Metabolic profiling of murine plasma reveals an
unexpected biomarker in rofecoxib-mediated
Jun-Yan Liua, Ning Lib, Jun Yanga, Nan Lic, Hong Qiub, Ding Aia,c, Nipavan Chiamvimonvatb, Yi Zhuc,
and Bruce D. Hammocka,1
aDepartment of Entomology and University of California-Davis Cancer Center andbDivision of Cardiovascular Medicine, University of California, Davis, CA
95616; andcDepartment of Physiology, Beijing University, Beijing 100083, People’s Republic of China
Contributed by Bruce D. Hammock, August 6, 2010 (sent for review June 16, 2010)
Chronic administration of high levels of selective COX-2 inhibitors
(coxibs), particularly rofecoxib, valdecoxib, and parecoxib, increases
risk for cardiovascular disease. Understanding the possibly multiple
incomplete. Using a metabolomics approach, we demonstrate that
which correlates with a significantly shorter tail bleeding time in
in 20-HETE is attributable to inhibition of its metabolism and that
the shortened bleeding time following rofecoxib administration is
attributable, in part, to this increase. The s.c. infusion of 20-HETE
shortened the tail bleeding time dramatically. Neither 20-HETE bio-
synthesis nor cytochrome P4A-like immune reactivity was increased
by rofecoxib administration, but 20-HETE production increased in
vitro with the addition of coxib. 20-HETE is significantly more potent
than its COX-mediated metabolites in shortening clotting time in
vitro. Furthermore, 20-HETE but not rofecoxib significantly increases
rat platelet aggregation in vitro in a dose-dependent manner. These
data suggest 20-HETE as a marker of rofecoxib exposure and that
inhibition of20-HETE’s degradationby rofecoxib is a partial explana-
tion for its dramatic increase, the shortened bleeding time, and, pos-
sibly, the adverse cardiovascular events associated with rofecoxib.
cardiovascular side effect|coxib|eicosanoids|metabolomics|Vioxx
tration to treat osteoarthritis, rheumatoid arthritis, acute pain,
dysmenorrhea, and migraine on May 20, 1999. Rofecoxib was
voluntarily withdrawn from the worldwide market by its manu-
facturer, Merck, on September 30, 2004 because it was associated
with a higher risk for adverse cardiovascular events and stroke in
arthritic patients compared with those on the control naproxen
(1, 2). This resulted in nearly 27,000 lawsuits involving an almost
$5 billion settlement (3). In addition, high doses of other coxibs
problems (1, 4).
The dominant theory proposed to explain the cardiovascular
Current theory is that rofecoxib reduces the production of the
much less effect on the production of the potent platelet activator
thromboxane (TX) A2(5, 6). Data generated in this laboratory
on PGI2/TXA2ratios in inflamed mice are consistent with this
hypothesis (7). This would be a common mechanism shared by
other selective COX-2 inhibitors. Based on this discovery, one
might expect that conventional nonselective nonsteroidal antiin-
even beneficial to the cardiovascular system. However, a signifi-
ofecoxib (Vioxx), a potent, orally active, and selective COX-2
inhibitor, was approved by the US Food and Drug Adminis-
infarction (MI), hypertension, and heart failure has also been ob-
served to be associated with the administration of the nonaspirin
conventional NSAIDs, including but not limited to diclofenac,
ibuprofen, naproxen, and indomethacin (8–12). In addition, there
could be rofecoxib-specific events such as the facile formation of
a cardiotoxic maleic anhydride derivative from rofecoxib that may
contribute to its adverse effects (13). This hypothesis fails to ex-
plain the increased risk in the cardiovascular system from other
nonaspirin NSAIDs. Thus, current mechanisms provide an in-
complete explanation for cardiovascular problems associated with
the use of NSAIDs.
To evaluate the risks and benefits of selective COX-2 inhibitors
and to develop safe coxibs or adjuvants to improve the safety of
existing coxibs, it is critical to understand the possibly multiple
mechanisms underlying the adverse cardiovascular events. Pre-
vious studies focused on the oxylipin mediators from arachidonic
cardiovascular events (5, 6). In contrast, in our current study, we
used a broader metabolomics approach to quantify the represen-
tative oxylipin mediators derivedfromARA and linoleic acid (LA)
mediated by COXs, lipoxygenases (LOXs), and cytochrome P450s
(CYP450s) using liquid chromatography-tandem mass spectrome-
try (LC-MS/MS) (Fig. S1). Metabolomics is a promising approach
that has been widely used as a powerful tool in disease diagnosis
(14), biomarker discovery (15), toxicity evaluation (16), gene func-
tion (17), and pharmacological research (18, 19). Here, we applied
that rofecoxib acts, in part, to accumulate the ARA metabolite 20-
thus increasing the risk for MI and stroke. This mechanism may be
shared with other nonaspirin NSAIDs.
Metabolomic Profiling of the Plasma from Mice Chronically Treated
with Rofecoxib. As expected, administration of rofecoxib to mice
for 3 mo resulted in a dramatically shortened bleeding time (Fig.
1A). Metabolomic profiling of the plasma from treated mice
resulted in the quantitative levels of 27 oxylipin mediators (Table
(the relatively stable metabolites of TXA2and PGI2, respectively)
were under the quantitation limits, as expected, in noninflamed
animals. However, an increase of greater than 120-fold was ob-
Author contributions: J.-Y.L., N.C., Y.Z., and B.D.H. designed research; J.-Y.L., Ning Li, J.Y.,
Nan Li, H.Q., and D.A. performed research; J.-Y.L., Ning Li, J.Y., Nan Li, H.Q., and D.A.
analyzed data; and J.-Y.L., N.C., Y.Z., and B.D.H. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| September 28, 2010
| vol. 107
| no. 39
served in the plasma concentration of 20-HETE in the mice trea-
ted with rofecoxib (Fig. 1 B and C, and Table S1).
Effect of 20-HETE Infusion on Murine Bleeding Time. The s.c. infusion
of 20-HETE to mice for 3 wk resulted in a dramatic decrease in
a significantlyhigherlevel of20-HETEin murineplasma(Fig. 2B).
Regulation of 20-HETE Production by Rofecoxib and Other COX
Inhibitors in Vitro. Incubation of rofecoxib with ARA in a murine
hepatic S-9 fraction shows that rofecoxib increases the production
of 20-HETE in a time- and dose-dependent manner (Fig. 3). In
addition, both the nonselective COX inhibitor indomethacin and
the selective COX-1 inhibitor SC-560 significantly increase the
production of 20-HETE (Fig. 3B).
Effects of 20-HETE and Its COX-Mediated Metabolites on Murine Blood
Clotting Time in Vitro. Fig. 4 illustrates the in vitro effects of 20-
HETE and two of its COX-mediated metabolites [20-hydroxyl
PGE2(20-OH PGE2) and 20-hydroxyl PGF2α(20-OH PGF2α)]
on murine blood clotting time (MBCT). 20-HETE significantly
shortens the MBCT when compared with a control. In compar-
ison to 20-HETE, 20-OH PGF2αsignificantly delays the MBCT
and 20-OH PGE2slightly delays MBCT (Fig. 4).
Effects of 20-HETE and Rofecoxib-Mediated Hemostatic Coagulation
on Murine Plasma in Vitro. To illustrate themechanism of thedelay
of MBCT by 20-HETE, prothrombin time (PT), thrombin time
(TT), activated partial thromboplastin time (APTT), and plasma
fibrinogen (FIB) levels were measured with fresh murine plasma
treated with different concentrations of 20-HETE and rofecoxib.
The international normalized ratio (INR) was also calculated. As
shown in Table S2, neither 20-HETE nor rofecoxib caused signif-
icant changes in those indexes of hemostatic coagulation in mice.
Effects of 20-HETE and Rofecoxib-Mediated Platelet Aggregation on
Rat Blood Platelet in Vitro. Fig. 5 shows the in vitro effects of 20-
HETE on ADP-induced platelet aggregation. Because of the lim-
itation of blood volume from mice, the platelet aggregation assay
20-HETE but not rofecoxib significantly increases the content of
ADP-induced platelet aggregation when compared with a control,
which is dose-dependent starting from 10−7M. In addition, 20-
HETE accelerates the ADP-induced platelet aggregation rate in
a time-related manner (Fig. 5B).
Bleeding time roughly reflects platelet aggregability. Increased
platelet aggregability has been found to be associated with the
pathogenesis of MI (21–23) and stroke (24). In addition, a signifi-
cantly shorter bleeding time was found in patients with diagnosed
MI when compared with patients without MI (25, 26). Thus, tail
risk for chronic administration of rofecoxib in the current study.
A randomized double-blind experiment was conducted in a mu-
rine model (protocol 1). After 3 mo of drug administration, as
expected, a dramatically shortened tail bleeding time was observed
in the animals administered rofecoxib compared with controls
(Fig. 1A). This increased clotting propensity may contribute to
a high incidence of MI and stroke. Many pharmaceutical agents,
including rofecoxib, inhibit enzymes in the arachidonate cascade.
Previous studies focused onanalysis oftheARA metabolites ofthe
COX pathway as markers of administration of rofecoxib and other
NSAIDs and on investigation of both the therapeutical effects and
side effects of these pharmaceutical agents (5, 6). In contrast,
a broader metabolomic profiling strategy was used in current study
CYP450 branches of the arachidonate and linoleate cascades (tar-
get analytes illustrated in Fig. S1). Among the detectable oxylipin
mediators derived from ARAand LA (Table S1), the most striking
in the plasma of the mice administrated rofecoxib (Fig. 1B).
the increase in 20-HETE was still observed when rofecoxib was
incubated with ARA and the hepatic S-9 fraction prepared from
the treated animals (Fig. 3). This is consistent with the in vivo
observation (Fig. 1B). 20-HETE is one of the metabolites of ARA
produced by CYP450s (CYP4As and CYP4Fs) (28). One could
ARA substrate available after COX inhibition. If this were the
cause, one could expect other CYP metabolites such as epox-
yeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids
(DHETs) to increase dramatically as well. However, increases in
EETs or DHETs were not observed (Table S1). Another expla-
level of 20-HETE, leading to a dramatically shortenedtail bleeding time. (A) Oral
administration of rofecoxib for 3 mo results in a dramatically shortened tail
killed. If the bleeding time was more than 10 min, the experiment was termi-
mo results in a dramatic increase in plasma levels of 20-HETE (n = 6; dotted line,
quantitative limit).(C)Structuresofrofecoxib and 20-HETE.Datarepresentthe
50 mg/L. The bleeding time is a parameter of blood aggregation. A shorter
bleeding time means that the blood is easier to aggregate, which can increase
the risk for cardiovascular events. Statistical significance was determined by
a two-sided unpaired t test and one-way ANOVA (**P < 0.01).
Chronic administration of rofecoxib significantly increases the blood
time. (A) Infusion (s.c.) of 20-HETE for 3 wk leads to a significantly shortened
tail bleeding time (n = 6). The tail bleeding time was measured before the
mice were killed. If the bleeding time was more than 10 min, the experiment
was terminated at 10 min and recorded as 10 min. (B) Infusion (s.c.) of 20-
HETE for 3 wk results in a higher plasma level of 20-HETE (n = 6). Data
represent the mean ± SD. 20-HETE was administered with s.c. infusion at
a flow rate of 250 ng/h. The bleeding time is a parameter of blood co-
agulation. A shorter bleeding time means that the blood is easier to ag-
gregate, which increases the risk for cardiovascular events. Statistical
significance was determined by a two-sided unpaired t test and one-way
ANOVA (**P < 0.01).
Infusion of 20-HETE significantly shortened the murine tail bleeding
| www.pnas.org/cgi/doi/10.1073/pnas.1011278107Liu et al.
nation could be that rofecoxib administration induces the enzymes
rat CYP4A antibody showed that rofecoxib does not alter the ex-
pression of related CYPs at the protein level in murine liver (Fig.
S2). The fact that the 20-HETE formation rate is not altered by in
20-HETE is not altered. 20-HETE has been demonstrated to be
20-OH-PGF2α) and other mediators (29–31) in in vitro studies.
Thus, the 20-HETE increase both in vivo and in vitro could be
explained by blocking one of its major routes of degradative me-
tabolism pathways using rofecoxib. This time- and dose-dependent
increase in the production of 20-HETE is demonstrated not only
with rofecoxib but with the selective COX-1 inhibitor SC-560 and
its metabolites such as maleic anhydride (13) could attenuate the
oxidization of 20-HETE, and thus result in the accumulation of
20-HETE, was not supported by the significant increase of 20-
HETE associated with the selective COX-1 inhibitor SC-560 and
the nonselective COX inhibitor indomethacin, which are structur-
ally different from rofecoxib. We propose the hypothesis that the
reduction in the metabolism of20-HETEby the inhibition of COX
enzymes that are important in the metabolism of 20-HETE.
the possible alteration in bleeding time (protocol 2). After 3 wk of
infusion, 20-HETE rose to 5 nM in the murine plasma and dra-
matically shortened bleeding time (Fig. 2). When compared with
chronic administration of rofecoxib, direct infusion of 20-HETE
results in a lower plasma level of 20-HETE but a similar effect on
shortening bleeding time, which may be attributable to the expo-
sure duration of 20-HETE. This also indicates more complicated
mechanism(s) underlying the chronic administration of rofecoxib
than those proposed. However, this demonstrates that an increase
in 20-HETE contributes to a shortening of bleeding time in vivo.
This hypothesis is supported by in vitro assays showing that 20-
HETE significantly shortens MBCT (Fig. 4A). In addition, we
measured the plasma level of FIB and platelet factor 4 (PF-4) for
the mice in protocol 2. The results show that infusion of 20-HETE
increases the plasma levels of FIB and PF-4 without statistic sig-
nificance (Fig. S3). In summary, both in vivo and in vitro studies
suggest that a higher level of 20-HETE contributes to the en-
hanced speed of blood clotting associated with chronic adminis-
tration of rofecoxib. This and other effects of increased 20-HETE
such as vasoconstriction may contribute to the occurrence of MI
and stroke (33).
An increase in 20-HETE has been documented to be associated
inhibition oftheproduction of 20-HETEdecreasestheinfarctsize
and ablates strokes in several animal models (34–37). To study the
mechanism by which 20-HETE enhances the speed of blood
clotting, we measured the function of coagulation and platelet
induced platelet aggregation but not coagulation. Thus, 20-HETE
caused the shortening of bleeding time and blood clotting time as
a result of the increase in platelet aggregation rather than dys-
function of coagulation via clotting factors. Previous studies show
that 20-HETE is released from human platelet phospholipids on
= 6). (B) Significant increase was observed in the production of 20-HETE following a 3-h incubation of normal murine liver S-9 fraction and ARA with rofecoxib,
SC-560, and indomethacin (1 μM each; n = 6), whereas a slight increase was observed with rofecoxib (100 nM; n = 6). Data represent the mean ± SD. Statistical
significance was determined by a two-sided unpaired t test and one-way ANOVA (*P < 0.05).
Accumulation of 20-HETE following treatment of murine hepatic S-9 fraction with rofecoxib, SC-560, and indomethacin. (A) Significant increase was
blood clotting time. Venous blood (25 μL) was collected from the tail of Swiss
Webster mice (male, 8 wk of age) to test blood clotting time using prescored
capillary tubes rinsed with DMSO, 20-HETE (0.5 mM in DMSO), 20-OH-PGE2
of 20-HETE, 20-OH-PGE2, and 20-OH-PGF2αwas calculated to be ≈13 μM. Each
compound was tested with bloodfrom six individual mice. Data represent the
mean ± SD (n = 6). Statistical significance was determined by a two-sided
unpaired t test and one-way ANOVA (*P < 0.05; **P < 0.01).
In vitro effects of 20-HETE and its COX-mediated metabolites on
Liu et al. PNAS
| September 28, 2010
| vol. 107
| no. 39
phospholipase treatment and inhibits the ARA A23187- and
of 10 and 30 μM, respectively, in a dose-dependent manner (29,
38). However, 20-HETE had no detectable agonist activity on
20-HETE was demonstrated to accelerate rat platelet aggrega-
tion significantly at a concentration between 100 nM and 10 μM
in a dose-dependent and time-related manner in vitro (Fig. 5),
whereas rofecoxib had no effect on platelet aggregation under the
same conditions. In addition, an in vitro assay of blood clotting
time showed that 20-HETE significantly increases the clotting
tendency, whereas its COX-mediated metabolites 20-OH-PGF2α
and 20-OH-PGE2are similar to the control platelet aggregation
(Fig. 4A). This indicates that 20-HETE is more detrimental to the
vascular system than its COX-mediated metabolites. These data
suggest that accumulation of 20-HETE contributes to the car-
diovascular problems associated with a high dose of rofecoxib.
This also cautions against the use of NSAIDs in patients with
cardiovascular diseases, which was indicated by the clinical facts
that NSAIDs, including rofecoxib, celecoxib, ibuprofen, diclofe-
nac, and naproxen, increase mortality and cardiovascular mor-
bidity in chronic heart failure (39).
The dramatic increase in 20-HETE observed with rofecoxib
treatment seems more attributable to decreased degradation of
20-HETE than to increased production. These data link the ac-
with rofecoxib and related compounds. This dramatic increase in
20-HETE may, in turn, be linked to the adverse cardiovascular
events of rofecoxib that may be shared with other nonaspirin
NSAIDs. This hypothesis suggests 20-HETE as a biomarker for
cardiovascular risk from coxibs as well as possible strategies for
attenuation of their adverse effects. For example, we predict that
inhibition or down-regulation of CYP4A and or CYP4F may ab-
late the cardiovascular events of coxibs. In addition, this study
exemplifies metabolomic profiling as a promising tool to gain
a more comprehensive understanding of biological processes.
Materials and Methods
All procedures and animal care were performed in accordance with the pro-
tocols approved by the Institutional Animal Care and Use Committee of the
University of California, Davis. Male C57BL/6 and Swiss Webster mice were
purchased from Charles River Laboratories. Rofecoxib was purchased from
Toronto Research Chemicals, Inc. PEG400, (2-hydroxypropyl)-β-cyclodextrin
(HPCD), β-NADP sodium salt, and monoclonal anti-β-actin antibody were from
Sigma–Aldrich. EDTA(K3) was purchased from Tyco Health Group LP. Water
(>18.0 MΩ) was purified by a NANO pure system (Barnstead). ARA was pur-
chased from Nu-Chek Prep, Inc. Glucose-6-phosphate dehydrogenase was
purchased from MP Biomedicals, LLC. Calibrated capillary tubes (25 μL) were
purchased from VWR International, LLC. Oxylipin standards and SC-560 were
purchased from Cayman Chemical or synthesized in-house. Polyclonal anti-rat
CYP4A antibody was purchased from Novus Biologicals. An Alzet miniosmotic
pump (model 2004) was purchased from DURECT Corporation.
Animal Protocol 1. After acclimation for 1 wk in a standard animal facility,
two equal groups (n = 6) and each mouse was housed in a separate cage with
food and water available ad libitum. One group was administered rofecoxib
(50 mg/L) that was suspended in drinking water containing 1% (vol/vol)
PEG400 and 0.5% (wt/vol) HPCD for 3 mo, whereas another group was ad-
ministered the drinking water containing 1% (vol/vol) PEG400 and 0.5%
(wt/vol) HPCD as the control. The body weight, food intake, and water con-
sumption of animals were monitored weekly during the investigation (results
presented in Fig. S4). Bleeding time was measured in mice 15 min before they
were killed. Mice were killed 3 mo after treatment. All samples were stored
at −80 °C until analysis. Blood collection and plasma separation were per-
formed for oxylipin analysis as described previously (40). The whole heart was
removed after blood collection and weighed (results presented in Fig. S5).
Measurement of tail bleeding time. The tail was placed in a horizontal position
and kept at room temperature, and it was amputated 3 mm from the tip.
Blood was blotted onto filter paper every 30 s. The time until no blood blot
appeared on the filter paper was recorded as the bleeding time.
Oxylipin profiling analysis. Oxylipin mediators were extracted from the murine
plasma using the method described by Liu et al. (40) and were analyzed with
LC-MS/MS as described previously (20).
Animal Protocol 2. After acclimation for 1 wk in a standard animal facility,
apparently healthy mice (C57BL/6, male, 20 wk of age) were randomly
assigned to two equal groups (n = 6) and each mouse was housed in a sep-
arate cage with food and water available ad libitum. One group was
implanted s.c. with a miniosmotic pump loaded with 20-HETE, which was
dissolved inamixture ofsolvents [PEG400(50%vol/vol)+ DMSO (40%vol/vol)
+ ethanol (10% vol/vol)]. The infusion flow rate of 20-HETE was designed as
250 ng/h. Another group was implanted s.c. with a miniosmotic pump loaded
with the vehicle alone. Bleeding time was measured in mice 30 min before
they were killed. Mice were killed 3 wk after treatment. Blood collection and
plasma separation were performed for oxylipin analysis as described by Liu
et al. (40). All samples were stored at −80 °C until analysis.
Animal Protocol 3: Measurement of Blood Clotting Time. Prescored capillary
tubes for blood collection were previously rinsed with DMSO, 20-HETE
(0.5 mM in DMSO), 20-OH-PGE2(0.5 mM in DMSO), or 20-OH-PGF2α(0.5 mM
in DMSO). The final concentration of 20-HETE, 20-OH-PGE2, and 20-OH-
PGF2αwas calculated to be approximately 13 μM. Twenty-five microliters of
venous blood was collected from the tail of Swiss Webster mice (male, 8 wk
of age) after wiping off the first drop of blood. The capillary tube filled with
the blood was immediately held between the palms to maintain it at body
temperature for 2 min. The capillary tube was then broken off 1 cm from one
end every 30 s and checked for the appearance of a thread of fibrin. The timer
rofecoxib or 20-HETE at 1 min after ADP was added. (B) Aggregation percentage tracing curve of ADP-induced platelet aggregation under 10−7M 20-HETE or
HETE. Each point represents the mean ± SD (n = 6). Statistical significance was determined by a two-sided unpaired t test and repeated measures one-way ANOVA
(*P < 0.05; **P < 0.01).
20-HETE but not rofecoxib accelerates ADP-induced rat platelet aggregation in vitro. (A) Aggregation percentage under a different concentration of
| www.pnas.org/cgi/doi/10.1073/pnas.1011278107 Liu et al.
was started when the blood was visible out of the tail and was stopped when
a thin string of fibrin was seen. The time this took was regarded as the blood
clotting time. Each compound was tested with blood from six individual mice.
Incubation of Murine Liver S-9 Fraction. Murineliverwasfromthepooledlivers
of the six mice in the control group (protocol 1). S-9 fractions were prepared
according to the method described previously (41). The incubation was
according to the modified method of Watanabe et al. (42). Specifically, 178 μL
and incubatedat 37 °C for5 min. Then,20 μL of theNADPH generating system
(4 mg/mL) and 2 μL of ARA (10-μM final concentration) with or without rofe-
coxib (1-μM final concentration) were added to the tube. The whole reaction
system was incubated at 37 °C for 2 h after 30 s of gentle mixing on a Vortex
mixer (n = 6). The reaction was quenched by the addition of 400 μL of meth-
anol, followed by vigorous mixing for 15 s. The resulting solution (100 μL) was
mixed with an equal volume of internal standard [200 nM 12-(3-cyclohexyl-
ureido)-dodecanoic acid in methanol]. After centrifugation at 20,800 × g for
5 min, 50 μL of supernatant was transferred to analytical vials containing
a 150-μL insert for oxylipin analysis, as described above.
The 20-HETE formation rate was determined by the incubation method
described above. The pooled livers of the six mice in the control group and
exposure to rofecoxib (protocol 1) were used. The incubation was terminated
at 0, 0.25, 0.5, 1, 1.5, 2, 2.5, and 3 h after adding a NADPH generating system.
Plasma Preparation and Hemostatic Coagulation Tests. Venous blood was
obtained from C57BL/6 mice (8–10 wk of age, male). The mice were anes-
thetized with sodium pentobarbital (1% water solution, 100 mg/kg admin-
istered i.p). About 0.7 mL of venous blood was collected from each mouse by
suctioning from the right ventricle using a syringe containing sodium citrate.
Coagulation was blocked in the blood sample with 109 mmol/L sodium
citrate (1:9 vol/vol), and the sample was then centrifuged at 2,400 × g for 15
min to get platelet-free plasma. In this experiment, only fresh blood samples
were used. All tests were completed within 2 h at room temperature. The
plasma was incubated with different concentrations of rofecoxib or 20-HETE
ranging from 10−9to 10−5M for exactly 3 min at 37 °C before being used for
all measurements, which were performed by modifications of the conven-
tional clinical procedures using a photoelectrical electromagnetism coagul-
For testing PT, 50 μL of pretreated plasma was mixed with 100 μL of acti-
vating reagent at 37 °C. Thecoagulation timewas then measured,and theINR
was calculated by the coagulometer automatically. To measure TT, 100 μL of
added, and coagulation time was measured. For the measurement of APTT,
50 μL of plasma and 50 μL of APTT reagent, which contained cephalin and
CaCl2solution (37 °C) was added and coagulation time was measured. To
measure the plasma FIB level, 90 μL of buffer and 10 μL of plasma were in-
coagulation time was measured. The plasma FIB concentration (g/L) was cal-
culated by the coagulometer automatically from a reference curve.
Platelet Preparation and Aggregation Assays. Venous blood was obtained
from male Sprague–Dawley rats (180 ± 10 g). Preparation of platelet-rich
plasma (PRP) and platelet-poor plasma (PPP) was performed as described
previously (43). For the platelet aggregation assay, PRP was incubated in 10−9
to 10−5M rofecoxib or 20-HETE at 37 °C in a photoelectrical turbidimetric
platelet aggregometer (STEELLEX). Aggregation was induced by 5 μM ADP.
Results were quantified by measurement of the rate or extent of change of
light transmittance through the sample cuvette, and baseline transmittance
was calibrated with the PPP.
Immunoblot Analysis. Western immunoblot analysis was performed using the
methods detailedby Schmelzer et al. (7). Specifically, the microsomal proteins
were isolated from the liver for CYP4A analysis. The proteins were separated
by electrophoresis using 10% (wt/vol) SDS/PAGE and then transferred onto
poly(vinylidene fluoride) membranes (Immobilon P; Millipore). CYP4A-like
proteins were detected with polyclonal antibodies raised to rat CYP4A from
Novus Biologicals, and β-actin was detected with monoclonal antibody from
Sigma–Aldrich.Themembraneswereincubated with anHRP-linked IgGwhole
secondary antibody (Amersham Pharmacia Biosciences) at 1:5,000 dilutions
and then washed. Secondaryantibodieswere visualizedbyaSuperSignalWest
autoradiography. The experiment was replicated withindependent livers.The
gels were scanned, and the CYP4A1 reactive bands were expressed as a ratio
with β-actin. Single bands were detected.
Measurement of Cytokines. Plasma levels of FIB and PF-4 were measured using
(RayBiotech) according to the manufacturers’ instructions. Briefly, the plasma
was thawed and added to 96-well plate precoated with immobilized antibody
and wasincubatedfor2.5hatroomtemperature.Thewellswerewashed, and
were incubated for 30 min with HRP-conjugated streptavidin. The wells were
washed again, and a tetramethylbenzidine substrate solution was added to
the wells and developed for 30 min. The enzyme reaction was stopped by the
nm. A linear standard calibration curve was constructed by plotting the ab-
sorbance of the standards. The FIB and PF-4 concentrations in each sample
were determined from the detectable ranges of the linear calibration curve.
unpaired t test and one-way ANOVA, and P < 0.05 was considered significant.
ACKNOWLEDGMENTS. This work was supported, in part, by National
Institute of Environmental Health Sciences Grant ES02710, National Institute
of Environmental Health Sciences Superfund P42 ES04699, National Heart,
Lung, and Blood Institute Grants HL85727 and HL85844, a Major National
Basic Research Grant of China (2010CB912504), the Elizabeth Nash Memorial
fellowship from the Cystic Fibrosis Foundation, Inc. (to J.Y.), a Veterans
Affairs merit review grant (to N.C.) and American Heart Association Western
Affiliates postdoctoral fellowship award (to H.Q.). B.D.H. is a George and
Judy Marcus Senior Fellow of the American Asthma Foundation.
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