Platelets, February 2012; 23(1): 26–35
Copyright � 2012 Informa UK Ltd.
ISSN: 0953-7104 print/1369-1635 online
DOI: 10.3109/09537104.2011.589543
ORIGINAL ARTICLE
Nitric oxide and peroxynitrite platelet levels in gestational hypertensionand preeclampsia
LAURA MAZZANTI1, FRANCESCA RAFFAELLI1, ARIANNA VIGNINI1,
LAURA NANETTI1, PAOLA VITALI2, VIRGINIA BOSCARATO2,
STEFANO R. GIANNUBILO2, & ANDREA L. TRANQUILLI2
1Department of Biochemistry, Biology and Genetics, Marche Polytechnic University, via Tronto 10 – 60128 Ancona
(Italy) and 2Department of Obstetrics and Gynaecology, Marche Polytechnic University, via Tronto 10 –
60128 Ancona (Italy)
AbstractThe aim of the study was to investigate platelet nitric oxide (NO) pathways in women with Gestational Hypertension (GH),Preeclampsia (PE) and Controls.Platelet NOx and peroxynitrite (ONOO�) levels, inducible (iNOS) and endothelial nitric oxide synthase (eNOS) andNitrotyrosine expression (N-Tyr) in 30 women with GH, 30 with PE and 30 healthy pregnant controls, age, parity andgestational age-matched, were assessed.Platelet NOx and ONOO� levels were significantly higher in GH and PE vs. Controls, with higher levels in GH vs. PE. At thesame way, iNOS and N-Tyr were significantly higher in GH and PE vs. Controls, with higher levels in GH vs. PE.Since GH expressed higher amount of NO metabolites and higher activation of iNOS compared to PE, we can hypothesizethat the severity of hypertensive pathology is almost not related to only NO metabolism, this research confirmed that GH andPE are associated with marked changes in NO pathways; it is not easy to understand if they could be interpreted as causes orconsequence of these pathologic states.
Keywords: Preeclampsia, gestational hypertension, nitric oxide, peroxynitrite, platelets
Introduction
Several conditions are grouped under the term
‘‘hypertensive disorders of pregnancy’’ such as ges-
tational hypertension (GH), preeclampsia (PE),
chronic hypertension and superimposed preeclamp-
sia on chronic hypertension; they represent 10–15 %
of all pregnancies [1].
PE, the hypertensive disorder specific to human
pregnancy, is a serious complication of pregnancy
and a leading cause of maternal-foetal morbidity
and mortality. It is a multisystem disorder affect-
ing 2% of all first pregnancies, that develops after
the twentieth week of pregnancy; PE has clinical
diagnostic features of hypertension and protein-
uria, but in its early stages, women often show no
symptoms [2]. When high blood pressure is not
accompanied by the proteinuria, it is referred to as
GH or Pregnancy-induced hypertension (PIH).
Currently, PE and GH are considered either
separate diseases affecting similar organs or differ-
ent severities of the same underlying disorder.
According to the latter hypothesis, gestational
hypertension is merely an early or mild stage of
preeclampsia, perhaps preceding renal involvement
and thus proteinuria [3]. GH may progress to PE,
so all women who develop high blood pressure
in pregnancy are monitored closely. Both PE
and GH are regarded as very serious conditions
and they require careful monitoring of mother and
foetus.
Although the aetiology of PE is unclear, there
exists accumulated evidence for a pathogenic model
whereby a deficiency in trophoblastic invasion of the
placental bed leads to a poorly perfused fetoplacental
unit. The clinical features, due to a combination of
endothelial damage, generalized vasoconstriction
and activation of coagulation, have been well
described [4].
Correspondence: Francesca Raffaelli, Faculty of Medicine, Marche Polytechnic University, Via Tronto 10, Ancona, Italy. Tel: þ 39 071 2206058.
Fax: þ 39 071 2206058. E-mail: [email protected]
(received 24 June 2010; revised 19 April 2011; accepted 16 May 2011)
To date, the analysis about nitric oxide pathway
and platelet function in PE and GH has been widely
studied but separately and unfortunately an overview
is still confused [5–7].
Little is known about the pathogenesis of PE, but
its relation with platelets is expressed in various
papers [8, 9]. In particular, increased platelet activa-
tion has been observed in preeclampsia [10, 11].
Because excessive platelet activation results in plate-
let adhesion, vasoconstriction and endothelial injury
[12] (all of which may contribute to the pathogenesis
of preeclampsia, [13]), there is considerable interest
in the role of platelets in the pathogenesis and
progression of PE.
Furthermore, there have been several recent
reviews on the role of NO in the control of placental
vascular tone [14–16] and in the establishment and
maintenance of the fetoplacental circulation [17, 18];
impairment of the L-arginine-NO system has been
suggested to be involved in the pathophysiology of
PE [19–21].
NO synthesis from terminal guanidino nitrogen of
L-arginine is catalysed by nitric oxide synthases
(NOSs). Two isoforms are constitutive and calcium/
calmodulin-dependent, releasing NO from endothe-
lium (eNOS) and neurons (nNOS); another NOS,
inducible and calcium/calmodulin-independent
(iNOS), is not normally found in large amounts
but is expressed after stimulation by endotoxins,
cytokines, INT-�, TNF-� and IL-1. Specifically, the
constitutive eNOS, activated by hemodynamic stim-
uli, produces small amounts of NO (basal level) for a
short time in response to transient increases of
intracellular Caþþ; thus, the eNOS is responsible of
beneficial effects of NO production related to several
tissue physiological homeostasis. The inducibile
iNOS, instead, activated by inflammatory stimuli,
produces high amounts of NO for a long time; thus,
the iNOS is responsible of harmful effects of NO
production related to inflammatory response and
tissue damage [22, 23].
In addition to being regulated by NO, platelets
have the capacity to synthesize and release NO
[24, 25]; then, further studies have shown that
human platelet cytosol possesses both constitutive
and inducible forms of NO synthase [26–29].
While the constitutive release of NO seems to
have an important physiologic role, large amounts of
NO released in response to inflammatory stimuli
may be cytotoxic. In platelets, as well as in other
tissues, the iNOS also play a role in pathologic states
such as thrombosis and atherosclerosis when plate-
lets, on stimulation by cytokines [30, 31] could
produce large amounts of NO and superoxide anions
and possibly perpetuate the cascade of events leading
to thrombosis.
NO overproduction may combine with superoxide
anion (O��2 ) to produce peroxynitrite (ONOO�),
which is involved in cellular dysfunction.
Peroxynitrite is formed when the two free radicals
react in a near diffusion-limited reaction [32]. N-Tyr
is widely used as a marker of oxidative stress induced
by peroxynitrite [33]. In fact, N-Tyr is not formed by
the action of hydrogen peroxide, superoxide or
hydroxyl radical [34]; therefore, its appearance is
presumably indicative of peroxynitrite formation and
activity in the vascular endothelial cells of the
placenta [35].
Moreover, there is considerable evidence that
peroxynitrite and oxidative stress state are important
mediators to vascular endothelial cell dysfunction in
PE [36]. Despite a large amount of information on
PE, there are no relevant studies exploring the
pathogenesis of GH. The mechanisms should be
similar, but there has been no explanation as to why a
hypertension developing during pregnancy, as a
result of the feto-placental unit, does or does not
progress to renal dysfunction and proteinuria. There
are not many studies designed trying to differentiate,
from the pathogenic point of view, the differences
between GH and PE [37–39]. Investigation of NO
activity, including expression of correlated enzymes,
concentration and effects of some NO products on
platelets, was undertaken in the present study aimed
to clarify NO pathways role in women suffering from
GH and PE.
Patients and methods
The study was performed on 30 women with
Preeclampsia, 30 women with GH and 30 healthy
pregnant controls matched for age, parity and ges-
tational age at recruitment and at blood sampling,
consecutively admitted at the Department of
Obstetrics and Gynaecology of Marche Polytechnic
University (Ancona, Italy) between May 2005 and
June 2007 (Table I). Subjects from the control group
were asked to participate during their first appoint-
ment in the foetal-maternal unit. A written informed
consent was subscribed by all women enrolled in the
study. The study was performed in accordance with
the principles contained in the Declaration of
Helsinki as revised in 2001 and it was approved by
the Bioethical Committee of Marche Polythecnic
University.
According to ACOG criteria [1], GH was defined
as a systolic blood pressure level of 140 mm Hg or
higher or a diastolic blood pressure level of 90 mm
Hg or higher, for at least two consecutive readings 6
or more h apart, found after 20 weeks of gestation in
women with previously normal blood pressure, and
in the absence of proteinuria.
Nitric oxide platelet levels in Preeclampsia 27
PE was defined as elevated blood pressure
(4140/90 mm Hg) for at least two consecutive read-
ings 6 or more h apart, in association with significant
proteinuria (4300 mg/24 h), at a gestational age of
over twenty weeks. Voided urine specimens were
collected for measurement of protein by dipstick.
Dipsticks that indicated proteinuria of at least 1þ
(300 mg/L) were confirmed in clean-catch, mid-
stream urine samples. A dipstick of zero or trace in
the confirmatory sample was considered negative.
Furthermore, a data confirmation of proteinuria in
24 h urine collection was obtained.
According to our departmental protocol, gesta-
tional age was determined by reference to the last
menstrual period and crown rump length measure-
ments between 8 and 12 weeks of gestation, and
confirmed by an early second trimester ultrasono-
graphic examination.
Specific exclusion criteria for the control group
included a history of smoking, a history of hyperten-
sion, renal disease, cardiac disease, diabetes mellitus,
collagen disease, anti-phospholipid syndrome, thy-
roid and immunologic diseases and congenital or
acquired thrombophilic disorders and the presence
of chromosomal and other foetal anomalies.
Moreover, no patient had HELLP syndrome.
Furthermore, none of the women were taking any
regular medication, which included antihypertensive
drugs or MgSO4 before and during the experiment,
and none of the control subjects had received oral
contraception.
All recruited women had a singleton pregnancy
and were of Caucasian race. From each entire groups
a maternal venous blood sample was drawn at 33
weeks gestation, at the Department of Obstetrics and
Gynaecology, after the diagnosis of PE and GH and
prior to any surgical intervention, after overnight
fasting in a resting state, between 08:00 and 09:00,
and after at least 24 h of nitrite-free diet to eliminate
any impact on nitric oxide content. Samples were
immediately processed: first, after samples
centrifugation, plasma was isolated and subsequently
platelets were extracted. Platelet samples were stored
at �80�C until use. Platelets from each samples were
assessed to determine nitric oxide and peroxynitrite
levels. Moreover, iNOS, eNOS and nitrotyrosine
(N-Tyr) expressions in the same samples were also
measured.
The study was designed to concomitantly measure
platelet count to address the possible impact of
thrombocytopenia or thrombocytosis on platelet
levels of nitric oxide metabolites; at the same way
platelets aggregation and platelet mean volume
analysis were performed.
Platelet isolation
Peripheral venous blood was drawn after overnight
fasting, and immediately mixed with Anticoagulant
Citrate Dextrose (ACD) (36 ml citric acid, 5 mM
KCl, 90 mM NaCl, 5 mM glucose, 10 mM EDTA,
pH 6.8). Platelets were isolated by differential
centrifugation in anti-aggregation buffer (Tris-HCl
10 mm; NaCl 150 mm; EDTA 1 mm; glucose 5 mm;
pH 7.4) according to Vignini et al. [40]. The method
involved a preliminary centrifugation step (200 x g
for 10 min) to obtain platelet-rich plasma (PRP). The
platelets were then washed three times in anti-
aggregation buffer and centrifuged as above in
order to remove any residual erythrocytes. A final
centrifugation at 2000 x g for 20 min was performed
to isolate the platelets. The platelet pellet was washed
twice in phosphate buffered saline PBS (containing
NaCl 135 mM, KCl 5 mM, EDTA 10 mM, Na2PO4
8 mM, NaH2PO4 H2O 2 mM, pH 7.2) and imme-
diately used for the experiments or stored at –80�C.
Platelet count, platelet aggregation and platelet
mean volume
The platelet count and mean platelet volume in whole
blood were measured immediately using a Coulter
Counter (Sysmex Ltd, Buckinghamshire, UK).
Table I. Maternal and neonatal characteristics. Data are presented as Mean�SD.
Controls
(n¼ 30)
GH
(n¼ 30)
PE
(n¼ 30)
Maternal age (years) 31.3� 4.5 30.9� 3.8 32.7� 5.6
Nulliparous (n) 2 2 2
Gestational age at enrolment (weeks) 31.1� 3.7 30.6� 1.7 30.3� 2.7
Platelet count (PLT/mL� 1000) 213.11� 58.6 203.48� 35.2 196.51� 28.6
Mean platelet volume (fL) 9.4� 0.6 9.7� 0.8 9.8� 0.9
Platelet aggregation with collagen (%) 72� 7.3 75� 7.9 76� 8.1
Systolic blood pressure at enrolment (mmHg) 103.5� 4.2 152.7� 6.2** 168.2� 6.1** y
Diastolic blood pressure at enrolment (mmHg) 64.8� 6.1 97.2� 4.4** 108.1� 5.9** y
Gestational age at delivery (weeks) 39.1� 0.8 38.9� 1.6 35.6� 3.9** y
Birthweight (g) 3326.7� 436.1 3042.2� 321.3* 2041.2� 447.1** y
Placental weight (g) 645.3� 182.6 523.6� 168.4* 394.2� 132.9** y
*p< 0.05, **p< 0.001 vs. Controls; yp< 0.05 vs. GH
28 L. Mazzanti et al.
In vitro platelet aggregation studies was evaluated in
PRP by optical densitometry.
The platelet concentration in PRP was adjusted
with platelet-poor plasma (PPP) to achieve a con-
stant count of 250� 109/L. Platelet-poor plasma was
obtained by centrifuging the leftover blood at 600 g
for 10 min. Aggregation was induced by increasing
concentration of collagen (0.5, 1, 2, 4 mg/mL) and
responses monitored for 6 min in a dual-channel
aggregometer (Chrono-Log, Havertown, PA, USA)
at 37�C with a continuous stirring speed of
900 r.p.m. Platelet aggregation was expressed as a
percentage of the light transmission at 6 min related
to the negative control (PPP).
NO production
Prior to performing the assay, platelets suspensions
were pre-incubated at 37�C for 30 min in the absence
or presence of L-arginine analogue, L-NG-
Monomethyl-Arginine (L-NMMA), at a concentra-
tion of 1 mmol/L.
NO platelet production was evaluated by Assay
DesignsTM Total nitric oxide Assay Kit (Catalog No.
917-020; 192 Determination Kit); this commercial
kit is a complete kit for the quantitative determina-
tion of total NO in biological fluids. The kit involves
the enzymatic conversion of nitrate to nitrite, by the
enzyme Nitrate Reductase, followed by the colori-
metric detection of nitrite as a colored azo dye
product by Griess reaction, adding sulfanilamide
(Griess reagent I) and N(1-naphthyl)ethylenedia-
mine (Griess Reagent II) [41, 42], that absorbs
visible light at 540 nm. The modified protocol which
employs deproteinization and reduction of nitrates to
nitrites in the presence of NADPH-sensitive reduc-
tase, prior to addition of the Griess reagent, allows
measurement of combined nitrite (NO�2 ), and nitrate
(NO�3 ) (recently named together NOx) and can be
successfully applied for measurement of NO levels in
human body fluids [43]. Since all products of NO
and its derived species ultimately give only NO�2 and
NO�3 , this modified Griess reaction allows a quan-
titative tally of all NO produced during a given
period [44, 45].
Blank (background) was determined in each
experiment utilizing medium incubated without the
sample. The amount of NOx in each sample was
determined using a standard curve generated with
known concentrations of NOx and expressed as nmol
NOx/mg protein. Protein concentration was deter-
mined by Bradford BioRad protein assay, using
serum albumin as a standard to normalize the NOx
concentration data [46].
This assay can be performed in a conventional
spectrophotometer using 96-well microtiter plates,
(provided with the kit) which require less sample
volume and offer increased speed, throughput, and
precision (due to simultaneous measurement of all
standards and unknowns).
Peroxynitrite production
Peroxynitrite production in platelets was evaluated
using the fluorescence probe 2,7-Dichlorofluorescein
diacetate (DCFDA) as previously described [47].
DCFDA free base was prepared daily, by mixing
0.05 ml of 10 mmol/L DCFDA with 2 ml of 0.01 N
NaOH, at room temperature for 30 min. The mix-
ture was neutralized with 18.0 ml of 25 mmol/L
phosphate-buffered saline (PBS) pH 7.4; this solu-
tion was maintained on ice in the dark until use [48].
Briefly, platelets were incubated for 15 min with
5mM DCFDA-free base at 37�C. Then the DCFDA
treated platelets were divided into two sets: one set
was incubated with the addition of a mixture of
L-arginine 100 mM and NG-monomethyl-L-arginine
(L-NMMA) 100 mM for 15 min at 37�C in the dark
while the other set was incubated the above men-
tioned mixture but only with PBS. After washing
platelets twice in PBS (25 mmol/L) pH 7.4, samples
were sonicated to brake platelet pellets. The mixture
was then centrifuged at 200 x g for 5 min and the
fluorescence was measured in the supernatant in a
Perkin-Elmer LS-50B spectrofluorometer, at an
excitation wavelength of 475 nm and emission wave-
length of 520 nm. Blank samples contained all
reagents except platelets. The fluorescence results
obtained after L-NMMA incubation were subtracted
from those without L-NMMA. ONOO� production
was corrected by protein concentration and it was
expressed in arbitrary fluorescence numbers/mg prt.
Western blotting
Washed platelets were lysed in RIPA lysis buffer
containing 1�PBS, 1% Igepal CA-630, 0.5%
sodium deoxycholate, 0.1% SDS, 10 mg/ml PMSF,
aprotinin, 100 mM sodium orthovanadate and 4%
protease inhibitor cocktails by microcentrifugation at
10 000� g for 10 min at 4�C. The supernatants were
collected and treated with an equal volume of sample
application buffer (125 mmol/L Tris–HCl, pH 6.8,
2% SDS, 5% glycerol, 0.003% bromophenol blue,
1% �-mercaptoethanol). The mixture was boiled for
5 min; 15 mL (50mg protein/lane) of each sample was
applied to each well of an 8% SDS polyacrylamide
gel and electrophoresed for 1 h at 130 V along with a
set of molecular weight markers (Broad Range,
Sigma Chemical Co., St. Louis, MO). The resolved
protein bands were then transferred onto PVDF
membranes at 100 V for 60 min using a transfer
buffer of 25 mmol/L Tris base, 192 mmol/L glycine,
and 20% methanol. The blots were blocked over-
night at 4�C with blocking buffer (5% non-fat milk in
Nitric oxide platelet levels in Preeclampsia 29
10 mmol/L Tris pH 7.5, 100 mmol/L NaCl, 0.1%
Tween 20). The blocking buffer was decanted and
blots were incubated for 1 h at room temperature
with primary antibody: rabbit anti-endothelial nitric
oxide synthase (eNOS, 1:1000 dilution, Chemicon,
CA, USA), rabbit anti-inducible NOS (iNOS,
1:1000 dilution, Chemicon, CA, USA) and rabbit
anti-itrotyrosine (N-Tyr, 1:2000 dilution,
Chemicon, CA, USA) diluted in blocking buffer.
Positive controls (eNOS: HuVEC lysate 50mg/lane,
iNOS: rat liver 50mg/lane) were included in all
experiments, as provided by the manufacturer, to
confirm antibody specificity. As an internal control,
to control and correct for loading errors, blots were
reprobed with an anti-�-actin antibody (1:5000;
Sigma Chemical Co., St. Louis, Mo). Blots were
then washed using TTBS washing buffer (10 mmol/L
Tris pH 7.5, 100 mmol/L NaCl, 0.1% Tween 20)
and incubated with horseradish peroxidase-conju-
gated anti-rabbit immunoglobulin G (IgG) (1:5000;
Sigma Chemical Co., St. Louis, Mo) for 1 h at room
temperature following washes in TTBS. Peroxidase
activity was revealed using 3,30-diaminobenzidine
(Sigma Chemical Co., St. Louis, Mo) as a substrate.
Densitometry was performed using software
AMERSHAM Image Master 1D. All densitometric
data are expressed as mean densities, defined as the
sum of the grey values of all pixels in a selection
divided by the number of pixels [48].
Statistical analysis
Statistical analysis was performed using the SAS
statistical package (Statistical Analysis System
Institute, Cary, NC). Results are expressed as
Means�SD. ANOVA was used to analyse the
difference of results obtained in different experimen-
tal conditions followed by Bonferroni t multiple
comparisons test to reduce the probability of signif-
icant differences arising by chance. Differences were
considered significant with p< 0.05.
Results
Patients and controls characteristics are shown in
Table I. Platelet count, platelet mean volume and
platelet aggregation did not show any significant
differences among GH, PE and control groups,
although GH and PE showed higher but not signif-
icant levels in respect to controls.
Platelet NOx production, measured as nitrite-
nitrate release into the medium, was significantly
higher in GH and PE groups than in Controls
(42.71� 4.09 nmol NOx/mg prot in GH, 35.55�
3.15 nmol NOx/mg prot in PE, 20.12� 2.35 nmol
NOx/mg prot in Controls, p< 0.05); among patho-
logic pregnancies, platelet NOx was significantly
higher in GH group versus PE (Figure 1). In platelets
incubated with L-NMMA, NOx levels were signifi-
cantly reduced in GH, PE and Controls showing,
however, the same trend than in non-inhibited
platelets (7.88� 0.91 nmol NOx/mg prot in GH,
6.28� 0.72 nmol NOx/mg prot in PE, 3.72�
0.51 nmol NOx/mg prot in Controls, p < 0.05).
Thus, L-NMMA was effective inhibitor of NO
synthesis and the increased NOx levels observed in
PE and GH were NOS derived.
ONOO� levels were significantly higher in GH and
PE groups compared to Controls (4.26� 0.52 arbi-
trary fluorescence numbers in GH, 3.82� 0.08 arbi-
trary fluorescence numbers in PE, 2.68� 0.31
arbitrary fluorescence numbers in Controls,
p< 0.05), and ONOO� levels in GH group were
significantly higher than in PE group (Figure 2).
In platelets incubated with L-NMMA, ONOO�
levels were significantly reduced in GH, PE and
Controls showing, however, the same trend than in
non inhibited platelets (0.94� 0.08 arbitrary fluores-
cence numbers in GH, 0.83� 0.06 arbitrary
fluorescence numbers in PE, 0.62� 0.05 arbitrary
fluorescence numbers in Controls, p< 0.05).
Arb
itrar
y fl
uore
scen
ce n
umbe
rs/m
g pr
ot
p < 0.05
L-NMMAL-NMMA L-NMMA
0
1
2
3
4
5
6
Controls GH PE
PEROXYNITRITE
Figure 2. Peroxynitrite production (Fluorescence arbitrary num-
bers/mg protein) in platelets obtained from Controls (C), GH and
PE subjects. Means�Standard Deviations are shown (p< 0.05).
nmol
NO
x/m
g pr
ot
p < 0.05
L-NMMAL-NMMA L-NMMA
0
5
10
15
20
25
30
35
40
45
50
Controls GH PE
NITRIC OXIDE
Figure 1. NOx production (nmol NOx/mg prot) in platelets
obtained from Controls (C), GH and PE subjects.
Means�Standard Deviations are shown (p< 0.05).
30 L. Mazzanti et al.
Thus, L-NMMA was an effective inhibitor of NO
synthesis and also the increased ONOO� levels
observed in PE and GH were related to the higher
NOx levels-NOS derived.
Western blot analysis using anti-iNOS and eNOS
monoclonal antibodies demonstrated that both iso-
forms were detectable in platelet lysates. Band
densitometric analysis showed significant higher
eNOS protein levels in GH versus PE, and no
differences vs. controls (Figure 3(a)). Densitometric
analysis of bands indicated that iNOS protein levels
were significantly higher in the GH and PE samples
compared to controls and they were significantly
higher in GH vs. PE (Figure 3(b)). The same
technique revealed the presence of nitrotyrosine
which was more pronounced in the cell lysates
from GH and PE women than in Controls with
significant higher levels in GH vs. PE (Figure 4).
Discussion
PE is a pregnancy-associated disease with maternal
symptoms, but of placental origin. Although clinical
symptoms are late, systemic and maternal, the origin
of PE is early, local and placental.
Information about NO synthesis and its action in
the feto-placental vasculature in PE pregnancies is
controversial mainly due to the use of different
methodological approaches, severity of the disease
and cell type that had been analysed [6]. Several
studies have tried to determine NO production by
assessing the levels of the major metabolites of NO,
nitrate and nitrite, in the serum or plasma of patients
with PE compared with normotensive pregnant
patients. PE patients were shown to have decreased
levels of nitrates [49, 50], similar levels of nitrates
[51, 52] and increased levels of nitrates [53–58]
compared to pregnant controls. Studying serum
nitrate levels to evaluate NO production can be
misleading for several reasons. Diet, some medica-
tions and urinary excretion can affect serum nitrate
concentrations. Investigations that have taken these
sources of error into consideration reported a
decrease in NO production [59], no change in NO
production [60], and an increase in NO production
[61], adding to the continuing controversy.
0
0,02
0,04
0,06
0,08
0,1
0,12
+ C GH PE
eNOS (130kD)
β-actin (42 kD)
0
0,05
0,1
0,15
0,2
0,25
+ C GH PE
iNOS (135 kD)
β-actin (42 kD)
(a)
(b)
p < 0.05 PE and GH vs C
p < 0.05 PE vs GH
p < 0.05 PE vs GH*
*
*
*
°
° °
+ C GH PE
eNOS 0.081 ± 0.004 0.090 ± 0.007 0.093 ± 0.005 0.089 ± 0.004
β-actin 0.074 ± 0.005 0.075 ± 0.005 0.075 ± 0.005 0.071 ± 0.005
+ C GH PE
iNOS 0.064 ± 0.003 0.082 ± 0.016 0.203 ± 0.016 0.163 ± 0.005
β-actin 0.079 ± 0.008 0.080 ± 0.009 0.083 ± 0.005 0.081 ± 0.007
Figure 3. Western Blot analysis of eNOS and iNOS protein expression, along with the internal control �-actin, in Controls (C), GH and PE.
þ Indicates positive control.
Nitric oxide platelet levels in Preeclampsia 31
Moreover, in established PE, production of NO is
higher in the uteroplacental, fetoplacental and
peripheral circulation than in normotensive pregnan-
cies [62]. Also, plasma from women with PE
stimulate endothelial cell synthesis of NO and greater
NO synthase expression than control plasma [63].
Although all the questions about the pathophys-
iology of PE have not been answered, and there is a
wide range of studies yet on this purpose, the novelty
of our research is about considering platelets as an
attractive model for the study of the pathophysiology
of preeclampsia, in order to find predictive markers
for PE. In fact, PE is associated with increased
activation of platelets, circulating cells primary
involved in NO pathway [64].
Endothelial dysfunction and oxidative stress are
observed in PE [65]; in addition, because in PE there
is an expressive superoxide generation, resulting
from inflammatory response associated to this dis-
ease, thus, O�2 reacts promptly with NO, producing
peroxynitrite [65]. ONOO� may be considered as a
marker of oxidative and nitrative stress and may
damage cells in various ways, including nitration of
protein tyrosine residues [66]. Previous studies
demonstrated that the intensity of nitrotyrosine
immunostaining in placental villous tissue of hyper-
tensive pregnancies, was significantly higher than in
normal pregnancies [23]. The presence of N-Tyr
residues, particularly in the endothelium, may
indicate ONOO� formation and action, resulting in
vascular damage that contributes to increased pla-
cental vascular resistance.
During hypertension in pregnancy, peroxynitrite
may also play an important role because NO
normally inhibits platelet activation but, at higher
concentrations, ONOO� increases the platelet acti-
vation as previously reported [67]. Subsequently, an
alteration of the equilibrium among oxidant and
antioxidant agents occurs, which leads to lipoperox-
idation, thus damaging the endothelial cell mem-
brane [68]. Oxidative stress probably determines NO
production in the attempt to compensate the vaso-
constriction. Oxidative stress represents one of the
most important factors that induce iNOS expression
and the consequent increase of NO levels which
augment oxidative damage. On the other hand, NO
synthesized from eNOS, as a compensatory mecha-
nism, might lead to an improvement of foetal
conditions, balancing the vasoconstrictive effects. If
NO production continues without interference, this
metabolic pathway could lead to an improvement;
however, if it does not occur, then the massive NO
production by iNOS activation determines the pro-
ductive cascade of oxidative compounds, such as
ONOO�, contributing to the physiopathological
damage.
Clinical studies suggest that alterations in mater-
nal and fetal vascular response are useful for
0
0,05
0,1
0,15
0,2
0,25
0,3
+ C GH PE
N-Tyr (130 kD)
β-actin (42 kD)
p < 0.05 PE and GH vs C
p < 0.05 PE vs GH*
°
*
°*
+ C GH PE
N-tyr 0.064 ± 0.014 0.093 ± 0.013 0.243 ± 0.026 0.176 ± 0.028
β-actin 0.071 ± 0.007 0.072 ± 0.008 0.076 ± 0.006 0.070 ± 0.007
Figure 4. Western Blot analysis of Nitrotyrosine protein (N-Tyr) expression, along with the internal control �-actin, in Controls (C), GH
and PE.
þ Indicates positive control.
32 L. Mazzanti et al.
diagnosis and become predictive markers for PE
[69]. Several mechanisms have been proposed to be
involved in the maternal vascular alterations, includ-
ing reduced synthesis [70], altered biological actions
or reduced bioavailability of NO [71, 72]. Some
studies have proposed the possibility of an exagger-
ated adaptation in PE compared to the expected
normal metabolic adaptations to pregnancy [73, 74].
These observations indicate the potential existence
and functionality of compensatory mechanisms that
could ameliorate the increased feto-placental vascu-
lar resistance characteristic of PE.
Our investigation highlighted an increase of plate-
let NO and ONOO� levels in GH and PE pregnant
women compared to controls. Platelets from hyper-
tensive GH and PE women expressed higher iNOS
than controls, while eNOS expression was not
significantly modified in both patients and controls.
In our study, no compensatory mechanism of NO
synthesized from eNOS was observed and NO,
produced by iNOS, could probably lead to an
increase of peroxynitrite production.
Moreover, first by our results, platelets from GH
women expressed higher levels of NO, peroxynitrite,
iNOS, eNOS and nitrotyrosine content, compared to
PE women. Our data are very interesting because a
low grade of maternal pathology is related to higher
levels of oxidative and nitrative stress markers, such
as peroxynitrite and nitrotyrosine. The amount of
reactive oxygen species, such as peroxynitrite, may
result by antioxidant depletion, (e.g. the oxygen
radical absorbance capacity and the ferric reducing
ability of plasma), individual antioxidant levels or the
enzyme activity of antioxidant enzymes. In this point
of view, since gestational hypertension expressed
higher amount of nitric oxide metabolites and higher
activation of iNOS compared to PE, we can hypoth-
esize that the severity of hypertensive pathology is
almost not only related to nitric oxide metabolism. It
could be related also to the antioxidant status during
or antecedent pregnancy. In fact, is still controversial
whether antioxidants can prevent PE and is not also
commonly accepted if GH is a lower grade of PE.
Probably there is an early programming of pregnancy
outcomes, when the micro pathway of hypoxia-
reperfusion may conduct to a partial failure of
spiral artery remodelling in the placental bed of PE
women. During early pregnancy, in fact, placentation
occurs in a relatively hypoxic environment which is
essential for appropriate trophoblast invasion and
adequate embryonic development [75].
Our data are likely to be a useful future resource in
the elucidation of the disease-process and in the
identification of novel markers for PE.
Understanding the proposed hypothesis requires
further studies to obtain whole and more detailed
information regarding the complicated
pathophysiology of GH and PE and related hyper-
tensive syndrome together with the complicated
physiology involving NO.
Acknowledgements
Laura Mazzanti and Francesca Raffaelli equally
contributed to this research. This work was sup-
ported by a COFIN (MIUR to L.M).
References
1. ACOG practice bulletin: Diagnosis and management of
preeclampsia and eclampsia. Number 33, January 2002.
American College of Obstetricians and Gynecologists.
International Journal of Gynecology and Obstetrics
2002;77:67–75.
2. Llurba E, Gratacos E, Martın-Gallan P, Cabero L,
Dominguez C. A comprehensive study of oxidative stress
and antioxidant status in preeclampsia and normal pregnancy.
Free Radical Biology and Medicine 2004;37:557–570.
3. Villar J, Carroli G, Wojdyla D, Abalos E, Giordano D,
Ba’aqeel H, Farnot U, Bergsjø P, Bakketeig L, Lumbiganon P,
et al. World Health Organization Antenatal Care Trial
Research Group. Preeclampsia, gestational hypertension and
intrauterine growth restriction, related or independent condi-
tions? American Journal of Obstetrics and Gynecology
2006;194(4):921–31.
4. Roberts JM, Redman CWG. Pre-eclampsia: More than
pregnancy-induced hypertension. Lancet 1993;341:
1446–1450.
5. Lopez-Jaramillo P, Arenas WD, Garcıa RG, Rincon MY,
Lopez M. The role of the L-arginine-nitric oxide pathway in
preeclampsia. Therapeutic Advances in Cardiovascular
Diseases 2008;2(4):261–75.
6. Escudero C, Sobrevia L. A hypothesis for preeclampsia:
Adenosine and inducible nitric oxide synthase in human
placental microvascular endothelium. Placenta 2008;29(6):
469–83.
7. Valera MC, Parant O, Vayssiere C, Arnal JF, Payrastre B.
Physiologic and pathologic changes of platelets in pregnancy.
Platelets 2010;21(8):587–95.
8. Ceyhan T, Beyan C, Bas er_I, Kaptan K, Gungor S, Ifran A.
The effect of preeclampsia on complete blood count, platelet
count and mean platelet volume. Annals of Hematology
2006;85:320–322.
9. Redman CW. Platelets and the beginnings of preeclampsia.
New England Journal of Medicine 1990;323:478–480.
10. Nisell H, Grunewald C, Berglud M, Karlberg KE, Lunell NO,
Sylven C. Platelet aggregation in vitro and ex vivo in normal
pregnancy, pregnancy-induced hypertension, and preeclamp-
sia. Hypertension and Pregnancy 1998;17:147–155.
11. Konijnenberg A, Stokkers EW, Van der Post JA, Schaap MC,
Boer K, Bleker OP, Sturk A. Extensive platelet activation in
preeclampsia compared with normal pregnancy: Enhanced
expression of cell adhesion molecules. American Journal of
Obstetrics and Gynecology 1997;176(2):461–469.
12. McCarthy AL, Woolfson RG, Raju SK, Poston L. Abnormal
endothelial cell function of resistance arteries from women
with preeclampsia. American Journal of Obstetrics and
Gynecology 1993;168:1323–1330.
13. Whigham KAE. Abnormal platelet function in preeclampsia.
British Journal of Obstetrics and Gynecology 1978;85:28–32.
Nitric oxide platelet levels in Preeclampsia 33
14. Hull AD, white CR, Pearce WJ. Endothelium-derived relax-
ing factor and cyclic GMP-dependent vasorelaxation in
human chorionic plate arteries. Placenta 1994;15:363–75.
15. Gude NM. Endothelium-derived relaxing factor [NO] and the
placenta. In: Rice GE and Breenecke SP, editors. Molecular
aspect of placenta and fetal membranes autacoids. Boca,
Raton, FL: CRC; 1993. pp 3–276.
16. Buttery LD, McCarthy A, Springall DR, Sullivan MH,
Elder MG, Michel T, Polak JM. Endothelial nitric oxide
synthase in the human placenta: Regional distribution and
proposed regulatory role at the feto-maternal interface.
Placenta 1994;15:257–265.
17. Gude NM. Endothelium-derived relaxing factor (nitric oxide)
and the placenta. In: Rice GE and Breenecke SP, editors.
Molecular aspects of placenta and fetal membranes autacoids.
Boca Raton, FL: CRC; 1993. pp 263–276.
18. Buttery LD, McCarthy A, Springall DR, Sullivan MH,
Elder MG, Michel T, Polak JM. Endothelial nitric oxide
synthase in the human placenta: Regional distribution and
proposed regulatory role at the feto–maternal interface.
Placenta 1994;15:257–265.
19. Cameron IT, Van Papendorp CL, Palmer RMJ, Smith SK,
Moncada S. Relation between nitric oxide synthesis and
increase in systolic blood pressure in women with hyperten-
sion in pregnancy. Hypertension and Pregnancy 1993;
12:85–92.
20. Yallampalli C, Garfield RE. Inhibition of nitric oxide synthesis
in rats during pregnancy produces signs similar to those of
preeclampsia. American Journal of Obstetrics and Gynecology
1993;169:1316–20.
21. Cameron IT, Van Papendorp CL, Palmer RMJ, Smith SK,
Moncada S. Relation between nitric oxide synthesis and
increase in systolic blood pressure in women with hyperten-
sion in pregnancy. Hypertension and Pregnancy 1993;
12:85–92.
22. Moncada S, Martin JF, Higgs A. Symposium on regression of
atherosclerosis. European Journal of Clinical Investigation
1993;23:385–398.
23. Moncada S, Higgs A. The L-arginine-nitric oxide pathway.
New England Journal of Medicine 1993;30:2002–2010.
24. Naseem KM, Riba R. Unresolved roles of platelet nitric oxide
synthase. Journal of Thrombosis and Haemostasis 2008;
6:10–19.
25. Radomski MW, Palmer RJ, Moncada S. An L-arginine to
nitric oxide pathway in human platelets regulates aggregation.
Proceedings of the National Academy of Sciences 1990;
87:5193–5197.
26. Mehta JL, Chen LY, Kone BC, Mehta P, Turner P.
Identification of constitutive and inducible forms of nitric
oxide synthase in human platelets. Journal of Laboratory and
Clinical Medicine 1995;125(3):370–377.
27. Fleming I, Schulz C, Fichtlscherer B, Kemp BE, Fisslthaler B,
Busse R. AMP-activated protein kinase (AMPK) regulates the
insulin-induced activation of the nitric oxide synthase in
human platelets. Thrombosis and Haemostasis 2003;90:
863–871.
28. Marjanovic JA, Stojanovic A, Brovkovych VM, Skidgel RA,
Du X. Signaling-mediated functional activation of inducible
nitric-oxide synthase and its role in stimulating platelet
activation. J Biol Chem. 2008 Oct 24;283(43):28827-34.
29. Chen LY, Mehta JL. Further evidence of the presence of
constitutive and inducible nitric oxide synthase isoforms in
human platelets. Journal of Cardiovascular Pharmocology
1996;27(1):154–158.
30. Vaddi K, Nicolini FA, Mehta P, Mehta JL. Increased
secretion of tumor necrosis factor-a and interferon-g by
mononuclear leukocytes in patients with ischemic heart
disease relevance in superoxide anion generation.
Circulation 1994;90:694–649.
31. Barath P, Fishbein MC, Cao J, Berenson J, Helfant RH,
Forrester JS. Detection and localization of tumor necrosis
factor in human atheroma. American Journal of Cardiology
1990;65:297–302.
32. Szabo C. Multiple pathway of peroxynitrite cytotoxicity.
Toxicology Letters 2003;140-141:105–112.
33. Halliwell B. What nitrates tyrosine? Is nitrotyrosine specific as
biomarker of peroxynitrite formation in vivo? FEBS Letters
1997;411:157–160.
34. Schiessl B, Strasburger CJ, Bidlingmaier M, Spannagl M,
Ugele B, Kainer F. Decreasing resistance in the mater-
nal uterine and peripheral arterial system is apparently
unrelated to plasma and urinary levels of nitrite/nitrate
and cyclic-guanosinmonophosohate during the course of
normal pregnancies. Journal of Perinatal Medicine 2003;31:
281–286.
35. Myatt L, Rosenfield RB, Eis AL, Brockman DE, Greer I,
Lyall F. Nitrotyrosine residues in placenta. Evidence of
peroxynitrite formation and action. Hypertension 1996;
28:488–493.
36. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and
peroxynitrite: The good, the bad, and ugly. American Journal
of Physiology 1996;271:1424–1437.
37. Kaaja RJ, Moore MP, Yandle TG, Ylikorkala O,
Frampton CM, Nicholls MG. Blood pressure and vasoactive
hormones in mild preeclampsia and normal pregnancy.
Hypertension and Pregnancy 1999;18:173–187.
38. Slowinski T, Neumayer HH, Stolze T, Gossing G, Halle H,
Hocher B. Endothelin system in normal and hypertensive
pregnancy. Clinical Science 2002;103:446S–449S.
39. Teran E, Escudero C, Vivero S, Enrıquez A, Calle A.
Intraplatelet cyclic guanosine-30,50-monophosphate levels
during pregnancy and preeclampsia. Hypertension and
Pregnancy 2004;23:303–308.
40. Vignini A, Nanetti L, Moroni C, Testa R, Sirolla C, Marra M,
Cenerelli S, Gregori A, Fumelli D, Olivieri F, et al. Platelet
nitric oxide production and IR: Relation with obesity and
hypertriglyceridemia. Nutrition, Metabolism and
Cardiovascular Diseases 2008;18:553–558.
41. Griess P. Bemerkungen zu der Abhandlung der HH. Weselsky
und Benedikt Ueber einige Azoverbindungen. Berichte der
Deutschen chemischen Gesellschaft. 1879;12(1):426428.
42. Sun J, Zhang X, Broderick M, Harry Fein. Measurement of
nitric oxide production in biological systems by using Griess
reaction assay. Sensors 2003;3(8):276–284.
43. Guevara I, Iwanejko J, Dembinska-Kiec A, Pankiewicz J,
Wanat A, Anna P, Gol�abek I, Bartus S, Malczewska-
Malec M, Szczudlik A. Determination of nitrite/nitrate in
human biological material by the simple Griess reaction.
Clinica Chimica Acta 1998;274(2):177–88.
44. Weissman BA, Gross SS. Measurement of NO and NO
synthase. Current Protocols in Neuroscience. 2001 Chapter
7:Unit7.13.
45. Moshage H, Kok B, Huizenga JR, Jansen PLM. Nitrite and
nitrate determinations in plasma: A critical evaluation.
Clinical Chemistry 1995;41/6:892–6.
46. Bradford M. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of
proteine-dye binding. Analytical Biochemistry
1976;72:248–254.
47. Vignini A, Nanetti L, Moroni C, Tanase L, Bartolini M,
Luzzi S, Provinciali L, Mazzanti L. Modifications of platelet
from Alzheimer disease patients: A possible relation between
membrane properties and NO metabolites. Neurobiology of
Aging 2007;28:987–994.
34 L. Mazzanti et al.
48. Nanetti L, Giannubilo SR, Raffaelli F, Curzi CM, Vignini A,
Moroni C, Tanase L, Carboni E, Turi A, Mazzanti L, et al.
Nitric oxide and peroxynitrite platelet levels in women with
small-for-gestational-age fetuses. BJOG 2008;115:14–21.
49. Seligman SP, Buyon JP, Clancy RM, Young BK,
Abramson SB. The role of nitric oxide in the pathogenesis
of preeclampsia. American Journal of Obstetrics and
Gynecology 1994;171:944–948.
50. Mutlu-Turkoglu U, Aykac-Toker G, Ibrahimoglu L,
Ademoglu E, Uysal M. Plasma nitric oxide metabolites and
lipid peroxide levels in preeclamptic pregnant women before
and after delivery. Gynecologic and Obstetric Investigation
1999;48:247–250.
51. Silver RK, Kupferminc MJ, Russell TL, Adler L, Mullen TA,
Caplan MS. Evaluation of nitric oxide as a mediator of severe
preeclampsia. American Journal of Obstetrics and Gynecology
1996;175:1013–1017.
52. Egerman RS, Andersen RN, Manejwala FM, Sibai BM.
Neuropeptide Y and nitrite levels in preeclamptic and
normotensive gravid women. American Journal of Obstetrics
and Gynecology 1999;181:921–923.
53. Smarason AK, Allman KG, Young D, Redman CW. Elevated
levels of serum nitrate, a stable end product of nitric oxide, in
women with pre-eclampsia. British Journal of Obstetrics and
Gynecology 1997;104:538–543.
54. Bartha JL, Comino-Delgado R, Bedoya FJ, Barahona M,
Lubian D, Garcia-Benasach F. Maternal serum nitric oxide
levels associated with biochemical and clinical parameters
in hypertension in pregnancy. European Journal of
Obstetrics, Gynecology and Reproductive Biology
1999;82:201–207.
55. Pathak N, Sawhney H, Vasishta K, Majumdar S. Estimation
of oxidative products of nitric oxide (nitrates, nitrites) in
preeclampsia. The Australian and New Zealand Journal of
Obstetrics and Gynecology 1999;39:484–487.
56. Shaamash AH, Elsnosy ED, Makhlouf AM, Zakhari MM,
Ibrahim OA, EL-dien HM. Maternal and fetal serum nitric
oxide (NO) concentrations in normal pregnancy, pre-eclamp-
sia and eclampsia. International Journal of Gynaecology and
Obstetrics 2000;68:207–214.
57. Nobunaga T, Tokugawa Y, Hashimoto K, Kimura T,
Matsuzaki N, Nitta Y, Fujita T, Kidoguchi KI, Azuma C,
Saji F. Plasma nitric oxide levels in pregnant patients with
preeclampsia and essential hypertension. Gynecologic and
Obstetric Investigation 1996;41:189–193.
58. Suzuki Y, Sawa S, Miura R, Doi A, Otsubo D, Araki Y, T.
Plasma nitric oxide levels and the expression of P-selectin on
platelets in preeclampsia. American Journal of Obstetrics and
Gynecology 2002;187:676–680.
59. Davidge ST, Stranko CP, Roberts JM. Urine but not plasma
nitric oxide metabolites are decreased in women with
preeclampsia. American Journal of Obstetrics and
Gynecology 1996;174:1008–1013.
60. Conrad KP, Kerchner LJ, Mosher MD. Plasma and 24-h
NO(x) and cGMP during normal pregnancy and preeclampsia
in women on a reduced NO(x) diet. American Journal of
Physiology 1999;277:F48–F57.
61. Ranta V, Viinikka L, Halmesmaki E, Ylikorkala O. Nitric
oxide production with preeclampsia. Obstetrics and
Gynecology 1999;93:442–445.
62. Norris LA, Higgins JR, Darling MR, Walshe JJ, Bonnar J.
Nitric oxide in the uteroplacental, fetoplacental, and periph-
eral circulations in preeclampsia. Obstetrics and Gynecology
1999;93:958–963.
63. Baker PN, Davidge ST, Robets JM. Plasma from women with
pre-eclampsia increases cell nitric oxide production.
Hypertension 1995;26:244–248.
64. Konijnenberg A, Stokkers EW, Van der Post JA, Schaap MC,
Boer K, Bleker OP, Sturk A. Extensive platelet activation in
preeclampsia compared with normal pregnancy: Enhanced
expression of cell adhesion molecules. American Journal of
Obstetrics and Gynecology 1997;176:461–469.
65. Redmann CWG, Sargent IL. Pre-eclampsia, the placenta and
the maternal systemic inflammatory response-a review.
Placenta 2003;24:S21–S27.
66. Koo JR, Vaziri ND. Effects of diabetes, insulin and
antioxidants on NO synthase abundance and NO interaction
with reactive oxygen species. Kidney International
2003;63:195–201.
67. Holthe MR, Staff AC, Berge LN, Lyberg T. Different levels of
platelet activation in preeclamptic, normotensive pregnant,
and nonpregnant women. American Journal of Obstetrics and
Gynecology 2004;190:1128–1134.
68. Cester N, Staffolani R, Rabini RA, Magnanelli R, Salvolini E,
Galassi R, Mazzanti L, Romanini C. Pregnancy induced
hypertension: A role for peroxidation in microvillus plasma
membranes. Molecular and Cellular Biochemistry
1994;131:151–155.
69. Papageorghiou AT, Yu CK, Erasmus IE, Cuckle HS,
Nicolaides KH. Assessment of risk for the development of
pre-eclampsia by maternal characteristics and uterine artery
Doppler. BJOG 2005;112:703–9.
70. Teran E, Escudero C, Vivero S, Calle A, Molina G. NO in
early pregnancy and development of preeclampsia.
Hypertension 2006;47:17.
71. Teran E, Escudero C, Vivero S, Enriquez A, Calle A.
Intraplatelet cyclic guanosine-30,50-monophosphate levels
during pregnancy and preeclampsia. Hypertension and
Pregnancy 2004;23:303–8.
72. Khan F, Belch JJ, MacLeod M, Mires G. Changes in
endothelial function precede the clinical disease in women
in whom preeclampsia develops. Hypertension
2005;46:1123–8.
73. Myatt L. Placental adaptive responses and fetal programming.
Journal of Physiology 2006;572:25–30.
74. von Versen-Hoeynck FM, Powers RW. Maternal-fetal meta-
bolism in normal pregnancy and preeclampsia. Frontiers in
Bioscience 2007;12:2457–70.
75. Caniggia I, Winter J, Lye SJ, Post M. Oxygen and placental
development during the first trimester: Implications for the
pathophysiology of pre-eclampsia. Placenta 2000;21:S25–S30.
Nitric oxide platelet levels in Preeclampsia 35
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