ArticlePDF AvailableLiterature Review

The Current and Future Landscape of Urinary Thromboxane Testing to Evaluate Atherothrombotic Risk

Authors:

Abstract and Figures

Biomarker testing for efficacy of therapy is an accepted way for clinicians to individualize dosing to genetic and/or environmental factors that may be influencing a treatment regimen. Aspirin is used by nearly 43 million Americans on a regular basis to reduce risks associated with various atherothrombotic diseases. Despite its widespread use, many clinicians are unaware of the link between suboptimal response to aspirin therapy and increased risk for inferior clinical outcomes in several disease states, and biomarker testing for efficacy of aspirin therapy is not performed as routinely as efficacy testing in other therapeutic areas. This article reviews the clinical and laboratory aspects of determining whole-body thromboxane production, particularly as it pertains to efficacy assessment of aspirin responsiveness.
Content may be subject to copyright.
Risk stRatification Update
The Current and Future Landscape
of Urinary Thromboxane Testing to
Evaluate Atherothrombotic Risk
Sean-Xavier Neath, MD, PhD, FACEP,1 John L. Jefferies, MD, MPH, FAAP, FACC, FAHA,2
Jeffrey S. Berger, MD, MS, FAHA, FACC,3 Alan H.B. Wu, PhD,4 Joseph P. McConnell, PhD, DABCC,5
Jeffrey L. Boone, MS, MD,6 Peter A. McCullough, MD, MPH, FACC, FACP, FCCP, FAHA, FNKF,7
Robert L. Jesse, MD, PhD,8 Alan S. Maisel, MD9
1Department of Emergency Medicine, University of California, San Diego, San Diego, CA; 2Advanced Heart
Failure and Cardiomyopathy Services, Pediatric Cardiology and Adult Cardiovascular Diseases, and Division
of Human Genetics, The Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH;
3Department of Medicine, and Marc and Ruti Bell Program in Vascular Biology, New York University School
of Medicine, New York, NY; 4Department of Laboratory Medicine, University of California, San Francisco, San
Francisco, CA; 5Health Diagnostic Laboratory, Richmond, VA; 6Boone Heart Institute, Denver, CO; 7Baylor Heart
and Vascular Institute, Dallas, TX; 8Departments of Internal Medicine and Cardiology, Virginia Commonwealth
University Health System, Richmond, VA, and Veterans Health Administration, Department of Veterans Affairs,
Washington, DC; 9Department of Medicine, Division of Cardiovascular Medicine, University of California, San
Diego and Director, Coronary Care Unit and Heart Failure Program, Veterans Affairs San Diego Healthcare
System, San Diego, CA
Biomarker testing for efficacy of therapy is an accepted way for clinicians to individual-
ize dosing to genetic and/or environmental factors that may be influencing a treatment
regimen. Aspirin is used by nearly 43 million Americans on a regular basis to reduce risks
associated with various atherothrombotic diseases. Despite its widespread use, many
clinicians are unaware of the link between suboptimal response to aspirin therapy and
increased risk for inferior clinical outcomes in several disease states, and biomarker
testing for efficacy of aspirin therapy is not performed as routinely as efficacy testing
in other therapeutic areas. This article reviews the clinical and laboratory aspects of
determining whole-body thromboxane production, particularly as it pertains to efficacy
assessment of aspirin responsiveness.
[Rev Cardiovasc Med. 2014;15(2):119-130 doi: 10.3909/ricm0739]
© 2014 MedReviews®, LLC
Key words
Aspirin • Urinary thromboxane • Atherothrombotic disease • Platelet function •
Personalized medicine
Vol. 15 No. 2 • 2014 • Reviews in Cardiovascular Medicine • 119
4170004_RICM0739.indd 119 24/06/14 3:10 PM
Biomarker testing for efficacy
of therapy is an accepted way
for clinicians to individualize
dosing to genetic and/or environ-
mental factors that may be influ-
encing a treatment regimen. Classic
examples include determining glu-
cose or hemoglobin A1C levels prior
to modulating diabetes therapy,1
determination of biomarkers such
as low-density lipoprotein choles-
terol or C-reactive protein when
making a decision to place certain
patients on statin drugs,2,3 and
measuring natriuretic peptides in
congestive heart failure.4 However,
biomarker testing for efficacy of
aspirin therapy is not performed as
routinely as efficacy testing in other
therapeutic areas. Aspirin is used
by nearly 43 million Americans on
a regular basis,5 prescribed by clini-
cians to reduce risks associated with
various atherothrombotic diseases.
Despite its widespread use, many
clinicians are unaware of the link
between suboptimal response to
aspirin therapy and increased risk
for inferior clinical outcomes in
several disease states.3,6 This article
reviews the clinical and laboratory
aspects of determining whole-body
thromboxane production, particu-
larly as it pertains to efficacy assess-
ment of aspirin responsiveness.
Platelet Pathobiology
in Atherothrombotic
Diseases: the Role of
Aspirin
Arterial thrombosis is the final
pathogenic mechanism of many
acute ischemic events, including
acute myocardial infarction (MI)
and sudden cardiac arrest. This
process involves complex interac-
tions within the atherosclerotic
artery, including endothelial injury,
vasospasm, and platelet activation.
Plaque rupture exposes thrombo-
genic subendothelial components,
which leads to platelet deposition
and further activation.7
Aspirin has several important
effects on the human body, most
notably the reduction of pain, fever,
and inflammation. Aspirin’s capac-
ity to prevent blood clotting as a
platelet function inhibitor has been
employed as an important treatment
and prevention modality in athero-
thrombotic disease. The ability of
aspirin to produce these myriad
effects is mediated by reducing the
production of prostaglandins and
thromboxane via the irreversible
inactivation of the cyclooxygenase
(COX) enzyme, which is required
for thromboxane synthesis. Aspirin
irreversibly acetylates a serine resi-
due in the COX-1 enzyme, a unique
function not served by other drugs
in the nonsteroidal anti-inflamma-
tory drug (NSAID) class.8
Aspirin therapy has been
reported to reduce cardiovascular
events by up to 40%. However, use
of aspirin is associated with higher
frequencies of gastrointestinal
bleeding and hemorrhagic stroke.
Moreover, in certain individuals,
the expected degree of aspirin
responsiveness is muted. Given
both these considerations there is
the potential for great clinical util-
ity in identifying individuals who
will most likely benefit from anti-
platelet therapy with aspirin versus
patients in whom aspirin therapy
may not be sufficient.
A substantial body of literature
exists examining aspirin-insen-
sitive thromboxane production
and its association with athero-
thrombotic risk. Various terms
have been used to describe in vivo
and in vitro phenomena, in which
the expected degree of aspirin
responsiveness is not manifested.
The in vivo clinical phenomenon
is variably called aspirin nonre-
sponse, aspirin treatment failure,
or aspirin resistance after the use
of aspirin, and has parallels (but
is not synonymous) with the in
vitro laboratory phenomenon of
aspirin resistance. The laboratory
endpoint addressed in this article
is physiologic aspirin-insensitive
thromboxane biosynthesis, a term
preferred by some authors.9-11
Thromboxane Metabolism:
Critical Links for Risk
Assessment
Antiplatelet medications are fre-
quently used in the prevention of
stroke, MI, and vascular throm-
botic diseases due to the funda-
mental role of platelet aggregation
in a variety of atherothrombotic
processes. Modulation of the pros-
taglandin thromboxane A2 (TxA2)
pathway is one of the pivotal routes
of activation involved in stimulat-
ing platelet aggregation. The COX
enzyme regulates the conversion of
arachidonic acid to thromboxane.
This enzyme exists in two forms:
COX-1, the constitutive form found
in all tissues, and COX-2, which
is induced during inflammatory
states.12,13 Both COX-1 and COX-2
metabolize arachidonic acid to
prostaglandin H2, the common sub-
strate for TxA2.Synthesis of TxA2 in
platelets is mediated by the COX-1
enzyme, which must be functional
for stimulating the production of
TxA2 from arachidonic acid. COX-2
has been identified in human ath-
erosclerotic plaques, cells associated
with chronic inflammation such as
monocytes/macrophages, and in
newly formed platelets.14,15 These
nonplatelet sources may contribute
to thromboxane production through
Arterial thrombosis is the nal pathogenic mechanism of many
acute ischemic events, including acute myocardial infarction and
sudden cardiac arrest.
120 • Vol. 15 No. 2 • 2014 • Reviews in Cardiovascular Medicine
Urinary Thromboxane Testing to Evaluate Atherothrombotic Risk
continued
4170004_RICM0739.indd 120 24/06/14 3:10 PM
production of prostaglandin H2,
which is considered a possible
bypass mechanism to the effects of
low-dose aspirin therapy (Figure1).
Additionally, it has been suggested
that the presence of naturally occur-
ring isoprostanes may contribute to
platelet activation and subsequent
irreversible aggregation when plate-
let agonists such as TxA2 occur in
subthreshold concentrations.16
The importance of TxA2 is dem-
onstrated by the reduction in risk
of acute MI or death in patients
with acute coronary syndrome
(ACS) following administration of
low-dose aspirin, which irrevers-
ibly inhibits the production of TxA2
in platelets. Production of platelet-
derived TxA2 has been completely
inhibited by daily doses of aspirin
as low as 100 mg.17
TxA2 has an extremely short half-
life, converting to two stable but
inactive metabolites: 11-dehydro-
thromboxane B2 (TxB2) and 2,3-
dinor-11-dehydro-TxB2. Excretion
of TxB2 in the urine has been
shown to reflect in vivo platelet
activation. Elevated concentra-
tions of TxB2 have been noted in
patients with various high-risk
phenotypes.18-20
Quantitation of urinary TxB2
(UTxB2) offers an advantage
over platelet activation markers
measured in plasma or blood
because urine measurements are not
subject to interference from in vitro
platelet activation, which commonly
occurs as a result of preanalytical
variables such as local vein trauma
or insufficient anticoagulation dur-
ing phlebotomy.21,22 Measurement
of UTxB2 may be performed in
patients to assess the effectiveness
of specific inhibition in TxA2 pro-
duction, along with identification of
patients’ ability to benefit from anti-
platelet therapy, and their associated
risk for developing future cardiovas-
cular events (Figure 1).
How Urinary Thromboxane
Testing Varies From Other
Forms of Platelet Testing
The laboratory
measurement of
aspirin “failure”
has been achieved
to date by examining one of two
pathways: (1) TxA2 production, or
(2) thromboxane- dependent plate-
let function. Though thrombox-
ane is central to both measurement
techniques, each method provides
a different facet of information that
may be complementary to the other.
For example, little or no serum
thromboxane may indicate that
aspirin is effectively eliminating
COX-1 production of thromboxane
by the platelet, whereas elevated
levels of urinary thromboxane in
The importance of TxA2 is demonstrated by the reduction in risk of
acute MI or death in patients with acute coronary syndrome follow-
ing administration of low-dose aspirin, which irreversibly inhibits
the production of TxA2 in platelets.
Figure 1. Pathway of thromboxane production and antiplatelet effects of aspirin. Reproduced with permission from Hankey and Eikelboom.23
Monocytes macrophages,
and other nonplatelet
sources of thromboxane
Platelet sources of
thromboxane
Blocked by
COX-2 inhibition
Urinary 11 dehydro-
thromboxane B2
Serum
thromboxane B2
Thromboxane-dependent
platelet function
Platelet activation and
aggregation
Blocked by
low-dose aspirin
COX-2 COX-1
Thromboxane
synthase
Thromboxane A2Thromboxane A2
Thromboxane
synthase
Arachidonic acid Arachidonic acid
Prostaglandin G2/H2Prostaglandin G2/H2
Vol. 15 No. 2 • 2014 • Reviews in Cardiovascular Medicine • 121
Urinary Thromboxane Testing to Evaluate Atherothrombotic Risk
4170004_RICM0739.indd 121 24/06/14 3:10 PM
the same individual may indicate
the continuing presence of elevated
risk due to systemic thromboxane
production not addressed by the
patient’s current aspirin dose.
Thromboxane A2 Production
TxA2 production can be deter-
mined by measuring stable metab-
olites of TxA2, such as TxB2 in the
serum (or plasma) and 11-dehydro-
TxB2 in the urine. The measurement
of serum or plasma thromboxane
is specific to the platelet’s capacity
to produce thromboxane, whereas
measurement of urinary throm-
boxane is nonspecific and is reflec-
tive of the entire body’s production
of thromboxane.
Thromboxane-dependent
Platelet Function
Tests of platelet function that are
dependent on platelet thromboxane
production include agonist-induced
platelet aggregation measured by
light or optical transmission (turbidi-
metric aggregometry in platelet-rich
plasma), electrical impedance (whole
blood platelet aggregometry), or
semiautomated platelet aggregome-
try assay (eg, VerifyNow P2Y12 Test;
Accumetrics, San Diego, CA; TEG–
Platelet Mapping; Mayo Medical
Laboratories, Rochester, MN;
Plateletworks; Helena Laboratories,
Beaumont, TX). Additionally, the
semiautomated PFA-100 (Siemens
Medical Solutions, Malvern, PA)
relies on thromboxane production;
however, unlike the previously men-
tioned platforms, it does not specifi-
cally activate the COX-1 pathway by
the addition of arachidonic acid.
Finally, the bleeding time is an in
vivo test of platelet function that is
also dependent, in part, on platelet
thromboxane production but is not
frequently used because it is highly
operator dependent and results are
poorly reproducible.
Urinary 11-dehydro-
TxB2: Stable Metabolite
to Assess Thromboxane
Metabolism
One approach to quantify the
activity of aspirin has been to
measure
the
levels of the byprod-
ucts of COX-1 enzyme activity.
Reduced levels of these products
would normally be expected as a
result of
aspirin
administration.
TxA2 cannot be used for measure-
ment because it is a highly unstable
compound with a short
half-life of
30 seconds.24
TxA2 is rapidly con-
verted in vivo to the more stable
TxB2
, which is subsequently con-
verted by the liver into two
major
metabolites:
2,3
-dinor-
TxB2
and
11-dehydro-
TxB2
. Both metabo-
lites, along with total
TxB2
, are
excreted unchanged in
the
urine.25
The small amount of unchanged
total
TxB2
in urine and
2,3
-dinor-
TxB2
levels are likely more reflec-
tive of renal
TxB2
biosynthesis than
of platelet activity. Additionally,
measurement of serum
TxB2
can
be prone to artifact. In contrast,
11-dehydro-
TxB2 is
a stable metab-
olite of
TxB2
that can
be
measured
noninvasively in the urine and can
thus serve as an indirect measure of
TxB2
activity in
vivo.26
Aspirin use has been shown to
cause a dose-dependent reduction
in urinary levels of 11-dehydro-
TxB2
. Use of standardized con-
trols circumvents the variability
of the assay among testing labora-
tories. The assay requires a 2-mL
urine sample and is normalized
to the patient’s urine creatinine
(therefore, results are expressed
as pg UTxB2/mg creatinine). This
assay is advantageous because
it is noninvasive and is normal-
ized with standard controls.27
Currently, a US Food and Drug
Administration (FDA)-cleared
and commercialized assay uses a
monoclonal-linked immunosor-
bent assay (ELISA) technology
(vs the previously available poly-
clonal assay used for research).
Both ELISAs utilize primary anti-
bodies that predominantly recog-
nize 11-dehydro-
TxB2
but exhibit
variable cross-reactivity with
other related
TxB2
species, such
as 11-dehydro-2,3-dinor-
TxB2
.28
DeFilippis and colleagues29 exam-
ined the two ELISA assays along
with a third method for UTxB2
detection (liquid chromatogra-
phy-mass spectrometry [LC-MS])
in a well-characterized cohort of
patients with acute MI. They found
that the newer-generation mono-
clonal ELISA (but not the older
polyclonal ELISA, nor LC-MS) was
capable of differentiating patients
with atherothrombotic acute MI
from those whose infarction was
not caused by atherothrombotic
disease.29 The authors concluded
that 11-dehydro-2,3-dinor-
TxB2
may play an important role in the
pathophysiology of atherothrom-
bosis, particularly in individu-
als on adequate aspirin therapy.
Moreover, they suggest that, “given
the great clinical importance of
the accurate diagnosis and treat-
ment of atherothrombosis, the
investigation of such an analyte is
warranted.”
The manufacturers of this
FDA-cleared assay studied appar-
ently healthy adults before and
after receiving controlled doses
of aspirin. Based on the result-
ing frequency of UTxB2 levels,
they established a cut-off value to
assess an adequate aspirin effect as
#1500 pg UTxB2/mg of creatinine.
The assay is indexed to urinary cre-
atinine concentration to control for
varying urine concentrations based
on hydration.30-31 This cut-off was
reconfirmed in subsequent studies
that investigated healthy and dis-
eased populations before and after
122 • Vol. 15 No. 2 • 2014 • Reviews in Cardiovascular Medicine
Urinary Thromboxane Testing to Evaluate Atherothrombotic Risk
continued
4170004_RICM0739.indd 122 24/06/14 3:10 PM
aspirin treatment. After aspirin
ingestion, UTxB2 levels below the
cutoff (,1500 pg/mg) were classi-
fied as good aspirin effect, whereas
levels above 1500 pg/mg were con-
sidered poor aspirin effect. Though
this established cutoff value was
used to determine the presence
or absence of an expected aspirin
effect, the actual concentrations of
the measured metabolite were used
to assess atherothrombotic risk in a
quartile fashion.
Patient Populations With
Potential to Benefit From
Urinary Thromboxane
Testing
Patients in Need of Cardiovas-
cular Disease Risk Reduction
There are strong suggestions from
three major clinical trials that
evaluation of 11-dehydro-
TxB2
production may be a more impor-
tant surrogate marker for clinical
response to aspirin therapy than
isolated arachidonic acid-induced
platelet aggregation. A nested case-
control study of 970 patients with
a history of aspirin use enrolled
in the Heart Outcomes Prevention
Evaluation (HOPE) trial20 had
baseline measurements taken of
urinary 11-dehydro-TxB2 levels as
a marker of overall thromboxane
generation. At the 5-year follow-
up, patients with lower response
to aspirin treatment (ie, levels in
the highest quartile) had a signifi-
cantly greater risk for a composite
endpoint of nonfatal MI, nonfa-
tal stroke, or cardiovascular death
compared with those in the lowest
quartile (adjusted odds ratio [OR]
1.8; 95% confidence interval [CI],
1.2-2.7) with significant increases
in acute MI and cardiovascular-
related death.
In the aspirin-treated patients
from the Clopidogrel for High
Atherothrombotic Risk and Ischemic
Stabilization, Management, and
Avoidance (CHARISMA) study,32
baseline urinary UTxB2 concentra-
tions in the highest quartile were
associated with an increased risk of
stroke, MI, or cardiovascular death
compared with results in the lowest
quartile (adjusted hazard ratio 1.7;
95% CI, 1.1-2.6).
The Reduction in Graft
Occlusion Rates (RIGOR)33 study
evaluated 229 subjects treated with
aspirin following coronary artery
bypass graft (CABG) surgery.
Although arachidonic acid-induced
platelet aggregation was inhib-
ited by . 99% at 6 months, 31% of
subjects still had elevated urinary
UTxB2 excretion. In multivariate
analysis, there was a statistically
significant association between
UTxB2 excretion $ 450 pg/mg
creatinine and graft occlusion
(OR2.59).
A more recent study tested 287
non–aspirin-treated patients who
presented with ACS and underwent
percutaneous coronary interven-
tions. Levels of UTxB2 were deter-
mined before and after aspirin
treatment to determine a possible
association of aspirin-free baseline
levels with adverse events. High
aspirin-free baseline levels were
associated with a higher incidence
of poor aspirin response after treat-
ment. Though aspirin-free baseline
levels did not predict adverse events
at 1 year, levels of UTxB2 in the upper
quartiles after aspirin treatment
were associated with a more than
twofold increased odds of adverse
events, consistent with the findings
from the CHARISMA study.34
Special Populations
Patients With Atrial Fibrillation
With Low CHADS2 or CHA2DS2-
VASc Score. There have been
many trials researching the optimi-
zation of therapy to prevent embo-
lization in atrial fibrillation due to
the high risk of disabling stroke in
patients who develop this increas-
ingly prevalent cardiac rhythm dis-
order. The recommendations from
these trials clearly point toward
anticoagulation or antiplatelet ther-
apy, depending on the individual’s
risk. However, the highest quality
evidence and recommendations
have been geared toward patients
with higher estimated risk based on
other comorbidities (as predicted
by CHADS2 [Congestive heart
failure, Hypertension, Age $ 75 y,
Diabetes mellitus, prior Stroke
or transient ischemic attack] or
CHA2DS2-VASc [Congestive heart
failure, Hypertension, Age $ 75 y,
Diabetes mellitus, prior Stroke or
transient ischemic attack, Vascular
disease, Age 65-74 y, female Sex]
scores).
For patients with CHADS2 scores
of $ 2, available evidence strongly
suggests treatment with antico-
agulation. Only the occasional
exception will be managed using
antiplatelet therapy such as aspirin.
Conversely, a good deal of clinical
uncertainty surrounds the manage-
ment of patients with atrial fibril-
lation and low risk scores. In these
patients, the benefits of anticoagu-
lation may not always outweigh
the lower risk of stroke in certain
categories.35 Determining which
patient subgroups may be more
responsive to aspirin therapy could
be beneficial to these patients.
Patients with atrial fibrillation
and a CHADS2 score of 1 are con-
sidered to be at intermediate risk of
stroke ( 2% per year) and, accord-
ing to recent guidelines, should
be treated with oral anticoagulant
therapy or aspirin (75-325 mg/d).
Anticoagulant therapy is generally
preferred to aspirin in most patients
with a CHADS2 score of 1, although
in certain groups (eg, patients with
a high risk of falling or of lower
Vol. 15 No. 2 • 2014 • Reviews in Cardiovascular Medicine • 123
Urinary Thromboxane Testing to Evaluate Atherothrombotic Risk
4170004_RICM0739.indd 123 24/06/14 3:10 PM
gastrointestinal bleeding) aspi-
rin may be the preferred therapy.
Determining which patients will be
best served by this strategy as well
as what dose of aspirin remains a
matter of controversy.
The issue of whether aspirin is the
preferred choice in low-risk patients
(CHADS2 5 0) has not been well
addressed. The individual trials
that compared aspirin with placebo
enrolled very few patients with a
CHADS2 of 0, but meta-analyses
of these trials suggest that the risk
of stroke was reduced by approxi-
mately 20% with aspirin use (but
the introduction of aspirin to these
patients included the potential for
harm).36 It is important to consider
that these studies have not been
done stratifying patients based on
their potential degree of respon-
siveness to aspirin using laboratory
testing, and there may therefore be
an opportunity to refine knowledge
and improve management in this
population
Type 2 Diabetes. Ames and
colleagues11 demonstrated that
patients with diabetes have a 50%
higher urinary 11-dehydro-TxB2
excretion (both baseline and after
administration of aspirin) than
healthy control subjects, suggesting
that suspected platelet hyperacti-
vation and/or alternative sources
of thromboxane generation may
contribute to the development of
more atherothrombotic sequelae
in this population.37,38 Patients who
are identified by systemic throm-
boxane generation as poor aspirin
responders are considered to be in
a state of enhanced oxidative stress
that may play an important role
in their maintaining active plate-
let function irrespective of COX-1
inhibition, perhaps by an increase
in systemic generation of throm-
boxane from nonplatelet sources.
Chronic Kidney Disease. There
is a paucity of data concerning
the efficacy of aspirin therapy
in patients with kidney disease.
Proper guidance for physicians
who manage renal patients is lack-
ing regarding which patients might
benefit from aspirin therapy. Some
observational studies have sug-
gested that aspirin use may be
associated with increased cardio-
vascular mortality or adverse car-
diovascular events, but these same
studies show a decrease in the risk
of stroke.39-41 A Cochrane meta-
analysis that included randomized
trials of nondialysis chronic kid-
ney disease (CKD) and end-stage
renal disease patients found that
antiplatelet agents, compared with
no treatment or placebo, reduced
the risk of MI but not all-cause
mortality, cardiovascular mortal-
ity, or stroke.42 The trials reviewed
consistently demonstrated that, in
CKD, antiplatelet agents increased
the risk of major and minor bleed-
ing. In a post hoc analysis of the
Hypertension Optimal Treatment
(HOT) trial, Jardine and col-
leagues43 suggested that aspirin
therapy produces greater absolute
reduction in major cardiovascular
events and mortality in hyperten-
sive patients with CKD than those
with normal kidney function, and
that the increased risk of major
bleeding appears to be outweighed
by significant benefits.
This is a sizeable at-risk popula-
tion about whom the experts are
undecided regarding when and
how best to deploy aspirin therapy.
Because the inhibition of prosta-
glandin synthesis by COX-1 inhibi-
tors may worsen elements of renal
function in patients with compro-
mised renal hemodynamics, there
is a reflex among nephrologists to
be wary of aspirin and NSAIDs as
a class. However, because the pro-
tective effects of aspirin against
various lethal nonrenal diseases
are so well appreciated, consensus
opinion from nephrologists still
recommends that “that individual
treatment decisions be based upon
consideration of patients’ indi-
vidual risks, potential benefits,
and preferences. The prescription
of low-dose aspirin (81 mg/d) is
probably safe in most patients with
CKD and those on chronic dialy-
sis. These recommendations are
consistent with the Kidney Disease
Outcomes Quality Initiative clini-
cal practice guidelines.”44 Knowing
the potential degree of aspirin
responsiveness of a given patient
might assist nephrologists and oth-
ers who manage patients with renal
disease to make better-informed
decisions on the optimization of
primary and secondary disease
prevention strategies in this high-
risk and growing population.
Cardiothoracic Surgery. CABG
is performed in patients with severe
coronary artery disease to reduce
risks of future atherothrombotic
disease, specifically fatal and non-
fatal MI. Paradoxically, however,
the CABG procedure is associated
with its own thrombotic risks of
perioperative MI, stroke, pulmo-
nary embolism, and bowel infarc-
tion.45 These risks are of concern to
the patient, the referring cardiolo-
gist and, of course, the operating
cardiothoracic surgeon. Although
antiplatelet drugs such as aspirin
and clopidogrel reduce throm-
botic events, they add to the risk of
excessive bleeding during and after
Patients who are identied by systemic thromboxane generation as
poor aspirin responders are considered to be in a state of enhanced
oxidative stress that may play an important role in their maintaining
active platelet function irrespective of COX-1 inhibition...
124 • Vol. 15 No. 2 • 2014 • Reviews in Cardiovascular Medicine
Urinary Thromboxane Testing to Evaluate Atherothrombotic Risk
continued
4170004_RICM0739.indd 124 24/06/14 3:10 PM
heart failure to provide both short-
and long-term hemodynamic sup-
port. Unfortunately, both bleeding
and thromboembolic complications
are introduced due to the severely
disturbed flow conditions gener-
ated by these devices. Patients sup-
ported by ventricular assist devices
are treated with systemic antico-
agulation and antiplatelet agents to
reduce the risk of thrombotic com-
plications such as device thrombo-
sis and embolic stroke. However,
initiating anticoagulation too early
or being too aggressive in this
approach increases the risk of bleed-
ing complications both in the early
perioperative period (exacerbated
by the coagulopathic effect of car-
diopulmonary bypass [CPB]) and
long-term postoperatively (via the
development of an acquired form of
von Willebrand disease). Rossi and
colleagues,50 among a set of recom-
mendations based on an extensive
review of the existing literature,
suggest that using patient labora-
tory results to titrate aspirin and
clopidogrel doses to a specifically
targeted percentage of inhibition,
permitted doses of aspirin to be low-
ered to as low as 25 mg/d while not
disturbing the bleeding/thrombosis
balance in these patients.
Human Immunodeficiency Virus.
Mechanisms for increased cardio-
vascular risk in human immuno-
deficiency virus (HIV)-1 infected
adults are not yet fully under-
stood, but platelet activation and
immune activation leading to
a prothrombotic state are pro-
posed as significant contributors.
However, aspirin therapy appears
to be underutilized in patients with
HIV.51 O’Brien and colleagues52
have shown in a pilot study that
low-dose aspirin may be a poten-
tial hot-spot for intervention for
surgery, with associated higher
rates of blood transfusion, postop-
erative tamponade, and reopera-
tion for bleeding.
Historically, bleeding concerns
have led to the recommendation
that aspirin be discontinued 3 to
5 days before surgery in patients
undergoing elective CABG.
However, this general approach is
no longer recommended according
to the 2011 American College of
Cardiology Foundation/American
Heart Association guideline for
CABG, which recommends that
aspirin be started or continued
preoperatively.46 Unfortunately, the
recommended dosing range for
aspirin is quite large (100-325 mg)
and there is no guidance in this
document or other consensus
guidelines on how best to choose
a specific dose of aspirin using
available clinical information or
laboratory testing. Clearly, the wide
threefold dose range in the admin-
istration of a drug recommended
as a Class I intervention represents
a pharmacologic missed opportu-
nity to reduce perioperative CABG
morbidity and mortality by identi-
fying the patients for whom aspirin
works and at what dose. This same
incongruity referring to wide aspi-
rin dose ranges continues through-
out the guidelines as they address
issues related to postoperative aspi-
rin administration in this high-risk
population.
We know from work by
Gluckman and colleagues33 that
we can identify aspirin-insensitive
individuals who may benefit from
alternate or more aggressive ther-
apy in the perioperative CABG
phases. This group’s analysis from
the RIGOR study showed that
TxA2
generation (as measured
by U
TxB2
) and shear-dependent
platelet hyper-reactivity (as mea-
sured by PFA-100 collagen and ade-
nosine diphosphate closure time)
act as independent risk factors for
early saphenous vein graft throm-
bosis. This indicates that there are
pathways that are independent of
platelet COX-1 activity that are not
inhibited effectively by aspirin. Yet
the timing, dosing and interrup-
tion of antiplatelet therapy periop-
eratively in CABG remain an area
of substantial debate and research,
with new concerns about bleed-
ing being raised in this generation
of dual antiplatelet antagonism.47
Fortunately, there are research-
ers who advocate a personalized
approach to the patient’s drug regi-
men, which uses measurements of
antiplatelet drug activity to predict
the risk of excessive postoperative
bleeding in patients.48
Heart Failure and Ventricular
Assist Devices. The decision of
whether to use antiplatelet and/or
antithrombotic therapy in heart
failure patients who do not have
coexisting atrial fibrillation is con-
troversial. The Warfarin and Aspirin
in Patients in Reduced Cardiac
Ejection Fraction (WARCEF) trial49
noted that among patients with
reduced left ventricular ejection
fraction who were in sinus rhythm,
there was no significant overall dif-
ference in the primary outcome
between treatment with warfa-
rin or aspirin. The reduced risk of
ischemic stroke that was seen in
those on warfarin was offset by an
increased risk of major hemorrhage.
The authors’ conclusion was that the
choice between warfarin and aspi-
rin should be individualized, once
again pointing toward the need to
assay whether aspirin works for an
individual patient. However, signifi-
cant guidance based on thrombox-
ane metabolite measurements has
yet to be reviewed in the literature.
Ventricular assist devices are
implanted in patients with end-stage
… aspirin therapy appears to be underutilized in patients with HIV.
Vol. 15 No. 2 • 2014 • Reviews in Cardiovascular Medicine • 125
Urinary Thromboxane Testing to Evaluate Atherothrombotic Risk
4170004_RICM0739.indd 125 24/06/14 3:10 PM
HIV-1-infected subjects on anti-
retroviral therapy in order to
blunt platelet and immune acti-
vation, as well as inflammation.
Their work has shown that the
basal level of median urinary con-
centration of 11-dehydro-TxB2
was significantly higher in
HIV-1-infected subjects compared
with control subjects (9626.8 vs
7295.2 pg/mL; P 5 .02). After
1 week of aspirin, 11- dehydro-TxB2
decreased significantly in both
groups but remained significantly
higher in the HIV-1-infected
group (2255.7 vs 1422.6 pg/mL;
P5.04).
Pediatric Heart Disease. In chil-
dren undergoing surgery for car-
diac defects, CPB is associated with
significant perioperative throm-
botic events.53 Additionally, sys-
temic arterial to pulmonary artery
shunt thrombosis is an uncommon
but life-threatening complication of
pediatric cardiac surgery. Despite
trials suggesting benefits, the pre-
cise role of aspirin in reduction of
occlusion of shunts unfortunately
is not fully defined in the litera-
ture.54 Children undergoing car-
diac surgery are also at increased
risk of venous thrombosis but the
ability of aspirin to prevent venous
thrombosis has not been formally
tested. Thus, there are many fea-
tures of pediatric cardiac surgery
that lend themselves to increased
knowledge about aspirin’s potential
to prevent critical arterial and/or
venous thromboses. Cholette and
colleagues54 investigated the issue
of thrombosis and potential aspi-
rin resistance in pediatric patients
undergoing cardiac surgery with
CPB. These investigators found
aspirin therapy effectively inhibits
ex vivo platelet function and sup-
presses in vivo TxA2 production
in pediatric patients undergoing
cardiac surgery. They noted that
aspirin resistance as measured by
aspirin resistance units was not
associated with a greater risk of
early symptomatic thrombosis but
that resistance, if measured using
a urinary
TxB2
assay, was indeed
predictive of thrombotic events
in all treated subjects, though this
finding only approached statisti-
cal significance in the high-risk
group. The authors suggested that
this trending of urinary
TxB2
may
prove to be predictive when larger
numbers of high-risk patients are
able to be tested.
Deep Vein Thrombosis and
Venous Thromboembolism
Prophylaxis
For the initial primary preven-
tion of venous thromboembolism,
anticoagulants are considered the
preferred first-line therapy for
both medical and surgical patients.
For extended prophylaxis (after
10 days of initial treatment with
a low molecular weight hepa-
rin), recent evidence suggests that
there is no difference in outcomes
between aspirin and dalteparin in
a surgical population,55 so it might
be reasonable to consider aspirin
therapy for extended prophylaxis,
especially when the use of antico-
agulants is contraindicated or not
feasible. For secondary prevention,
aspirin might be considered after
completion of initial anticoagulant
therapy for venous thromboembo-
lism.56-58 Clarification of the aspi-
rin effectiveness of an individual
patient may help the clinician make
a more structured decision in these
cases where protracted prophylaxis
is required and injected or oral
anticoagulation is not a preferred
option for the patient (Table 1).
Forward Thinking: Future
Implementation and
Research
A key consideration in advancing
broader use of urinary thrombox-
ane testing is prospective trials
that demonstrate improved clini-
cal outcomes with testing and sub-
sequent therapeutic adjustments.
Building on the encouraging data
from well-characterized retrospec-
tive cohorts, prospective data will
allow for integration of urinary
thromboxane testing into practice
standards as a noninvasive, read-
ily accessible means of identifying
at-risk individuals and titrating
antiplatelet therapy, and even opti-
mizing non-antiplatelet therapy in
those with poor responsiveness to
aspirin or other antiplatelet agents.
Also important to the incorpora-
tion of any diagnostic test into
clinical practice are subanalyses of
future studies that demonstrate an
economic advantage conferred by
testing, using cost or quality-of-
life adjustments, now commonly
accepted as cost-justification mod-
els. The optimal frequency of mon-
itoring for aspirin response should
be determined, with consideration
made for the natural progression
of disease states and the retooling
of therapy that advancing disease
requires.
Discussion
The subject of aspirin response is
more complex than a simple expla-
nation of how aspirin functions
and determining if it has achieved
its target acetylation, blocking sub-
sequent thromboxane production.
… aspirin therapy effectively inhibits ex vivo platelet function and
suppresses in vivo TxA2 production in pediatric patients undergoing
cardiac surgery.
126 • Vol. 15 No. 2 • 2014 • Reviews in Cardiovascular Medicine
Urinary Thromboxane Testing to Evaluate Atherothrombotic Risk
continued
4170004_RICM0739.indd 126 24/06/14 3:10 PM
Aspirin response in a clinical set-
ting has become synonymous
with the risk of the aspirin-treated
patient having an atherothrom-
botic event. Is an individual’s aspi-
rin treatment enough to protect
that individual from an athero-
thrombotic event, or is he or she at
continued risk of an event despite
aspirin treatment? Aspirin, at doses
that almost completely suppress
thromboxane production in most
may be useful in addressing com-
pliance issues or adjusting the
aspirin dosage.” As several studies
reviewed in this paper have shown,
testing of UTxB2 has indeed been
able to identify individuals on aspi-
rin treatment who are at increased
risk for an atherothrombotic event.
Would more complete throm-
boxane suppression be beneficial
in these disease states? If so, what
approaches can be taken to reduce
healthy patients, does not always
achieve the same degree of throm-
boxane suppression in various dis-
ease states. Albeit to date, review
articles on aspirin effect do not
provide clear direction for diagnos-
tic testing to adjust for individual
aspirin response, Hennekens and
colleagues59 suggested that “if the
in vitro response to arachidonic
acid is robust (demonstrating lack
of aspirin effect), this information
TABLe 1
Disease State Unmet Clinical Need
Benefit of Detection of
Aspirin-insensitive
Thromboxane Production Study
Atrial fibrillation
with low CHADS2
score
Thromboprevention without
increased bleeding side
effects in low-risk patients
Reducing bleeding risks in patient at
very low risk for stroke
Turagam MK
et al,35 Hart RG
et al36
Type 2 diabetes Higher risk of CVD but
apparent underprescription
of aspirin therapy
Determining which diabetes subpopula-
tions might benefit from personalized
aspirin dosing or other therapies
Ames PR et al11
CKD Higher risk of CVD but also
higher risks of NSAIDs on
marginal renal function
Studies needed to determine which CKD
patient overall benefit of aspirin will
outweigh risks
Cardiothoracic
surgery
Early SVG thrombosis Urinary thromboxane levels serve as
predictor of early SVG thrombosis
Gluckman TJ
et al33
Heart failure/
ventricular assist
devices
High frequency of bleeding
and thrombosis
Further optimization of aspirin dosing
and/or interval
HIV High risk of CVD with
apparent underutilization
of aspirin
Reduction of prothrombotic and
inflammatory states
Burkholder GA
et al,51 O’Brien
M et al52
Pediatric cardiac
surgery
Significant perioperative
thrombosis
Identify aspirin nonresponders and
adjust surveillance accordingly
Cholette JM
et al54
DVT prophylaxis Questionable efficacy of
aspirin for the primary
prevention of VTE in medical
and surgical patients
May allow for determination of
subpopulations for whom this therapy
is reasonable or preferred over
anticoagulation, such as patients
undergoing extended prophylaxis
CHADS2, Congestive heart failure, Hypertension, Age $ 75 y, Diabetes mellitus, prior Stroke or transient ischemic attack; CKD, chronic kidney disease; CVD, cardio-
vascular disease; DVT, deep vein thrombosis; HIV, human immunodeficiency virus; NSAIDs, nonsteroidal anti-inflammatory drugs; SVG, saphenous vein graft; VTE,
venous thromboembolism.
Unmet Clinical Needs in Disease States: Potential Benefits Conferred by Assessment of Individual
Patients’ Aspirin Responsiveness
Vol. 15 No. 2 • 2014 • Reviews in Cardiovascular Medicine • 127
Urinary Thromboxane Testing to Evaluate Atherothrombotic Risk
4170004_RICM0739.indd 127 24/06/14 3:10 PM
overall whole-body thromboxane
burden? Dragani and colleagues60
have shown that, in patients with
essential thrombocythemia who
are taking daily aspirin, the addi-
tion of a COX-2 inhibitor to aspirin
decreases both urinary excretion
of a thromboxane metabolite
(marker of endogenous produc-
tion) and ex vivo thromboxane
production in serum (measure of
total platelet synthetic capacity) by
approximately 30%. These studies
corroborate that COX-2 contrib-
utes to endogenous thromboxane
production in this disease state.
However, it is important to note,
the addition of the COX-2 inhibitor
did not abolish thromboxane pro-
duction, suggesting the role of still
additional mechanisms contribut-
ing to thromboxane production.
Strategies to optimize sensi-
tivity of diagnosis and therapy
remain an intriguing area of
research. Aspirin in the primary
and secondary prevention of ath-
erothrombotic disease states is
customarily prescribed taken once
daily. Therefore, for the treatment-
refractory patient, the first treat-
ment modification made is often a
change in drug dose. However, we
know that, traditionally, the aspi-
rin-dosing interval for most indica-
tions (such as fever) is much more
frequent, with doses sometimes as
close as 4 hours apart. Should our
focus therefore be on a different
element of aspirin pharmacother-
apy? Pascale and colleagues10 have
shown, using the disease model of
essential thrombocytopenia, that
thromboxane biosynthesis is bet-
ter controlled by modulating the
aspirin interval as opposed to the
dose or formulation. Modulation
of thromboxane outside of aspirin
dosing has also been demonstrated;
Lev and colleagues61 showed that
the introduction of omega-3 fatty
acids effectively reduced plate-
let reactivity and improved the
response to aspirin. In the Indian
Polycap Study,62 the investigators
demonstrated that statin treatment
lowers levels of thromboxane pro-
duction independent of aspirin
treatment. Clinicians may express
doubt about the phenomenon of
aspirin resistance but when you
look at their clinical behavior they
clearly acknowledge its existence by
providing incremental antiplatelet
therapy to their patients’ regimens.
However, if daily low-dose aspi-
rin really does remain effective pre-
vention for most, it would be unwise
to broadly and blindly recom-
mend either an increased quantity
or increased frequency of dosing
due to the well-known side effects
of aspirin. Therefore, determina-
tion of individual responsiveness
to aspirin represents an important
advance in personalized medicine
for its potential to more effectively
address long-term prevention of
atherothrombotic events.
Because not all patients receive
the same protective benefits from
low-dose aspirin therapy, guidance
should be developed to help opti-
mize antiplatelet therapy in those
Figure 2. Clinical interpretation of 11-dehydro-thromboxane B2 (11dhTxB2) results. ASA, aspirin; COX, cyclooxygenase; CVD, cardiovascular disease; TxA2, thromboxane A2.
Not taking ASA and
11dhTxB2 is:
Taking ASA and 11dhTxB2
is:
2500 2500–4000 4000
OK • Consider
prescribing ASA
for 1 week to
assess ASA
response.
• Consider other
CVD risk factors
before
continuing ASA
regimen.
• If no underlying
CVD condition
present, re-test
after 2 weeks.
• If underlying
condition present,
prescribe ASA for
1 week and re-test
for ASA response.
• Consider other
CVD risk factors
before continuing
ASA regimen.
1500
Good response
1500
Abnormal response
OK “Increased
Risk”
• Incomplete COX-1 TxA2 inhibition—modify
ASA dose or add other anti-platelet drug.
• Nonplatelet COX-2 TxA2 production from
pro-atherogenic oxidative inflammation—
modify/treat atherosclerosis risk factors.
128 • Vol. 15 No. 2 • 2014 • Reviews in Cardiovascular Medicine
Urinary Thromboxane Testing to Evaluate Atherothrombotic Risk
continued
4170004_RICM0739.indd 128 24/06/14 3:10 PM
with poor aspirin responsiveness.
Although large-scale prospective
clinical trials have not yet been
performed, some clinicians have
proposed an algorithm to aid in tai-
loring patient therapy based on the
clinical literature presently available.
Using urinary 11-dehydro-TxB2
testing, one could assess patients
prior to, and after, initiation of
aspirin therapy to assess for degree
of inhibition of thromboxane
biosynthesis, and, if suboptimal,
adjust therapy strategies accord-
ingly to minimize downstream
risks (Figure 2).
The major clinical impact of this
protocol is that consistently high
baseline 11-dehydro-TxB2 levels in
subjects not taking aspirin may jus-
tify further investigation for under-
lying cardiovascular risk factors
including poor aspirin response. In
addition, the presence of high post-
aspirin 11-dehydro-TxB2 levels will
identify patients with increased
risk of atherothrombotic disease.
This suggests the need of a com-
prehensive multifaceted manage-
ment plan that would include both
antiplatelet as well as antiathero-
sclerotic treatments.
Conclusions
The world faces growing epidemics
of coronary artery disease, type 2
diabetes, and CKD, all of which
present unique challenges in the
prevention and management of the
cardiovascular risks associated with
these conditions. Improving patient
care globally in the next decade
must address methods to reduce
atherothrombosis without increas-
ing morbidity and mortality from
bleeding risks. Assessing patients
for their potential to respond well to
preventative treatments such as
aspirin therapy can reduce this dis-
ease burden by individualizing ther-
apy to the appropriate patients.
Though future clinical investigation
is necessary to strengthen this argu-
ment, the present scientific litera-
ture suggests that testing UTxB2
levels is an appropriate first step for
identifying individuals at increased
risk for downstream atherothrom-
botic events associated with cardio-
vascular disease.
The authors received Scientific Advisory Board
honoraria from Corgenix (Broomfield, CO).
References
1. Inzucchi SE, Bergenstal RM, Buse JB, et al; American
Diabetes Association (ADA); European Association
for the Study of Diabetes (EASD). Management of
hyperglycemia in type 2 diabetes: a patient-centered
approach: position statement of the American Diabe-
tes Association (ADA) and the European Association
for the Study of Diabetes (EASD). Diabetes Care.
2012;35:1364-1379.
2. Wang TJ. Assessing the role of circulating, genetic, and
imaging biomarkers in cardiovascular risk prediction.
Circulation. 2011;123:551-565.
3. Perk J, De Backer G, Gohlke H, et al; European As-
sociation for Cardiovascular Prevention & Rehabilita-
tion (EACPR); ESC Committee for Practice Guide-
lines (CPG). European Guidelines on cardiovascular
disease prevention in clinical practice (version 2012).
The Fifth Joint Task Force of the European S ociety of
Cardiology and Other Societies on Cardiovascular
Disease Prevention in Clinical Practice (constituted
by representatives of nine societies and by invited
experts). Eur Heart J. 2012;33:1635-1701.
4. Maisel A, Mueller C, Adams K Jr, et al., State of the art:
using natriuretic peptide levels in clinical practice. Eur
J Heart Fail. 2008;10:824-839.
5. Zhou Y, Boudreau DM, Freedman AN. Trends in the
use of aspirin and nonsteroidal anti-inflammatory
drugs in the general US population. Pharmacoepide-
miol Drug Saf. 2014;23:43-50.
6. Thun MJ, Jacobs EJ, Patrono C. The role of aspirin in
cancer prevention. Nat Rev Clin Oncol. 2012;9:259-267.
7. Freed man JE. Molecu lar regulat ion of platelet-
depend ent th rombosis. Circulation. 2005;112:
2725-2734.
8. Fuster V, Sweeny JM. Aspi rin: a historica l and
contemporary ther apeutic overview. Circul ation.
2011;123:768-778.
9. Eikelb oom JW, Hankey GJ. Failure of aspirin to
prevent atherothrombosis. Am J Cardiovasc Drugs.
2004;4:57-67.
MAin PoinTs
• Arterial thrombosis is the final pathogenic mechanism of many acute ischemic events, including acute
myocardial infarction (MI) and sudden cardiac arrest. Aspirin has several important effects on the human
body, most notably the reduction of pain, fever, and inflammation. Aspirin’s capacity to prevent blood clotting
as a platelet function inhibitor has been employed as an important treatment and prevention modality in
atherothrombotic disease.
• Antiplatelet medications are frequently used in the prevention of stroke, MI, and vascular thrombotic diseases
due to the fundamental role of platelet aggregation in a variety of atherothrombotic processes. Modulation
of the prostaglandin thromboxane A2 (TxA2) pathway is one of the pivotal routes of activation involved in
stimulating platelet aggregation. The importance of TxA2 is demonstrated by the reduction in risk of acute MI or
death in patients with acute coronary syndrome following administration of low-dose aspirin, which irreversibly
inhibits the production of TxA2 in platelets.
• There are strong suggestions from three major clinical trials that evaluation of 11-dehydro-TxB2 production may
be a more important surrogate marker for clinical response to aspirin therapy than isolated arachidonic acid-
induced platelet aggregation.
• The current scientific literature suggests that testing urinary TxB2 levels is an appropriate first step for identifying
individuals at increased risk for downstream atherothrombotic events associated with cardiovascular disease.
Vol. 15 No. 2 • 2014 • Reviews in Cardiovascular Medicine • 129
Urinary Thromboxane Testing to Evaluate Atherothrombotic Risk
4170004_RICM0739.indd 129 24/06/14 3:10 PM
Force on Practice Guidelines. Developed in collabo-
ration with the American Association for Thoracic
Surgery, Society of Cardiovascular Anesthesiologists,
and Society of Thoracic Surgeons. J Am Coll Cardiol.
2011;58:e123-e210.
47. Kremke M, Tang M, Bak M, et al. Antiplatelet therapy
at the time of coronary artery bypass grafting: a
multicentre cohort study. Eur J Cardiothorac Surg.
2013;44:e133-e140.
48. Petricevic M, Biocina B, Lekic A, Gabelica R. Anti-
platelet therapy at the time of coronary artery surgery:
can a personalized approach improve outcomes? Eur J
Cardiothorac Surg. 2014;45:951-952.
49. Homma S, Thompson JL, Pullicino PM, et al; WAR-
CEF Investigators. Warfarin and aspirin in patients
with heart failure and sinus rhythm. N Engl J Med.
2012;366:1859-1869.
50. Rossi M, Serraino GF, Jiritano F, Renzulli A. What
is the optimal anticoagulation in patients with a left
ventricular assist device? Interact Cardiovasc Thorac
Surg. 2012;15:733-740.
51. Burkholder GA, Tamhane AR , Salinas JL, et al. Un-
derutilization of aspirin for primary prevention of
cardiovascular dis ease among HIV-infected patients.
Clin Infect Dis. 2012;55:1550-1557.
52. O’Brien M, Montenont E, Hu L, et al. Aspirin at-
tenuates platelet activation and immune ac tivation
in HIV-1-infected subjects on antiretroviral therapy:
a pilot study. J Acquir Immune Defic Syndr. 2013;63:
280-288.
53. Manlhiot C, Menjak IB, Brandão LR, et al. Risk,
clinical features, and outcomes of thrombosis as-
sociated with pediatric cardiac surgery. Circulation.
2011;124:1511-1519.
54. Cholette JM, Mamikonian L, Alfieris GM, et al. As-
pirin resistance following pediatric cardiac surgery.
Thromb Res. 2010;126:200-206.
55. Anderson DR, Dunbar MJ, Bohm ER, et al. Aspirin
versus low-molecular-weight heparin for extended
venous thromboembolism prophylaxis after total hip
arthroplasty: a randomized trial. Ann Intern Med.
2013;158:800-806.
56. Steinhubl SR, Eike lboom JW, Hylek EM, et al.
Antiplatelet therapy in prevention of cardio-
and venous thromb oembolic events. J T hromb
Thrombolysis. 2014;37:362-371.
57. Becattini C, Agnelli G, Schenone A, et al; WAR-
FASA Investigators. Aspirin for preventing the recur-
rence of venous thromboembolism. N Engl J Med.
2012;366:1959-1967.
58. Brighton TA, Eikelboom JW, Mann K, et al; ASPIRE
Investigators. Low-dose aspirin for preventing re-
current venous thromboembolism. N Engl J Med.
2012;367:1979-1987.
59. Hennekens CH, Cutlip D, Zehnder JL. Nonresponse
and resistance to aspirin. UpToDate. http://www.up-
todate.com/contents/nonresponse-and-resistance-to-
aspirin?source=search_result&search=Nonresponse+
and+Resistance+to+Aspirin&selectedTitle=1%7E150.
Accessed April 30, 2014.
60. Dragani A, Pascale S, Recchiuti A, et al. The con-
tribution of c yclooxygenase-1 and -2 to persistent
thromboxane biosynthesis in aspirin-treated ess en-
tial thrombocyt hemia: implications for antiplatelet
therapy. Blood. 2010;115:1054-1061.
61. Lev EI, Solo dky A, Harel N, et al. Treatment of
aspirin-resistant patients with omega-3 fatty acids
versus aspirin dose escalation. J Am Coll Cardiol.
2010;55:114-121.
62. Yusuf S, Pais P, Afzal R, et al; Indian Polycap Study
(TIPS). Effects of a polypill (Polycap) on risk factors
in middle-aged individuals without cardiovascular
disease (TIPS): a phase II, double-blind, randomised
trial. Lancet. 2009;373:1341.
30. Corgenix 11dhTxB2 Test Kit (11-Dehydro Thrombox-
ane B2) [package insert]. Broomfield, CO: C orgenix,
Inc; 2008.
31. Geske FJ, Lopez LR, Muncy IJ, Tew DJ. An ELISA
for deter mining aspirin effect from urine. I VD
Technology. http:/ /www.ivdtechnology.com/ar ticle/
elisa-determining-aspirin-effect-urine. Accessed
April30, 2014.
32. Eikelboom JW, Hankey GJ, Thom J, et al; Clopidogrel
for High Atherothrombotic Risk and Ischemic Stabi-
lization, Management, and Avoidance (CHARISMA)
Investigators. Incomplete inhibition of thrombox-
ane biosynthesis by ace tylsalicylic acid: dete rmi-
nants and effect on cardiovascular risk. Circulation.
2008;118:1705-1712.
33. Gluckman TJ, McLean RC, Schulman SP, et al. Effects
of aspirin responsiveness and platelet reactivity on early
vein graft thrombosis after coronary artery bypass graft
surgery. J Am Coll Cardiol. 2011;57:1069-1077.
34. Matsuura E, Guyer K, Yamamoto H, et al. On aspirin
treatment but not baseline thromboxane B2 levels pre-
dict adverse outcomes in patients with acute coronary
syndromes. J Thromb Haemost. 2012;10:1949-1951.
35. Turagam MK, Velagapudi P, Leal MA, Kocheril AG.
Aspirin in stroke prevention in nonvalvular atrial
fibrillation and stable vascular disease: an era of
new anticoagulants. E xpert Rev Cardiovasc Ther.
2012;10:433-439.
36. Hart RG, Pearce LA, Aguilar MI. Meta-analysis: anti-
thrombotic therapy to prevent stroke in patients who
have nonvalvular atrial fibrillation. Ann Intern Med.
2007;146:857-867.
37. Santilli F, Davì G, Consoli A, et al. Thromboxane-
dependent CD40 ligand release in type 2 diabetes
mellitus. J Am Coll Cardiol. 2006;47:391-397.
38. Gonçalves LH, Duss e LM, Fer nandes AP, et al.
Urinary 11-dehydro thromboxane B2 levels in type 2
diabetic patients before and during aspirin intake. Clin
Chim Acta. 2011;412:1366-1370.
39. Ethier J, Bragg-Gresham JL, Piera L, et al. Aspirin
prescription and outcomes in hemodialysis patients:
the Dialysis Outcomes and Practice Patterns Study
(DOPPS). Am J Kidney Dis. 2007;50:602-611.
40. Chan KE, L azarus JM, Thadhani R , Hakim RM.
Anticoagulant and antiplatelet usage associates with
mortality among hemodialysis p atients. J Am Soc
Nephrol. 2009;20:872-881.
41. Hiremath S, Holden RM, Fergusson D, Zimmerman
DL. Antiplatelet medications in hemodialysis patients:
a systematic review of bleeding rates. Clin J Am Soc
Nephrol. 2009;4:1347-1355.
42. Palmer SC, Di Micco L, Razavian M, et al. Antiplatelet
agents for chronic kidney disease. Cochrane Database
Syst Rev. 2013;2:CD008834.
43. Jardine MJ, Ninomiya T, Perkovic V, et al. Aspirin
is beneficial in hypertensive patients with chronic
kidney disease: a post-hoc subgroup analysis of a
randomized controlled trial. J Am Coll Cardiol. 2010;
56:956-965.
44. Cheung AK, Henrich WL. Secondary prevention of
cardiovascular disease in end-stage renal disease (dial-
ysis). UpToDate. http://www.uptodate.com/contents/
secondary -preventio n-of-cardi ovascu lar-disease-
in-end-stage-renal-disease-dialysis?source=search_
result&search=Secondary+prevention+of+cardiovasc
ular+disease+in+end-stage+renal+disease+%28dialys
is&selectedTitle=1%7E150. Accessed April 30, 2014.
45. Myles PS. Stopping aspirin before coronary artery
surgery between the devil and the deep blue sea. Cir-
culation. 2011;123:571-573.
46. Hillis LD, Smith PK, Anderson JL, et al. 2011 ACCF/
AHA Guideline for Coronary Artery Bypass Graft
Surgery. A report of the American College of Cardiol-
ogy Foundation/American Heart Association Task
10. Pascale S, Petrucci G, Dragani A, et al. Aspirin-insen-
sitive thromboxane biosynthesis in essential throm-
bocythemia is explained by accelerated renewal of the
drug target. Blood. 2012;119:3595-3603.
11. Ames PR, Batuca JR, Muncy IJ, et al. Aspirin insensi-
tive thromboxane generation is associated with oxida-
tive stress in type 2 diabetes mellitus. Thromb Res.
2012;130:350-354.
12. O’Neill GP, Ford-Hutchinson AW. Expression of
mRNA for cyclooxygenase-1 and cyclooxygenase-2 in
human tissues. FEBS Lett. 1993;330:157-160.
13. McAdam BF, Catella-Lawson F, Mardini IA, et al.
Systemic biosynthesis of prostacyclin by c yclooxy-
genase (COX)-2: the human pharmacology of a selec-
tive inhibitor of COX-2. Proc Natl Acad Sci U S A.
1999;96:272-277.
14. Schönbeck U, Sukhova GK, Graber P, et al. Aug-
mented expression of c yclooxygenase-2 in human
atherosclero tic le sions. Am J Pathol. 1999;155:
1281-1291.
15. Weber AA, Zimmerm ann KC, Meyer-Kirchrat h
J, Schrör K. Cyclooxygenase-2 in human platelets
as a possible factor in aspirin resistance. Lancet.
1999;353:900.
16. Ting HJ, Khasawneh FT. Platelet function and iso-
prostane biology. Should isoprostanes be t he newest
member of the orphan-ligand family? J Biomed Sci.
2010;17:24.
17. Patrignani P, Filabozzi P, Patrono C. Selective cumula-
tive inhibition of platelet thromboxane production
by low-dose aspirin in healthy subjects. J Clin Invest.
1982;69:1366.
18. McConnell JP, Cheryk LA, Durocher A, et al. Urinary
11-dehydro-thromboxane B(2) and coagulation acti-
vation markers measured within 24 h of human acute
ischemic stroke. Neurosci Lett. 2001;313:88-92.
19. Foegh ML, Zhao Y, Madren L, et al. Urinary throm-
boxane A2 metabolites in patients presenting in the
emergency room with acute chest pain. J Intern Med.
1994;235:153-161.
20. Eikelboom JW, Hirsh J, Weitz JI, et al. Aspirin-
resistant thromboxane biosynthesis and the risk of
myocardial infarction, stroke, or cardiovascular death
in patients at high risk for cardiovascular events.
Circulation. 2002;105:1650-1655.
21. Mani H, Kirchmayr K, Kläffling C, et al. Influence
of blood collection techniques on platelet function.
Platelets. 2004;15:315-318.
22. Breddin HK. Can platelet aggregometry be standard-
ized? Platelets. 2005;16:151-158.
23. Hankey GJ, Eikelboom JW. Aspirin resistance. Lancet.
2006;367:606-617.
24. FitzGerald GA, Pedersen AK, Patrono C. Analysis of
prostacyclin and thromboxane biosynthesis in cardio-
vascular disease. Circulation. 1983;67:1174-1177.
25. Roberts LJ 2nd, Sweetman BJ, Oates JA. Metabolism
of thromboxane B2 in man. Identification of twenty
urinary metabolites. J Biol Chem. 1981;256:8384-8393.
26. Catella F, Healy D, Lawson JA, FitzGerald GA. 11-
Dehydrothromboxane B 2: a quantitative index of
thromboxane A2 formation in the human circulation.
Proc Natl Acad Sci U S A. 1986;83:5861-5865.
27. Martin CP, Talbert RL. Aspirin resistance: an evalua-
tion of current evidence and measurement methods.
Pharmacotherapy. 2005;25:942-953.
28. Olson MT, Kickler TS, Lawson JA, et al. Effect of assay
specificity on the association of urine 11-dehydro
thromboxane B2 determination with cardiovascular
risk. J Thromb Haemost. 2012;10:2462-2469.
29. DeFilippis AP, Oloyede OS, Andrikopoulou E, et al.
Thromboxane A2 generation, in the absence of
platelet COX-1 activity, in patients with and with-
out atherothrombotic myocardial infarction. Circ J.
2013;77:2786-2792.
130 • Vol. 15 No. 2 • 2014 • Reviews in Cardiovascular Medicine
Urinary Thromboxane Testing to Evaluate Atherothrombotic Risk
continued
4170004_RICM0739.indd 130 24/06/14 3:10 PM
... Samples then were stored at À80 C for later batch analysis. Previous studies have shown no significant change in u11-dTXB concentrations with storage at room temperature for up to 6 days, 51 and long-term stability at À20 to À80 C. 30 2.6 | ELISA protocol Urinary 11-dTXB concentrations were quantified using a commercially available monoclonal ELISA validated for use with canine urine. 48 Urine samples were defrosted to room temperature and centrifuged at 2060g for 5 minutes to remove precipitated proteins before dilution. ...
... It has been found to be increased in several prothrombotic conditions including stroke, 40,59 myocardial infarction, 39,41,60 and atherosclerosis, 38,52 and has been widely investigated as a surrogate marker for clinical response to aspirin. 51 As such, our findings provide additional evidence that IMHA is a prothrombotic condition and introduce u11-dTXB:Cr as a potentially useful biomarker for assessing thrombotic risk in dogs with IMHA. ...
Article
Full-text available
Background Thromboembolic disease is a major cause of mortality in dogs with immune-mediated hemolytic anemia (IMHA). At present, no reliable biomarkers of individual patient thrombotic risk are available. In human medicine, increased urinary thromboxane concentrations have utility as markers of prothrombotic tendency in various situations. Hypothesis/Objectives First, to determine if urinary 11-dehydrothromboxane B2 (u11-dTXB) concentrations are increased in dogs with primary IMHA compared to normal dogs; second, to assess whether u11-dTXB concentration is associated with survival, known prognostic indicators, or frequency of thrombosis in dogs with IMHA. Animals Twenty client-owned dogs diagnosed with primary IMHA and 17 healthy dogs volunteered by hospital staff. Methods Prospective case-control study. A previously validated ELISA was used to measure urine 11-dTXB concentrations, which were normalized to urine creatinine concentration (u11-dTXB:Cr). Samples were obtained at presentation from patients with primary IMHA. Standard clincopathological data at baseline and survival data were collected. Urinary 11-dTXB:Cr was compared between outcome subgroups, and correlated with known markers of disease severity. Results Baseline u11-dTXB:Cr was significantly higher in dogs with IMHA than in healthy dogs (median, 3.75; range, 0.83-25.36 vs 0.65; 0.24-2.57; P = .003) but did not differ between dogs with IMHA that survived and did not survive to 30 days after presentation, nor between dogs with and without clinical suspicion of thrombotic disease. Conclusions and Clinical Importance Urinary 11-dTXB:Cr is increased in dogs with IMHA compared to healthy controls, consistent with a prothrombotic state. However, in this IMHA population u11-dTXB:Cr was not associated with survival or suspected thrombosis.
... This proportion increased to two-thirds when the data were interpreted using the algorithm proposed by Lopez et al., in which an 11-dehydro-TXB2 level of 2500 pg/mg creatinine was established as an upper normal limit for aspirin-naive healthy subjects [2]. The same level was adopted by Neath et al. to evaluate atherothrombotic risk [22]. ...
Article
Urine 11-dehydro-thromboxane B2 (11-dehydro-TXB2), an indirect measure of platelet activity, is elevated in cardiovascular diseases and diabetes. The purpose of our study was to determine whether urine 11-dehydro-TXB2 is elevated in aspirin-naive males with metabolic syndrome (MS) and to determine predictors of 11-dehydro-TXB2 levels. The secondary aim was to evaluate whether these MS patients could be potential candidates for the aspirin-mediated prevention of atherosclerotic cardiovascular diseases (ASCVDs). In 82 males with MS (76 hypertensive), anthropometric measures, urine 11-dehydro-TXB2, platelet count, creatinine, glucose, insulin, estimated insulin resistance, lipid parameters, high-sensitivity C-reactive protein (hs-CRP), adiponectin, homocysteine, and ten-year risk of fatal cardiovascular disease (SCORE) were assessed. Urine 11-dehydro-TXB2 levels were elevated (≥2500 pg/mg creatinine) in two-thirds of patients, including 11 high-risk patients (SCORE ≥ 5%). Homocysteine, adiponectin, hs-CRP, waist-to-hip ratio, and total cholesterol were found to be predictors of urine 11-dehydro-TXB2. In conclusion, there is a high incidence of elevated urine 11-dehydro-TXB2 in males with MS, including in some patients who are at a high or very high risk of ASCVDs. 11-dehydro-TXB2 levels are associated with hyperhomocysteinemia, inflammation, fat distribution, hypercholesterolemia, and adiponectin concentrations. Elevated 11-dehydro-TXB2 levels may support the use of personalised aspirin ASCVD prevention in high-risk males with MS. Giuseppe Patti.
Article
Full-text available
Anti-platelet therapy with aspirin has been shown to reduce adverse outcomes in patients with coronary artery disease (CAD). Aspirin irreversibly inhibits platelet cyclooxygenase-1 (COX-1) and attenuates thromboxane A2-mediated platelet aggregation, but there is variable suppression of COX-1. From a cohort of patients with stable coronary artery disease we performed blinded, detailed chart abstraction and measured urinary 11-dehydro-thromboxane B2 (11dhTxB2), an inactive metabolite of TxA2 from frozen samples. There were 327 (73%) men and 122 (27%) women with a mean age (± SD) of 67±10 and 65±10 years, respectively. A positive linear trend for age was observed among tertiles of 11dhTxB2 (p-trend=0.01). Higher proportions of women (p=0.001), chronic obstructive pulmonary disease (p-trend=0.0003), and heart failure (p-trend=0.003) were observed in the upper tertile of 11dhTxB2. Sixty-seven (14.9%) patients died over a median follow-up of 1149 days and 87.5% of the deaths were due to cardiovascular causes. Twenty-six (38.8%) non-survivors were treated with P2Y12 receptor antagonists vs. 161 (42.2%) survivors (p=0.61). By stepwise Cox proportional hazards analysis, we identified that patients in the middle (HR=7.14; 95% CI: 2.46-20.68) and upper tertiles (HR=9.91; 95% CI: 3.45-28.50) had higher risks for mortality after adjusting for age and comorbidities. In conclusion, urinary concentration of 11dhTxB2 was a strong independent risk factor for all-cause mortality among stable CAD patients on aspirin therapy and may be a marker for CAD patients who require more intensive secondary prevention measures.
Article
Lipid mediators (LM) of inflammation are a class of compounds derived from ω-3 and ω-6 fatty acids that play a wide role in modulating inflammatory responses. Some LM possess pro-inflammatory properties, while others possess pro-resolving characteristics, and the class switch from pro-inflammatory to pro-resolving is crucial for tissue homeostasis. In this article, we review the major classes of LM, focusing on their biosynthesis and signaling pathways, and their role in systemic and, especially, oral health and disease. We discuss the detection of these LM in various body fluids, focusing on diagnostic and therapeutic applications. We also present data showing gender-related differences in salivary LM levels in healthy controls, leading to a hypothesis on the etiology of inflammatory diseases, particularly, Sjögren's Syndrome. We conclude by enumerating open areas of research where further investigation of LM is likely to result in therapeutic and diagnostic advances. This article is protected by copyright. All rights reserved.
Article
Full-text available
Background: Aspirin's therapeutic action is via inhibition of platelet cyclooxygenase 1 (COX-1) thromboxane A2 (TxA2) production. The aim of this study was to evaluate TxA2 production, in the absence of platelet COX-1 activity, in coronary atherosclerotic heart disease patients with and without atherothrombotic myocardial infarction (MI). Methods and results: TxA2 production, in the absence of platelet COX-1 activity, was evaluated in 44 patients taking aspirin on 3 commercially available assays that detect metabolites of TxA2 in the urine. Two assays measure urine 11-dehydro-thromboxane B2 (TxB2) alone and 1 measures urine 11-dehydro-TxB2 plus 11-dehydro-2,3-dinor-TxB2. Platelet COX-1 inhibition was confirmed on <10% platelet aggregation in response to ≥1 mmol/L arachidonic acid. Median urine 11-dehydro-TxB2 was no different in those with and without a diagnosis of atherothrombotic MI (325 vs. 311 pg/mg creatinine, P=0.59 via polyclonal ELISA) and (312 vs. 244 pg/mg creatinine, P=0.11 via LC-MS/MS). Median urine 11-dehydro-TxB2 plus 11-dehydro-2,3-dinor-TxB2, however, was higher in those with vs. those without a diagnosis of atherothrombotic MI (1,035 vs. 606 pg/mg creatinine, P=0.03 via monoclonal ELISA). Conclusions: Differences in TxA2 production, in the absence of platelet COX-1 activity, between those with vs. without atherothrombotic MI were not observed when TxA2 generation was assessed on 11-dehydro-TxB2 production alone (polyclonal ELISA or LC-MS/MS), but differences were observed when TxA2 generation was assessed using 11-dehydro-TxB2 plus 11-dehydro-2,3-dinor-TxB2 (monoclonal ELISA). These findings highlight important differences between different commercially available assays for TxA2 generation and suggest that 11-dehydro-2,3-dinor-TxB2 may be critical to the biology of atherothrombosis.
Article
The rate-limiting step in the formation of prostanoids is the conversion of arachidonic acid to prostaglandin H-2 by cyclooxygenase, also known as prostaglandin G/H synthase/cyclooxygenase. Two forms of cyclooxygenase have been characterized: a ubiquitously expressed form (COX-1) and a recently described second form (COX-2) inducible by various factors including mitogens, hormones, serum and cytokines. Here we quantitate by the reverse transcriptase-polymerase chain reaction (RT-PCR) the expression of COX-1 and COX-2 mRNA in human tissues including lung, uterus, testis, brain, pancreas, kidney, liver, thymus, prostate, mammary gland, stomach and small intestine. All tissues examined contained both COX-1 and COX-2 mRNA and could be grouped according to the level of COX mRNA expression. The highest levels of COX mRNAs were detected in the prostate where approximately equal levels of COX-1 and COX-2 transcripts were present. In the lung high levels of COX-2 were observed whereas COX-1 mRNA levels were about 2-fold lower. An intermediate level of expression of both COX-1 and COX-2 mRNA was observed in the mammary gland, stomach, small intestine, and uterus. The lowest levels of COX-1 and COX-2 mRNA were observed in the testis, pancreas, kidney, liver, thymus, and brain.
Article
Aspirin (acetylsalicylic acid) reduces the odds of serious atherothrombotic vascular events and death in a broad category of high risk patients by about one-quarter. The mechanism is believed to be inhibition of thromboxane biosynthesis by inactivation of platelet cyclo-oxygenase-1 enzyme. However, aspirin is not that effective; it still fails to prevent the majority of serious vascular events. Mechanisms that may account for the failure of aspirin to prevent vascular events include non-atherothrombotic causes of vascular disease, non-adherence to aspirin therapy, an inadequate dosage, alternative ‘upstream’ pathways of platelet activation (e.g. via stimulation of the ADP, collagen or thrombin receptors on platelets), aspirin-insensitive thromboxane biosynthesis (e.g. via monocyte cyclo-oxygenase-2), or drugs that interfere with the antiplatelet effects of aspirin. Genetic or acquired factors may further modify the inhibitory effects of aspirin on platelets (e.g. polymorphisms involving platelet-associated proteins, increased platelet turnover states). Identification and treatment of the potential causes of aspirin failure could prevent at least another 20% of serious vascular events (i.e. over and above those that are currently prevented by aspirin). There is currently no role for routine laboratory testing to measure the antiplatelet effects of aspirin. Clinicians should ensure that patients at high risk of atherothrombosis (>3% risk over 5 years) are compliant with aspirin therapy and are taking the correct dosage (75–150 mg/day). Patients who cannot tolerate aspirin, are allergic to aspirin, or have experienced recurrent serious atherothrombotic events whilst taking aspirin, should be treated with clopidogrel, and patients with acute coronary syndromes benefit from the combination of clopidogrel plus aspirin. Future research is required to standardize and validate laboratory testing of the antiplatelet effects of aspirin and to identify treatments that can both improve these laboratory measures and reduce the risk of future atherothrombotic events.
Article
The contribution of platelets in the pathophysiology of low-shear thrombosis-specifically, in atrial fibrillation (AF) and venous thromboembolic events (VTE)-remains less clear than for arterial thrombosis. AF itself appears to lead to platelet activation, offering a potential target for aspirin and other antiplatelet agents. Randomized trial results suggest a small benefit of aspirin over placebo, and of dual antiplatelet therapy (aspirin plus clopidogrel) over aspirin alone, for prevention of cardioembolic events in AF. Antiplatelet therapy thus can represent an option for patients with AF who are unsuitable for therapy with warfarin or novel oral anticoagulant agents. For VTE, the rationale for antiplatelet therapy reflects the venous response to disrupted blood flow-interactions among monocytes, neutrophil extracellular traps, and platelets. Early randomized trials generally showed poorer performance of aspirin relative to heparins and danaparoid sodium in prevention of VTE. However, results from large placebo- and dalteparin-controlled randomized trials have spurred changes in the most recent practice guidelines-aspirin is now recommended after major orthopedic surgery for patients who cannot receive other antithrombotic therapies.