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: raffi.3@virgilio.it

(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).

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