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Hydrogen peroxide and peroxynitrite enhance Ca2+ mobilization and aggregation in platelets from type 2 diabetic patients
BBRC Biochemical and Biophysical Research Communications 333 (2005) 794–802 www.elsevier.com/locate/ybbrc
Hydrogen peroxide and peroxynitrite enhance Ca2+ mobilization and aggregation in platelets from type 2 diabetic patients q Pedro C. Redondo a,1, Isaac Jardin a,1, Juan M. Herna´ndez-Cruz b, Jose´ A. Pariente a, Gine´s M. Salido a, Juan A. Rosado a,* a
Department of Physiology, University of Extremadura, Ca´ceres, Spain b Clinical Analysis Laboratory, Ca´ceres, Spain Received 25 May 2005 Available online 13 June 2005
Abstract Cytosolic Ca2+ mobilization, especially Ca2+ entry, is enhanced in platelets from type 2 diabetic individuals, which might result in platelet hyperaggregability. In the present study, we report an increased oxidant production in resting and stimulated platelets from diabetic donors. Pretreatment of platelets with catalase or trolox, an analog of vitamin E, reversed the enhanced Ca2+ entry, evoked by thapsigargin plus ionomycin or thrombin, observed in platelets from diabetic subjects, so that in the presence of these scavengers Ca2+ entry was similar in platelets from healthy and diabetic subjects. In contrast, mannitol was without effect on Ca2+ mobilization. Catalase and trolox reduced thrombin-induced aggregation in platelets from type 2 diabetic subjects, while mannitol did not modify thrombin-induced platelet hyperaggregability. We conclude that H2O2 and ONOO are likely involved in the enhanced Ca2+ mobilization observed in platelets from type 2 diabetic patients, which might lead to platelet hyperactivity and hyperaggregability. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Type 2 diabetes mellitus; Thrombin; Store-operated calcium entry; Platelets; Antioxidant
Increasing evidence suggests that reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and hydroxyl radicals (OH), and reactive nitrogen species (RNS), such as peroxynitrite (ONOO), are both involved in cellular functions [1–3] and in the pathogenesis of a number of diseases [4–6] depending on the oxidant/ antioxidant equilibrium. In diabetes mellitus, oxidative stress has been reported to impair glucose uptake in muscle cells and adipocytes [7–9] leading to chronic q Abbreviations: ROS, reactive oxygen species; RNS, reactive nitrogen species; SOCE, store-operated calcium entry; [Ca2+]c, cytosolic free calcium concentration; TG, thapsigargin; Iono, ionomycin; HBS, Hepes-buffered saline; SERCA, sarcoendoplasmic reticulum Ca2+ ATPase. * Corresponding author. Fax: +34 927 257154. E-mail address: [email protected] (J.A. Rosado). 1 These authors contributed equally to this work.
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.05.178
hyperglycemia, which, in turn, causes glucotoxic alterations in many cell types, such as pancreatic b cells, resulting in a decrease in insulin content, secretion, and activity [10,11]. In addition, diabetes mellitus leads to several cardiovascular disorders, including angiopathy, which are the major cause of morbidity and mortality in type 2 diabetes mellitus (type 2 DM) [12]. Among the pathological processes involved in the development of micro- and macroangiopathy in diabetic patients, platelet hyperactivity has been presented as the main candidate [13,14]. Platelets from type 2 diabetic patients are hypersensitive and show an increased adhesiveness and spontaneous aggregation [15,16]. Although the intracellular mechanisms involved in the above-mentioned platelet dysfunction remain unclear, recent studies have reported an altered Ca2+ mobilization in platelets from type 2 DM [17,18]. Cytosolic
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free Ca2+ concentration ([Ca2+]c) controls a number of platelet functions, including aggregation. Platelet agonists, such as thrombin, elevate [Ca2+]c by releasing Ca2+ from intracellular stores and allowing Ca2+ entry. In contrast, removal of Ca2+ from the cytosol is mainly mediated by Ca2+ sequestration into the stores and Ca2+ extrusion mostly via the plasma membrane Ca2+-ATPase (PMCA) [19]. We have recently shown that agonist-induced Ca2+ release and entry are significantly enhanced in platelets from type 2 diabetic patients [18]. In addition, Ca2+ extrusion by PMCA is reduced by tyrosine phosphorylation of the pump and a lower PMCA expression [17]. However, the mechanisms responsible for the altered Ca2+ homeostasis in platelets from diabetic patients remain uncertain. In the present study, we describe further progress in the involvement of oxidant in the pathogenesis of platelet-derived cardiovascular disorders associated to type 2 DM. We demonstrate that platelet treatment with antioxidant, including scavengers of H2O2 and ONOO, reverses both the enhanced Ca2+ mobilization observed in these cells and the hyperaggregability described in platelets from diabetic patients, supporting that certain antioxidant may prove useful for therapeutic procedures to reduce cardiovascular complications in diabetic patients.
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340 and 380 nm and emission at 505 nm. Changes in [Ca2+]c were monitored using the fura-2 340/380 fluorescence ratio and calibrated according to the method of Grynkiewicz et al [21]. Ca2+ entry in TG + Iono or thrombin-treated cells was estimated using the integral of the rise in [Ca2+]c for 3 min after addition of CaCl2 [20]. Ca2+ release was estimated using the integral of the rise in [Ca2+]c for 3 min after agonist addition. Platelet aggregation. Platelets prepared as described above were suspended in HBS supplemented with 0.1% w/v bovine serum albumin. Platelet aggregation was monitored in a Chronolog (Havertown, Pa, USA) aggregometer at 37 °C under stirring at 1200 rpm [22]. Intracellular oxidant production in human platelets. CMH2DCFDA is a fluorescent probe that can be used to monitor oxidant production in living cells [23]. It passively diffuses into cells, where its acetate groups are cleaved by intracellular esterases, releasing the corresponding dichlorodihydrofluorescein derivative. CM-H2DCFDA oxidation yields a fluorescent adduct, dichlorofluorescein (DCF) that is trapped inside the cell [24]. Cells were incubated at 37 °C with 10 lM CM-H2DCFDA acetyl ester for 30 min, then centrifuged, and the pellet was resuspended in fresh HBS. Fluorescence was recorded from 2 ml aliquots using a Fluorescence Spectrophotometer (Varian, Madrid, Spain). Samples were excited at 488 nm and the resulting fluorescence was measured at 530 nm. Oxidant production after treatment of cells with different agonists in the absence or presence of antioxidants was quantified as the integral of the rise in DCF fluorescence for 5 min after the addition of the agent. Statistical analysis. Analysis of statistical significance was performed using StudentÕs t test. p < 0.05 was considered to be significant for a difference.
Results Materials and methods Materials. Fura-2 acetoxymethyl ester (fura-2/AM) and 5-(and-6)chloromethyl-2 0 ,7 0 -dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) were from Molecular Probes (Leiden, The Netherlands). Apyrase (grade VII), EGTA, aspirin, bovine serum albumin, thrombin, thapsigargin (TG), ionomycin (Iono), and catalase were from Sigma (Madrid, Spain). Trolox and mannitol were from Calbiochem (Madrid, Spain). All other reagents were purchased from Panreac (Barcelona, Spain). Inactive catalase was denatured by treatment with 3-amino-1,2,4-triazole as previously described [3]. Subjects. Patients with type 2 DM and not showing other pathologies were randomly obtained from normotensive patients of the Clinical Analysis Laboratory, Ca´ceres, Spain. Informed consent was obtained from every subject. Blood was obtained at 9:00 AM, in accordance with the principles of the Declaration of Helsinki, from 12 healthy (control) and 20 diabetic donors. Blood glucose concentration in diabetic patients was in the range of 180–240 mg/dl. The glycosylated Hb levels (HbA1c) were used as an index of metabolic control. Only patients with a level of HbA1c >6% were selected. The control subjects were normal age- and gender-matched healthy people that had HbA1c levels in the normal range (3.5–5%). Platelet preparation. Fura-2-loaded platelets were prepared as described previously [20]. For measurements of [Ca2+]c platelet-rich plasma was incubated at 37 °C with 2 lM fura-2/AM for 45 min. Cells were collected by centrifugation and resuspended in Hepes-buffered saline (HBS) containing (in mM): 145 NaCl, 10 Hepes, 10 D-glucose, 5 KCl, 1 MgSO4, pH 7.45, supplemented with 0.1% w/v bovine serum albumin, and 40 lg/ml apyrase. Measurement of intracellular free calcium concentration ([Ca2+]c). Fluorescence was recorded from 2 ml aliquots of magnetically stirred cell suspensions (108 cells/ml) at 37 °C using a Fluorescence Spectrophotometer (Varian, Madrid, Spain) with excitation wavelengths of
Altered Ca2+ mobilization induced by TG + Iono and thrombin in platelets from type 2 DM and control subjects In a Ca2+-free medium, treatment of platelets with 1 lM TG, a specific inhibitor of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) [25], plus a low concentration of ionomycin (50 nM [3]; required for extensive depletion of the intracellular Ca2+ stores in platelets where two agonist-sensitive stores have been suggested [20]) resulted in a transient increase in [Ca2+]c due to Ca2+ release from the intracellular stores. As we have previously reported [18], the peak response induced by treatment with TG + Iono was not significantly different in both subject groups (146 ± 18 and 154 ± 15 nM in platelets from control and type 2 diabetic subjects, respectively (means ± SEM; Fig. 1A; n = 8– 10)), suggesting that the amount of Ca2+ accumulated in the stores is similar. The subsequent addition of Ca2+ (300 lM) to the external medium induced a sustained elevation in [Ca2+]c indicative of store-operated Ca2+ entry (SOCE). As shown in Fig. 1A, SOCE was significantly higher in platelets from type 2 diabetic patients (155.7% of control; p < 0.05; n = 8–10). Similar results were obtained after platelet stimulation with the physiological agonist thrombin. Treatment with 0.1 U/ml thrombin resulted in a rapid and transient increase in [Ca2+]c. We found that the initial peak [Ca2+]c elevation above basal in platelets from type 2
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Fig. 1. Altered Ca2+ mobilization and aggregation in platelets from diabetic patients. (A,B) Fura-2-loaded human platelets from healthy donors (control) or type 2 diabetic patients (DM 2) were stimulated with TG (1 lM) + Iono (50 nM) (A) or thrombin (0.1 U/ml; B) in a Ca2+-free medium (100 lM EGTA was added). CaCl2 (final concentration 300 lM) was added to the medium 5 min later to initiate Ca2+ entry. Elevations in [Ca2+]c were monitored using the 340/380 nm ratio and traces were calibrated in terms of [Ca2+]c. Traces shown are representative of 8–10 separate experiments. (C) Aggregation of washed human platelets from healthy donors (Control) or diabetic patients (DM 2) was produced in an aggregometer by thrombin (0.1 U/ml), as indicated.
diabetic subjects was significantly greater (302 ± 29 nM) than in controls (231 ± 21 nM; Fig. 1B; p < 0.05; n = 8– 10). In addition, thrombin-induced Ca2+ entry was significantly higher in platelets from type 2 diabetic patients than in their respective controls (142% of control; p < 0.01; n = 8–10). We have further investigated platelet aggregation in type 2 diabetic patients compared to healthy donors. In our experiments, we used thrombin, which is a potent stimulator of platelet aggregation. As depicted in Fig. 1C, treatment of human platelets with 0.1 U/ml thrombin induced an initial shape change and then a rapid aggregation. In platelets from type 2 diabetic patients aggregation occurred faster and the amplitude was greater (85 ± 4% in type 2 diabetic patients and 72 ± 3% in controls; p < 0.01; n = 6). Oxidant generation induced by thrombin or store depletion by TG + Iono in platelets from healthy and type 2 diabetic donors The amount of intracellular oxidant was estimated using CM-H2DCFDA, which is sensitive to H2O2, OH, and ONOO generation. Treatment of human platelets
Fig. 2. Oxidant generation by thrombin and TG + Iono in platelets from diabetic and healthy donors. Human platelets from healthy donors (control) or type 2 diabetic subjects (DM 2) were loaded with CM-H2DCFDA and then stimulated with 1 lM TG + 50 nM Iono (A) or 0.1 U/ml thrombin (B) in a Ca2+-free medium (100 lM EGTA was added). Traces are representative of 5–10 independent experiments.
from healthy donors with 1 lM TG + 50 nM Iono (Fig. 2A; black trace) or with 0.1 U/ml thrombin (Fig. 2B; black trace) resulted in a rapid increase in the DCF fluorescence, reaching a maximum after 112 min of treatment and fluorescence was maintained for at least 5 min. In platelets from type 2 diabetic donors, DCF fluorescence at resting conditions was greater than in normal platelets (Figs. 2A and B, grey traces). In addition, stimulation with TG + Iono or thrombin in a Ca2+-free medium resulted in a significantly greater increase in DCF fluorescence compared to controls (1.7 ± 0.2 a.u. vs 2.4 ± 0.3 a.u. in control and diabetic platelets, respectively, stimulated with TG + Iono, and 2.1 ± 0.2 a.u. vs 2.6 ± 0.2 a.u. in control and diabetic platelets, respectively, stimulated with thrombin; p < 0.05), which reflects that the concentration of oxidant is increased in these cells (Figs. 2A and B, grey traces). Effect of catalase on the activation of TG + Iono- and thrombin-induced Ca2+ mobilization in platelets from diabetic and healthy donors To determine whether H2O2 produced after store depletion using TG + Iono or by thrombin is involved
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in the altered Ca2+ mobilization observed in platelets from type 2 diabetic patients, we examined the effect of catalase. In control platelets, pretreatment for 10 min at 37 °C with 300 U/ml catalase in a Ca2+-free medium had no effect on TG + Iono-induced Ca2+ release in the intracellular stores, suggesting that this treatment did not affect the ability of platelets to store Ca2+ in intracellular compartments; however, catalase significantly reduced the elevation in [Ca2+]c observed after the addition of Ca2+ (300 lM) to the extracellular medium by 34 ± 6% (Fig. 3A; p < 0.05), which indicates that H2O2 is required for SOCE in normal human platelets. When cells were treated with inactive catalase (300 U/ml) no modifications in SOCE were found (data not shown), indicating that catalase does not have non-
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specific effects as a Ca2+ chelator or Ca2+ channel blocker. The effect of catalase on Ca2+ mobilization induced by TG + Iono in diabetic platelets was qualitatively similar; however, the inhibitory effect of catalase on TG + Iono-induced SOCE was greater in platelets from type 2 diabetic subjects (catalase reduced SOCE by 49 ± 8%). As a result, in the presence of catalase 300 U/ml the difference in SOCE induced by TG + Iono between platelets from healthy and diabetic patients was significantly reduced (Fig. 3A vs 3B). Similar results were observed when the effect of catalase on thrombin-induced Ca2+ entry was tested. Treatment of control platelets for 10 min at 37 °C with 300 U/ml catalase in a Ca2+-free medium significantly reduced thrombin-evoked Ca2+ entry by 30 ± 4
Fig. 3. Effect of catalase on Ca2+ mobilization in platelets from diabetic and healthy donors. Fura-2-loaded human platelets from healthy (A,C) or type 2 diabetic donors (B,D) were pretreated with catalase (300 U/ml) for 10 min at 37 °C and then stimulated with TG (1 lM) + Iono (50 nM) (A,B) or thrombin (0.1 U/ml; C,D) in a Ca2+-free medium (100 lM EGTA was added). CaCl2 (final concentration 300 lM) was added to the medium 5 min later to initiate Ca2+ entry. (E) Fura-2-loaded human platelets from healthy donors were treated with 10 lM H2O2 in a medium containing 1 mM Ca2+. Elevations in [Ca2+]c were monitored using the 340/380 nm ratio and traces were calibrated in terms of [Ca2+]c. Traces shown are representative of six to eight separate experiments.
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(p < 0.001; n = 6), without having any effect on thrombin-evoked Ca2+ release (Fig. 3C). In platelets from diabetic donors treatment with catalase reduced both thrombin-evoked Ca2+ release and entry (thrombin-induced Ca2+ release and entry were reduced to 86% and 66%, respectively; Fig. 3D). As reported above for TG + Iono, in the presence of catalase thrombin-induced Ca2+ mobilization was qualitatively similar in platelets from diabetic and healthy subjects (Fig. 3C vs 3D). Therefore, our results suggest that H2O2 is involved in the altered Ca2+ entry observed in platelets from type 2 diabetic patients. Hence, we have further investigated the ability of H2O2 to increase [Ca2+]c in platelets. Treatment of platelets, suspended in a medium containing 1 mM CaCl2, with 10 lM H2O2 resulted in a rapid and sustained increase in [Ca2+]c (Fig. 3E), which clearly
demonstrates that H2O2 is able to mobilize Ca2+ in human platelets, as previously described [3,26]. As observed by the use of catalase, H2O2 generation is necessary for a normal Ca2+ entry in human platelets; the different influx of Ca2+ in platelets from healthy and diabetic donors might be explained by a greater H2O2 production or by an enhanced sensitivity of Ca2+ entry to H2O2 in diabetic platelets. Effect of trolox on TG + Iono- and thrombin-induced Ca2+ mobilization in platelets from diabetic and healthy donors We have further investigated whether ONOO is involved in the abnormal Ca2+ signaling observed in platelets from type 2 diabetic patients by using trolox,
Fig. 4. Effect of trolox on Ca2+ mobilization in platelets from diabetic and healthy donors. Fura-2-loaded human platelets from healthy (A,C) or type 2 diabetic donors (B,D) were pretreated with trolox (1 mM) for 10 min at 37 °C and then stimulated with TG (1 lM) + Iono (50 nM) (A,B) or thrombin (0.1 U/ml; C,D) in a Ca2+-free medium (100 lM EGTA was added). CaCl2 (final concentration 300 lM) was added to the medium 5 min later to initiate Ca2+ entry. (E) Fura-2-loaded human platelets from healthy donors were treated with 10 lM ONOO in a medium containing 1 mM Ca2+. Elevations in [Ca2+]c were monitored using the 340/380 nm ratio and traces were calibrated in terms of [Ca2+]c. Traces shown are representative of six separate experiments.
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a vitamin E analog, which prevents ONOO-mediated oxidative stress [27]. Preincubation of control platelets for 10 min at 37 °C with 1 mM trolox in a Ca2+-free medium did not alter either TG + Iono or thrombin-induced Ca2+ release in the intracellular stores or Ca2+ entry, suggesting that ONOO is not necessary for Ca2+ mobilization in platelets from healthy donors (Figs. 4A and C). In contrast, in platelets from diabetic donors trolox significantly reduced TG + Iono-evoked Ca2+ entry from 155% to 104% of control (healthy subjects) in the absence and presence of trolox (Ca2+ entry in platelets from healthy donors not treated with trolox was considered as 100%; Fig. 4B; p < 0.05; n = 6). In addition, trolox reduced thrombin-stimulated Ca2+ entry from 142% to 88% of control (healthy subjects) in the absence and presence of trolox, respectively (Fig. 4D; p < 0.05; n = 6), which indicates that ONOO is also involved in the enhanced Ca2+ entry observed in platelets from diabetic patients. In order to test this possibility, we have further investigated the ability of ONOO to increase [Ca2+]c in platelets. As shown in Fig. 4E, treatment of platelets, suspended in a medium containing 1 mM CaCl2 with 10 lM ONOO resulted in a rapid and sustained increase in [Ca2+]c. These findings demonstrate that ONOO is able to mobilize Ca2+ in human platelets. Therefore, in addition to the role of H2O2 on the enhanced Ca2+ entry in platelets from type
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2 diabetic patients compared to controls, this event also depends on the level of ONOO production, which, according to the results obtained with trolox, might be negligible in control platelets and more elevated in platelets from diabetic patients. Mannitol did not alter TG + Iono- and thrombin-induced Ca2+ mobilization in platelets from diabetic and healthy donors Mannitol is a selective scavenger of OH over ONOO and other ROS [28]. In order to investigate whether OH is also involved in the abnormal Ca2+ homeostasis observed in platelets from type 2 diabetic patients, cells were preincubated with 5 mM mannitol for 10 min at 37 °C. As shown in Fig. 5, treatment with mannitol did not modify either TG + Iono- or thrombin-induced Ca2+ release or entry, both in diabetic and control platelets. These observations suggest that OH is not involved in the abnormal Ca2+ mobilization observed in platelets from type 2 diabetic patients. Effect of oxidant scavengers on platelet aggregation in platelets from type 2 diabetic donors Fig. 6 shows that treatment of platelets from type 2 diabetic donors with 0.1 U/ml thrombin induced rapid
Fig. 5. Effect of mannitol on Ca2+ mobilization in platelets from diabetic and healthy donors. Fura-2-loaded human platelets from healthy (A,C) or type 2 diabetic donors (B,D) were pretreated with mannitol (5 mM) for 10 min at 37 °C and then stimulated with TG (1 lM) + Iono (50 nM) (A,B) or thrombin (0.1 U/ml; C,D) in a Ca2+-free medium (100 lM EGTA was added). CaCl2 (final concentration 300 lM) was added to the medium 5 min later to initiate Ca2+ entry. Elevations in [Ca2+]c were monitored using the 340/380 nm ratio and traces were calibrated in terms of [Ca2+]c. Traces shown are representative of six separate experiments.
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Fig. 6. Effect of catalase, mannitol, and trolox on aggregation in platelets from diabetic patients. Aggregation of washed human platelets from type 2 diabetic patients was induced for 3 min at a shear rate of 1200 rpm following preincubation for 10 min at 37 °C with mannitol (5 mM), catalase (300 U/ml), trolox (1 mM), or the vehicle (control). Aggregation was produced in an aggregometer by thrombin (0.1 U/ml) added, as indicated by the arrows.
aggregation with an amplitude of 84 ± 4% and an average slope of 68 ± 4% light transmission/min. In our experimental conditions, the OH scavenger mannitol (5 mM) failed to inhibit platelet aggregation by thrombin. In contrast, pretreatment of platelets with catalase (300 U/ml), which decomposes H2O2, significantly reduced both the amplitude (69 ± 5%; p < 0.05) and the average slope (54 ± 4% light transmission/min; p < 0.05) of thrombin-induced aggregation. Similarly, the ONOO scavenger trolox (1 mM) reduced thrombin-induced aggregation (in the presence of trolox thrombin-activated aggregation presented amplitude of 67 ± 5% and an average slope of 58 ± 4% light transmission/min; p < 0.05; n = 4). Thrombin-induced aggregation in platelets from type 2 diabetic subjects in the presence of catalase or trolox was similar to that observed in platelets from healthy donors not treated with inhibitors (Fig. 6 vs 1C).
Discussion Increasing evidence suggests that oxidant such as H2O2 and ONOO are involved in a wide range of both physiological and pathological processes. Several sources of ROS and RNS have been suggested in platelets and other cells, including the NADH/NADPH oxidase, superoxide-dismutase, the activation of arachidonic acid metabolism, and the metabolism of phosphoinositides [29–31]. Oxidant play major roles in the initiation and progression of cardiovascular dysfunctions associated with diseases such as diabetes mellitus [32,33]. The majority of ischemic coronary and cerebrovascular
events in diabetic patients are precipitated by vessel occlusion caused by atherosclerotic plaque disruption, platelet hyperaggregability and adhesion, resulting in intravascular thrombosis [32,34]. Although the mechanisms underlying platelet dysfunction remain unclear, an altered cellular Ca2+ homeostasis has been presented as a candidate to mediate platelet function disorders [17,18,23,35]. [Ca2+]c is an important factor involved in the regulation of a large number of cellular functions, including platelet aggregation [19]. Platelets and other cells increase [Ca2+]c by two mechanisms: Ca2+ release from intracellular compartments and Ca2+ entry across the plasma membrane, a process that is required for full platelet activation [19]. Non-excitable cells, such as platelets, lack voltage-activated Ca2+ channels, and the major mechanism for Ca2+ influx is store-operated Ca2+ entry (SOCE), which is regulated by the filling state of the intracellular Ca2+ stores [19,36]. Our results show that SOCE, stimulated by the physiological agonist thrombin or by direct depletion of the stores using TG + Iono, in platelets from type 2 diabetic patients is significantly higher than in healthy controls, which is the most remarkable difference observed in Ca2+ mobilization between both types of donors. Our results are in agreement with previous studies reporting an abnormal Ca2+ mobilization in platelets from patients with type 2 DM [17,18,23,37]. Since the extracellular medium is the major source of Ca2+, SOCE dysfunction is of great relevance in platelet function. Recent studies have presented evidence for the involvement of ROS in the activation of SOCE in different cell types, including platelets [3,38]. Hence, we looked for an increase in oxidant production in platelets from type 2 diabetic patients. The results presented here demonstrate that both resting and stimulated oxidant productions are elevated in platelets from diabetic patients compared to those in controls. The parallels between these two processes suggest that the increased oxidant generation might be involved in the elevated SOCE in diabetic platelets. To explore this possibility, we use scavengers of different ROS and RNS, such as catalase, which decomposes H2O2 into water and oxygen [24], trolox, a ONOO scavenger [27], and mannitol, which was directed to remove OH [28]. Our results indicate that catalase reduced SOCE in both healthy and diabetic platelets, which is in agreement with the reported role of H2O2 in the activation of SOCE (Fig. 3E) [3,38]. However, catalase was more effective inhibiting SOCE in platelets from patients with type 2 DM, as a consequence, the difference observed in SOCE in platelets from healthy and diabetic donors was significantly reduced in the presence of catalase. These findings clearly indicate that H2O2 is involved in the enhanced Ca2+ entry observed in platelets from type 2 diabetic patients. H2O2-dependent enhanced Ca2+ entry in diabetic
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donors might be explained by an increased amount of H2O2 or a greater sensitivity to oxidation by this ROS in diabetic platelets. The former might be due to an enhanced production of H2O2 or to reduced antioxidant levels [32] in platelets from type 2 diabetic platelets. Similar results were obtained with trolox, which had no effect on Ca2+ mobilization in control platelets. This finding suggests that ONOO is not involved in the physiological mechanisms involved in Ca2+ homeostasis. The lack of effect of trolox makes easier the interpretation of the results obtained in diabetic platelets, where trolox reduced SOCE reaching a level that was similar to that found in control platelets (see Figs. 4C and D). Our results indicate that ONOO is able to elevate [Ca2+]c in control platelets, therefore, the lack of effect of trolox on SOCE in platelets from healthy donors indicates that ONOO is not produced after treatment with TG + Iono or thrombin. The inhibitory effect of trolox on TG + Iono- or thrombin-induced Ca2+ entry in platelets from diabetic donors is likely to be mediated by an increased ONOO production in these cells. Interestingly, treatment of human platelets from healthy or diabetic donors with mannitol, which is more specific for OH [28], did not modify Ca2+ mobilization induced by TG + Iono or thrombin, suggesting that OH is not involved in the altered Ca2+ homeostasis observed in platelets from type 2 diabetic donors. Catalase and trolox almost completely reverse the differences in Ca2+ entry observed in platelets from diabetic and healthy donors, suggesting that both H2O2 and ONOO might be components of the same intracellular cascade inducing these effects; in fact, H2O2 represents the common final path of all oxidant in the cells [39]. We have recently reported that pp60src phosphorylation and then its activity is increased in platelets from diabetic donors, an event that may be responsible for the enhanced Ca2+ entry in these cells [18]. The results presented here are consistent with this study since oxidative stress has been shown to reduce protein tyrosine phosphatase activity [40], a process that is likely to be mediated by S-nitrosylation [41]. Since Ca2+ entry is essential for aggregation, we have investigated whether the enhanced oxidant production is also involved in platelet hyperaggregability in diabetic individuals. We have found that thrombin-induced aggregation is significantly increased in platelets from patients with type 2 DM, an effect that was reduced by treatment with catalase and trolox. In contrast, mannitol did not significantly modify this response. These findings are consistent with the results reported above and suggest that a reduction in the influx of Ca2+ is reflected in a reduction in platelet hyperaggregability. Our results indicate for the first time that the oxidant scavengers, catalase and the vitamin E analog, trolox, reverse the enhanced Ca2+ entry and hyperaggregability observed in platelets from type 2 diabetic donors com-
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pared with controls. Platelet function is altered in several ways in patients with diabetes mellitus, including an increased release of TXA2 [42], accelerated platelet turnover [43], and an increase in platelet aggregation [44]. Platelet dysfunction in type 2 DM might be mediated by an enhanced level of cytosolic oxidant, specially H2O2 and ONOO, which is likely to be due to an increased production or a reduced antioxidant capacity. The processes that maintain the integrity of the cardiovascular system are impaired in diabetes mellitus, including platelet and endothelial function, coagulation, and fibrinolysis [32]. Thus, the balance in hemostasis is shifted to favor thrombosis, increasing cardiovascular risk. The present study provides evidence that antioxidants, such as vitamin E, might be used as an antiplatelet therapy in order to reduce the risk of ischemic events in diabetic patients.
Acknowledgments This work was supported by Junta de ExtremaduraConsejerı´a de Sanidad y Consumo Grant SCSS0405 and DGI-MEC Grant BFU2004-00165. P.C.R. is supported by a DGESIC fellowship (BFI2001-0624). We thank Mercedes Go´mez Bla´zquez for her technical assistance.
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P.C. Redondo et al. / Biochemical and Biophysical Research Communications 333 (2005) 794–802 GLUT4 translocation in 3T3-L1 adipocytes, Diabetes 47 (1998) 1562–1569. R.C. Cooksey, H.A. Jouihan, R.S. Ajioka, M.W. Hazel, D.L. Jones, J.P. Kushner, D.A. McClain, Oxidative stress, beta-cell apoptosis, and decreased insulin secretory capacity in mouse models of hemochromatosis, Endocrinology 145 (2004) 5305– 5312. R.P. Robertson, H.J. Zhang, K.L. Pyzdrowski, T.F. Walseth, Preservation of insulin mRNA levels and insulin secretion in HIT cells by avoidance of chronic exposure to high glucose concentrations, J. Clin. Invest. 90 (1992) 320–325. C. Cimminiello, M. Milani, Diabetes mellitus and peripheral vascular disease: is aspirin effective in preventing vascular events? Diabetologia 39 (1996) 1402–1404. P.J. Guillausseau, E. Dupuy, C. Guillausseau, A. Warnet, J. Caen, J. Lubetzki, Hemostasis disorders in diabetes mellitus, Horm. Metab. Res. Suppl. 15 (1985) 60–62. A.B. Sobol, C. Watala, The role of platelets in diabetes-related vascular complications, Diab. Res. Clin. Pract. 50 (2000) 1–16. H. Knobler, N. Savion, B. Shenkman, S. Kotev-Emeth, D. Varon, Shear-induced platelet adhesion and aggregation on subendothelium are increased in diabetic patients, Thromb. Res. 90 (1998) 181–190. A.I. Vinik, T. Erbas, T.S. Park, R. Nolan, G.L. Pittenger, Platelet dysfunction in type 2 diabetes, Diab. Care 24 (2001) 1476–1485. J.A. Rosado, F.R. Saavedra, P.C. Redondo, J.M. HernandezCruz, G.M. Salido, J.A. Pariente, Reduced plasma membrane Ca2+-ATPase function in platelets from patients with non-insulindependent diabetes mellitus, Haematologica 89 (2004) 1142–1144. F.R. Saavedra, P.C. Redondo, J.M. Hernandez-Cruz, G.M. Salido, J.A. Pariente, J.A. Rosado, Store-operated Ca2+ entry and tyrosine kinase pp60src hyperactivity are modulated by hyperglycemia in platelets from patients with non insulin-dependent diabetes mellitus, Arch. Biochem. Biophys. 432 (2004) 261– 268. T.J. Rink, S.O. Sage, Calcium signaling in human platelets, Annu. Rev. Physiol. 52 (1990) 431–449. J.A. Rosado, J.J. Lopez, A.G. Harper, M.T. Harper, P.C. Redondo, J.A. Pariente, S.O. Sage, G.M. Salido, Two pathways for store-mediated calcium entry differentially dependent on the actin cytoskeleton in human platelets, J. Biol. Chem. 279 (2004) 29231–29235. G. Grynkiewicz, M. Poenie, R.Y. Tsien, A new generation of Ca2+ indicators with greatly improved fluorescence properties, J. Biol. Chem. 260 (1985) 3440–3450. J.W.M. Heemskerk, R.W. Farndale, S.O. Sage, Effects of U73122 and U73343 on human platelet calcium signaling and protein tyrosine phosphorylation, Biochim. Biophys. Acta 1355 (1997) 81–88. Y. Li, V. Woo, R. Bose, Platelet hyperactivity and abnormal Ca2+ homeostasis in diabetes mellitus, Am. J. Physiol. Heart Circ. Physiol. 280 (2001) H1480–H1489. Y. Luo, H. Umegaki, X. Wang, R. Abe, G.S. Roth, Dopamine induces apoptosis through an oxidation-involved SAPK/JNK activation pathway, J. Biol. Chem. 273 (1998) 3756–3764. O. Thastrup, A.P. Dawson, O. Scharff, B. Foder, P.J. Cullen, B.K. Drobak, P.J. Bjerrum, S.B. Christensen, M.R. Hanley, Thapsigargin, a novel molecular probe for studying intracellular calcium release and storage, Agents Actions 27 (1989) 17–23.
[26] P.C. Redondo, G.M. Salido, J.A. Pariente, J.A. Rosado, Dual effect of hydrogen peroxide on store-mediated calcium entry in human platelets, Biochem. Pharmacol. 67 (2004) 1065–1076. [27] M.G. Salgo, W.A. Pryor, Trolox inhibits peroxynitrite-mediated oxidative stress and apoptosis in rat thymocytes, Arch. Biochem. Biophys. 333 (1996) 482–488. [28] R. Leo, D. Pratico, L. Iuliano, F.M. Pulcinelli, A. Ghiselli, P. Pignatelli, A.R. Colavita, G.A. FitzGerald, F. Violi, Platelet activation by superoxide anion and hydroxyl radicals intrinsically generated by platelets that had undergone anoxia and then reoxygenated, Circulation 95 (1997) 885–891. [29] L. Iuliano, A.R. Colavita, R. Leo, D. Practico, F. Violi, Oxygen free radicals and platelet activation, Free Radic. Biol. Med. 22 (1997) 999–1006. [30] T. Seno, N. Inoue, D. Gao, M. Okuda, Y. Sumi, K. Matsui, S. Yamada, K. Hirata, S. Kawashima, R. Tawa, S. Imajoh-Ohmi, H. Sakurai, M. Yokoyama, Involvement of NADH/NADPH oxidase in human platelet ROS production, Thromb. Res. 103 (2001) 399–409. [31] B. Wachowicz, B. Olas, H.M. Zbikowska, A. Buczynski, Generation of reactive oxygen species in blood platelets, Platelets 13 (2002) 175–182. [32] J.A. Colwell, R.W. Nesto, The platelet in diabetes: focus on prevention of ischemic events, Diab. Care 26 (2003) 2181–2188. [33] Y. Taniyama, K.K. Griendling, Reactive oxygen species in the vasculature: molecular and cellular mechanisms, Hypertension 42 (2003) 1075–1081. [34] H. Hu, P. Hjemdahl, N. Li, Effects of insulin on platelet and leukocyte activity in whole blood, Thromb. Res. 107 (2002) 209– 215. [35] J. Levy, Abnormal cell calcium homeostasis in type 2 diabetes mellitus: a new look on old disease, Endocrine 10 (1999) 1–6. [36] J.A. Rosado, S.O. Sage, The actin cytoskeleton in store-mediated calcium entry, J. Physiol. 526 (2000) 221–229. [37] T. Yamaguchi, K. Kadono, T. Tetsutani, K. Yasunaga, Platelet free Ca2+ concentration in non-insulin-dependent diabetes mellitus, Diab. Res. 18 (1991) 89–94. [38] Y. Suzuki, T. Yoshimaru, T. Matsui, T. Inoue, O. Niide, S. Nunomura, C. Ra, Fc epsilon RI signaling of mast cells activates intracellular production of hydrogen peroxide: role in the regulation of calcium signals, J. Immunol. 171 (2003) 6119–6127. [39] C. Gonzalez, G. Sanz-Alfayate, A. Obeso, M.T. Agapito, Role of glutathione redox state in oxygen sensing by carotid body chemoreceptor cells, Methods Enzymol. 381 (2004) 40–71. [40] G. Poli, G. Leonarduzzi, F. Biasi, E. Chiarpotto, Oxidative stress and cell signaling, Curr. Med. Chem. 11 (2004) 1163–1182. [41] D.M. Barrett, S.M. Black, H. Todor, R.K. Schmidt-Ullrich, K.S. Dawson, R.B. Mikkelsen, Inhibition of protein tyrosine phosphatases by mild oxidative stresses is dependent on S-nitrosylation, J. Biol. Chem. 280 (2005) 14453–14461. [42] P.V. Halushka, R.C. Rogers, C.B. Loadholt, J.A. Colwell, Increased platelet thromboxane synthesis in diabetes mellitus, J. Lab. Clin. Med. 97 (1981) 87–96. [43] P.D. Winocour, M. Laimins, J.A. Colwell, Platelet survival in streptozotocin-induced diabetic rats, Thromb. Haemost. 51 (1984) 307–312. [44] J. Sagel, J.A. Colwell, L. Crook, M. Laimins, Increased platelet aggregation in early diabetes mellitus, Ann. Intern. Med. 82 (1975) 733–738.
Hydrogen peroxide and peroxynitrite enhance Ca2+ mobilization and aggregation in platelets from type 2 diabetic patients q Pedro C. Redondo a,1, Isaac Jardin a,1, Juan M. Herna´ndez-Cruz b, Jose´ A. Pariente a, Gine´s M. Salido a, Juan A. Rosado a,* a
Department of Physiology, University of Extremadura, Ca´ceres, Spain b Clinical Analysis Laboratory, Ca´ceres, Spain Received 25 May 2005 Available online 13 June 2005
Abstract Cytosolic Ca2+ mobilization, especially Ca2+ entry, is enhanced in platelets from type 2 diabetic individuals, which might result in platelet hyperaggregability. In the present study, we report an increased oxidant production in resting and stimulated platelets from diabetic donors. Pretreatment of platelets with catalase or trolox, an analog of vitamin E, reversed the enhanced Ca2+ entry, evoked by thapsigargin plus ionomycin or thrombin, observed in platelets from diabetic subjects, so that in the presence of these scavengers Ca2+ entry was similar in platelets from healthy and diabetic subjects. In contrast, mannitol was without effect on Ca2+ mobilization. Catalase and trolox reduced thrombin-induced aggregation in platelets from type 2 diabetic subjects, while mannitol did not modify thrombin-induced platelet hyperaggregability. We conclude that H2O2 and ONOO are likely involved in the enhanced Ca2+ mobilization observed in platelets from type 2 diabetic patients, which might lead to platelet hyperactivity and hyperaggregability. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Type 2 diabetes mellitus; Thrombin; Store-operated calcium entry; Platelets; Antioxidant
Increasing evidence suggests that reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and hydroxyl radicals (OH), and reactive nitrogen species (RNS), such as peroxynitrite (ONOO), are both involved in cellular functions [1–3] and in the pathogenesis of a number of diseases [4–6] depending on the oxidant/ antioxidant equilibrium. In diabetes mellitus, oxidative stress has been reported to impair glucose uptake in muscle cells and adipocytes [7–9] leading to chronic q Abbreviations: ROS, reactive oxygen species; RNS, reactive nitrogen species; SOCE, store-operated calcium entry; [Ca2+]c, cytosolic free calcium concentration; TG, thapsigargin; Iono, ionomycin; HBS, Hepes-buffered saline; SERCA, sarcoendoplasmic reticulum Ca2+ ATPase. * Corresponding author. Fax: +34 927 257154. E-mail address: [email protected] (J.A. Rosado). 1 These authors contributed equally to this work.
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.05.178
hyperglycemia, which, in turn, causes glucotoxic alterations in many cell types, such as pancreatic b cells, resulting in a decrease in insulin content, secretion, and activity [10,11]. In addition, diabetes mellitus leads to several cardiovascular disorders, including angiopathy, which are the major cause of morbidity and mortality in type 2 diabetes mellitus (type 2 DM) [12]. Among the pathological processes involved in the development of micro- and macroangiopathy in diabetic patients, platelet hyperactivity has been presented as the main candidate [13,14]. Platelets from type 2 diabetic patients are hypersensitive and show an increased adhesiveness and spontaneous aggregation [15,16]. Although the intracellular mechanisms involved in the above-mentioned platelet dysfunction remain unclear, recent studies have reported an altered Ca2+ mobilization in platelets from type 2 DM [17,18]. Cytosolic
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free Ca2+ concentration ([Ca2+]c) controls a number of platelet functions, including aggregation. Platelet agonists, such as thrombin, elevate [Ca2+]c by releasing Ca2+ from intracellular stores and allowing Ca2+ entry. In contrast, removal of Ca2+ from the cytosol is mainly mediated by Ca2+ sequestration into the stores and Ca2+ extrusion mostly via the plasma membrane Ca2+-ATPase (PMCA) [19]. We have recently shown that agonist-induced Ca2+ release and entry are significantly enhanced in platelets from type 2 diabetic patients [18]. In addition, Ca2+ extrusion by PMCA is reduced by tyrosine phosphorylation of the pump and a lower PMCA expression [17]. However, the mechanisms responsible for the altered Ca2+ homeostasis in platelets from diabetic patients remain uncertain. In the present study, we describe further progress in the involvement of oxidant in the pathogenesis of platelet-derived cardiovascular disorders associated to type 2 DM. We demonstrate that platelet treatment with antioxidant, including scavengers of H2O2 and ONOO, reverses both the enhanced Ca2+ mobilization observed in these cells and the hyperaggregability described in platelets from diabetic patients, supporting that certain antioxidant may prove useful for therapeutic procedures to reduce cardiovascular complications in diabetic patients.
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340 and 380 nm and emission at 505 nm. Changes in [Ca2+]c were monitored using the fura-2 340/380 fluorescence ratio and calibrated according to the method of Grynkiewicz et al [21]. Ca2+ entry in TG + Iono or thrombin-treated cells was estimated using the integral of the rise in [Ca2+]c for 3 min after addition of CaCl2 [20]. Ca2+ release was estimated using the integral of the rise in [Ca2+]c for 3 min after agonist addition. Platelet aggregation. Platelets prepared as described above were suspended in HBS supplemented with 0.1% w/v bovine serum albumin. Platelet aggregation was monitored in a Chronolog (Havertown, Pa, USA) aggregometer at 37 °C under stirring at 1200 rpm [22]. Intracellular oxidant production in human platelets. CMH2DCFDA is a fluorescent probe that can be used to monitor oxidant production in living cells [23]. It passively diffuses into cells, where its acetate groups are cleaved by intracellular esterases, releasing the corresponding dichlorodihydrofluorescein derivative. CM-H2DCFDA oxidation yields a fluorescent adduct, dichlorofluorescein (DCF) that is trapped inside the cell [24]. Cells were incubated at 37 °C with 10 lM CM-H2DCFDA acetyl ester for 30 min, then centrifuged, and the pellet was resuspended in fresh HBS. Fluorescence was recorded from 2 ml aliquots using a Fluorescence Spectrophotometer (Varian, Madrid, Spain). Samples were excited at 488 nm and the resulting fluorescence was measured at 530 nm. Oxidant production after treatment of cells with different agonists in the absence or presence of antioxidants was quantified as the integral of the rise in DCF fluorescence for 5 min after the addition of the agent. Statistical analysis. Analysis of statistical significance was performed using StudentÕs t test. p < 0.05 was considered to be significant for a difference.
Results Materials and methods Materials. Fura-2 acetoxymethyl ester (fura-2/AM) and 5-(and-6)chloromethyl-2 0 ,7 0 -dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) were from Molecular Probes (Leiden, The Netherlands). Apyrase (grade VII), EGTA, aspirin, bovine serum albumin, thrombin, thapsigargin (TG), ionomycin (Iono), and catalase were from Sigma (Madrid, Spain). Trolox and mannitol were from Calbiochem (Madrid, Spain). All other reagents were purchased from Panreac (Barcelona, Spain). Inactive catalase was denatured by treatment with 3-amino-1,2,4-triazole as previously described [3]. Subjects. Patients with type 2 DM and not showing other pathologies were randomly obtained from normotensive patients of the Clinical Analysis Laboratory, Ca´ceres, Spain. Informed consent was obtained from every subject. Blood was obtained at 9:00 AM, in accordance with the principles of the Declaration of Helsinki, from 12 healthy (control) and 20 diabetic donors. Blood glucose concentration in diabetic patients was in the range of 180–240 mg/dl. The glycosylated Hb levels (HbA1c) were used as an index of metabolic control. Only patients with a level of HbA1c >6% were selected. The control subjects were normal age- and gender-matched healthy people that had HbA1c levels in the normal range (3.5–5%). Platelet preparation. Fura-2-loaded platelets were prepared as described previously [20]. For measurements of [Ca2+]c platelet-rich plasma was incubated at 37 °C with 2 lM fura-2/AM for 45 min. Cells were collected by centrifugation and resuspended in Hepes-buffered saline (HBS) containing (in mM): 145 NaCl, 10 Hepes, 10 D-glucose, 5 KCl, 1 MgSO4, pH 7.45, supplemented with 0.1% w/v bovine serum albumin, and 40 lg/ml apyrase. Measurement of intracellular free calcium concentration ([Ca2+]c). Fluorescence was recorded from 2 ml aliquots of magnetically stirred cell suspensions (108 cells/ml) at 37 °C using a Fluorescence Spectrophotometer (Varian, Madrid, Spain) with excitation wavelengths of
Altered Ca2+ mobilization induced by TG + Iono and thrombin in platelets from type 2 DM and control subjects In a Ca2+-free medium, treatment of platelets with 1 lM TG, a specific inhibitor of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) [25], plus a low concentration of ionomycin (50 nM [3]; required for extensive depletion of the intracellular Ca2+ stores in platelets where two agonist-sensitive stores have been suggested [20]) resulted in a transient increase in [Ca2+]c due to Ca2+ release from the intracellular stores. As we have previously reported [18], the peak response induced by treatment with TG + Iono was not significantly different in both subject groups (146 ± 18 and 154 ± 15 nM in platelets from control and type 2 diabetic subjects, respectively (means ± SEM; Fig. 1A; n = 8– 10)), suggesting that the amount of Ca2+ accumulated in the stores is similar. The subsequent addition of Ca2+ (300 lM) to the external medium induced a sustained elevation in [Ca2+]c indicative of store-operated Ca2+ entry (SOCE). As shown in Fig. 1A, SOCE was significantly higher in platelets from type 2 diabetic patients (155.7% of control; p < 0.05; n = 8–10). Similar results were obtained after platelet stimulation with the physiological agonist thrombin. Treatment with 0.1 U/ml thrombin resulted in a rapid and transient increase in [Ca2+]c. We found that the initial peak [Ca2+]c elevation above basal in platelets from type 2
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Fig. 1. Altered Ca2+ mobilization and aggregation in platelets from diabetic patients. (A,B) Fura-2-loaded human platelets from healthy donors (control) or type 2 diabetic patients (DM 2) were stimulated with TG (1 lM) + Iono (50 nM) (A) or thrombin (0.1 U/ml; B) in a Ca2+-free medium (100 lM EGTA was added). CaCl2 (final concentration 300 lM) was added to the medium 5 min later to initiate Ca2+ entry. Elevations in [Ca2+]c were monitored using the 340/380 nm ratio and traces were calibrated in terms of [Ca2+]c. Traces shown are representative of 8–10 separate experiments. (C) Aggregation of washed human platelets from healthy donors (Control) or diabetic patients (DM 2) was produced in an aggregometer by thrombin (0.1 U/ml), as indicated.
diabetic subjects was significantly greater (302 ± 29 nM) than in controls (231 ± 21 nM; Fig. 1B; p < 0.05; n = 8– 10). In addition, thrombin-induced Ca2+ entry was significantly higher in platelets from type 2 diabetic patients than in their respective controls (142% of control; p < 0.01; n = 8–10). We have further investigated platelet aggregation in type 2 diabetic patients compared to healthy donors. In our experiments, we used thrombin, which is a potent stimulator of platelet aggregation. As depicted in Fig. 1C, treatment of human platelets with 0.1 U/ml thrombin induced an initial shape change and then a rapid aggregation. In platelets from type 2 diabetic patients aggregation occurred faster and the amplitude was greater (85 ± 4% in type 2 diabetic patients and 72 ± 3% in controls; p < 0.01; n = 6). Oxidant generation induced by thrombin or store depletion by TG + Iono in platelets from healthy and type 2 diabetic donors The amount of intracellular oxidant was estimated using CM-H2DCFDA, which is sensitive to H2O2, OH, and ONOO generation. Treatment of human platelets
Fig. 2. Oxidant generation by thrombin and TG + Iono in platelets from diabetic and healthy donors. Human platelets from healthy donors (control) or type 2 diabetic subjects (DM 2) were loaded with CM-H2DCFDA and then stimulated with 1 lM TG + 50 nM Iono (A) or 0.1 U/ml thrombin (B) in a Ca2+-free medium (100 lM EGTA was added). Traces are representative of 5–10 independent experiments.
from healthy donors with 1 lM TG + 50 nM Iono (Fig. 2A; black trace) or with 0.1 U/ml thrombin (Fig. 2B; black trace) resulted in a rapid increase in the DCF fluorescence, reaching a maximum after 112 min of treatment and fluorescence was maintained for at least 5 min. In platelets from type 2 diabetic donors, DCF fluorescence at resting conditions was greater than in normal platelets (Figs. 2A and B, grey traces). In addition, stimulation with TG + Iono or thrombin in a Ca2+-free medium resulted in a significantly greater increase in DCF fluorescence compared to controls (1.7 ± 0.2 a.u. vs 2.4 ± 0.3 a.u. in control and diabetic platelets, respectively, stimulated with TG + Iono, and 2.1 ± 0.2 a.u. vs 2.6 ± 0.2 a.u. in control and diabetic platelets, respectively, stimulated with thrombin; p < 0.05), which reflects that the concentration of oxidant is increased in these cells (Figs. 2A and B, grey traces). Effect of catalase on the activation of TG + Iono- and thrombin-induced Ca2+ mobilization in platelets from diabetic and healthy donors To determine whether H2O2 produced after store depletion using TG + Iono or by thrombin is involved
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in the altered Ca2+ mobilization observed in platelets from type 2 diabetic patients, we examined the effect of catalase. In control platelets, pretreatment for 10 min at 37 °C with 300 U/ml catalase in a Ca2+-free medium had no effect on TG + Iono-induced Ca2+ release in the intracellular stores, suggesting that this treatment did not affect the ability of platelets to store Ca2+ in intracellular compartments; however, catalase significantly reduced the elevation in [Ca2+]c observed after the addition of Ca2+ (300 lM) to the extracellular medium by 34 ± 6% (Fig. 3A; p < 0.05), which indicates that H2O2 is required for SOCE in normal human platelets. When cells were treated with inactive catalase (300 U/ml) no modifications in SOCE were found (data not shown), indicating that catalase does not have non-
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specific effects as a Ca2+ chelator or Ca2+ channel blocker. The effect of catalase on Ca2+ mobilization induced by TG + Iono in diabetic platelets was qualitatively similar; however, the inhibitory effect of catalase on TG + Iono-induced SOCE was greater in platelets from type 2 diabetic subjects (catalase reduced SOCE by 49 ± 8%). As a result, in the presence of catalase 300 U/ml the difference in SOCE induced by TG + Iono between platelets from healthy and diabetic patients was significantly reduced (Fig. 3A vs 3B). Similar results were observed when the effect of catalase on thrombin-induced Ca2+ entry was tested. Treatment of control platelets for 10 min at 37 °C with 300 U/ml catalase in a Ca2+-free medium significantly reduced thrombin-evoked Ca2+ entry by 30 ± 4
Fig. 3. Effect of catalase on Ca2+ mobilization in platelets from diabetic and healthy donors. Fura-2-loaded human platelets from healthy (A,C) or type 2 diabetic donors (B,D) were pretreated with catalase (300 U/ml) for 10 min at 37 °C and then stimulated with TG (1 lM) + Iono (50 nM) (A,B) or thrombin (0.1 U/ml; C,D) in a Ca2+-free medium (100 lM EGTA was added). CaCl2 (final concentration 300 lM) was added to the medium 5 min later to initiate Ca2+ entry. (E) Fura-2-loaded human platelets from healthy donors were treated with 10 lM H2O2 in a medium containing 1 mM Ca2+. Elevations in [Ca2+]c were monitored using the 340/380 nm ratio and traces were calibrated in terms of [Ca2+]c. Traces shown are representative of six to eight separate experiments.
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(p < 0.001; n = 6), without having any effect on thrombin-evoked Ca2+ release (Fig. 3C). In platelets from diabetic donors treatment with catalase reduced both thrombin-evoked Ca2+ release and entry (thrombin-induced Ca2+ release and entry were reduced to 86% and 66%, respectively; Fig. 3D). As reported above for TG + Iono, in the presence of catalase thrombin-induced Ca2+ mobilization was qualitatively similar in platelets from diabetic and healthy subjects (Fig. 3C vs 3D). Therefore, our results suggest that H2O2 is involved in the altered Ca2+ entry observed in platelets from type 2 diabetic patients. Hence, we have further investigated the ability of H2O2 to increase [Ca2+]c in platelets. Treatment of platelets, suspended in a medium containing 1 mM CaCl2, with 10 lM H2O2 resulted in a rapid and sustained increase in [Ca2+]c (Fig. 3E), which clearly
demonstrates that H2O2 is able to mobilize Ca2+ in human platelets, as previously described [3,26]. As observed by the use of catalase, H2O2 generation is necessary for a normal Ca2+ entry in human platelets; the different influx of Ca2+ in platelets from healthy and diabetic donors might be explained by a greater H2O2 production or by an enhanced sensitivity of Ca2+ entry to H2O2 in diabetic platelets. Effect of trolox on TG + Iono- and thrombin-induced Ca2+ mobilization in platelets from diabetic and healthy donors We have further investigated whether ONOO is involved in the abnormal Ca2+ signaling observed in platelets from type 2 diabetic patients by using trolox,
Fig. 4. Effect of trolox on Ca2+ mobilization in platelets from diabetic and healthy donors. Fura-2-loaded human platelets from healthy (A,C) or type 2 diabetic donors (B,D) were pretreated with trolox (1 mM) for 10 min at 37 °C and then stimulated with TG (1 lM) + Iono (50 nM) (A,B) or thrombin (0.1 U/ml; C,D) in a Ca2+-free medium (100 lM EGTA was added). CaCl2 (final concentration 300 lM) was added to the medium 5 min later to initiate Ca2+ entry. (E) Fura-2-loaded human platelets from healthy donors were treated with 10 lM ONOO in a medium containing 1 mM Ca2+. Elevations in [Ca2+]c were monitored using the 340/380 nm ratio and traces were calibrated in terms of [Ca2+]c. Traces shown are representative of six separate experiments.
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a vitamin E analog, which prevents ONOO-mediated oxidative stress [27]. Preincubation of control platelets for 10 min at 37 °C with 1 mM trolox in a Ca2+-free medium did not alter either TG + Iono or thrombin-induced Ca2+ release in the intracellular stores or Ca2+ entry, suggesting that ONOO is not necessary for Ca2+ mobilization in platelets from healthy donors (Figs. 4A and C). In contrast, in platelets from diabetic donors trolox significantly reduced TG + Iono-evoked Ca2+ entry from 155% to 104% of control (healthy subjects) in the absence and presence of trolox (Ca2+ entry in platelets from healthy donors not treated with trolox was considered as 100%; Fig. 4B; p < 0.05; n = 6). In addition, trolox reduced thrombin-stimulated Ca2+ entry from 142% to 88% of control (healthy subjects) in the absence and presence of trolox, respectively (Fig. 4D; p < 0.05; n = 6), which indicates that ONOO is also involved in the enhanced Ca2+ entry observed in platelets from diabetic patients. In order to test this possibility, we have further investigated the ability of ONOO to increase [Ca2+]c in platelets. As shown in Fig. 4E, treatment of platelets, suspended in a medium containing 1 mM CaCl2 with 10 lM ONOO resulted in a rapid and sustained increase in [Ca2+]c. These findings demonstrate that ONOO is able to mobilize Ca2+ in human platelets. Therefore, in addition to the role of H2O2 on the enhanced Ca2+ entry in platelets from type
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2 diabetic patients compared to controls, this event also depends on the level of ONOO production, which, according to the results obtained with trolox, might be negligible in control platelets and more elevated in platelets from diabetic patients. Mannitol did not alter TG + Iono- and thrombin-induced Ca2+ mobilization in platelets from diabetic and healthy donors Mannitol is a selective scavenger of OH over ONOO and other ROS [28]. In order to investigate whether OH is also involved in the abnormal Ca2+ homeostasis observed in platelets from type 2 diabetic patients, cells were preincubated with 5 mM mannitol for 10 min at 37 °C. As shown in Fig. 5, treatment with mannitol did not modify either TG + Iono- or thrombin-induced Ca2+ release or entry, both in diabetic and control platelets. These observations suggest that OH is not involved in the abnormal Ca2+ mobilization observed in platelets from type 2 diabetic patients. Effect of oxidant scavengers on platelet aggregation in platelets from type 2 diabetic donors Fig. 6 shows that treatment of platelets from type 2 diabetic donors with 0.1 U/ml thrombin induced rapid
Fig. 5. Effect of mannitol on Ca2+ mobilization in platelets from diabetic and healthy donors. Fura-2-loaded human platelets from healthy (A,C) or type 2 diabetic donors (B,D) were pretreated with mannitol (5 mM) for 10 min at 37 °C and then stimulated with TG (1 lM) + Iono (50 nM) (A,B) or thrombin (0.1 U/ml; C,D) in a Ca2+-free medium (100 lM EGTA was added). CaCl2 (final concentration 300 lM) was added to the medium 5 min later to initiate Ca2+ entry. Elevations in [Ca2+]c were monitored using the 340/380 nm ratio and traces were calibrated in terms of [Ca2+]c. Traces shown are representative of six separate experiments.
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Fig. 6. Effect of catalase, mannitol, and trolox on aggregation in platelets from diabetic patients. Aggregation of washed human platelets from type 2 diabetic patients was induced for 3 min at a shear rate of 1200 rpm following preincubation for 10 min at 37 °C with mannitol (5 mM), catalase (300 U/ml), trolox (1 mM), or the vehicle (control). Aggregation was produced in an aggregometer by thrombin (0.1 U/ml) added, as indicated by the arrows.
aggregation with an amplitude of 84 ± 4% and an average slope of 68 ± 4% light transmission/min. In our experimental conditions, the OH scavenger mannitol (5 mM) failed to inhibit platelet aggregation by thrombin. In contrast, pretreatment of platelets with catalase (300 U/ml), which decomposes H2O2, significantly reduced both the amplitude (69 ± 5%; p < 0.05) and the average slope (54 ± 4% light transmission/min; p < 0.05) of thrombin-induced aggregation. Similarly, the ONOO scavenger trolox (1 mM) reduced thrombin-induced aggregation (in the presence of trolox thrombin-activated aggregation presented amplitude of 67 ± 5% and an average slope of 58 ± 4% light transmission/min; p < 0.05; n = 4). Thrombin-induced aggregation in platelets from type 2 diabetic subjects in the presence of catalase or trolox was similar to that observed in platelets from healthy donors not treated with inhibitors (Fig. 6 vs 1C).
Discussion Increasing evidence suggests that oxidant such as H2O2 and ONOO are involved in a wide range of both physiological and pathological processes. Several sources of ROS and RNS have been suggested in platelets and other cells, including the NADH/NADPH oxidase, superoxide-dismutase, the activation of arachidonic acid metabolism, and the metabolism of phosphoinositides [29–31]. Oxidant play major roles in the initiation and progression of cardiovascular dysfunctions associated with diseases such as diabetes mellitus [32,33]. The majority of ischemic coronary and cerebrovascular
events in diabetic patients are precipitated by vessel occlusion caused by atherosclerotic plaque disruption, platelet hyperaggregability and adhesion, resulting in intravascular thrombosis [32,34]. Although the mechanisms underlying platelet dysfunction remain unclear, an altered cellular Ca2+ homeostasis has been presented as a candidate to mediate platelet function disorders [17,18,23,35]. [Ca2+]c is an important factor involved in the regulation of a large number of cellular functions, including platelet aggregation [19]. Platelets and other cells increase [Ca2+]c by two mechanisms: Ca2+ release from intracellular compartments and Ca2+ entry across the plasma membrane, a process that is required for full platelet activation [19]. Non-excitable cells, such as platelets, lack voltage-activated Ca2+ channels, and the major mechanism for Ca2+ influx is store-operated Ca2+ entry (SOCE), which is regulated by the filling state of the intracellular Ca2+ stores [19,36]. Our results show that SOCE, stimulated by the physiological agonist thrombin or by direct depletion of the stores using TG + Iono, in platelets from type 2 diabetic patients is significantly higher than in healthy controls, which is the most remarkable difference observed in Ca2+ mobilization between both types of donors. Our results are in agreement with previous studies reporting an abnormal Ca2+ mobilization in platelets from patients with type 2 DM [17,18,23,37]. Since the extracellular medium is the major source of Ca2+, SOCE dysfunction is of great relevance in platelet function. Recent studies have presented evidence for the involvement of ROS in the activation of SOCE in different cell types, including platelets [3,38]. Hence, we looked for an increase in oxidant production in platelets from type 2 diabetic patients. The results presented here demonstrate that both resting and stimulated oxidant productions are elevated in platelets from diabetic patients compared to those in controls. The parallels between these two processes suggest that the increased oxidant generation might be involved in the elevated SOCE in diabetic platelets. To explore this possibility, we use scavengers of different ROS and RNS, such as catalase, which decomposes H2O2 into water and oxygen [24], trolox, a ONOO scavenger [27], and mannitol, which was directed to remove OH [28]. Our results indicate that catalase reduced SOCE in both healthy and diabetic platelets, which is in agreement with the reported role of H2O2 in the activation of SOCE (Fig. 3E) [3,38]. However, catalase was more effective inhibiting SOCE in platelets from patients with type 2 DM, as a consequence, the difference observed in SOCE in platelets from healthy and diabetic donors was significantly reduced in the presence of catalase. These findings clearly indicate that H2O2 is involved in the enhanced Ca2+ entry observed in platelets from type 2 diabetic patients. H2O2-dependent enhanced Ca2+ entry in diabetic
P.C. Redondo et al. / Biochemical and Biophysical Research Communications 333 (2005) 794–802
donors might be explained by an increased amount of H2O2 or a greater sensitivity to oxidation by this ROS in diabetic platelets. The former might be due to an enhanced production of H2O2 or to reduced antioxidant levels [32] in platelets from type 2 diabetic platelets. Similar results were obtained with trolox, which had no effect on Ca2+ mobilization in control platelets. This finding suggests that ONOO is not involved in the physiological mechanisms involved in Ca2+ homeostasis. The lack of effect of trolox makes easier the interpretation of the results obtained in diabetic platelets, where trolox reduced SOCE reaching a level that was similar to that found in control platelets (see Figs. 4C and D). Our results indicate that ONOO is able to elevate [Ca2+]c in control platelets, therefore, the lack of effect of trolox on SOCE in platelets from healthy donors indicates that ONOO is not produced after treatment with TG + Iono or thrombin. The inhibitory effect of trolox on TG + Iono- or thrombin-induced Ca2+ entry in platelets from diabetic donors is likely to be mediated by an increased ONOO production in these cells. Interestingly, treatment of human platelets from healthy or diabetic donors with mannitol, which is more specific for OH [28], did not modify Ca2+ mobilization induced by TG + Iono or thrombin, suggesting that OH is not involved in the altered Ca2+ homeostasis observed in platelets from type 2 diabetic donors. Catalase and trolox almost completely reverse the differences in Ca2+ entry observed in platelets from diabetic and healthy donors, suggesting that both H2O2 and ONOO might be components of the same intracellular cascade inducing these effects; in fact, H2O2 represents the common final path of all oxidant in the cells [39]. We have recently reported that pp60src phosphorylation and then its activity is increased in platelets from diabetic donors, an event that may be responsible for the enhanced Ca2+ entry in these cells [18]. The results presented here are consistent with this study since oxidative stress has been shown to reduce protein tyrosine phosphatase activity [40], a process that is likely to be mediated by S-nitrosylation [41]. Since Ca2+ entry is essential for aggregation, we have investigated whether the enhanced oxidant production is also involved in platelet hyperaggregability in diabetic individuals. We have found that thrombin-induced aggregation is significantly increased in platelets from patients with type 2 DM, an effect that was reduced by treatment with catalase and trolox. In contrast, mannitol did not significantly modify this response. These findings are consistent with the results reported above and suggest that a reduction in the influx of Ca2+ is reflected in a reduction in platelet hyperaggregability. Our results indicate for the first time that the oxidant scavengers, catalase and the vitamin E analog, trolox, reverse the enhanced Ca2+ entry and hyperaggregability observed in platelets from type 2 diabetic donors com-
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pared with controls. Platelet function is altered in several ways in patients with diabetes mellitus, including an increased release of TXA2 [42], accelerated platelet turnover [43], and an increase in platelet aggregation [44]. Platelet dysfunction in type 2 DM might be mediated by an enhanced level of cytosolic oxidant, specially H2O2 and ONOO, which is likely to be due to an increased production or a reduced antioxidant capacity. The processes that maintain the integrity of the cardiovascular system are impaired in diabetes mellitus, including platelet and endothelial function, coagulation, and fibrinolysis [32]. Thus, the balance in hemostasis is shifted to favor thrombosis, increasing cardiovascular risk. The present study provides evidence that antioxidants, such as vitamin E, might be used as an antiplatelet therapy in order to reduce the risk of ischemic events in diabetic patients.
Acknowledgments This work was supported by Junta de ExtremaduraConsejerı´a de Sanidad y Consumo Grant SCSS0405 and DGI-MEC Grant BFU2004-00165. P.C.R. is supported by a DGESIC fellowship (BFI2001-0624). We thank Mercedes Go´mez Bla´zquez for her technical assistance.
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