Enzymesthathydrolyzeadeninenucleotidesof patients
with hypercholesterolemiaandinflammatory processes
Marta Medeiros Frescura Duarte
1
,Va
ˆ
nia L. Loro
1
, Joa
˜
o B. T. Rocha
1
, Daniela B. R. Leal
2
,
Andreza F. de Bem
1
, Arace
´
li Dorneles
1
, Vera M. Morsch
1
and Maria R. C. Schetinger
1
1 Departamento de Quı
´
mica, Centro de Cie
ˆ
ncias Naturais e Exatas, Programa de Pos-Graduac¸a˜o em Bioquı
´
mica Toxicolo
´
gica, Universidade
Federal de Santa Maria, Brazil
2 Hospital Universita
´
rio de Santa Maria, Santa Maria, RS, Brazil
Hypercholesterolemia is widely accepted as one of the
major risk factors for the development of ischemic heart
disease, angina, and myocardial infarction. Although
the risks imposed by hypercholesterolemia seem to be
manifold, most attention has been devoted to its role in
atherosclerosis [1]. Atherosclerosis is an inflammatory
disease that is associated with endothelial cell activation,
oxidative stress, and the accumulation of leukocytes in
the walls of arteries [2]. The inflammatory process
induced by hypercholesterolemia is not limited to large
arteries. Endothelial cell adhesion molecule expression,
enhanced oxidant production and leukocyte–endothelial
cell adhesion have been demonstrated in postcapillary
venules of different tissues of hypercholesterolemic
animals [3–6].
Low-density lipoprotein is a major carrier of
cholesterol in the circulation, and can play an impor-
tant role in atherogenesis if it undergoes oxidative
Keywords
cholesterol; highly sensitive C-reactive
protein; NTPDase; oxidized low-density
lipoprotein; oxidized low-density lipoprotein
autoantibodies
Correspondence
M. R. C. Schetinger, Departamento de
Quı
´
mica, Centro de Cie
ˆ
ncias Naturais e
Exatas, Universidade Federal de Santa
Maria, Av. Roraima 1000, 97105-900, Santa
Maria, RS, Brazil
Fax: +55 5532 208978
Tel: +55 5532 208665
E-mail: mariaschetinger@gmail.com
(Received 18 August 2006, revised 29
January 2007, accepted 19 March 2007)
doi:10.1111/j.1742-4658.2007.05805.x
The activity of NTPDase (EC 3.6.1.5, apyrase, CD39) was verified in plate-
lets from patientswith increasing cholesterol levels. A possible association
between cholesterol levels andinflammatory markers, such as oxidized low-
density lipoprotein, highly sensitive C-reactive protein and oxidized
low-density lipoprotein autoantibodies, was also investigated. Lipid per-
oxidation was estimated by measurement of thiobarbituric acid reactive
substances in serum. The following groups were studied: group I,
< 150 mgÆdL
)1
cholesterol; group II, 151–200 mgÆdL
)1
cholesterol; group -
III, 201–250 mgÆdL
)1
cholesterol; and group IV, > 251 mgÆdL
)1
choles-
terol. The results demonstrated that both ATP hydrolysis and ADP
hydrolysis were enhanced as a function of cholesterol level. Low-density
lipoprotein levels increased concomitantly with total cholesterol levels. Tri-
glyceride levels were increased in the groups with total cholesterol above
251 mgÆdL
)1
. Oxidized low-density lipoprotein levels were elevated in
groups II, III, and IV. Highly sensitive C-reactive protein was elevated in
the group with cholesterol levels higher than 251 mgÆdL
)1
. Oxidized low-
density lipoprotein autoantibodies were elevated in groups III and IV.
Thiobarbituric acid reactive substance content was enhanced as a function
of cholesterol level. In summary, hypercholesterolemia is associated with
enhancement ofinflammatory response, oxidative stress, and ATP and
ADP hydrolysis. The increased ATP and ADP hydrolysis in group IV was
confirmed by an increase in CD39 expression on its surface. The increase
in CD39 activity is possibly related to a compensatory response to the
inflammatory and pro-oxidative state associated with hypercholesterolemia.
Abbreviations
hsCRP, highly sensitive C-reactive protein; OxLDL, oxidized low-density lipoprotein; PRP, platelet-rich plasma.
FEBS Journal 274 (2007) 2707–2714 ª 2007 The Authors Journal compilation ª 2007 FEBS 2707
modification by endothelial cells, vascular smooth
muscle, or macrophages within the arterial wall [7].
Oxidized low-density lipoprotein (OxLDL) as well as
OxLDL autoantibodies have been found in several
studies in atherosclerotic lesions [8]. The highly
sensitive C-reactive protein (hsCRP) enhances the
binding of OxLDL to monocytic ⁄ macrophage-like
cells through Fcc receptors [9].
Platelets also accumulate within atherosclerotic
lesions, and can recruit additional platelets to form a
thrombus, indicating that the arterial wall can assume
both an inflammatoryand prothrombogenic phenotype
when blood cholesterol levels are elevated [10,11].
Platelets are one of the most important blood compo-
nents that participate in and regulate thrombus forma-
tion by releasing active substances, such as ADP [12].
Micromolar concentrations of ADP are sufficient to
induce human platelet aggregation, and in the coagula-
tion cascade, AMP is hydrolyzed to adenosine, which
has an important function in the regulation of platelet
aggregation [13–15]. Furthermore, the roles of nuc-
leotides and nucleosides as extracellular signaling
molecules have been well established. Extracellular nu-
cleotides have become recognized for the significant
role that they play in modulating a variety of processes
related to vascular inflammation and thrombosis
[16–19]. In this vein, recent data from our laboratory
have indicated changes in nucleotide hydrolysis by
platelets from patients carrying diseases generally asso-
ciated with changes in coagulation ⁄ homeostasis
[13–15]. More recently, there has been growing interest
in the long-term effects of extracellular nucleotides and
nucleosides on cell growth, proliferation, and death
[16–19].
NTPDase (CD39, ecto-apyrase, ATP diphospho-
hydrolase) is a glycosylated, membrane-bound enzyme
that hydrolyzes ATP and ADP to AMP, which is sub-
sequently converted to adenosine by 5¢-nucleotidase
(EC 3.1.3.5, CD73). NTPDases are located in various
tissues, including the platelet membrane [20,21]. NTP-
Dase and CD73 play an important role in the regula-
tion of blood flow and thrombogenesis by regulating
ADP catabolism [22]. Another aspect that must be
emphasized here is the fact that NTPDase1 rapidly
metabolizes ADP released during platelet activation.
This event is very important, because ADP is the final
mediator of platelet recruitment and thrombus forma-
tion [23]. In fact, NTPDase1 has been recognized to
play an important role in thromboregulation by hydro-
lyzing and lowering extracellular ADP, which inhibits
platelet aggregation. In a recent study, Papanikolaou
et al. [24] have shown that depletion of membrane cho-
lesterol results in strong inhibition of NTPDase, and
that this is reversed by purified cholesterol.
Thus, one question that could be asked is whether
the different cholesterol levels observed in human
blood could affect NTPDase1 activity. This study was
performed in an attempt to answer this question. The
activity of NTPDase1 was measured on platelets of
human donors with cholesterol levels ranging from less
than 150 to more than 251 mgÆdL
)1
. Furthermore,
we studied whether cholesterol levels were associated
with inflammatory markers, e.g. hsCRP, andwith lipid
peroxidation.
Results
Patient characteristics are shown in Table 1. Glucose
levels were in the normal range in all the groups
studied, and ranged from 88 to 99 mgÆdL
)1
. No signi-
ficant differences were observed in the high-density
lipoprotein cholesterol levels among the groups.
Conversely, low-density lipoprotein levels increased
concomitantly with the increase in total cholesterol
levels, and were significantly higher in groups III and
IV. Triglyceride levels were increased in group IV (the
Table 1. Characteristics of the four groups: age (years), sex (male ⁄ female), smoking, hypolipemic medication, and anti-inflammatory treat-
ment. Age is represented by mean ± SE.
Patients
Cholesterol groups
I (< 150 mgÆdL
)1
) II (151–200 mgÆdL
)1
) III (201–250 mgÆdL
)1
) IV (> 251 mgÆdL
)1
)
Age 48 ± 5 55 ± 8 65 ± 5 56 ± 10
Sex
Male 20 20 20 20
Female 20 20 20 20
Smoking (yes or no) No No No No
Hypolipemic
medication
Not receiving Not receiving Not receiving Not receiving
Anti-inflammatory
treatment
Not receiving Not receiving Not receiving Not receiving
Cholesterol and ectonucleotidase activities M. Medeiros Frescura Duarte et al.
2708 FEBS Journal 274 (2007) 2707–2714 ª 2007 The Authors Journal compilation ª 2007 FEBS
group with total cholesterol above 251 mgÆdL
)1
;
Table 2).
OxLDL was elevated in groups II, III, and IV
(Table 3), whereas hsCRP was elevated only in
group IV (cholesterol higher than 251 mgÆdL
)1
). Ox-
LDL autoantibodies were elevated in groups III and
IV (Table 3).
A statistically significant positive correlation was
found between the cholesterol levels and thiobarbituric
acid reactive substance production (r ¼ 0.7438,
P < 0.01), which indicates an association between
plasma ⁄ serum ⁄ blood and oxidative stress (Fig. 1).
ATP hydrolysis was modified by cholesterol levels
above 151 mgÆL
)1
, and post hoc comparisons by
Duncan’s test revealed that it was significantly higher
in patients from groups II, III, and IV. Furthermore,
these groups were significantly different from each
other (Fig. 2A). Similar results were observed for
ADP hydrolysis. Post hoc comparisons by Duncan’s
multiple range test revealed thatpatients from
groups II, III and IV presented higher levels of ADP
hydrolysis andthat these groups were significantly
different from each other (Fig. 2B). Correlation ana-
lysis indicated a positive correlation between
increased cholesterol levels and platelet ATP and
ADP hydrolysis (Fig. 3A,3B).
Statistical analysis of the content of CD39-positive
cells by flow cytometry using labeled antibody against
NTPDase1 revealed that group IV (cholesterol higher
than 251 mgÆdL
)1
) had a significant increase in the
expression of NTPDase1, when compared to the other
groups (P<0.05, post hoc comparisons by Duncan
multiple test; Fig. 4).
There was a statistically significant correlation
between ATP and ADP hydrolysis with OxLDL
(r ¼ 0.82, P < 0.01; r ¼ 0.91, P < 0.001), hsCRP
(r ¼ 0.82, P < 0.01; r ¼ 0.91, P < 0.001), and
OxLDL autoantibodies (r ¼ 0.85, P < 0.01; r ¼ 0.96,
P < 0.001). ATP and ADP hydrolysis were also corre-
lated with tryglyceride levels (r ¼ 0.819, P < 0.001;
r ¼ 0.92, P < 0.001; Table 4).
Table 2. High-density lipoprotein (HDL), low-density lipoprotein (LDL), triglyceride and glucose (mg ⁄ dL) levels ofpatientswith different cho-
lesterol levels. Results are expressed as the mean ± SE (n ¼ 40 for each group).
Blood parameters
Cholesterol groups
I (< 150 mgÆdL
)1
) II (151–200 mgÆdL
)1
) III (201–250 mgÆdL
)1
) IV (> 251 mgÆdL
)1
)
LDL cholesterol 70.8 ± 13.2 105.5 ± 17.5 134.3 ± 48.3* 209 ± 46*
HDL cholesterol 44.8 ± 10.3 48.8 ± 10.4 57.3 ± 14.8 54 ± 14.8
Triglycerides 88.2 ± 25 121 ± 46 124 ± 54.5 286 ± 33.9*
Glucose 88 ± 15.5 98 ± 27.8 93 ± 27 98 ± 32
* Indicates significant difference at P<0.05 between groups.
Table 3. OxLDL (mg ⁄ dL), hsCRP (mg ⁄ L) and OxLDL autoantibodies (mg ⁄ L) in patientswith different cholesterol levels. Results are
expressed as the mean ± SE (n ¼ 40 for each group).
Parameters
Cholesterol levels
I (< 150 mgÆdL
)1
) II (151–200 mgÆdL
)1
) III (201–250 mgÆdL
)1
) IV (> 251 mgÆdL
)1
)
OxLDL 0.082 ± 0.21 0.42 ± 0.25* 0.48 ± 0.26* 0.78 ± 0.08*
hsCRP 0.42 ± 0.12 0.46 ± 0.06 0.68 ± 0.16 2.06 ± 0.08*
Anti-OxLDL 6.2 ± 1.5 7.26 ± 2.5 21.1 ± 3.5* 38.66 ± 8.5*
* Indicates significant difference at P<0.05 between groups.
35
30
25
20
15
10
nmol MDA/mg protein
Cholesterol (mg/dl)
5
0
0 50 100 150 200 250 300 350 400 450
Fig. 1. Correlation between cholesterol levels and thiobarbituric
acid reactive substances (n ¼ 40) (r ¼ 0.74, P < 0.01). y ¼
4.37 + 0.077x, where y ¼ malondialdehyde production (nmolÆmL
)1
)
and x ¼ cholesterol levels.
M. Medeiros Frescura Duarte et al. Cholesterol and ectonucleotidase activities
FEBS Journal 274 (2007) 2707–2714 ª 2007 The Authors Journal compilation ª 2007 FEBS 2709
Discussion
The present study clearly indicated that cholesterol lev-
els are associated with increased OxLDL, OxLDL
autoantibody formation, andinflammatory markers.
The results of this study reveal thatpatientswith high
cholesterol may have a predisposition to atheroma pla-
que development. The results of the present study also
show a positive correlation between cholesterol levels
and OxLDL and hsCRP production, which is in
accordance with the literature data [7]. Our study con-
firmed the hypothesis that increased levels of choles-
terol may be associated with hsCRP and OxLDL
autoantibodies, which are good indicators of increased
risk for atherosclerosis development.
The present results show for the first time that nucleo-
tide hydrolysis is enhanced in platelets of patients
with hypercholesterolemia. Probably, high cholesterol
levels induce an increase in platelet ATP and ADP
hydrolysis as a compensatory mechanism to inhibit
platelet aggregation and limit thrombus formation in
patients with a predisposition to atheroma development.
30
A
B
< 150
> 251
*
*
*
*
*
*
201-250
151-200
< 150
> 251
201-250
151-200
20
10
0
10.0
7.5
5.0
2.5
0.0
< 150
NTPDase Specific Activity
NTPDase Specific Activity
151-200
Cholesterol (mg/dl)
201-250
> 251
< 150
151-200
Cholesterol (m
g
/dl)
201-250
> 251
Fig. 2. ATP (A) and ADP (B) hydrolysis in platelets from patients
with hypercholesterolemia (n ¼ 40). Results are expressed as
nmol P
i
Æmin
)1
Æmg
)1
of protein. Different letters indicate a significant
difference at P<0.05 between groups.
A
40
30
20
10
0
0 50 100 150 200
Cholesterol (mg/dl)
ATP hydrolysis
ADP hydrolysis
250 300 350 400
80
10
8
6
4
2
0
120 160 200 240 280 320 360 400
450
B
Fig. 3. Correlation analysis between cholesterol levels and ATP (A)
and ADP (B) hydrolysis (n ¼ 40) (r ¼ 0.75, P < 0.01). y ¼
2.06 + 0.078x, where y ¼ ATP hydrolysis and x ¼ cholesterol levels.
< 150
> 251
*
201-250
151-200
0
5
10
15
20
25
30
35
< 150
% of CD39 positive cells
151-200
Cholesterol (mg/dl)
201-250 > 251
Fig. 4. CD39 expression on platelets. The analysis was done by
flow cytometry (see Experimental procedures). Data represent
the mean ± SE of 10 individuals. Data were analyzed statistically
by Duncan’s multiple range test. *Significantly different from the
others (P<0.05).
Cholesterol and ectonucleotidase activities M. Medeiros Frescura Duarte et al.
2710 FEBS Journal 274 (2007) 2707–2714 ª 2007 The Authors Journal compilation ª 2007 FEBS
Data from nucleotide hydrolysis and NTPDase1
(CD39) expression in platelet membranes indicated that
the mechanisms involved in ATP and ADP hydrolysis
vary with the cholesterol level. In fact, up to
250 mgÆdL
)1
, there was no increase in CD39 expression
in platelets, but patientswith cholesterol higher than
251 mgÆdL
)1
exhibited a significant increase in the
expression of this complement. This may indicate that
cholesterol up to 250 mgÆdL
)1
causes an increase in
ATP and ADP hydrolysis by changing the plasma mem-
brane properties of platelets.
In a previous study, our laboratory demonstrated
that diabetic, hypertensive and diabetic ⁄ hypertensive
patients presented elevated nucleotide hydrolysis by
platelets [13,15]. Taken together, we can suppose that
these associated pathologic conditions may change
the rate of platelet nucleotide hydrolysis. Platelets
comprise one of the components of the thrombus
microenvironment andof the process of thrombo-
regulation. It is known that the rate of platelet nuc-
leotide hydrolysis is lower in platelets than in
endothelium. However, considering the function and
the mobility of platelets, we can understand their
active role in this process and the importance of
such hydrolysis. In line with this, literature data indi-
cate thatadeninenucleotidesand adenosine are
important modulators of atherosclerosis [18], and
that upregulation of CD39 ⁄ NTPDase1 on platelets
has beneficial effects on endothelial cell activation;
this may be observed in vascular inflammation [19].
It is of importance that Papanikolaou et al. [24]
showed that the reduction of cholesterol levels by
drugs results in strong inhibition of the enzymatic
and antiplatelet activities of NTPDase. The results
presented here are in agreement with this, and we
have shown that circulating cholesterol positively
modulates platelet NTPDase1 activity. Papanikolaou
et al. [24] suggested that cholesterol may affect the
ability of this enzyme to undergo conformational
changes required for nucleotide hydrolysis. Probably,
it enhances the interaction between the enzyme and
its substrates.
In the literature, there are some studies showing that
the levels of cholesterol or its oxidation status can
affect ATPase activities [25,26]. However, a direct role
of plasma cholesterol levels on platelet ecto-CD39 has
never been established. Lijnen et al. [27] suggested that
cholesterol lowering in hypercholesterolemic patients
may result in a significant decrease in erythrocyte and
platelet membrane cholesterol content. Perhaps this
occurred in our study. When the cholesterol level is
increased, the platelet membrane cholesterol content is
enhanced, increasing the conformational stability of
the transmembrane NTPDase1 protein, promoting
activation.
Taking together the importance of the physiologic
(lipid bilayer component) or pathologic (factor related
to atherogenesis) functions of cholesterol, as well as
the importance of the platelets and NTPDase, we con-
cluded that cholesterol levels can modulate platelet
NTPDase activity in vivo.
In conclusion, our study demonstrated that the
hydrolysis ofadeninenucleotides is modified in plate-
lets from hypercholesterolemic patients, and we suggest
that this may play a beneficial role by preventing
thrombus formation. However, the modulation of nuc-
leotide hydrolysis is possibly not sufficient to inhibit
thrombus formation. Indeed, clinical evidence indicates
with clarity that the hypercholesterolemia is a risk fac-
tor for future fatal events (unstable angina and myo-
cardial infarction).
Experimental procedures
Chemicals
Nucleotides, sodium azide, Hepes and Trizma base were pur-
chased from Sigma (St Louis, MO, USA). Antibodies for
flow cytometry analysis [R-phycoerythrin-conjugated mouse
monoclonal antibody against human CD39, and fluorescein
isothiocyanate-conjugated mouse monoclonal antibody
against human CD61] were purchased from Serotec Ltd
(Kidlington, Oxford, UK) and BD PharMingen (San Jose,
CA, USA), respectively. All other reagents used in the experi-
ments were of analytical grade andof the highest purity.
Patients
The sample consisted ofpatientswith different cholesterol
levels and ages ranging from 40 to 70 years, from LABI-
MED (Santa Maria, RS, Brazil). We chose nonsmoking
patients not undergoing hypolipemic or anti-inflammatory
treatment, with glucose levels ranging from 70 to
95 mgÆdL
)1
. The sample was divided into four groups cons-
siting of 50% female and 50% male, as follows: group 1,
Table 4. Correlation between ATP and ADP hydrolysis and inflam-
matory markers (OxLDL, hsCRP and OxLDL autoantibodies, and
triglycerides.
OxLDL hsCRP
OxLDL
autoantibodies Triglycerides
ATP r ¼ 0.82 r ¼ 0.82 r ¼ 0.85 r ¼ 0.819
P<0.01 P<0.01 P<0.01 P<0.001
ADP r ¼ 0.91 r ¼ 0.92 r ¼ 0.96 r ¼ 0.92
P<0.001 P<0.001 P<0.001 P<0.001
M. Medeiros Frescura Duarte et al. Cholesterol and ectonucleotidase activities
FEBS Journal 274 (2007) 2707–2714 ª 2007 The Authors Journal compilation ª 2007 FEBS 2711
cholesterol levels < 150 mgÆdL
)1
(n ¼ 40); group 2, choles-
terol levels ranging from 151 to 200 mgÆdL
)1
(n ¼ 40);
group 3, cholesterol levels ranging from 201 to 250 mgÆdL
)1
(n ¼ 40); and group 4, cholesterol levels > 251 mgÆdL
)1
(n ¼ 40). These variations in the cholesterol values (50–
50 mg) were selected to correspond approximately to the
actual clinical interval criterion used to evaluate risk
factors. The separation of the subjects into the groups was
based on clinical considerations. We consider that a
cholesterol level below 150 mgÆdL
)1
is low, and a level
between 151 and 200 mgÆdL
)1
is clinically acceptable, but
close to the limit of 200 mgÆdL
)1
. A level between 201 and
250 mgÆdL
)1
is just above the limit, and a level higher than
251 mgÆdL
)1
is well above the safe limit for risk of cardio-
vascular disease. All subjects gave written informed consent
to participate in the study. The protocol was approved by
the Human Ethics Committee of the Health Science Center,
Federal University of Santa Maria (Protocol number:
015 ⁄ 2004). Eight milliliters of blood was obtained from
each participant and used for biochemical and hematologic
determinations and platelet-rich plasma (PRP) preparation.
Biochemical determinations
Serum total cholesterol and triglyceride concentrations were
measured using standard enzymatic methods with the use of
Ortho-Clinical Diagnostics reagents, and a fully automated
analyzer (Vitros 950, dry chemistry; Johnson & Johnson,
Rochester, NY, USA). High-density lipoprotein cholesterol
was measured in the supernatant plasma after precipitation
of apolipoprotein B-containing lipoproteins with dextran
sulfate and magnesium chloride according to Bachorik &
Albers [28]. Low-density lipoprotein cholesterol was estima-
ted with the Friedewald equation [29]. hsCRP was measured
by immunoluminometry (IMMULITE 2000; Diagnostic
Products Corporation, Los Angeles, CA, USA). OxLDL was
determined by a capture ELISA according to the manufac-
turer’s instructions (Mercodia AB, Uppsala, Sweden), as
described by Holvoet et al. [30]. OxLDL autoantibodies were
determined using ELISA as described by Wu & Lefvert [31].
PRP preparation
PRP was prepared from human donors by the methods of
Pilla et al. [21] and Lunkes et al. [13]. Briefly, blood was
collected into 0.129 m citrate and centrifuged at 160 g for
10 min. The PRP was centrifuged at 1400 g for 15 min
and washed twice with 3.5 mm Hepes isosmolar buffer
containing 142 mm NaCl, 2.5 mm KCl, and 5.5 mm glu-
cose. The washed platelets were resuspended in Hepes buf-
fer, and protein was adjusted to 0.3–0.5 mgÆmL
)1
, where
6–10 lg of protein was used per tube to ensure linearity
in the enzyme assay. NTPDase is an ectoenzyme, and
thus platelet viability and integrity were confirmed by the
measurement of lactate dehydrogenase activity using the
enzymatic Vitros 950 (Ortho-Clinical Diagnostics; Johnson
& Johnson).
NTPDase activity
Twenty microliters of the PRP preparation (10–15 l g
protein) was added to the reaction mixture of NTPDase
and preincubated for 10 min at 37 °C in a final volume
of 200 lL. NTPDase activity was determined by the
method of Pilla et al. [21], in a reaction medium contain-
ing 5.0 mm CaCl
2
, 100 mm NaCl, 4.0 mm KCl, 6 mm
glucose, and 50 mm Tris ⁄ HCl buffer (pH 7.4). The reac-
tion was started by the addition of ATP or ADP as sub-
strate at a final concentration of 1.0 mm. The reaction
was stopped by the addition of 200 lL of 10% trichloro-
acetic acid to provide a final concentration of 5%. The
inorganic phosphate (P
i
) released by ATP and ADP
hydrolysis was measured by the method of Chan et al.
[32], using KH
2
PO
4
as a standard. Controls were pre-
pared to correct for nonenzymatic hydrolysis by adding
PRP after trichloroacetic acid addition. All samples were
run in triplicate. Enzyme activities are reported as nmol
P
i
releasedÆmin
)1
Æmg
)1
protein.
Flow cytometry analysis
Peripheral blood cells were incubated with anti-CD39 and
anti-CD61 (20 lL per 10
6
cells) for 25 min, lysed with
fluorescence activated cell sorter (FACS) reagent, and incu-
bated again for 15 min in the dark. Cells were washed twice
in NaCl ⁄ P
i
buffer (pH 7.4) containing 0.02% (w ⁄ v) sodium
azide and 0.2% (w ⁄ v) BSA. The cells were then resuspend-
ed in NaCl ⁄ P
i
buffer (pH 7.4) and immediately analyzed
with a FACScalibur flow cytometer using cellquest
software (Becton Dickinson, San Jose, CA, USA), without
fixation.
Hematologic determinations
Quantitative determinations of platelets obtained by veni-
puncture were performed using a Pentra 120 analyzer
(ABX, Montpellier, France). Platelet aggregation was per-
formed by the technique of Biggs [33], consisting of the
in vitro macroscopic visualization of aggregates at intervals
of 15–50 s by the addition of ADP to PRP.
Determination of lipid peroxidation
Lipid peroxidation was estimated by the measurement of
thiobarbituric acid reactive substances in serum samples by
modifications of the method of Jentzsch et al. [34]. Briefly,
0.2 mL of serum was added to the reaction mixture con-
taining 1 mL of 1% orthophosphoric acid, and 0.25 mL of
an alkaline solution of thiobarbituric acid (final volume
Cholesterol and ectonucleotidase activities M. Medeiros Frescura Duarte et al.
2712 FEBS Journal 274 (2007) 2707–2714 ª 2007 The Authors Journal compilation ª 2007 FEBS
2.0 mL), and this was followed by 45 min of heating at
95 °C. After cooling, samples and standards of malondial-
dehyde were read at 532 nm against the blank of the stand-
ard curve. The results were expressed as nmol
malondialdehydeÆmL
)1
.
Protein determination
Protein was determined by the Coomassie blue method
using BSA as standard [35].
Statistical analysis
Data were analyzed statistically by one-way anova fol-
lowed by the Duncan test. Differences between groups were
considered to be significant when P < 0.05. Linear correla-
tion between variables was also carried out.
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2714 FEBS Journal 274 (2007) 2707–2714 ª 2007 The Authors Journal compilation ª 2007 FEBS
. Enzymes that hydrolyze adenine nucleotides of patients
with hypercholesterolemia and inflammatory processes
Marta Medeiros Frescura. understand their
active role in this process and the importance of
such hydrolysis. In line with this, literature data indi-
cate that adenine nucleotides and