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Enzymes that hydrolyze adenine nucleotides in diabetes and associated pathologies
Thrombosis Research 109 (2003) 189 – 194
Regular Article
Enzymes that hydrolyze adenine nucleotides in diabetes and associated pathologies Gilberto Ina´cio Lunkes, Daniela Lunkes, Francieli Stefanello, Andre´ Morsch, Vera Maria Morsch, Cinthia Melazzo Mazzanti, Maria Rosa Chitolina Schetinger* Departamento de Quı´mica, Centro de Cieˆncias Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil Received 21 October 2002; received in revised form 17 January 2003; accepted 11 March 2003
Abstract The activities of the enzymes NTPDase (E.C. 3.6.1.5, apyrase, ATP diphosphohydrolase, ecto-CD39) and 5 V- nucleotidase (E.C. 3.1.3.5, CD73) were analyzed in platelets of type 2 diabetic, hypertensive and type 2 diabetic/hypertensive patients. The results showed an increase in platelet NTPDase activity in type 2 diabetic (34% and 72%), hypertensive (32% and 70%) and type 2 diabetic/hypertensive patients (30% and 55%) when compared to control ( P < .01) with ATP and ADP as substrate, respectively. 5 V-Nucleotidase activity was elevated in the hypertensive (60%) and type 2 diabetic/hypertensive (53%) groups when compared to the control and type 2 diabetic group ( P < .01). No differences in sensitivity to inhibitors was detected between the platelets of controls and type 2 diabetic/hypertensive patients. No effects on the enzyme activities were observed when pharmacological doses of propranolol, captopril, furosemide, chlorpropamide, acetylsalicylic acid and glibenclamide were administered. Furthermore, changes in platelet adhesiveness and reactivity were found in all groups tested. In conclusion, we may postulate that NTPDase and 5 V- nucleotidase from platelets are altered in patients with type 2 diabetes and hypertension. Probably, such alterations are involved in compensatory physiological responses in these diseases and are related to other important mechanisms of thromboregulation. D 2003 Elsevier Science Ltd. All rights reserved. Keywords: Type 2 diabetes; Hypertension; NTPDase; 5 V- nucleotidase; Platelets
1. Introduction Chronic diseases, such as diabetes and hypertension, present high prevalence indexes in the community [1,2]. Resistance to insulin and compensatory hyperinsulinemia are decisive factors for predisposition to the cardiovascular disease, independent of other risk factors commonly associated with cardiovascular pathology such as high levels of triglycerides and low HDL concentrations [3 –5]. Platelets play a fundamental role in hemostasis, and investigation of their functioning in diabetic and hypertensive patients can contribute to a better understanding of the pathophysiology of vascular disease [1,6]. On this basis, alterations in components of the platelet membrane seem to represent a decisive factor in the hypersensitivity and platelet function of diabetic patients [1,6].
* Corresponding author. Fax: +55-552208031. E-mail address: [email protected] (M.R.C. Schetinger).
NTPDases are highly distributed in animals tissues [7– 9] and represent the main ectonucleotidases expressed by endothelial and smooth muscle cells of the circulatory system [10], representing an essential mechanism for the maintenance of blood fluidity [11]. The enzymes NTPDase (E.C. 3.6.1.5, ecto-CD39, ecto-apyrase, ATP diphosphohydrolase) and ecto-5 V- nucleotidase (E.C. 3.1.3.5, CD73) are located in the platelet membrane and complete the hydrolysis of ATP to its nucleoside, adenosine, in the extracellular medium [12,13]. In the coagulation cascade, the enzymes NTPDase and 5 Vnucleotidase have an important function in the regulation of platelet aggregation [10 –15]. There are conclusive lines of evidence showing that CD39 could be a potential therapeutic agent for inhibition of platelet-mediated thrombotic diatheses [11,16– 19]. Of particular interest, platelets normally liberate ATP, ADP and UTP into the blood stream. Thus, the metabolism of extracellular nucleotides plays an important regulatory role in the control of adequate hemostasis, mainly by regulating platelet coagulant status [20]. ADP is the most important platelet agonist and recruiting agent present in the
0049-3848/03/$ - see front matter D 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0049-3848(03)00178-6
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microenvironment of the thrombus [21]. In contrast, adenosine, the final product of the nucleotide chain enzymatic reactions, is a modulator of vascular tone and a well-known inhibitor of platelet aggregation [22]. As pointed out above, the association of NTPDase and 5 V-nucleotidase can promote the hydrolysis of ATP, ADP and AMP, leading to the formation of the anti-aggregant metabolite adenosine [15]. In addition, NTPDase in tandem with ecto-5 V-nucleotidase facilitates the salvage of nucleotides by the ultimate generation of dephosphorylated forms that are taken up by cells via specific transporters [23]. Considering the importance of ecto-CD39 and ecto-5 Vnucleotidase for normal physiological hemostasis, we examined the activity of these enzymes on platelets obtained from normal, diabetic and diabetic/hypertensive patients. In addition, some complementary studies were performed in an attempt to understand platelet functionality in such disorders.
patients aged 58.2 F 6.2, 50% males and 50% females, with type 2 diabetes mellitus plus hypertension who received appropriate medication for the associated diseases. Ten milliliters of blood was obtained from each participant and used for platelet-rich plasma preparations, biochemical determinations and hematological determinations. 2.3. Platelet-rich plasma preparation Platelet-rich plasma was prepared from human donors according to Pilla et al. [12]. Briefly, blood was collected with 0.129 M citrate, was pooled and was centrifuged at 160 g for 10 min. The platelet-rich plasma was centrifuged at 1400 g for 15 min and washed twice by centrifugation at 1400 g with 3.5 mM HEPES isosmolar buffer containing 142 mM NaCl, 2.5 mM KCl, and 5.5 mM glucose. The washed platelets were resuspended in HEPES isosmolar buffer and protein was adjusted to 0.3 –0.5 mg/ml.
2. Patients and methods
2.4. NTPDase and 5 V-nucleotidase determinations
2.1. Materials
NTPDase activity was determined by the method of Pilla et al. [12] in a reaction medium containing 5.0 mM CaCl2, 100 mM NaCl, 4.0 mM KCl, 50 mM glucose, and 50 mM Tris – HCl buffer, pH 7.4, in a final volume of 200 Al. The reaction was started by the addition of ATP or ADP as substrate at a final concentration of 1.0 mM. 5 V- nucleotidase activity was determined by the method of Heymann et al. [24] in a reaction medium containing 10 mM MgCl2, 100 mM Tris – HCl buffer, pH 7.4, in a final volume of 200 Al.
Nucleotides, sodium azide, HEPES, and Trizma base were purchased from Sigma (St. Louis, MO). Captopril, furosemide, acetylsalicylic acid, glibenclamide, chlorpropamide and propranolol were obtained from Galena (Campinas, SP, Brazil). All other reagents used in the experiments were of analytical grade and of the highest purity. 2.2. Patients
2.5. Hematological determinations The sample consisted of patients from the Program of Attendance to Diabetic, Hypertensive and Diabetic-Hypertensive Patients associated with the Municipal Secretary’s Office of Health and Environment of Cruz Alta (RS, Brazil) and of healthy volunteers. 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 from the Federal University of Santa Maria. The sample was divided into four groups. The control group consisted of 26 individuals aged 49.8 F 13.2 years, 50% males and 50% females, who did not present any disease and who had not been submitted to any pharmacological therapy during the last month. Controls were carefully selected by clinical evaluation, matched by sex, age and body mass index similar to that of the patients. The type 2 diabetic group consisted of 16 patients aged 54.5 F 4.9 years, 50% males and 50% females, with type 2 diabetes mellitus treated with chlorpropamide (250 mg/day) or glibenclamide (5 mg/day). The hypertensive group was formed by 12 patients aged 55.8 F 7.5, 50% males and 50% females, with different hypertension levels treated with captopril (25 mg/day), furosemide (40 mg/day), acetylsalicylic acid (100 mg/day) or propranolol (40 mg/day). The type 2 diabetic/hypertensive group was formed by 26
Quantitative and qualitative determinations of platelets, and time of coagulation (by venipuncture) were performed by the method of Vallada [25]. Platelet aggregation was determined by the technique of Biggs [26], consisting of the in vitro macroscopic visualization of aggregates between intervals 15 to 50 s by the addition of ADP to PRP. Bleeding time was determined by the method of Lee et al. [27]. This procedure consists of evaluating the time needed to stop blood flow at a standard small incision made with a lancet at a depth of 3 mm. The arm pressure of the patients was maintained at 40 mm Hg during the procedure. Coagulation time was determined by the method of Vallada [25] by measuring in vitro the coagulation under standard conditions.
3. Results 3.1. Biochemical parameters The control and the hypertensive groups presented normal blood glucose levels (70 – 110 mg/dl), whereas the type 2 diabetic and the type 2 diabetic/hypertensive groups presented elevated blood glucose levels (above 140 mg/
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dl). The lipid profile showed that only the control group presented desirable cholesterol values (less than 200 mg/dl), whereas the other groups presented higher values (200 –240 mg/dl). Triglyceride measurement revealed that the control group presented normal values (down to 200 mg/dl), whereas the other groups presented higher values. Similarly, cholesterol-HDL was acceptable only in the control group (higher than 55 mg/dl), whereas type 2 diabetic, hypertensive and type 2 diabetic/hypertensive patients presented low values ranging from 35 to 40 mg/dl. 3.2. Platelet aggregation, coagulation and bleeding time of the control, type 2 diabetic, hypertensive and type 2 diabetic/hypertensive groups Qualitative analysis demonstrated that platelets obtained from all patients were within normal limits, with values ranging from 200,000 to 400,000 platelets/mm3. Microscopic analysis of platelet size and shape revealed a typical pattern (data not shown). In addition, platelet integrity was determined by the lactate dehydrogenase activity assay. The activity was verified with disrupted and nondisrupted platelets and less than 10% of platelets were found to be disrupted (data not shown), indicating that the preparation of PRP was adequate. For analysis of platelet aggregation, bleeding and coagulation time, the control group was used as reference. Results are listed in Table 1. Statistical analysis showed a difference between the control and other groups in platelet aggregation [ F(3,76) = 18.1, P < .0001], bleeding [ F(3,72) = 51.8, P < .0001] and coagulation [ F(3,76) = 107.3, P < .0001] times.
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Table 2 Platelet NTPDase and 5 -Vnucleotidase activities from controls, type 2 diabetic, hypertensive and type 2 diabetic/hypertensive patients Groups
NTPDase ATP
NTPDase ADP
5 -Vnucleotidase AMP
Control (n = 22) Type 2 diabetic (n = 14) Hypertensive (n = 10) Type 2 diabetic/ hypertensive (n = 24)
11.7 F 1.2a 15.7 F 1.5
6.7 F 0.73a 11.5 F 1.03
3.8 F 0.18a 3.7 F 0.12a
15.5 F 1.6
11.4 F 1.16
4.8 F 0.18
15.2 F 2.04
10.4 F 2.35
4.6 F 0.2
Values represent mean F standard deviation. a Different from the others in the same column ( P < .01) Tukey – Kramer test.
(34%), hypertension (32%) and type 2 diabetes/hypertension (30%) with ATP [ F(3,76) = 29,26, P < .0001] as substrate. ADP hydrolysis was also increased [ F(3,76) = 46.25, P < .0001] in the patients with type 2 diabetes (72%), Table 3 Effect of drugs on NTPDase and 5 V- nucleotidase activities from controls NTPDase ATP
NTPDase ADP
5 -Vnucleotidase AMP
Propranolol 0 15 30 40.5
16.5 F 0.70 15.0 F 1.73 17.0 F 0.90 17.0 F 1.5
8.3 F 1.00 7.0 F 0.8 8.1 F 0.7 8.2 F 0.65
4.4 F 0.50 3.5 F 0.60 3.7 F 0.50 3.6 F 0.3
Captopril 0 26 54 72
16.7 F 0.58 17.1 F 0.58 16.5 F 0.00 16.0 F 0.58
8.4 F 0.88 7.5 F 1.40 7.5 F 0.55 7.7 F 0.60
3.5 F 0.40 3.7 F 0.45 3.5 F 0.75 3.4 F 0.50
The results obtained with NTPDase and 5V-nucleotidase are shown in Table 2. As can be observed, NTPDase activity was enhanced in the groups of patients with type 2 diabetes
Acetylsalicylic acid 0 15.8 F 0.70 30 15.2 F 2.45 45 15.5 F 2.10 60 15.3 F 0.85
7.0 F 0.9 6.5 F 1.1 7.2 F 0.5 6.5 F 0.30
4.0 F 0.20 3.8 F 0.42 3.7 F 0.40 3.7 F 0.60
Table 1 Coagulation parameters obtained from controls, type 2 diabetic, hypertensive and type 2 diabetic/hypertensive patients
Furosemide 0 10 15 25
16.5 F 1.2 16.5 F 1.3 17.1 F 0.5 16.7 F 2.2
8.2 F 0.80 8.0 F 0.60 8.4 F 0.85 7.8 F 0.65
3.5 F 0.70 3.1 F 0.60 3.4 F 0.40 3.2 F 0.58
Chlorpropamide 0 34 67.5 135
15.3 F 1.2 15.0 F 1.4 17.0 F 1.1 17.1 F 2.8
7.5 F 0.9 7.5 F 1.1 8.0 F 0.8 7.7 F 0.9
3.4 F 0.4 3.4 F 0.6 3.8 F 0.5 3.7 F 0.35
Gliblenclamide 0 0.5 1.0 1.5
13.0 F 1.7 13.5 F 1.4 13.0 F 0.8 14.0 F 1.8
7.0 F 0.8 7.3 F 1.1 7.1 F 0.4 7.1 F 0.7
4.0 F 0.4 3.9 F 0.45 3.5 F 0.40 3.8 F 0.60
3.3. Effects of diabetes and hypertension on platelet nucleotide hydrolysis
Groups
Control (n = 26) Type 2 diabetic (n = 16) Hypertensive (n = 12) Type 2 diabetic/ hypertensive (n = 26)
Blood parameters Platelets aggregation (s)
Bleeding time (min)
Time of coagulation (min)
35 F 7.66* 25 F 3.73
3.2 F 0.67* 2.5 F 0.44
6.6 F 0.80* 4.5 F 0.98
26 F 6.02
2.2 F 0.58
4.5 F 0.86
28 F 4.54
2.3 F 0.47
4.1 F 0.47
Values represent mean F standard deviation. * Different from the others in the same column ( P < .01) Tukey – Kramer test.
Drug (AM)
Values represent mean F standard deviation from three individual experiments.
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hypertension (70%) and type 2 diabetes/hypertension (55%) compared to control. 5 V- nucleotidase activity was also altered, with the control and type 2 diabetes groups differing from the hypertensive and type 2 diabetes/hypertensive groups [ F(3,76) = 7.05, P < .0003] which presented 60% and 53% activation, respectively.
mal subjects and from patients with type 2 diabetes and hypertension (group 4). The results demonstrated that NTPDase and 5 V-nucleotidase, when tested in the presence of azide (5 –20 mM), NaF (5 – 20 mM), SUR (30 – 300 AM) and TFP (50 – 200 AM), exhibited the same sensitivity to inhibitors in normal and type 2 diabetic/hypertensive patients, being inhibited by all compounds (Table 4).
3.4. Effects of drugs used in the treatment of diabetes and hypertension on nucleotide hydrolysis 4. Discussion In vitro concentrations ranged from zero to 40.5 AM for propranolol, from zero to 72 AM for captopril, from zero to 60 AM for acetylsalicylic acid, from zero to 72 AM for furosemide, from zero to 135 AM for chlorpropamide, and from zero to 15 AM for glibenclamide. All concentrations used in vitro represented, approximately, the mean plasma values of the medications. The results obtained demonstrate that neither NTPDase nor 5 V- nucleotidase activity was affected by the presence of the medications at the concentrations cited above (Table 3). As the medications did not alter NTPDase or 5 V-nucleotidase activity, we asked: Could the platelet NTPDase and 5 V- nucleotidase obtained from diabetic and hypertensive patients have a different sensitivity to inhibitors? To test this hypothesis, we examined the effect of classical inhibitors such as azide, sodium fluoride (NaF), suramin (SUR) and trifluorperazine (TFP) on platelets obtained from nor-
Multiple studies from different laboratories offer evidence of enhanced activation or increased platelet activity in patients with diabetes [6,28 –30]. It is known that the hypercoagulable state of patients with diabetes arise as a substantial factor accelerating atherosclerosis development and the incidence of arterial thrombotic pathogenesis [28]. Here, we observed changes in platelets aggregation, coagulation and bleeding time which are consistent with an enhanced activity of their coagulation cascade (Table 1). In line with this, platelets obtained from diabetic subjects show increased adhesiveness and an exaggerated aggregation both spontaneously and in response to stimulating agents [6,28,30]. Interestingly, diabetic patients with an additional risk factor such as hypertension (diabetic type 2/hypertensive patients) did not show exacerbation of this tendency. Recently, Ouvin˜a et al. [31] found significant
Table 4 Effect of inhibitors on NTPDase and 5 V- nucleotidase activities from controls and type 2 diabetic/hypertensive patients Inhibitor
Control
Type 2 diabetic/hypertensive
NTPDase ATP
NTPDase ADP
5 -Vnucleotidase AMP
NTPDase ATP
NTPDase ADP
5V - nucleotidase AMP
Azide (mM) 0 5 10 20
11.5 F 0.67 11.00 F 1.73 10.00 F 1.00 8.68 F 0.38*
7.33 F 0.88 6.00 F 0.9 5.66 F 0.58* 4.43 F 0.60*
3.33 F 0.58* 2.10 F 0.58 1.89 F 0.58 1.00 F 0.00
15.1 F 0.9* 11.33 F 0.58 9.67 F 1.52 8.01 F 1.00
10.00 F 1.00* 8.15 F 0.60 7.01 F 1.53 6.34 F 1.00
4.79 F 0.56* 3.12 F 0.49 2.03 F 0.62 1.53 F 0.31
Fluoride (mM) 0 5 10 20
13.79 F 0.58* 12.01 F 0.58 10.82 F 0.00 8.66 F 0.58
6.67 F 0.58* 5.34 F 0.40 4.21 F 0.50 3.01 F 0.58
4.33 F 0.54* 2.98 F 0.40 2.03 F 0.58 1.01 F 0.42
17.33 F 0.49* 11.81 F 0.12 10.78 F 0.12 9.03 F 0.12
12.00 F 1.00* 9.33 F 0.12 8.12 F 0.12 7.04 F 0.12
5.00 F 1.00* 3.66 F 1.15 2.55 F 0.58 1.34 F 0.60
Suramin (AM) 0 30 150 300
13.21 F 0.65* 11.75 F 0.45 8.89 F 1.00 6.34 F 0.58
6.33 F 0.58* 5.12 F 0.38 4.06 F 0.58 2.35 F 0.65
4.38 F 0.50* 3.12 F 0.32 2.07 F 0.40 1.34 F 0.39
16.33 F 0.58* 13.00 F 1.00 11.45 F 1.12 9.88 F 1.00
11.03 F 0.58* 9.33 F 2.01 8.33 F 1.52 7.00 F 1.00
5.15 F 0.68* 3.98 F 0.58 2.78 F 0.60 1.45 F 0.58
7.67 F 0.38* 5.65 F 0.61 4.89 F 0.58 3.63 F 0.60
4.89 F 0.70* 3.11 F 0.38 2.05 F 0.58 1.01 F 0.48
14.67 F 0.65* 12.74 F 0.50 10.89 F 0.71 9.13 F 0.60
10.8 F 0.90* 8.79 F 0.76 7.65 F 0.60 5.98 F 0.65
6.13 F 0.6* 4.18 F 0.59 3.22 F 0.75 1.78 F 0.8
Trifluorperazine (AM) 0 13.8 F 1.0* 50 11.2 F 1.0 100 9.89 F 0.58 200 8.0 F 0.61
Values represent mean F standard deviation from three individual experiments. * Different from others in the same column (ANOVA, Tukey – Kramer test, P < .05).
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differences in platelet activation between hypertensive diabetic type 2 and hypertensive patients by measuring platelet expression of CD62P (GMP-140, PADGEM, P-selectin). NTPDase and 5 V-nucleotidase activity in platelets from the control group was compatible with the data reported by Pilla et al. [12]. Here, for the first time, we showed that platelet NTPDase activity was enhanced in type 2 diabetic, hypertensive and type 2 diabetic/hypertensive patients. The increase in NTPDase activity could be related to a compensatory organic response. An explanation could be that the rapid ATP and ADP hydrolysis favors adenosine production. It is known that ATP promotes vasoconstriction in the vascular endothelium and ADP activates platelet aggregation, whereas adenosine promotes vasodilatation and the inhibition of platelet aggregation. Consequently, the organism could be avoiding thrombotic processes by compensatory ADP depletion and adenosine production. Recently, the role of NTPDase activity on thromboregulation have been investigated by different laboratories [11,19]. Se´vigny et al. [19] demonstrated the role of two NTPDases, one is NTPDase1 that faces the blood circulation and abrogates platelet aggregation, and another is NTPDase2 that is expressed in supporting cells of the vasculature and facilitates platelet aggregation, showing that NTPDase1 and NTPDase2 have opposing effects on platelet aggregation in vitro. NTPDase1 (ecto-CD39) hydrolyzes both nucleoside triphosphates and diphosphates, whereas NTPDase2 preferentially hydrolyzes ATP [32, 33]. In addition, Pinsky et al. [11] demonstrated the thromboregulatory role of CD39/ectoapyrase in the ischemic brain and verified that SolCD39 (soluble CD39) disaggregates platelets under recruitment, but it does not have a harmful consequence on primary hemostasis. These publications reinforce the crescent importance of NTPDase in thromboregulation. Taking the results obtained in this work, perhaps, in plasma-rich platelets the enhancement in ATP and ADP hydrolysis is due to different NTPDase isoenzymes or by another enzymatic activities that promote extracellular nucleotide hydrolysis. Recently, Birk et al. [34] also reported the presence of a plasma nucleotidase, nucleoside-5V-monophosphate phosphoanhydrolase phosphodiesterase (NMPP) that hydrolyzes ATP directly to AMP avoiding ADP production, being important in the regulation of nucleotide concentrations in the circulation. We can speculate that endothelial and platelet NTPDase together with plasma NMPP could regulate platelet reactivity. In type 2 diabetes and hypertension, there is a predisposition to thrombus formation. Normally, platelets maintain an intrinsic contact with the endothelium and no pathological conditions are generated. However, in the microenvironment of the thrombus, there is an intrinsic cell – cell interaction between platelets, neutrophils, erythrocytes and endothelial cells [15]. It is unusual to relate the enhancement in platelet NTPDase activity to thrombus formation. Although this is a speculative assumption, we may propose that the activation of platelet CD39 is not
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sufficient to prevent thrombus formation in the microenvironment of such pathological condition. In a conceptual definition, thromboregulation is a process or group of processes by which circulating blood cells and cells of the vessel wall interact to regulate or inhibit thrombus formation [15]. Perhaps thromboregulation is not occurring efficiently in type 2 diabetes and hypertension. In order to exclude a direct effect of drugs commonly used for the treatment of diabetes, hypertension and hyperglycemia, we investigated the influence of propranolol, captopril, acetylsalicylic acid, furosemide, chlorpropamide and glibenclamide. At concentration even higher than that found therapeutically, they do not alter NTPDase or 5 Vnucleotidase activities (Table 3). The enzymes NTPDase and 5 V- nucleotidase from type 2 diabetic/hypertensive patients responded in a similar way to inhibitors such as azide, NAF, SUR and TFP. Consequently, we may propose that the increase observed in NTPDase and 5V- nucleotidase activity is not due to the medications used by the patients or to some specific characteristic such as a differential sensitivity to inhibitors. Also, these findings support the argument that it is the pathological condition that generates the increase in these enzyme activities. Interestingly, the classical NTPDase inhibitors affect also platelet 5V- nucleotidase activity. These findings were unexpected and were not related in the literature, but are being investigated with more details. Taken together, our results indicate that probably there are many factors that could be acting in the context of thromboregulation. Although type 2 diabetic and hypertensive patients present a predisposition to thrombus formation, they also present an enhancement in NTPDase and 5 V-nucleotidase activity. How could this be explained as it is postulated that NTPDase is a potent inhibitor of platelet recruitment and aggregation? Perhaps, we should observe not only platelets and NTPDase but all factors involved in thrombus formation; we must keep in mind that a compensatory physiological response is not always sufficient to prevent a pathophysiological event. Acknowledgements The authors wish to thank Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and Fundacß a˜o de Amparo a` Pesquisa do Rio Grande do Sul (FAPERGS) for financial support (00/2169.1). References [1] Vinik AI, Erbas T, Park TS, Nolan R, Pittenger GL. Platelet dysfunction in type 2 diabetes. Diabetes Care 2001;24:1476 – 85. [2] Sowers JR, Epstein M, Frohlich ED. Diabetes, hypertensive, and cardiovascular disease: an update. Hypertension 2001;37:1053 – 9. [3] Yip J, Facchini FS, Reaven GM. Resistance to insulin-mediated glucose disposal as a predictor of cardiovascular disease. J Clin Endocrinol Metab 1998;83:2773 – 6.
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Qawi I, et al. Differential catalytic properties and vascular topography of murine nucleoside triphosphate diphosphohydrolase 1 (NTPDase1) and NTPDase2 have implications for thromboregulation. Blood 2002;99:2801 – 9. Kas-Deelen AM, Bakker WW, Olinga P, Visser J, Maar EF, Son WJ, et al. Cytomegalovirus infection increases the expression and activity of ecto-ATPase (CD39) and ecto-5 V- nucleotidase (CD73) on endothelial cells. FEBS Lett 2001;491:21 – 5. Marcus AJ, Safier LB. Thromboregulation: multicellular modulation of platelet reactivity in homeostasis and thrombosis. FASEB J 1997; 7:516 – 22. Kawashima Y, Nagasawa T, Ninomiya H. Contribution of ecto-5 Vnucleotidase to the inhibition of platelet aggregation by human endothelial cells. Blood 2000;96:2157 – 62. Che MX, Gatmaitan Z, Arias IM. Ectonucleotidases, purine nucleoside transporter, and function of the bile canalicular plasma membrane of the hepatocyte. FASEB J 1997;11:101 – 8. Heymann D, Reddington M, Kreutzberg GW. Subcellular localization of 5 V- nucleotidase in rat brain. J Neurochem 1984;43:263 – 73. Vallada EP. Manual de te´cnicas hematolo´gicas. Sa˜o Paulo: Editora Atheneu; 1988. p. 423. Biggs R. In: Millan L, editor. Coagulacio´n sanguı´nea, hemostası´a y trombosis. Barcelona: Editorial Jims; 1975. p. 595. Lee GR, Bithell TC, Foerster J, Athens JW, Lukens JN. Wintrobe Hematologia Clinica. Sa˜o Paulo: Editora Manole; 1998. p. 2623. Carr ME. Diabetes mellitus: a hypercoagulable state. J Diabetes Its Complicat 2001;15:44 – 54. Garcia Frade LJ, de la Calle H, Alava I, Navarro JL, Creighton L, Gaffney PJ. Diabetes mellitus as a hypercoagulable state: its relationship with fibrin fragments and vascular damage. Thromb Res 1987; 47:533 – 40. Rosove MH, Frank HJ, Harwig SS. Plasma beta-thromboglobulin, platelet factor 4, fibrinopeptide A, and other hemostatic functions during improved, shortterm glycemic control in diabetes mellitus. Diabetes Care 1984;7:174 – 9. Ouvin˜a SM, La Greca RD, Zanaro NL, Palmer L, Sassetti B. Endothelial dysfunction, nitric oxide and platelet activation in hypertensive and diabetic type II patients. Thromb Res 2001;102:107 – 14. Kegel B, Braun N, Heine P, Maliszewski CR, Zimmermann H. An ecto-ATPase and an ecto-ATP diphosphohydrolase are expressed in rat brain. Neuropharmacology 1997;38:1189 – 200. Zimmermann H. Ectonucleotidases: some recent developments and a note on nomenclature. Drug Dev Res 2001;52:44 – 56. Birk AV, Bubman D, Broekman MJ, Robertson HD, Drosopoulos JHF, Marcus AJ, et al. Role of a novel soluble nucleotide phosphohydrolase from sheep plasma in inhibition of platelet reactivity: hemostasis, thrombosis, and vascular biology. J Lab Clin Med 2002;139: 116 – 24.
Regular Article
Enzymes that hydrolyze adenine nucleotides in diabetes and associated pathologies Gilberto Ina´cio Lunkes, Daniela Lunkes, Francieli Stefanello, Andre´ Morsch, Vera Maria Morsch, Cinthia Melazzo Mazzanti, Maria Rosa Chitolina Schetinger* Departamento de Quı´mica, Centro de Cieˆncias Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil Received 21 October 2002; received in revised form 17 January 2003; accepted 11 March 2003
Abstract The activities of the enzymes NTPDase (E.C. 3.6.1.5, apyrase, ATP diphosphohydrolase, ecto-CD39) and 5 V- nucleotidase (E.C. 3.1.3.5, CD73) were analyzed in platelets of type 2 diabetic, hypertensive and type 2 diabetic/hypertensive patients. The results showed an increase in platelet NTPDase activity in type 2 diabetic (34% and 72%), hypertensive (32% and 70%) and type 2 diabetic/hypertensive patients (30% and 55%) when compared to control ( P < .01) with ATP and ADP as substrate, respectively. 5 V-Nucleotidase activity was elevated in the hypertensive (60%) and type 2 diabetic/hypertensive (53%) groups when compared to the control and type 2 diabetic group ( P < .01). No differences in sensitivity to inhibitors was detected between the platelets of controls and type 2 diabetic/hypertensive patients. No effects on the enzyme activities were observed when pharmacological doses of propranolol, captopril, furosemide, chlorpropamide, acetylsalicylic acid and glibenclamide were administered. Furthermore, changes in platelet adhesiveness and reactivity were found in all groups tested. In conclusion, we may postulate that NTPDase and 5 V- nucleotidase from platelets are altered in patients with type 2 diabetes and hypertension. Probably, such alterations are involved in compensatory physiological responses in these diseases and are related to other important mechanisms of thromboregulation. D 2003 Elsevier Science Ltd. All rights reserved. Keywords: Type 2 diabetes; Hypertension; NTPDase; 5 V- nucleotidase; Platelets
1. Introduction Chronic diseases, such as diabetes and hypertension, present high prevalence indexes in the community [1,2]. Resistance to insulin and compensatory hyperinsulinemia are decisive factors for predisposition to the cardiovascular disease, independent of other risk factors commonly associated with cardiovascular pathology such as high levels of triglycerides and low HDL concentrations [3 –5]. Platelets play a fundamental role in hemostasis, and investigation of their functioning in diabetic and hypertensive patients can contribute to a better understanding of the pathophysiology of vascular disease [1,6]. On this basis, alterations in components of the platelet membrane seem to represent a decisive factor in the hypersensitivity and platelet function of diabetic patients [1,6].
* Corresponding author. Fax: +55-552208031. E-mail address: [email protected] (M.R.C. Schetinger).
NTPDases are highly distributed in animals tissues [7– 9] and represent the main ectonucleotidases expressed by endothelial and smooth muscle cells of the circulatory system [10], representing an essential mechanism for the maintenance of blood fluidity [11]. The enzymes NTPDase (E.C. 3.6.1.5, ecto-CD39, ecto-apyrase, ATP diphosphohydrolase) and ecto-5 V- nucleotidase (E.C. 3.1.3.5, CD73) are located in the platelet membrane and complete the hydrolysis of ATP to its nucleoside, adenosine, in the extracellular medium [12,13]. In the coagulation cascade, the enzymes NTPDase and 5 Vnucleotidase have an important function in the regulation of platelet aggregation [10 –15]. There are conclusive lines of evidence showing that CD39 could be a potential therapeutic agent for inhibition of platelet-mediated thrombotic diatheses [11,16– 19]. Of particular interest, platelets normally liberate ATP, ADP and UTP into the blood stream. Thus, the metabolism of extracellular nucleotides plays an important regulatory role in the control of adequate hemostasis, mainly by regulating platelet coagulant status [20]. ADP is the most important platelet agonist and recruiting agent present in the
0049-3848/03/$ - see front matter D 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0049-3848(03)00178-6
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microenvironment of the thrombus [21]. In contrast, adenosine, the final product of the nucleotide chain enzymatic reactions, is a modulator of vascular tone and a well-known inhibitor of platelet aggregation [22]. As pointed out above, the association of NTPDase and 5 V-nucleotidase can promote the hydrolysis of ATP, ADP and AMP, leading to the formation of the anti-aggregant metabolite adenosine [15]. In addition, NTPDase in tandem with ecto-5 V-nucleotidase facilitates the salvage of nucleotides by the ultimate generation of dephosphorylated forms that are taken up by cells via specific transporters [23]. Considering the importance of ecto-CD39 and ecto-5 Vnucleotidase for normal physiological hemostasis, we examined the activity of these enzymes on platelets obtained from normal, diabetic and diabetic/hypertensive patients. In addition, some complementary studies were performed in an attempt to understand platelet functionality in such disorders.
patients aged 58.2 F 6.2, 50% males and 50% females, with type 2 diabetes mellitus plus hypertension who received appropriate medication for the associated diseases. Ten milliliters of blood was obtained from each participant and used for platelet-rich plasma preparations, biochemical determinations and hematological determinations. 2.3. Platelet-rich plasma preparation Platelet-rich plasma was prepared from human donors according to Pilla et al. [12]. Briefly, blood was collected with 0.129 M citrate, was pooled and was centrifuged at 160 g for 10 min. The platelet-rich plasma was centrifuged at 1400 g for 15 min and washed twice by centrifugation at 1400 g with 3.5 mM HEPES isosmolar buffer containing 142 mM NaCl, 2.5 mM KCl, and 5.5 mM glucose. The washed platelets were resuspended in HEPES isosmolar buffer and protein was adjusted to 0.3 –0.5 mg/ml.
2. Patients and methods
2.4. NTPDase and 5 V-nucleotidase determinations
2.1. Materials
NTPDase activity was determined by the method of Pilla et al. [12] in a reaction medium containing 5.0 mM CaCl2, 100 mM NaCl, 4.0 mM KCl, 50 mM glucose, and 50 mM Tris – HCl buffer, pH 7.4, in a final volume of 200 Al. The reaction was started by the addition of ATP or ADP as substrate at a final concentration of 1.0 mM. 5 V- nucleotidase activity was determined by the method of Heymann et al. [24] in a reaction medium containing 10 mM MgCl2, 100 mM Tris – HCl buffer, pH 7.4, in a final volume of 200 Al.
Nucleotides, sodium azide, HEPES, and Trizma base were purchased from Sigma (St. Louis, MO). Captopril, furosemide, acetylsalicylic acid, glibenclamide, chlorpropamide and propranolol were obtained from Galena (Campinas, SP, Brazil). All other reagents used in the experiments were of analytical grade and of the highest purity. 2.2. Patients
2.5. Hematological determinations The sample consisted of patients from the Program of Attendance to Diabetic, Hypertensive and Diabetic-Hypertensive Patients associated with the Municipal Secretary’s Office of Health and Environment of Cruz Alta (RS, Brazil) and of healthy volunteers. 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 from the Federal University of Santa Maria. The sample was divided into four groups. The control group consisted of 26 individuals aged 49.8 F 13.2 years, 50% males and 50% females, who did not present any disease and who had not been submitted to any pharmacological therapy during the last month. Controls were carefully selected by clinical evaluation, matched by sex, age and body mass index similar to that of the patients. The type 2 diabetic group consisted of 16 patients aged 54.5 F 4.9 years, 50% males and 50% females, with type 2 diabetes mellitus treated with chlorpropamide (250 mg/day) or glibenclamide (5 mg/day). The hypertensive group was formed by 12 patients aged 55.8 F 7.5, 50% males and 50% females, with different hypertension levels treated with captopril (25 mg/day), furosemide (40 mg/day), acetylsalicylic acid (100 mg/day) or propranolol (40 mg/day). The type 2 diabetic/hypertensive group was formed by 26
Quantitative and qualitative determinations of platelets, and time of coagulation (by venipuncture) were performed by the method of Vallada [25]. Platelet aggregation was determined by the technique of Biggs [26], consisting of the in vitro macroscopic visualization of aggregates between intervals 15 to 50 s by the addition of ADP to PRP. Bleeding time was determined by the method of Lee et al. [27]. This procedure consists of evaluating the time needed to stop blood flow at a standard small incision made with a lancet at a depth of 3 mm. The arm pressure of the patients was maintained at 40 mm Hg during the procedure. Coagulation time was determined by the method of Vallada [25] by measuring in vitro the coagulation under standard conditions.
3. Results 3.1. Biochemical parameters The control and the hypertensive groups presented normal blood glucose levels (70 – 110 mg/dl), whereas the type 2 diabetic and the type 2 diabetic/hypertensive groups presented elevated blood glucose levels (above 140 mg/
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dl). The lipid profile showed that only the control group presented desirable cholesterol values (less than 200 mg/dl), whereas the other groups presented higher values (200 –240 mg/dl). Triglyceride measurement revealed that the control group presented normal values (down to 200 mg/dl), whereas the other groups presented higher values. Similarly, cholesterol-HDL was acceptable only in the control group (higher than 55 mg/dl), whereas type 2 diabetic, hypertensive and type 2 diabetic/hypertensive patients presented low values ranging from 35 to 40 mg/dl. 3.2. Platelet aggregation, coagulation and bleeding time of the control, type 2 diabetic, hypertensive and type 2 diabetic/hypertensive groups Qualitative analysis demonstrated that platelets obtained from all patients were within normal limits, with values ranging from 200,000 to 400,000 platelets/mm3. Microscopic analysis of platelet size and shape revealed a typical pattern (data not shown). In addition, platelet integrity was determined by the lactate dehydrogenase activity assay. The activity was verified with disrupted and nondisrupted platelets and less than 10% of platelets were found to be disrupted (data not shown), indicating that the preparation of PRP was adequate. For analysis of platelet aggregation, bleeding and coagulation time, the control group was used as reference. Results are listed in Table 1. Statistical analysis showed a difference between the control and other groups in platelet aggregation [ F(3,76) = 18.1, P < .0001], bleeding [ F(3,72) = 51.8, P < .0001] and coagulation [ F(3,76) = 107.3, P < .0001] times.
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Table 2 Platelet NTPDase and 5 -Vnucleotidase activities from controls, type 2 diabetic, hypertensive and type 2 diabetic/hypertensive patients Groups
NTPDase ATP
NTPDase ADP
5 -Vnucleotidase AMP
Control (n = 22) Type 2 diabetic (n = 14) Hypertensive (n = 10) Type 2 diabetic/ hypertensive (n = 24)
11.7 F 1.2a 15.7 F 1.5
6.7 F 0.73a 11.5 F 1.03
3.8 F 0.18a 3.7 F 0.12a
15.5 F 1.6
11.4 F 1.16
4.8 F 0.18
15.2 F 2.04
10.4 F 2.35
4.6 F 0.2
Values represent mean F standard deviation. a Different from the others in the same column ( P < .01) Tukey – Kramer test.
(34%), hypertension (32%) and type 2 diabetes/hypertension (30%) with ATP [ F(3,76) = 29,26, P < .0001] as substrate. ADP hydrolysis was also increased [ F(3,76) = 46.25, P < .0001] in the patients with type 2 diabetes (72%), Table 3 Effect of drugs on NTPDase and 5 V- nucleotidase activities from controls NTPDase ATP
NTPDase ADP
5 -Vnucleotidase AMP
Propranolol 0 15 30 40.5
16.5 F 0.70 15.0 F 1.73 17.0 F 0.90 17.0 F 1.5
8.3 F 1.00 7.0 F 0.8 8.1 F 0.7 8.2 F 0.65
4.4 F 0.50 3.5 F 0.60 3.7 F 0.50 3.6 F 0.3
Captopril 0 26 54 72
16.7 F 0.58 17.1 F 0.58 16.5 F 0.00 16.0 F 0.58
8.4 F 0.88 7.5 F 1.40 7.5 F 0.55 7.7 F 0.60
3.5 F 0.40 3.7 F 0.45 3.5 F 0.75 3.4 F 0.50
The results obtained with NTPDase and 5V-nucleotidase are shown in Table 2. As can be observed, NTPDase activity was enhanced in the groups of patients with type 2 diabetes
Acetylsalicylic acid 0 15.8 F 0.70 30 15.2 F 2.45 45 15.5 F 2.10 60 15.3 F 0.85
7.0 F 0.9 6.5 F 1.1 7.2 F 0.5 6.5 F 0.30
4.0 F 0.20 3.8 F 0.42 3.7 F 0.40 3.7 F 0.60
Table 1 Coagulation parameters obtained from controls, type 2 diabetic, hypertensive and type 2 diabetic/hypertensive patients
Furosemide 0 10 15 25
16.5 F 1.2 16.5 F 1.3 17.1 F 0.5 16.7 F 2.2
8.2 F 0.80 8.0 F 0.60 8.4 F 0.85 7.8 F 0.65
3.5 F 0.70 3.1 F 0.60 3.4 F 0.40 3.2 F 0.58
Chlorpropamide 0 34 67.5 135
15.3 F 1.2 15.0 F 1.4 17.0 F 1.1 17.1 F 2.8
7.5 F 0.9 7.5 F 1.1 8.0 F 0.8 7.7 F 0.9
3.4 F 0.4 3.4 F 0.6 3.8 F 0.5 3.7 F 0.35
Gliblenclamide 0 0.5 1.0 1.5
13.0 F 1.7 13.5 F 1.4 13.0 F 0.8 14.0 F 1.8
7.0 F 0.8 7.3 F 1.1 7.1 F 0.4 7.1 F 0.7
4.0 F 0.4 3.9 F 0.45 3.5 F 0.40 3.8 F 0.60
3.3. Effects of diabetes and hypertension on platelet nucleotide hydrolysis
Groups
Control (n = 26) Type 2 diabetic (n = 16) Hypertensive (n = 12) Type 2 diabetic/ hypertensive (n = 26)
Blood parameters Platelets aggregation (s)
Bleeding time (min)
Time of coagulation (min)
35 F 7.66* 25 F 3.73
3.2 F 0.67* 2.5 F 0.44
6.6 F 0.80* 4.5 F 0.98
26 F 6.02
2.2 F 0.58
4.5 F 0.86
28 F 4.54
2.3 F 0.47
4.1 F 0.47
Values represent mean F standard deviation. * Different from the others in the same column ( P < .01) Tukey – Kramer test.
Drug (AM)
Values represent mean F standard deviation from three individual experiments.
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hypertension (70%) and type 2 diabetes/hypertension (55%) compared to control. 5 V- nucleotidase activity was also altered, with the control and type 2 diabetes groups differing from the hypertensive and type 2 diabetes/hypertensive groups [ F(3,76) = 7.05, P < .0003] which presented 60% and 53% activation, respectively.
mal subjects and from patients with type 2 diabetes and hypertension (group 4). The results demonstrated that NTPDase and 5 V-nucleotidase, when tested in the presence of azide (5 –20 mM), NaF (5 – 20 mM), SUR (30 – 300 AM) and TFP (50 – 200 AM), exhibited the same sensitivity to inhibitors in normal and type 2 diabetic/hypertensive patients, being inhibited by all compounds (Table 4).
3.4. Effects of drugs used in the treatment of diabetes and hypertension on nucleotide hydrolysis 4. Discussion In vitro concentrations ranged from zero to 40.5 AM for propranolol, from zero to 72 AM for captopril, from zero to 60 AM for acetylsalicylic acid, from zero to 72 AM for furosemide, from zero to 135 AM for chlorpropamide, and from zero to 15 AM for glibenclamide. All concentrations used in vitro represented, approximately, the mean plasma values of the medications. The results obtained demonstrate that neither NTPDase nor 5 V- nucleotidase activity was affected by the presence of the medications at the concentrations cited above (Table 3). As the medications did not alter NTPDase or 5 V-nucleotidase activity, we asked: Could the platelet NTPDase and 5 V- nucleotidase obtained from diabetic and hypertensive patients have a different sensitivity to inhibitors? To test this hypothesis, we examined the effect of classical inhibitors such as azide, sodium fluoride (NaF), suramin (SUR) and trifluorperazine (TFP) on platelets obtained from nor-
Multiple studies from different laboratories offer evidence of enhanced activation or increased platelet activity in patients with diabetes [6,28 –30]. It is known that the hypercoagulable state of patients with diabetes arise as a substantial factor accelerating atherosclerosis development and the incidence of arterial thrombotic pathogenesis [28]. Here, we observed changes in platelets aggregation, coagulation and bleeding time which are consistent with an enhanced activity of their coagulation cascade (Table 1). In line with this, platelets obtained from diabetic subjects show increased adhesiveness and an exaggerated aggregation both spontaneously and in response to stimulating agents [6,28,30]. Interestingly, diabetic patients with an additional risk factor such as hypertension (diabetic type 2/hypertensive patients) did not show exacerbation of this tendency. Recently, Ouvin˜a et al. [31] found significant
Table 4 Effect of inhibitors on NTPDase and 5 V- nucleotidase activities from controls and type 2 diabetic/hypertensive patients Inhibitor
Control
Type 2 diabetic/hypertensive
NTPDase ATP
NTPDase ADP
5 -Vnucleotidase AMP
NTPDase ATP
NTPDase ADP
5V - nucleotidase AMP
Azide (mM) 0 5 10 20
11.5 F 0.67 11.00 F 1.73 10.00 F 1.00 8.68 F 0.38*
7.33 F 0.88 6.00 F 0.9 5.66 F 0.58* 4.43 F 0.60*
3.33 F 0.58* 2.10 F 0.58 1.89 F 0.58 1.00 F 0.00
15.1 F 0.9* 11.33 F 0.58 9.67 F 1.52 8.01 F 1.00
10.00 F 1.00* 8.15 F 0.60 7.01 F 1.53 6.34 F 1.00
4.79 F 0.56* 3.12 F 0.49 2.03 F 0.62 1.53 F 0.31
Fluoride (mM) 0 5 10 20
13.79 F 0.58* 12.01 F 0.58 10.82 F 0.00 8.66 F 0.58
6.67 F 0.58* 5.34 F 0.40 4.21 F 0.50 3.01 F 0.58
4.33 F 0.54* 2.98 F 0.40 2.03 F 0.58 1.01 F 0.42
17.33 F 0.49* 11.81 F 0.12 10.78 F 0.12 9.03 F 0.12
12.00 F 1.00* 9.33 F 0.12 8.12 F 0.12 7.04 F 0.12
5.00 F 1.00* 3.66 F 1.15 2.55 F 0.58 1.34 F 0.60
Suramin (AM) 0 30 150 300
13.21 F 0.65* 11.75 F 0.45 8.89 F 1.00 6.34 F 0.58
6.33 F 0.58* 5.12 F 0.38 4.06 F 0.58 2.35 F 0.65
4.38 F 0.50* 3.12 F 0.32 2.07 F 0.40 1.34 F 0.39
16.33 F 0.58* 13.00 F 1.00 11.45 F 1.12 9.88 F 1.00
11.03 F 0.58* 9.33 F 2.01 8.33 F 1.52 7.00 F 1.00
5.15 F 0.68* 3.98 F 0.58 2.78 F 0.60 1.45 F 0.58
7.67 F 0.38* 5.65 F 0.61 4.89 F 0.58 3.63 F 0.60
4.89 F 0.70* 3.11 F 0.38 2.05 F 0.58 1.01 F 0.48
14.67 F 0.65* 12.74 F 0.50 10.89 F 0.71 9.13 F 0.60
10.8 F 0.90* 8.79 F 0.76 7.65 F 0.60 5.98 F 0.65
6.13 F 0.6* 4.18 F 0.59 3.22 F 0.75 1.78 F 0.8
Trifluorperazine (AM) 0 13.8 F 1.0* 50 11.2 F 1.0 100 9.89 F 0.58 200 8.0 F 0.61
Values represent mean F standard deviation from three individual experiments. * Different from others in the same column (ANOVA, Tukey – Kramer test, P < .05).
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differences in platelet activation between hypertensive diabetic type 2 and hypertensive patients by measuring platelet expression of CD62P (GMP-140, PADGEM, P-selectin). NTPDase and 5 V-nucleotidase activity in platelets from the control group was compatible with the data reported by Pilla et al. [12]. Here, for the first time, we showed that platelet NTPDase activity was enhanced in type 2 diabetic, hypertensive and type 2 diabetic/hypertensive patients. The increase in NTPDase activity could be related to a compensatory organic response. An explanation could be that the rapid ATP and ADP hydrolysis favors adenosine production. It is known that ATP promotes vasoconstriction in the vascular endothelium and ADP activates platelet aggregation, whereas adenosine promotes vasodilatation and the inhibition of platelet aggregation. Consequently, the organism could be avoiding thrombotic processes by compensatory ADP depletion and adenosine production. Recently, the role of NTPDase activity on thromboregulation have been investigated by different laboratories [11,19]. Se´vigny et al. [19] demonstrated the role of two NTPDases, one is NTPDase1 that faces the blood circulation and abrogates platelet aggregation, and another is NTPDase2 that is expressed in supporting cells of the vasculature and facilitates platelet aggregation, showing that NTPDase1 and NTPDase2 have opposing effects on platelet aggregation in vitro. NTPDase1 (ecto-CD39) hydrolyzes both nucleoside triphosphates and diphosphates, whereas NTPDase2 preferentially hydrolyzes ATP [32, 33]. In addition, Pinsky et al. [11] demonstrated the thromboregulatory role of CD39/ectoapyrase in the ischemic brain and verified that SolCD39 (soluble CD39) disaggregates platelets under recruitment, but it does not have a harmful consequence on primary hemostasis. These publications reinforce the crescent importance of NTPDase in thromboregulation. Taking the results obtained in this work, perhaps, in plasma-rich platelets the enhancement in ATP and ADP hydrolysis is due to different NTPDase isoenzymes or by another enzymatic activities that promote extracellular nucleotide hydrolysis. Recently, Birk et al. [34] also reported the presence of a plasma nucleotidase, nucleoside-5V-monophosphate phosphoanhydrolase phosphodiesterase (NMPP) that hydrolyzes ATP directly to AMP avoiding ADP production, being important in the regulation of nucleotide concentrations in the circulation. We can speculate that endothelial and platelet NTPDase together with plasma NMPP could regulate platelet reactivity. In type 2 diabetes and hypertension, there is a predisposition to thrombus formation. Normally, platelets maintain an intrinsic contact with the endothelium and no pathological conditions are generated. However, in the microenvironment of the thrombus, there is an intrinsic cell – cell interaction between platelets, neutrophils, erythrocytes and endothelial cells [15]. It is unusual to relate the enhancement in platelet NTPDase activity to thrombus formation. Although this is a speculative assumption, we may propose that the activation of platelet CD39 is not
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sufficient to prevent thrombus formation in the microenvironment of such pathological condition. In a conceptual definition, thromboregulation is a process or group of processes by which circulating blood cells and cells of the vessel wall interact to regulate or inhibit thrombus formation [15]. Perhaps thromboregulation is not occurring efficiently in type 2 diabetes and hypertension. In order to exclude a direct effect of drugs commonly used for the treatment of diabetes, hypertension and hyperglycemia, we investigated the influence of propranolol, captopril, acetylsalicylic acid, furosemide, chlorpropamide and glibenclamide. At concentration even higher than that found therapeutically, they do not alter NTPDase or 5 Vnucleotidase activities (Table 3). The enzymes NTPDase and 5 V- nucleotidase from type 2 diabetic/hypertensive patients responded in a similar way to inhibitors such as azide, NAF, SUR and TFP. Consequently, we may propose that the increase observed in NTPDase and 5V- nucleotidase activity is not due to the medications used by the patients or to some specific characteristic such as a differential sensitivity to inhibitors. Also, these findings support the argument that it is the pathological condition that generates the increase in these enzyme activities. Interestingly, the classical NTPDase inhibitors affect also platelet 5V- nucleotidase activity. These findings were unexpected and were not related in the literature, but are being investigated with more details. Taken together, our results indicate that probably there are many factors that could be acting in the context of thromboregulation. Although type 2 diabetic and hypertensive patients present a predisposition to thrombus formation, they also present an enhancement in NTPDase and 5 V-nucleotidase activity. How could this be explained as it is postulated that NTPDase is a potent inhibitor of platelet recruitment and aggregation? Perhaps, we should observe not only platelets and NTPDase but all factors involved in thrombus formation; we must keep in mind that a compensatory physiological response is not always sufficient to prevent a pathophysiological event. Acknowledgements The authors wish to thank Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and Fundacß a˜o de Amparo a` Pesquisa do Rio Grande do Sul (FAPERGS) for financial support (00/2169.1). References [1] Vinik AI, Erbas T, Park TS, Nolan R, Pittenger GL. Platelet dysfunction in type 2 diabetes. Diabetes Care 2001;24:1476 – 85. [2] Sowers JR, Epstein M, Frohlich ED. Diabetes, hypertensive, and cardiovascular disease: an update. Hypertension 2001;37:1053 – 9. [3] Yip J, Facchini FS, Reaven GM. Resistance to insulin-mediated glucose disposal as a predictor of cardiovascular disease. J Clin Endocrinol Metab 1998;83:2773 – 6.
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