals, how-
ever, found that VWF reproducibility is similar to blood
Figure 1. Relationship between left atrial appendage size and
function and whole blood stasis. The association between mea-
sures of whole blood stasis (spontaneous echo contrast [SEC])
up to the development of left atrial appendage thrombus (LAAT)
and either left atrial appendage emptying velocity (LAAEV) (A) or
left atrial volume index (LAVI) (B) were assessed for patients
with nonvalvular atrial brillation. Measures are presented as
meanstandard deviation.
Figure 2. Relationship between components of the von Wille-
brand system and whole blood stasis. Progression of whole
blood stasis (spontaneous echo contrast [SEC]) up to the devel-
opment of left atrial appendage thrombus (LAAT) correlated with
both von Willebrand factor (VWF) antigen () and VWF activity
(E). In contrast, measures of ADAMTS13 activity () were simi-
lar for subgroups of patients with nonvalvular atrial brillation
(AF) and control subjects with normal sinus rhythm (NSR). Mea-
sures are presented as meanstandard deviation.
2764 Arterioscler Thromb Vasc Biol November 2011
by guest on April 25, 2014 http://atvb.ahajournals.org/ Downloaded from
pressure and serum cholesterol.
38
This suggests that VWF is
sufficiently stable for potential use in the long-term predic-
tion of cardiovascular disease. Moreover, VWF levels are not
influenced by warfarin or aspirin, common therapies in AF.
10
Several study limitations deserve comment. First, SEC is a
dynamic process that is subjectively quantified. To improve
reproducibility of this measure, assessment was limited to one
cardiologist blinded to both clinical and laboratory data.
Furthermore, although the TEE was often not repeated
sequentially, the measures obtained may not adequately
capture the severity of atrial stasis over time. Second, we
cannot exclude referral bias for the AF patients enrolled. Only
AF patients who had a TEE requested by their primary health
care provider were approached for recruitment. The clinical
characteristics of these patients, however, did not differ from
those of typical AF patients at our institution
21,22
or partici-
pants of large multicenter registries.
3,4,9
Third, the blood type
was not measured. Blood type O is associated with lower than
normal values of circulating VWF.
In summary, we now provide evidence supporting a direct
relationship between VWF and measures of atrial whole
blood stasis. The direct correlation between VWF levels and
the severity of left atrial whole blood stasis may help to
explain the thrombotic propensity among AF patients. Ele-
vated VWF may identify AF patients at risk for LAAT. There
is a temptation to provide a cutoff value for VWF levels
discriminating stroke risk or lack thereof. Whereas stroke in
AF is a complex and poorly understood phenomenon, defin-
ing critical values of one of the pieces of this puzzle
represents a willingness to overly simply a complex process.
Similar paradigms have been proposed for hypertension and
cholesterol. Although undisputed in the risk of coronary
disease, the risk and treatment targets are indistinct and
constantly changing. Thrombosis in AF is no less complex.
Sources of Funding
This work was funded, in part, by Grant 17896 CR 20 from the
Department of Internal Medicine, Mayo Clinic and Foundation for
Medical Education and Research.
Disclosures
None.
References
1. Aberg H. Atrial fibrillation: I: a study of atrial thrombosis and systemic
embolism in a necropsy material. Acta Med Scand. 1969;185:373379.
2. Manning WJ, Silverman DI, Waksmonski CA, Oettgen P, Douglas PS.
Prevalence of residual left atrial thrombi among patients with acute
thromboembolism and newly recognized atrial fibrillation. Arch Intern
Med. 1995;155:21932198.
3. Klein AL, Grimm RA, Murray RD, Apperson-Hansen C, Asinger RW,
Black IW, Davidoff R, Erbel R, Halperin JL, Orsinelli DA, Porter TR,
Stoddard MF; Assessment of Cardioversion Using Transesophageal
Echocardiography Investigators. Use of transesophageal echocardiogra-
phy to guide cardioversion in patients with atrial fibrillation N Engl
J Med. 2001;344:14111420.
4. Handke M, Harloff A, Hetzel A, Olschewski M, Bode C, Geibel A. Left
atrial appendage flow velocity as a quantitative surrogate parameter for
thromboembolic risk: determinants and relationship to spontaneous echo-
contrast and thrombus formation: a transesophageal echocardiographic
study in 500 patients with cerebral ischemia. J Am Soc Echocardiogr.
2005;18:13661372.
5. Goldman ME, Pearce LA, Hart RG, Zabalgoitia M, Asinger RW, Safford
R, Halperin JL. Pathophysiologic correlates of thromboembolism in non-
valvular atrial fibrillation: I: reduced flow velocity in the left atrial
appendage (the Stroke Prevention in Atrial Fibrillation [SPAF-III] study).
J Am Soc Echocardiogr. 1999;12:10801087.
6. Bernhardt P, Schmidt H, Hammerstingl C, Luderitz B, Omran H. Patients
with atrial fibrillation and dense spontaneous echo contrast at high risk a
prospective and serial follow-up over 12 months with transesophageal
echocardiography and cerebral magnetic resonance imaging. J Am Coll
Cardiol. 2005;45:18131814.
7. Zabalgoitia M, Halperin JL, Pearce LA, Blackshear JL, Asinger RW, Hart
RG. Transesophageal echocardiographic correlates of clinical risk of
thromboembolism in nonvalvular atrial fibrillation. Stroke Prevention in
Atrial Fibrillation III Investigators. J Am Coll Cardiol. 1998;31:
16221626.
8. Ruggeri ZM, Savage B. Biological functions of von Willebrand factor. In:
Ruggeri ZM, ed. Von Willebrand Factor and the Mechanisms of Platelet
Function. Berlin, Germany: Springer; 1998:79109.
9. Feinberg WM, Pearce LA, Hart RG, Cushman M. Cornell ES, Lip GY,
Bovill EG. Markers of thrombin and platelet activity in patients with atrial
fibrillation: correlation with stroke among 1531 participants in the stroke
prevention in atrial fibrillation III study. Stroke. 1999;30:25472553.
10. Conway DSG, Pearce LA, Chin BS, Hart RG, Lip GY. Plasma von
Willebrand factor and soluble P-selectin as indices of endothelial damage
and platelet activation in 1321 patients with non-valvular atrial fibril-
lation: relationship to stroke risk factors. Circulation. 2002;106:
19621967.
Figure 3. Plasma von Willebrand factor (VWF) multimers. Examples of plasma VWF multimer electrophoresis are presented for normal
pool plasma (NPP), inversed VWF:Act/VWF:Ag ratio (patient A), and reduced VWF:Act/VWF:Ag ratio 0.7 (patient B) (A). Electrophoretic
densitometry highlights the differences in the molecular weight distribution of VWF multimers between these examples (B).
Ammash et al VWF and Stasis in Fibrillating Atrium 2765
by guest on April 25, 2014 http://atvb.ahajournals.org/ Downloaded from
11. Conway DS, Pearce LA, Chin BS, Hart RG, Lip GY. Prognostic value of
plasma von Willebrand factor and soluble P-selectin as indices of endo-
thelial damage and platelet activation in 994 patients with nonvalvular
atrial fibrillation. Circulation. 2003;107:31413145.
12. Watson T, Arya A, Sulke N, Lip GY. Relationship of indices of inflam-
mation and thrombogenesis to arrhythmia burden in paroxysmal atrial
fibrillation. Chest. 2010;137:869876.
13. Uemura T, Kaikita K, Yamabe H, Soejima K, Matsukawa M, Fuchigami
S, Tanaka Y, Morihisa K, Enomoto K, Sumida H, Sugiyama S, Ogawa H.
Changes in plasma von Willebrand factor and ADAMTS13 levels asso-
ciated with left atrial remodeling in atrial fibrillation. Thromb Res. 2009;
124:2832.
14. Freestone B, Gustafsson F, Chong AY, Corell P, Kistorp C, Hildebrandt
P, Lip GY. Influence of atrial fibrillation on plasma von Willebrand
factor, soluble E-selectin, and N-terminal pro B-type natriuretic peptide
levels in systolic heart failure. Chest. 2008;133:12031208.
15. Fu R, Wu S, Wu P, Qiu J. A study of blood soluble P-selectin, fibrinogen,
and von Willebrand factor levels in idiopathic and lone atrial fibrillation.
Europace. 2011;13:3136.
16. Hou J, Liang Y, Gai X, Zhang H, Yang X, Lan X, Zheng W, Huang M.
The impact of acute atrial fibrillation on the prothrombotic state in
patients with essential hypertension. Clin Biochem. 2010;43:12121215.
17. Fischer BE, Thomas KB, Dorner F. Von Willebrand factor: measuring
antigen or function? Correlation between the level of antigen, activity,
and multimer size using various detection systems. Thromb Haemost.
1998;91:3943.
18. Zheng X, Chung D, Takayama TK, Majerus EM, Sadler JE, Fujikawa K.
Structure of von Willebrand factor-cleaving protease (ADAMTS13), a
metalloprotease involved in thrombotic thrombocytopenic purpura. J Biol
Chem. 2001;276:4105941063.
19. Fukuchi M, Watanabe J, Kumagai K, Katori Y, Baba S, Fukuda K, Yagi
T, Iguchi A, Yokoyama H, Miura M, Kagaya Y, Sato S, Tabayashi K,
Shirato K. Increased von Willebrand factor in the endocardium as a local
predisposing factor for thrombogenesis in overloaded human atrial
appendage. J Am Coll Cardiol. 2001;37:14361442.
20. Lopez JA, Dong JF. Cleavage of von Willebrand factor by ADAMTS13
on endothelial cells. Semin Hematol. 2004;41:1523.
21. Melduni RM, Chandrasekaran K, Friedman PA, White RD, Malouf JF,
Hodge DO, Seward JB, Oh JK, Ammash NM. Does left atrial appendage
peak emptying flow velocity predict the electrical energy required to
achieve successful direct-current cardioversion in patients with persistent
atrial fibrillation? J Am Soc Echocardiogr. 2007;20:10041008.
22. Wysokinski WE, Ammash N, Sobande F, Kalsi H, Hodge D, McBane
RD. Predicting left atrial thrombi in atrial fibrillation. Am Heart J.
2010;159:665671.
23. Fatkin D, Kelly RP, Feneley MP. Relations between left atrial appendage
blood flow velocity, spontaneous echocardiographic contrast and throm-
boembolic risk in vivo, J Am Coll Cardiol. 1994;23:961969.
24. Oh JK, Seward JB, Tajik AJ. The Echo Manual, 3rd ed. Philadelphia, PA:
Lippincott Williams and Wilkins; 1994:109119.
25. Gage BF, Waterman AD, Shannon W, Boechler M, Rich MW, Radford
MJ. Validation of clinical classification schemes for predicting stroke:
results from the National Registry of Atrial Fibrillation. JAMA. 2001;
285:28642870.
26. Genuth S, Alberti KG, Bennett P, Buse J, Defronzo R, Kahn R, Kitzmiller
J, Knowler WC, Lebovitz H, Lernmark A, Nathan D, Palmer J, Rizza R,
Saudek C, Shaw J, Steffes M, Stern M, Tuomilehto J, Zimmet P; Expert
Committee on the Diagnosis and Classification of Diabetes Mellitus.
Follow-up report on the diagnosis of diabetes mellitus. Diabetes Care
2003;26:3160.
27. Gillum RF, Fortmann SP, Prineas RJ, Kottke TE. International diagnostic
criteria for acute myocardial infarction and acute stroke. Am Heart J.
1984;108:150158.
28. Fuster V, Ryden LE, Cannom DS, Crijns HJ, Curtis AB, Ellenbogen KA,
Halperin JL, Le Heuzey JY, Kay GN, Lowe JE, Olsson SB, Prystowsky
EN, Tamargo JL, Wann S, Smith SC Jr, Jacobs AK, Adams CD,
Anderson JL, Antman EM, Halperin JL, Hunt SA, Nishimura R, Ornato
JP, Page RL, Riegel B, Priori SG, Blanc JJ, Budaj A, Camm AJ, Dean V,
Deckers JW, Despres C, Dickstein K, Lekakis J, McGregor K, Metra M,
Morais J, Osterspey A, Tamargo JL, Zamorano JL; American College of
Cardiology/American Heart Association Task Force on Practice Guide-
lines; European Society of Cardiology Committee for Practice Guide-
lines; European Heart Rhythm Association; Heart Rhythm Society. ACC/
AHA/ESC 2006 guidelines for the management of patients with atrial
fibrillation: a report of the American College of Cardiology/American
Heart Association Task Force on Practice Guidelines and the European
Society of Cardiology Committee for Practice Guidelines. Circulation.
2006;15:114:e257e354.
29. Salem RO, Van Cott EMA. New automated screening assay for the
diagnosis of von Willebrand disease. Am J Clin Pathol. 2007;127:
730735.
30. Kokame K, Nobe Y, Kokubo Y, Okayama A, Miyata T. FRETS-VWF73,
a first fluorogenic substrate for ADAMTS13 assay. Br J Haematol.
2005;129:93100.
31. Pruthi RK, Daniels TM, Heit JA, Chen D, Owen WG, Nichols WL.
Plasma von Willebrand factor multimer quantitative analysis by in-gel
immunostaining and infrared fluorescent imaging. Thromb Res. 2010;
126:543549.
32. Rastegar R, Harnick DJ, Weidemann P, Fuster V, Coller B, Badimon JJ,
Chesebro J, Goldman ME. Spontaneous echo contrast videodensity is
flow-related and is dependent on the relative concentrations of fibrinogen
and red blood cells. J Am Coll Cardiol. 2003;41:603610.
33. Hart RG, Pearce LA, Miller VT, Anderson DC, Rothrock JF, Albers GW,
Nasco E. Cardioembolic vs. noncardioembolic strokes in atrial fibril-
lation: frequency and effect of antithrombotic agents in the stroke pre-
vention in atrial fibrillation studies. Cerebrovasc Dis. 2000;10:3943.
34. Siedlecki CA, Lestini BJ, Kottke-Marchant KK, Eppell SJ, Wilson DL,
Marchant RE. Shear-dependent changes in the three-dimensional
structure of human von Willebrand factor. Blood. 1996;88:29392950.
35. Lip GY, Lane D, Van Walraven C, Hart RG. Additive role of plasma von
Willebrand factor levels to clinical factors for risk stratification of
patients with atrial fibrillation. Stroke. 2006;37:22942300; Erratum in:
Stroke. 2006;37:2444.
36. Heppell RM, Berkin KE, McLenachan JM, Davies JA. Haemostatic and
haemodynamic abnormalities associated with left atrial thrombosis in
non-rheumatic atrial fibrillation. Heart. 1997;77:407411.
37. Kumagai K, Fukuchi M, Ohta J, Baba S, Oda K, Akimoto H, Kagaya Y,
Watanabe J, Tabayashi K, Shirato K. Expression of the von Willebrand
factor in atrial endocardium is increased in atrial fibrillation depending on
the extent of structural remodeling. Circ J. 2004;68:321327.
38. Danesh J, Wheeler JG, Hirschfield GM, Eda S, Eiriksdottir G, Rumley A,
Lowe GD, Pepys MB, Gudnason V. C-reactive protein and other circu-
lating markers of inflammation in the prediction of coronary heart
disease. N Engl J Med. 2004;350:13871397.
2766 Arterioscler Thromb Vasc Biol November 2011
by guest on April 25, 2014 http://atvb.ahajournals.org/ Downloaded from