Enzymes that hydrolyze adenine nucleotides of patients with hypercholesterolemia and inflammatory 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 patients with increasing cholesterol levels. A possible association between cholesterol levels and inflammatory 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 of inflammatory 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 inflammatory and 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, and with 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 that patients from groups II, III and IV presented higher levels of ADP hydrolysis and that 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 of patients with 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 patients with 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, and inflammatory markers. The results of this study reveal that patients with 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 patients with 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 and of 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 that adenine nucleotides and 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 of adenine nucleotides 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 and of the highest purity. Patients The sample consisted of patients with 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. References 1 Tailor A & Granger DN (2003) Hypercholesterolemia promotes P-selectin-dependent platelet–endothelial cell adhesion in postcapillary venules. Arterioscler Thromb Vasc Biol 23, 675–680. 2 Libby P (2000) Changing concepts of atherogenesis. J Int Med 247, 349–358. 3 Scalia R & Appel JZ 3rd & Lefer AM (1998) Leuko- cyte–endothelium interaction during the early stages of hypercholesterolemia in the rabbit: role of P-selectin, ICAM-1, and VCAM-1. Arterioscler Thromb Vasc Biol 18, 1093–1100. 4 Stokes KY, Clanton EC, Russell JM, Ross CR & Granger DN (2001) NAD(P)H oxidase-derived superoxide mediates hypercholesterolemia-induced leukocyte–endothelial cell adhesion. Circ Res 88 , 499–505. 5 Song L, Leung C & Schindler C (2001) Lymphocytes are important in early atherosclerosis. J Clin Invest 108, 251–259. 6 Zhao Z, Beer MC, Cai L, Asmis R, Beer FC, Villiers WJS & Westhuyzen DRV (2005) Low-density lipopro- tein from apolipoprotein E-deficient mice induces macrophage lipid accumulation in a CD36 and scaven- ger receptor class A-dependent manner. Arterioscler Thromb Vasc Biol 25, 168–173. 7 Hulthe J & Fagerberg B (2002) Circulating oxidized LDL is associated with subclinical atherosclerosis devel- opment and inflammatory cytokines (AIR Study). Arter- ioscler Thromb Vasc Biol 22, 62–167. 8 Yla-Herttuala S, Palinski W, Butler S, Picard S, Steinberg D & Witztum J (1994) Rabbit and human atherosclerotic lesions contain IgG that recognizes epitopes of oxidized LDL. Arterioscler Thromb 14, 32–40. 9 Ridker PM & Haughie P (1998) Prospective studies of C-reactive protein as a risk factor for cardiovascular disease. J Invest Med 46, 391–395. 10 Massberg S, Brand K, Gruner S, Page S, Muller E, Muller I, Bergmeier W, Richter T, Loren M, Konrad I et al. (2002) A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation. J Exp Med 196, 887–896. 11 Wagner DD & Burger PC (2003) Platelets in inflamma- tion and thrombosis. Arterioscler Thromb Vasc Biol 23, 2131–2137. 12 Marcus AJ, Broekman MJ, Drosopoulos JFH, Islam N, Pisnky DJ, Sesti C & Levi R (2003) Heterologous cell– cell interactions: thromboregulation, cerebroprotection and cardioprotection by CD39 (NTPDase-1). J Thromb Haemost 1, 2497–2509. 13 Lunkes GI, Lunkes D, Stefanello F, Morsch A, Morsch VM, Mazzantti CM & Schetinger MRC (2003) Enzymes that hydrolyze adenine nucleotides in diabetes and associated pathologies. Thromb Res 109, 189–194. 14 Arau´ jo MC, Rocha JBT, Morsch A, Zanin R, Bauchspiess R, Morsch VM & Schetinger MRC (2004) Enzymes that hydrolyze adenine nucleotides in platelets from breast cancer patients. Biochem Biophys Acta 1740, 421–426. 15 Lunkes GI, Lunkes D, Morsch VM, Mazzantti CM, Morsch A, Miron VR & Schetinger MRC (2004) NTPDase and 5¢-nucleotidase activities in rats with all- oxan-induced diabetes. Diabetes Res Clin Pract 65, 1–6. 16 Burnstock G (2002) Purinergic signaling and vascular cell proliferation and death. Arterioscler Thromb Vasc Biol 22, 364–373. 17 Atkinson B, Dwyer K, Enjyoji K & Robson SC (2006) Ecto-nucleotidases of the CD39 ⁄ NTPDase family modulate platelet activation and thrombus formation: potential as therapeutic targets. Blood Cells Mol Dis 36, 217–222. 18 Seye CI, Kong Q, Erb L, Garrad RC, Krugh B, Wang M, Turner JT, Sturek M, Gonza ´ lez FA & Weisman GA (2002) Functional P2y 2 nucleotide recep- tors mediate uridine 5¢-triphosphate-induced intimal hyperplasia in collared rabbit carotid arteries. Circula- tion 106, 2720–2726. 19 Seye CI, Yu N, Jain R, Kong Q, Minor T, Newton J, Erb L, Gonza ´ lez FA & Weisman GA (2003) The P2Y 2 nucleotide receptor mediates UTP-induced vascular cell adhesion molecule-1 expression in coronary artery endothelial cells. J Biol Chem 278(27), 24960–24965. 20 Zimmermann H (2001) Ectonucleotidases: some recent developments and note on nomenclature. Drug Dev Res 52, 44–56. 21 Pilla C, Emanuelli T, Frassetto SS, Battastini AMO, Dias RD & Sarkis JJF (1996) ATP diphosphohydrolase 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 2713 activity (Apyrase, EC 3.6.1.5.) in human blood platelets. Platelets 7, 225–230. 22 Kawashina Y, Nagasawa T & Ninomiya H (2000) Con- tribution of ecto-5¢ nucleotidase to the inhibition of platelet aggregation by human endothelial cells. Blood 96, 2157–2162. 23 Marcus AJ, Broekman MJ, Drosopoulos JHF, Pinsky DJ, Islam N, Gayle RB III & Maliszewski CR (2001) Thromboregulation by endothelial cells: significance for occlusive vascular diseases. Arterioscler Thromb Vasc Biol 21, 178–182. 24 Papanikolaou A, Papafotika A, Murphy C, Papamarc- aki T, Tsolas O, Drab M, Kurzchalia TV, Kasper M & Christoforidis S (2005) Cholesterol-dependent lipid assemblies regulate the activity of the ecto-nucleotidase CD39. J Biol Chem 280, 26404–26414. 25 Wood WG, Igbavboa U, Rao AM, Schroeder F & Avdulov NA (1995) Cholesterol oxidation reduces Ca 2+ +Mg 2+ -ATPase activity, interdigitation, and increase fluidity of brain synaptic plasma membranes. Brain Res 683, 36–42. 26 Ortega A, Santiago-Garcı ´ a J, Mas-Oliva J & Lepock JR (1996) Cholesterol increases the thermal stability of the Ca 2+ ⁄ Mg 2+ -ATPase of cardiac microsomes. Biochim Biophys Acta 1283, 45–50. 27 Lijnen P, Echevaria-Vazquez D & Petrov V (1996) Influence of cholesterol-lowering on plasma membrane lipids and function. Methods Find Exp Clin Pharmacol 18, 123–126. 28 Bachorik PS & Albers JJ (1986) Precipitation methods for quantification of lipoproteins. Methods Enzymol Pharmacogenetics Genomics 129, 78–100. 29 Friedewald WT & Levy RI, Fredrickson DS (1972) Esti- mation of the concentration of low-density lipoprotein cholesterol in plasma, without the use of preparative ultracentrifuge. Clin Chem 18, 499–502. 30 Holvoet P, Stassen JM, Van Cleemput J, Collen D & Vanhaecke J (1998) Oxidized low density lipoproteins in patients with transplant-associated coronary artery dis- ease. Arterioscler Thromb Vasc Biol 18, 100–107. 31 Wu R & Lefvert AK (1995) Autoantibodies against oxi- dized low density lipoprotein (OxLDL): characterization of antibody isotype, subclass, affinity and effect on the macrophage uptake of oxLDL. Clin Exp Immunol 102, 174–180. 32 Chan K, Delfret D & Junges K (1986) A direct colori- metric assay for Ca 2+ ATPase activity. Anal Biochem 157, 375–380. 33 Biggs R (1975) In Coagulacio ´ n Sanguı ´ nea, hemostasia y trombosis (Millan L, ed.), p. 606. Editorial JIMS S95, Barcelona. 34 Jentzsch AM, Bachmann H, Furst P & Biesalski H (1996) Improved analysis of malondialdehyde in human body fluids. Free Radic Biol Med 2, 251–256. 35 Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Bio- chem 72, 218–254. Cholesterol and ectonucleotidase activities M. Medeiros Frescura Duarte et al. 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