- Email: [email protected]
Role of ascorbic acid in the modulation of inhibition of platelet aggregation by polymorphonuclear leukocytes
Thrombosis Research 110 (2003) 117 – 126
Regular Article
Role of ascorbic acid in the modulation of inhibition of platelet aggregation by polymorphonuclear leukocytes S.A.V. Raghavan, P. Sharma, M. Dikshit* Division of Pharmacology, Central Drug Research Institute, P.O. Box 173, Lucknow 226001, India Received 3 November 2002; received in revised form 28 May 2003; accepted 3 June 2003
Abstract Objectives: We investigated the modulatory effect of ascorbate on the inhibition of platelet aggregation response by polymorphonuclear leukocytes (PMNs) and characterized the mechanism of the inhibitory response. Background: PMNs have been reported to play a significant role in vascular homeostasis by releasing various factors including short-lived reactive oxygen species (ROS) and nitric oxide (NO). NO prevents the activation of circulating platelets and plays a significant role in hemostasis. In addition, PMNs also have the capacity to store very high concentrations of ascorbate. The physiological implications of storing such high concentrations of an antioxidant by a cell-releasing free radicals is unknown, viz. a viz. hemostatic regulation. Methods: ADP-induced aggregation in human, monkey and rat platelet-rich plasma (PRP) was monitored in the presence of PMNs treated with varying concentrations of ascorbate/dehydroascorbate. NO generation from rat and human PMNs treated with ascorbate was monitored on a FACS Calibur flow cytometer and intraplatelet cyclic guanosine 3V,5V-monophosphate (cGMP) levels was also measured. Results: PMNs induced a cell number and time-dependent inhibition of ADP-induced aggregation. The PMNs dependent inhibition was enhanced significantly at 30 min by ascorbate (300 AM). Ascorbate seemed to exert its effects through its oxidized product, dehydroascorbate, as the effects was prevented in the presence of D-glucose (10 mM). Dehydroascorbate elicited significant potentiation of the PMNs induced inhibitory responses and these effects were mediated by the release of NO and subsequent activation of platelet guanylyl cyclase. Flow cytometry experiments with human and rat PMNs confirmed the release of NO and the elevated platelet cGMP levels confirmed NO-mediated activation of guanylyl cyclase. Conclusions: Ascorbate in circulation seems to prevent the activation of platelets by enhancing the release of antiaggregatory NO, from neighbouring or cohabitant PMNs. The ascorbate effect is mediated through its conversion to dehydroascorbate, subsequently, gets taken up by the cell and converted back to ascorbate. Intracellular ascorbate potentiates the release of NO from the PMNs and subsequently activates guanylyl cyclase in the platelets. Condensed Abstract The thrombotic process involves platelets and polymorphonuclear leukocytes (PMNs). PMNs release both reactive oxygen species (ROS) and nitric oxide (NO). The present study was conducted to investigate the physiological significance of the ascorbate in circulation, by using rat, human and monkey platelets and PMNs. Ascorbate significantly potentiated the PMNs-mediated inhibition of aggregation. Ascorbate-mediated effects were mediated by dehydroascorbate and subsequently releases NO. NO, then activates the guanylyl cyclase in the platelets and elevates the platelet cGMP levels. It seems likely that ascorbic acid in PMNs play a significant role in vascular homeostasis by elevating levels of NO and subsequently maintaining the platelet in an inactivated state, preventing the initiation of thrombotic process. D 2003 Elsevier Ltd. All rights reserved. Keywords: Platelet aggregation; Polymorphonuclear leukocytes; Ascorbic acid; Dehydroascorbic acid; Nitric oxide; cGMP
Abbreviations: PMNs, polymorphonuclear leukocytes; ROS, reactive oxygen species; NO, nitric oxide; cGMP, cyclic guanosine 5V-monophosphate; PRP, platelet-rich plasma; DAF, diaminofluorescein diacetate; DHA, dehydroascorbate; ADP, adenosine-5V-diphosphate; PTIO, 2-phenyl4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide; ODQ, 1H-[1,2,4]oxadiazolo[4,3]quinoxalin-1-one; cAMP, cyclic adenosine 5V-monophosphate; HBSS, Hanks balanced salt solution. * Corresponding author. Tel.: +91-522-2212411 to 18; fax: +91-5222223405, +91-522-2223938. E-mail address: [email protected] (M. Dikshit). 0049-3848/03/$ - see front matter D 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0049-3848(03)00312-8
1. Introduction The thrombotic process is a multicellular phenomenon in which not only platelets but neutrophils are also involved. Coexistence of polymorphonuclear leukocytes (PMNs) with platelets in the hemostatic plug has been observed repeatedly [1,2] and their interaction with each other under different conditions has been demonstrated to influence the course of thrombosis [3– 7].
118
S.A.V. Raghavan et al. / Thrombosis Research 110 (2003) 117–126
PMNs have also been reported to release the potent antiaggregatory molecule, nitric oxide (NO) from the semi-essential amino acid L-arginine [2,8,9]. NO is known to inhibit free radical generation from PMNs [10,11] and plays a significant role in vascular homeostasis [12]. Previous work from our laboratory has demonstrated the presence of an inhibitory factor from the supernatant from PMNs on platelet aggregation [13]. Further work characterized this factor to be a h-globin related protein (unpublished observations) and the mechanism of inhibition of aggregation is under characterisation. PMNs, by their ability to release reactive oxygen species (ROS) and NO, performs the important function of phagocytosing small organisms especially bacteria. However, PMNs have the capacity to store high concentrations of ascorbic acid [14,15], a potent antioxidant. Vitamin C has been known to scavenge a wide variety of reactive nitrogen and oxygen species, mainly superoxide radicals [16]. Vitamin C increased the production of Lcitrulline and cyclic GMP, a marker of NO bioactivity, in endothelial cells and arterial cultures [17]. However, role of ascorbate in the PMNs, which has the potential to release both superoxide and NO, is not completely elucidated viz. a viz. its physiology and in vascular homeostasis. Hence, the present study was carried out to investigate the effects of ascorbate on PMNs-mediated inhibition of platelet aggregation. Results from the present study indicate that addition of ascorbate potentiated the inhibition of platelet aggregation by PMNs, by augmenting the formation of NO and related species, subsequently elevating the cGMP levels. It seems likely that ascorbic acid from PMNs play a significant role in vascular homeostasis by supplementing the levels of the relaxing and antiaggregatory factor, NO. This subsequently maintains the platelet in an inactivated state and prevents the initiation of thrombotic process.
revised 1985) and all the experiments were approved by the committee on the study of humans and ethics committee of CDRI. 2.2. Preparation of polymorphonuclear leukocytes Ten male healthy, nonsmoking, non-fasted volunteers (mean age: 31.4 F 3.1 years) were recruited for participation in this study. All the subjects included in the study were normotensive with no evidence of anemia, renal or hepatic dysfunction. Nutritional status of all the subjects included were normal (measured by skin fold thickness). None of the subjects had taken antioxidants, vitamins or nutritional supplements for at least 4 weeks prior to enrollment for the study. Blood was taken from the cubital vein in sodium citrate (3.8% w/v; 9:1 v/v). Blood was also collected from the femoral vein of Rhesus monkeys in sodium citrate (3.8% w/v; 9:1 v/v). While, rat (male, Sprague –Dawley, 200– 225 g) blood was collected by cardiac puncture under ether anaesthesia in sodium citrate (3.8%, 9:1). PMNs were isolated from the blood as reported earlier [13,18]. The PMNs were suspended in calcium and magnesium free Hank’s balanced salt solution (HBSS, pH 7.4). Cell viability was tested by Trypan blue exclusion test, which was never less than 95%. 2.3. Platelet aggregation Blood from human volunteers, monkeys or rat was collected in sodium citrate (3.8% w/v for humans and monkeys and 1.9% w/v for rats, 9:1 v/v) and centrifuged at 200 –250 g for 15 min to obtain the platelet-rich plasma (PRP). Platelet counts were adjusted to 2 108 cells/ml with platelet-poor plasma, which was obtained on further centrifugation of the blood at 2000 g for 20 min at 20 jC. Aggregation was monitored on a dual channel Lumi aggregometer (Chronolog, Haverton, CA, USA) with constant stirring at 1000 rpm at 37 jC.
2. Materials and methods
2.4. Effect of PMNs on ADP-induced platelet aggregation
2.1. Materials
To investigate the effect of PMNs on platelet aggregation, PRP was suspended with PMNs (106 cells) for 5, 15, 30 min and subsequently aggregation was monitored with ADP (5 AM). In these experiments, autologus PMNs were used to assess the effect of PMNs on ADP-induced platelet aggregation. All the interventions used in the study were also evaluated for their per se effect on ADP-induced platelet aggregation. PRP was incubated with PMNs (106 cells to 5 106 cells) for 5, 15 or 30 min prior to aggregation with ADP (5 AM). Control experiments were performed by incubating PRP with vehicle (HBSS) for the same incubation period prior to ADP-induced aggregation. Autologus PMNs were used for evaluating the effect on ADP-induced platelet aggregation.
Ascorbic acid, adenosine 5V-diphosphate (ADP), catalase, dehydroascorbic acid, 4,5-diaminofluorescein diacetate, Dglucose, 1H-[1,2,4]-oxadiazolo[4,3]quinoxalin-1-one (ODQ), hemoglobin (Hb), methylene blue, 2-phenyl4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), sodium citrate, superoxide dismutase were obtained from Sigma (St. Louis, USA). All other chemicals were obtained from E. Merck, Mumbai, India. Cyclic GMP enzyme immunoassay kit was obtained from Cayman Chemical (USA). Animals were obtained from the animal colony of CDRI, Lucknow (India) and the animal experiments conducted were in accordance of ‘‘Principles of laboratory animal care’’ (NIH publication No. 85 – 23,
S.A.V. Raghavan et al. / Thrombosis Research 110 (2003) 117–126
2.5. Effect of ascorbate or dehydroascorbate on the PMNs response Autologus PMNs were used through out for evaluating the effect of ascorbate/dehydroascorbate on ADP-induced platelet aggregation. PMNs was treated with ascorbate (100 or 300 AM) or dehydroascorbate (100 or 300 AM) for 5, 15 or 30 min prior to ADP-induced aggregation in rat, human or monkey platelets. Control experiments were performed by incubating PRP with ascorbate or dehydroascorbate. To characterise the mechanism of ascorbate-induced modulation of PMNs response on platelet aggregation, PMNs was pretreated with D-glucose (10 mM), an inhibitor of dehydroascorbate uptake prior to addition of ascorbate (300 AM) and subsequently ADP-induced aggregation was evaluated in the presence of these PMNs. PMNs and ascorbate effect was also investigated in the presence of reactive oxygen scavengers (ROS) scavengers, SOD (100 U/ml) and catalase (500 U/ml) for 30 min prior to addition of ascorbate (300 AM) and subsequently ADPinduced aggregation was induced. To investigate the involvement of NO/cGMP, rat or human PRP was incubated with NO scavenger, PTIO (100 AM) or inhibitor of soluble guanylyl cyclase (ODQ, 10 AM) for 15 min prior to the addition of PMNs treated with ascorbate or dehydroascorbate and subsequently ADP-induced aggregation was evaluated. While, monkey PRP was incubated with haemoglobin (10 AM), a NO scavenger or with methylene blue (10 AM), an inhibitor of soluble guanylyl cyclase for 15 min prior to addition of PMNs treated with ascorbate (300 AM) and subsequently ADPinduced aggregation was evaluated. Aggregation to ADP (5 AM) was considered as 100% and aggregation to ADP in the presence of various interventions are represented as percentage of the ADP-induced aggregation [18].
119
action was stopped with 6% (final concentration) ice-cold trichloroacetic acid (TCA). Precipitated proteins were removed by centrifugation at 5000 g for 10 min at 4 jC. The supernatant was separated and TCA was extracted with 4 volumes of water saturated diethylether (five times). The ethereal layer was discarded and aqueous layer was heated at 55 – 60 jC for 5 min to remove residual ether. The samples were then lyophilised and cGMP was estimated on acetylation by enzyme immunoassay according to the manufacturer’s instructions (Cayman Chemical). Platelet cGMP values are expressed as nM/106 platelets. 2.8. Statistical analysis All results are expressed as mean F S.E.M. of at least five experiments. The statistical significance between two columns was calculated by using unpaired Student’s t-test and multiple columns were compared by one-way ANOVA followed by Student – Newman– Keuls test. The difference between two groups was considered significant at p < 0.05.
2.6. Estimation of NO generation by flow cytometry PMNs from rats and humans were used for assessing the release of NO by flow cytometry. PMNs (2 106 cells/ml) were incubated for 5 min with 4,5-diaminofluorescein diacetate (DAF-2 DA, 10 AM) [19] and were subsequently treated with ascorbate or DHA for 30 min at 37 jC. Each sample was evaluated on FACS Calibur (Becton Dickinson, USA). The increase in fluorescence were analyzed by Cell Quest program (Becton Dickinson) and results are represented as shifts in the histogram corresponding to the increased NO generation [20,21]. 2.7. Estimation of cyclic GMP levels As the trend in the ascorbate/dehydroascorbate-mediated response on platelets was similar in PMNs, mechanism of inhibition of ADP-induced aggregation was elucidated in rat PMNs. At the end of platelet aggregation, the platelet re-
Fig. 1. (A) Inhibition of ADP (5 AM)-induced platelet aggregation by PMNs (106 to 5 106 cells) in rat PRP. PMNs-mediated inhibition were evaluated from the same rat PRP (n = 5 in each set). *P < 0.001 in the presence of PMNs compared to vehicle treatment. #P < 0.05 compared to 106 PMNs. (B) Time-dependent inhibition of ADP (5 AM)-induced aggregation in PRP from human (n = 5), rat (n = 10) and monkey (n = 5). PMNs and PRP were obtained from the same animal/human. *P < 0.05, **P < 0.01, ***P < 0.001 in PMNs (106 cells) at different time intervals compared to their respective group at 5 min of incubation.
120
S.A.V. Raghavan et al. / Thrombosis Research 110 (2003) 117–126
Data were analyzed using Graph Pad Prism 3.0 statistical package (San Diego, CA, USA).
3. Results 3.1. Effect of PMNs on ADP-induced platelet aggregation ADP (5 AM) produced an irreversible aggregation ( f 60%) over a duration of 5 –7 min in all the three species under study. Addition of rat PMNs to platelet-rich plasma produced a cell number (1 106 to 5 106 PMNs) dependent inhibition of ADP-induced aggregation. Significant inhibition of aggregation was observed with 106 PMNs after 5 min of incubation with PRP (Fig. 1A) and the same cell number was used for the subsequent experiments for different time intervals in all the species under study. ADP-
Fig. 3. Time-dependent effects of dehydroascorbate (100 AM and 300 AM) on the PMNs (106 cells)-induced inhibition of ADP (5 AM) in rat PRP. *P < 0.05, **P < 0.01, ***P < 0.001 at 15 and 30 min of incubation compared to their respective treatments at 5 min of incubation. #P < 0.05 in the presence of dehydroascorbate (300 AM) compared to their absence at 30 min of incubation (n = 10 in each group). @P < 0.05 in the presence of 300 AM compared to 100 AM dehydroascorbate at 30 min of incubation (n = 10 in each group).
induced aggregation by rat, human or monkey PMNs (Fig. 1B) was also inhibited in a time-dependent manner. The maximal inhibition of aggregation was observed at the end of 30 min and further incubation did not produce any significant enhancement of the inhibitory response. 3.2. Effect of ascorbate on the PMNs-mediated response
Fig. 2. (A) Effect of ascorbate (100 AM and 300 AM) on PMNs (106 cells)mediated inhibition of ADP (5 AM)-induced aggregation in rat PRP at 5, 15 and 30 min of incubation. *P < 0.01, **P < 0.001 in different groups at 15 and 30 min compared to their respective treatments at 5 min of incubation (n = 10 in each group). (B) Effect of scavengers of ROS, SOD (100 U/ ml) + Catalase (500 U/ml) on ascorbate (300 AM)-induced augmentation of PMNs (106 cells)-mediated inhibition of ADP (5 AM)-induced aggregation in rat PRP. *P < 0.001 at 15 and 30 min of incubation compared to their respective treatments at 5 min of incubation (n = 10 in each group). # P < 0.05 in the presence of scavengers of ROS compared to their absence at 30 min of incubation.
Incubation of rat PRP with different concentrations of ascorbate (1 AM – 1 mM) did not produce significant changes in the ADP-induced aggregations (data not shown). However, pretreatment of rat PMNs (106 cells) with ascorbate augmented the inhibition of ADP induced aggregation in a concentration- and time-dependent manner. Significant attenuation of ADP-induced aggregation was observed only after 30 min of incubation of 100 AM ascorbate with PRP, whereas 300 AM ascorbate showed significant inhibition at 15 min of incubation (Fig. 2A). Treatment of PMNs with concentrations greater than 300 AM ascorbate produced significant inhibitions of ADP-induced aggregation even at lesser incubation intervals (maximal aggregation at 1 mM ascorbate being, 28 F 2.5% (n = 3) and 14 F 3.8% (n = 3) after 15 and 30 min of incubation, respectively). Pretreatment of the rat PMNs with scavengers of ROS, i.e., combination of superoxide dismutase (SOD) and catalase, augmented the inhibitory responses of ascorbate (Fig. 2B). The inhibitory response was potentiated at 30 min of incubation with SOD and catalase (Fig. 2B). 3.3. Effect of dehydroascorbate on the PMNs-mediated response Effect of dehydroascorbate was evaluated on the cosuspension of PMNs and PRP in rats. The inhibition of
S.A.V. Raghavan et al. / Thrombosis Research 110 (2003) 117–126
aggregation of PMNs treated with dehydroascorbate was more than that observed on PMNs treated with ascorbate, and DHA (300 AM)-mediated effects was comparable to that of ascorbate (1 – 2 mM). Similar to ascorbate, dehydroascorbate at 300 AM showed significant inhibition at lesser incubation intervals compared to 100 AM dehydroascorbate (Fig. 3). In all the species studied, dehydroascorbate (300 AM) produced significant potentiation of PMNs-mediated inhibition at 30 min of incubation compared to ascorbate (300 AM) (Fig. 4A and B). Ascorbate by itself possesses minimal capacity to enter PMNs. Ascorbate enters the cell as its oxidized product, dehydroascorbic acid, through the GLUT1 transporter, and is inhibited by high D-glucose [22]. Hence, we performed experiments to ascertain the hypothesis that the effects of ascorbic acid are indeed mediated by dehydroascorbic acid. Rat PRP was preincubated with glucose (10 mM), which inhibited the uptake of dehydroascorbic acid into the PMNs. Pretreatment with glucose significantly inhibited the atten-
121
Fig. 5. Effect of glucose (10 mM) on the ascorbate (100 AM and 300 AM)induced augmentation of PMNs (106 cells)-mediated inhibition of ADP (5 AM)-induced aggregation in rat PRP. *P < 0.05, **P < 0.01 in the presence of glucose compared to their respective treatments (n = 10 and n = 5 in the absence and presence of glucose, respectively).
uation in aggregation responses by PMNs alone or PMNs incubated with ascorbate (Fig. 5), confirming that ascorbic acid-induced effects on platelet aggregation was indeed mediated by its oxidized product, dehydroascorbic acid. Ascorbate in the suspension medium was spontaneously oxidized to dehydroascorbate by molecular oxygen and hence entered the PMNs to elicit the inhibitory effects. Subsequently, experiments were performed to characterize the mechanism behind the ascorbate/DHA-mediated augmentation of inhibition of aggregation. 3.4. Involvement of NO/cGMP in ascorbate- or dehydroascorbate-mediated response
Fig. 4. (A) Time-dependent effects of ascorbate (300 AM) and DHA (300 AM) on the PMNs (106 cells)-induced inhibition of ADP (5 AM)-induced aggregation in human PRP. *P < 0.05, **P < 0.01 in the respective treatments at 15 and 30 min of incubation compared to the same treatment at 5 min of incubation. #P < 0.05 in the ascorbate- and DHA-treated group in comparison to PMNs alone at 30 min of incubation. (B) Timedependent effects of ascorbate (300 AM) and DHA (300 AM) on the PMNs (106 cells)-induced inhibition of ADP (5 AM)-induced aggregation in human PRP. *P < 0.05, **P < 0.01 in the respective treatments at 30 min of incubation compared to the same treatment at 5 min of incubation. # P < 0.05 in the DHA-treated group in comparison to PMNs alone at 30 min of incubation.
Dehydroascorbate like ascorbate did not inhibit ADPinduced aggregations similar to ascorbate. Since no significant alterations in the aggregation responses was observed on treatment of PRP with varying concentrations of ascorbate/ dehydroascorbate, it seemed likely that the effects of ascorbate were due to the release/production of some intermediates from PMNs, which exerted antiaggregatory effects on the platelets. Previous work from our lab has indicated towards the involvement of the potent antiaggregatory factor, NO. To confirm the involvement of NO, PRP was incubated with NO scavenger, PTIO or hemoglobin, and NO-sensitive guanylyl cyclase inhibitor, ODQ or methylene blue, before treatment with ascorbate- or dehydroascorbate-treated PMNs. Pretreatment of PRP with PTIO (to scavenge NO) and ODQ (to inhibit soluble guanylyl cyclase) significantly inhibited the potentiation of inhibition by PMNs at all time intervals in rats. Human PRP incubated with PTIO or ODQ also showed similar trend at 30 min of incubation with either ascorbate or dehydroascorbate. Monkey PRP incubated with hemoglobin (another scavenger of NO) or methylene blue (guanylyl cyclase inhibitor), also reversed the potentiation of inhibition
122
S.A.V. Raghavan et al. / Thrombosis Research 110 (2003) 117–126
by ascorbate (300 AM; 30 min) or dehydroascorbate (300 AM; 30 min). NO scavengers and guanylyl cyclase inhibitors reversed the inhibition of aggregation, confirming the involvement of NO-mediated activation of soluble guanylyl cyclase (Fig. 6A – C). 3.5. NO generation in ascorbate-treated PMNs In the present study, it seemed that NO released from the PMNs was responsible for the observed effects with ascor-
Fig. 6. (A) Effect of NO scavenger, PTIO (100 AM) and guanylyl cyclase inhibitor, ODQ (10 AM) on the ascorbate (300 AM)- and dehydroascorbate (300 AM)-induced effects on PMNs (106 cells)-mediated inhibition of ADP (5 AM)-induced aggregation in rat PRP. **P < 0.01 in the PTIO and ODQ pretreated group in comparison to their respective controls. #P < 0.05 at 30 min compared to the same treatment at 15 min of incubation. (B) Effect of NO scavenger, PTIO (100 AM) and guanylyl cyclase inhibitor, ODQ (10 AM) on the ascorbate (300 AM)- and dehydroascorbate (DHA, 300 AM)induced augmentation of PMNs (106 cells)-induced inhibition of ADP (5 AM)-induced aggregation in human PRP. *P < 0.01 in the PTIO and ODQ pretreated group in comparison to their respective controls. (C) Effect of NO scavenger, hemoglobin (10 AM) and guanylyl cyclase inhibitor, methylene blue (10 AM) on the ascorbate (300 AM)-induced augmentation of PMNs (106 cells)-mediated inhibition of ADP (5 AM)-induced aggregation in monkey PRP. *P < 0.01 in the PTIO and ODQ pretreated group in comparison to their respective controls.
Fig. 7. (A) Representative histogram indicating increased fluorescence to 4V,5V-diaminofluorescein diacetate from rat PMNs (2 106 cells/ml) treated with ascorbate 300 AM after 30 min of incubation. Shifts indicated in figure are representations of one out of five independent experiments. (B) Representative histogram indicating increased fluorescence to 4V,5Vdiaminofluorescein diacetate from human PMNs (2 106 cells/ml) treated with ascorbate 300 AM after 30 min of incubation. Shifts indicated in figure are representations of one out of three independent experiments.
S.A.V. Raghavan et al. / Thrombosis Research 110 (2003) 117–126
Fig. 8. Bar diagram depicting increased platelet cGMP levels estimated by enzyme immunoassay from PMNs (106 cells) alone or from PMNs treated with ascorbate (300 AM; 30 min)/dehydroascorbate (DHA, 300 AM; 30 min) on the ADP (5 AM)-induced aggregation in rat PRP. *P < 0.05 in the DHA-treated group compared to PMNs receiving no treatment.
bate and dehydroascorbate. PMNs loaded with 4,5-diaminofluorescein diacetate, a dye specific for nitric oxide measurement, prior to treatment with ascorbate also exhibited formation of NO. Mean fluorescence was significantly increased in both the species after 30 min of incubation, indicating that PMNs treated with ascorbate released significant amounts of NO (Fig. 7A and B). 3.6. Effect of ascorbate/dehydroascorbate on platelet cGMP levels To further ascertain the activation of guanylyl cyclase by NO, platelet cGMP levels were estimated in presence of PMNs treated with either ascorbate or dehydroascorbate. Incubation of PRP with PMNs (106 cells) elevated the cGMP levels significantly (Fig. 8). Platelet cGMP levels were further increased in the presence of PMNs incubated with ascorbate/dehydroascorbate, suggesting that incubation of PMNs with ascorbate or dehydroascorbate potentiated the inhibitory responses to ADP-induced aggregation by elevating cGMP levels.
4. Discussion L-Ascorbic acid has been described to exert multiple beneficial effects in cardiovascular disorders associated with impaired nitric oxide/cGMP signalling. Thus, endothelial dysfunction in the course of essential hypertension was improved by supplementation of vitamin C [23]. Others described the preventive effect on the development of tolerance during long-term administration of organic nitrates [24]. Recently, stimulation of NO biosynthesis by L-ascorbic acid has been observed in human endothelial cells [17] and
123
the antioxidant was shown to sensitize isolated coronary arteries towards NO-induced vasodilation [25]. Oral administration of vitamin C, reaching up to a plasma concentration of 100 AM, has been reported to reduce arterial stiffness and platelet aggregation [26]. However, the mechanism involving protection of NO from inactivation in this study by vitamin C was not confirmed. Moreover, the mechanism behind this antiaggregatory effect has not been explained to date. Vitamin C has been reported to exert a pro-oxidant effect in addition to its antioxidant effect [27,28] and particularly increases the nitrosative stress in cells [29,30]. Hence, it is unlikely that the antiaggregatory effects of this vitamin could be mainly due to the antioxidant effect, associated with this vitamin. In the present study, we did not observe any significant inhibition in vitro of ADP-induced aggregation with ascorbate (1 AM to 1 mM). The present study was a part of the study which was aimed to evaluate the role of ascorbic acid in regulating PMNs function. We initially tested the idea in rat cells, and the outcome of the study prompted us to confirm it in primates and humans. PMNs, the co-habitant also synthesize NO in substantial amounts [8,18,31] and like endothelial cells, also store high concentrations of ascorbate [14]. Role of ascorbate in PMNs is not so well defined. Previous work from our lab has already demonstrated inhibition of ADP-induced aggregation by the factors released from PMNs [13]. One of the most potent inhibitory factor released by PMNs is NO [8,18,31]. Moreover, PMNs also play a significant role in hemostasis and thrombosis [3,4,18]. Hence, we found it worthwhile to investigate the role of ascorbate in PMNs and platelet interactions. Co-suspension of PMNs with platelets showed augmented inhibition of aggregation on treatment with ascorbate. As ascorbate by itself did not elicit any effect on platelet aggregation, it seemed likely that ascorbate-mediated inhibition of aggregation were due to its effect on PMNs. Ascorbate potentiated the PMNs-mediated inhibition of aggregation in a time- and concentration-dependent manner (Fig. 2A). Pretreatment of PRP with scavengers of ROS indicated that the effects of ascorbate were not mediated due to its antioxidant action (Fig. 2B). Had ascorbate exerted its antioxidant effect, such high concentration of ascorbate used in this study, should have been sufficient to scavenge superoxide radicals, and further addition of scavengers would not have any additional effect. Vitamin C exists primarily as ascorbic acid (reduced form) in human plasma and enters the cells slowly by a Na-dependent transport. Transiently produced dehydroascorbic acid from the activated PMNs is transported into cells readily by the GLUT1 transporter. No measurable dehydroascorbic acid is detected in human blood [32,33]. Oxidants in circulation have the capacity to oxidize extracellular ascorbate to dehydroascorbic acid. Dehydroascorbic acid is more rapidly transported than ascorbate and is immediately reduced intracellularly to ascorbate [14]. This observation
124
S.A.V. Raghavan et al. / Thrombosis Research 110 (2003) 117–126
was confirmed in our studies as pretreatment of PRP with Dglucose (10 mM) inhibited the anti aggregatory effects observed with PMNs treated with either ascorbate/dehydroascorbate. Moreover, extracellular administration of dehydroascorbate elicited higher inhibition in comparison to ascorbate indicating the ready transport and immediate effect (Figs. 3 and 4A,B). Also, Takajo et al. [34] observed that following oral administration of vitamin C, concentrations of vitamin C were higher in the platelets than plasma suggesting the transport of vitamin C into the cells. Treatment of the co-suspension of platelets and PMNs with ascorbate, though reduces the oxidative stress, however, seems to increase the nitrosative stress. The nitrosative stress is of particular interest since this can generate NO donors, based upon modification of polyhydroxylated compounds and thiols [35 –38]. The chemical identity of the NO donor(s) generated is likely to involve the formation of either S-nitrosothiols or organic nitrites [35,36]. Synthesis of NO from PMNs has been well reported and the NO modulates release of other free radicals from PMNs [11,20,39]. Recently, Sharma et al. [21] have observed that increased oxidative stress in the PMNs seem to favor release of other free radicals over NO. Results from the present study also suggest to this conclusion, as ascorbate augmented the release of NO. The mechanism of release of NO from PMNs was not explored in detail in the present study, but, could probably involve one or more of the mechanism suggested for endothelial cells which is presently under investigation in our lab. Ascorbate acts intracellularly, [40] and could improve the availability for NOS of tetrahydrobiopterin [17], or sparing intracellular thiols [41,42]. To investigate the involvement of NO, we used DAF, specific probe for NO, in the flow cytometry experiments. Increase in the DAF fluorescence in the PMNs treated with ascorbate indicated the involvement of NO (Fig. 7A and B). This result was confirmed on preincubation of PRP with scavengers of NO (Fig. 6A –C). NO subsequently activates soluble guanylyl cyclase elevating platelet cGMP levels. The reversal of inhibition with guanylyl cyclase inhibitors (Fig. 6A –C) and elevated cGMP levels from rat platelets (Fig. 8) confirmed the involvement of NO – sGC system in the potentiation of PMNs induced inhibition of aggregation by both ascorbate and dehydroascorbate. The NO-mediated antiaggregatory effect on platelet has been reported to be mediated by an increase in cGMP levels [31,43,44]. However, cGMP-independent mechanisms of inhibition of platelet aggregation by NO and related species have been documented recently [45 – 47]. A possible explanation of the role of cGMP in the inhibition of platelet activation could be due to the nitration of platelet proteins by the formation of NO and NO donors, subsequently forming S-nitrosothiols and consequent inhibition of platelet aggregation [47]. An increase in cAMP in the platelets cannot be dismissed in the inhibitory effects observed in the present study. However, NO-mediated increase of intraplatelet levels of cAMP have been observed in many studies
where elevation of cGMP [48 – 51] is explained due to the reduction of cGMP-inhibited cAMP phosphodiesterase [52,53]. We however did not investigate involvement of cGMP-independent effect in the ascorbate-mediated effects. In hyperglycemia, where there is a decreased cellular content of L-arginine [54,55], supplementation of ascorbate could enhance the synthesis of NO by increasing the intracellular tetrahydrobiopterin levels [56,57]. High glucose concentration might also interfere with ascorbate/dehydroascorbate uptake and thereby down regulating NO bioavailability. A prothrombotic state is very common in diabetic patients. Ascorbate could also enhance the release of NO in circulation and could control the renin – angiotensin activation [58], preventing the progression of diabetic nephropathy. Though the role of ascorbate in platelet function has been enumerated, the results of the present study give a better understanding of ascorbate in hemostasis. In circulation, ascorbate prevents the activation of platelets not only by acting as an antioxidant, but also by potentiating the release of NO from the PMNs. The NO released from the PMNs subsequently activates the guanylate cyclase, elevating cGMP levels in the platelets and inhibits the aggregation of platelets. The present study is yet another confirmation of the regulatory role of the potent relaxing and antiaggregatory factor, nitric oxide. Acknowledgements Financial support in form of grant-in-aid to MD from the Department of Biotechnology, New Delhi, India is gratefully acknowledged. SAVR and PS acknowledge CSIR, New Delhi for the Senior Research Fellowship. Technical assistance of Mr. A.L. Viswakarma during the flow cytometry experiments is acknowledged. References [1] Marcus AJ. Recent progress in the role of platelets in occlusive vascular disease. Stroke 1983;14:475 – 9. [2] Dikshit M, Kumari R. Modulation of platelet aggregation response by factors released from polymorphonuclear leukocytes. Hematology 1997;2:39 – 53. [3] Marcus AJ. Thrombosis and inflammation as multicellular process: pathophysiologic significance of transcellular metabolism. Blood 1990;66:1093 – 7. [4] Cerletti C, Evangelista V, de Gaetono G. Polymorphonuclear leukocyte-dependent modulation of platelet function: relevance to the pathogenesis of thrombosis. Pharmacol Res 1992;26:168 – 261. [5] Bazzoni G, Dejana E, Delmaschio A. Platelet – neutrophil interactions—possible relevance in the pathogenesis of thrombosis and inflammation. Haematologica 1991;76:491 – 9. [6] Faint RW. Platelet neutrophil interactions: their significance. Blood Rev 1992;6:83 – 91. [7] Stewart BJ. Neutrophils and deep venous thrombosis. Haemostasis 1993;23:127 – 40. [8] Salvemini D, de Nucci G, Sneddon JM, Vane JR. Human neutrophils and mononuclear cells inhibit platelet aggregation by releasing a nitric oxide-like factor. Proc Natl Acad Sci U S A 1989;86:6328 – 32.
S.A.V. Raghavan et al. / Thrombosis Research 110 (2003) 117–126 [9] Yoshimoto H, Suchiro A, Kakishita E. Exogenous nitric oxide inhibits platelet activation in whole blood. J Cardiovasc Pharmacol 1999;33: 109 – 15. [10] Rubanyi GM, Ho EH, Cantor EH, et al. Cytoprotective function of nitric oxide: Inactivation of superoxide radicals produced by human leukocytes. Biochem Biophys Res Commun 1991;181:1392 – 7. [11] Seth P, Kumari R, Dikshit M, Srimal RC. Modulation of rat peripheral polymorphonuclear leukocyte response by nitric oxide and arginine. Blood 1994;84:2741 – 8. [12] Sethi S, Dikshit M. Modulation of polymorphonuclear leukocytes function by nitric oxide. Thromb Res 2000;100:223 – 47. [13] Kumari R, Singh MP, Seth P, Dikshit M. Inhibition of platelet aggregation by a protein factor present in rat neutrophil supernatant. Thromb Res 1998;91:75 – 82. [14] Welch RW, Wang Y, Crossman A, et al. Accumulation of vitamin C (ascorbate) and its oxidized metabolite dehydroascorbic acid occurs by separate mechanisms. J Biol Chem 1995;21:12584 – 92. [15] Park JB, Levine M. Purification, cloning and expression of dehydroascorbic acid reducing activity from human neutrophils: identification as glutaredoxin. Biochem J 1996;315:931 – 8. [16] Gotoh N, Niki E. Rates of interactions of superoxide with vitamin E, vitamin C and related compounds as measured by chemiluminescence. Biochim Biophys Acta 1992;1115:201 – 7. [17] Heller R, Munscher-Paulig F, Grabner R, Till U. L-ascorbic acid potentiates nitric oxide synthesis in endothelial cells. J Biol Chem 1999;274:8254 – 60. [18] Dikshit M, Kumari R, Srimal RC. Pulmonary thromboembolism induced alterations in nitric oxide release from rat circulating neutrophils. J Pharmacol Exp Ther 1993;265:1369 – 72. [19] Qiu W, Kass DA, Hu Q, Ziegelstein RC. Determination of shear stress-stimulated endothelial NO production assessed in real time by 4,5-diaminofluorescein fluorescence. Biochem Biophys Res Commun 2001;286:328 – 35. [20] Sethi S, Sharma P, Dikshit M. Nitric oxide and oxygen derived free radical generation from control and lipopolysaccharide treated rat polymorphonuclear leukocyte. Nitric Oxide 2001;5:482 – 93. [21] Sharma P, Barthwal MK, Dikshit M. NO synthesis and its regulation in the arachidonic acid stimulated polymorphonuclear leukocytes. Nitric Oxide 2002;7:119 – 26. [22] Washko P, Levine M. Inhibition of ascorbic acid transport in human neutrophils by glucose. J Biol Chem 1992;267:23568 – 74. [23] Taddei S, Virdis A, Ghiadoni L, et al. Vitamin C improves endothelium-dependent vasodilation by restoring nitric oxide activity in essential hypertension. Circulation 1998;97:2222 – 9. [24] Hinz B, Schroder H. Vitamin C attenuates nitrate tolerance independently of its antioxidant effect. FEBS Lett 1998;428:97 – 9. [25] Murphy ME. Ascorbate and dehydroascorbate modulate nitric oxideinduced vasodilations of rat coronary arteries. J Cardiovasc Pharmacol 1999;34:295 – 303. [26] Wilkinson IB, Megson IL, MacCallum H, et al. Oral vitamin C reduces arterial stiffness and platelet aggregation in humans. J Cardiovasc Pharmacol 1999;34:690 – 3. [27] Halliwell B, Gutteridge JMC. Free radicals in biology and medicine. New York: Clarendon Press; Oxford, UK: Oxford Univ. Press; 1999. [28] Roginsky VA, Stegmann HB. Ascorbyl radical as natural indicator of oxidative stress: quantitative regularities. Free Radic Biol Med 1994; 17:93 – 103. [29] Childs A, Jacobs C, Kaminski T, et al. Supplementation with vitamin C and N-acetylcysteine increases oxidative stress in humans after an acute muscle injury induced by eccentric exercise. Free Radic Biol Med 2001;31:745 – 53. [30] Brown AS, Moro MA, Masse JM, et al. Nitric oxide-dependent and independent effects on human platelets treated with peroxynitrite. Cardiovasc Res 1998;40:380 – 8. [31] Faint RW, Mackie IJ, Machin SJ. Platelet aggregation is inhibited by a nitric oxide like factor released from human neutrophils in vitro. Br J Hematol 1991;77:539 – 45.
125
[32] Koshiishi I, Mamura Y, Liu J, Lmanari T. Evaluation of an acidic deproteinization for the measurement of ascorbate and dehydroascorbate in plasma samples. Clin Chem 1998;44:863 – 8. [33] Levine M, Dhariwal KR, Wang Y, et al. In: Frei B, editor. Natural antioxidants in human health and disease. San Diego, CA: Academic Press; 1994. p. 469 – 84. [34] Takajo Y, Ikeda H, Haramaki N, et al. Augmented oxidative stress of platelets in oxidative stress in chronic smokers. J Am Coll Cardiol 2001;38:1320 – 7. [35] Moro MA, Darley-Usmar VM, Goodwin DA, et al. Paradoxical and biological action of peroxynitrite on human platelets. Proc Natl Acad Sci U S A 1994;91:6702 – 6. [36] Moro MA, Darley-Usmar VM, Lizasoain J, et al. The formation of nitric oxide donors from peroxynitrite. Br J Pharmacol 1995;116: 1999 – 2004. [37] White CR, Moellering D, Patel R, et al. Formation of the NO donors glyceryl mononitrate and glyceryl mononitrite from the reaction of peroxynitrite with glycerol. Biochem J 1997;328:517 – 24. [38] Mayer B, Schrammel A, Klatt P, et al. Peroxynitrite-induced accumulation of cyclic GMP in endothelial cells and stimulation of purified soluble guanylyl cyclase. Dependence on glutathione and possible role of S-nitrosation. J Biol Chem 1995;270:17355 – 60. [39] Sethi S, Singh MP, Dikshit M. Nitric oxide mediated augmentation of polymorphonuclear leukocyte free radical generation after hypoxiareoxygenation. Blood 1999;9:333 – 40. [40] Martin A, Frei B. Both intracellular and extracellular vitamin C inhibit atherogenic modification of LDL by human vascular endothelial cells. Arterioscler Thromb Vasc Biol 1997;17:1583 – 90. [41] Carr AC, Frei B. Toward a new recommended dietary allowance for vitamin C based on antioxidant and health effects in humans. Am J Clin Nutr 1999;69:1086 – 107. [42] Carr A, Frei B. The role of natural antioxidants in preserving the biological activity of endothelium-derived nitric oxide. Free Radic Biol Med 2000;28:1806 – 14. [43] Gerzer R, Karrenbrock B, Siess W, Heim J-M. Direct comparison of the effects of nitroprusside, SIN-1, and various nitrates on platelet aggregation and soluble guanylyl cyclase activity. Thromb Res 1988;52: 11 – 21. [44] Ivanova K, Schaefer M, Drummer C, Gerzer R. Effects of nitric oxidecontaining compounds on increases in cytosolic ionized Ca2 + and on aggregation of human platelets. Eur J Pharmacol 1993;244:37 – 47. [45] Sogo N, Magid KS, Shaw CA, et al. Inhibition of human platelet aggregation by nitric oxide donor drugs: Relative contribution of cGMP-independent mechanisms. Biochem Biophys Res Commun 2000;279:412 – 9. [46] Tsikas D, Ikic M, Tewes KS, et al. Inhibition of platelet aggregation by S-nitrosocysteine via cGMP-independent mechanisms: evidence of inhibition of thromboxane A2 synthesis in human blood platelets. FEBS Lett 1999;442:162 – 6. [47] Low SY, Sabetkar M, Bruckdorfer KR, Naseem KM. The role of protein nitration in the inhibition of platelet activation by peroxynitrite. FEBS Lett 2002;511:59 – 64. [48] Macdonald PS, Read MA, Dusting GJ. Synergistic inhibition of platelet aggregation by endothelium-derived relaxing factor and prostacyclin. Thromb Res 1988;49:437 – 49. [49] Anfossi G, Massucco P, Mularoni E, et al. Organic nitrates and compounds that increase intraplatelet guanosine monophosphate (cGMP) levels enhance the antiaggregating effects of the stable prostacyclin analogue iloprost. Prostaglandins Leukot Essent Fatty Acids 1993;49: 439 – 45. [50] Anfossi G, Massucco P, Mularoni E, et al. Effects of forskolin and organic nitrate on aggregation and intracellular cyclic nucleotide content in human platelets. Gen Pharmacol 1994;25:1093 – 100. [51] Cavallini L, Coassin MG, Borean A, Alexandre A. Prostacyclin and sodium nitroprusside inhibit the activity of the platelet inositol-1,4,5triphosphate receptor and promote its phosphorylation. J Biol Chem 1996;8:5545 – 51.
126
S.A.V. Raghavan et al. / Thrombosis Research 110 (2003) 117–126
[52] Maruice DH, Haslam RJ. Molecular basis of the synergistic inhibition of platelet function by nitrovasodilators and activators of adenylate cyclase: inhibition of cyclic AMP breakdown by cyclic GMP. Mol Pharmacol 1990;37:671 – 81. [53] Anfossi G, Russo I, Massucco P, et al. Studies on inhibition of human platelet function by sodium nitroprusside. Kinetic evaluation of the effect on aggregation and cyclic nucleotide content. Thromb Res 1999;102:319 – 20. [54] Haneda M, Kikkawa R, Horide N, et al. Glucose enhances type IV collagen production in cultured rat glomerular mesangial cells. Diabetologia 1991;34:198 – 200. [55] Prabhakar SS. Tetrahydrobiopterin reverses the inhibition of nitric
oxide by high glucose in cultured endothelial cells. Am J Physiol Renal Physiol 2001;281:F179 – 88. [56] Ting HH, Timimi FK, Boles KS, et al. Vitamin C improves endothelial dependent vasodilatation in non insulin dependent diabetes mellitus. J Clin Invest 1996;97:22 – 8. [57] Huang A, Vita JA, Venema RC, Keany JF. Ascorbic acid enhances endothelial nitric oxide synthase activity by increasing intracellular tetrahydrobiopterin. J Biol Chem 2000;275:17399 – 406. [58] Lewis EJ, Hunsicker LG, Brain RP, Rohde RD. The effect of angiotensin converting enzyme inhibition on diabetic nephropathy. N Engl J Med 1993;329:1456 – 62.
Regular Article
Role of ascorbic acid in the modulation of inhibition of platelet aggregation by polymorphonuclear leukocytes S.A.V. Raghavan, P. Sharma, M. Dikshit* Division of Pharmacology, Central Drug Research Institute, P.O. Box 173, Lucknow 226001, India Received 3 November 2002; received in revised form 28 May 2003; accepted 3 June 2003
Abstract Objectives: We investigated the modulatory effect of ascorbate on the inhibition of platelet aggregation response by polymorphonuclear leukocytes (PMNs) and characterized the mechanism of the inhibitory response. Background: PMNs have been reported to play a significant role in vascular homeostasis by releasing various factors including short-lived reactive oxygen species (ROS) and nitric oxide (NO). NO prevents the activation of circulating platelets and plays a significant role in hemostasis. In addition, PMNs also have the capacity to store very high concentrations of ascorbate. The physiological implications of storing such high concentrations of an antioxidant by a cell-releasing free radicals is unknown, viz. a viz. hemostatic regulation. Methods: ADP-induced aggregation in human, monkey and rat platelet-rich plasma (PRP) was monitored in the presence of PMNs treated with varying concentrations of ascorbate/dehydroascorbate. NO generation from rat and human PMNs treated with ascorbate was monitored on a FACS Calibur flow cytometer and intraplatelet cyclic guanosine 3V,5V-monophosphate (cGMP) levels was also measured. Results: PMNs induced a cell number and time-dependent inhibition of ADP-induced aggregation. The PMNs dependent inhibition was enhanced significantly at 30 min by ascorbate (300 AM). Ascorbate seemed to exert its effects through its oxidized product, dehydroascorbate, as the effects was prevented in the presence of D-glucose (10 mM). Dehydroascorbate elicited significant potentiation of the PMNs induced inhibitory responses and these effects were mediated by the release of NO and subsequent activation of platelet guanylyl cyclase. Flow cytometry experiments with human and rat PMNs confirmed the release of NO and the elevated platelet cGMP levels confirmed NO-mediated activation of guanylyl cyclase. Conclusions: Ascorbate in circulation seems to prevent the activation of platelets by enhancing the release of antiaggregatory NO, from neighbouring or cohabitant PMNs. The ascorbate effect is mediated through its conversion to dehydroascorbate, subsequently, gets taken up by the cell and converted back to ascorbate. Intracellular ascorbate potentiates the release of NO from the PMNs and subsequently activates guanylyl cyclase in the platelets. Condensed Abstract The thrombotic process involves platelets and polymorphonuclear leukocytes (PMNs). PMNs release both reactive oxygen species (ROS) and nitric oxide (NO). The present study was conducted to investigate the physiological significance of the ascorbate in circulation, by using rat, human and monkey platelets and PMNs. Ascorbate significantly potentiated the PMNs-mediated inhibition of aggregation. Ascorbate-mediated effects were mediated by dehydroascorbate and subsequently releases NO. NO, then activates the guanylyl cyclase in the platelets and elevates the platelet cGMP levels. It seems likely that ascorbic acid in PMNs play a significant role in vascular homeostasis by elevating levels of NO and subsequently maintaining the platelet in an inactivated state, preventing the initiation of thrombotic process. D 2003 Elsevier Ltd. All rights reserved. Keywords: Platelet aggregation; Polymorphonuclear leukocytes; Ascorbic acid; Dehydroascorbic acid; Nitric oxide; cGMP
Abbreviations: PMNs, polymorphonuclear leukocytes; ROS, reactive oxygen species; NO, nitric oxide; cGMP, cyclic guanosine 5V-monophosphate; PRP, platelet-rich plasma; DAF, diaminofluorescein diacetate; DHA, dehydroascorbate; ADP, adenosine-5V-diphosphate; PTIO, 2-phenyl4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide; ODQ, 1H-[1,2,4]oxadiazolo[4,3]quinoxalin-1-one; cAMP, cyclic adenosine 5V-monophosphate; HBSS, Hanks balanced salt solution. * Corresponding author. Tel.: +91-522-2212411 to 18; fax: +91-5222223405, +91-522-2223938. E-mail address: [email protected] (M. Dikshit). 0049-3848/03/$ - see front matter D 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0049-3848(03)00312-8
1. Introduction The thrombotic process is a multicellular phenomenon in which not only platelets but neutrophils are also involved. Coexistence of polymorphonuclear leukocytes (PMNs) with platelets in the hemostatic plug has been observed repeatedly [1,2] and their interaction with each other under different conditions has been demonstrated to influence the course of thrombosis [3– 7].
118
S.A.V. Raghavan et al. / Thrombosis Research 110 (2003) 117–126
PMNs have also been reported to release the potent antiaggregatory molecule, nitric oxide (NO) from the semi-essential amino acid L-arginine [2,8,9]. NO is known to inhibit free radical generation from PMNs [10,11] and plays a significant role in vascular homeostasis [12]. Previous work from our laboratory has demonstrated the presence of an inhibitory factor from the supernatant from PMNs on platelet aggregation [13]. Further work characterized this factor to be a h-globin related protein (unpublished observations) and the mechanism of inhibition of aggregation is under characterisation. PMNs, by their ability to release reactive oxygen species (ROS) and NO, performs the important function of phagocytosing small organisms especially bacteria. However, PMNs have the capacity to store high concentrations of ascorbic acid [14,15], a potent antioxidant. Vitamin C has been known to scavenge a wide variety of reactive nitrogen and oxygen species, mainly superoxide radicals [16]. Vitamin C increased the production of Lcitrulline and cyclic GMP, a marker of NO bioactivity, in endothelial cells and arterial cultures [17]. However, role of ascorbate in the PMNs, which has the potential to release both superoxide and NO, is not completely elucidated viz. a viz. its physiology and in vascular homeostasis. Hence, the present study was carried out to investigate the effects of ascorbate on PMNs-mediated inhibition of platelet aggregation. Results from the present study indicate that addition of ascorbate potentiated the inhibition of platelet aggregation by PMNs, by augmenting the formation of NO and related species, subsequently elevating the cGMP levels. It seems likely that ascorbic acid from PMNs play a significant role in vascular homeostasis by supplementing the levels of the relaxing and antiaggregatory factor, NO. This subsequently maintains the platelet in an inactivated state and prevents the initiation of thrombotic process.
revised 1985) and all the experiments were approved by the committee on the study of humans and ethics committee of CDRI. 2.2. Preparation of polymorphonuclear leukocytes Ten male healthy, nonsmoking, non-fasted volunteers (mean age: 31.4 F 3.1 years) were recruited for participation in this study. All the subjects included in the study were normotensive with no evidence of anemia, renal or hepatic dysfunction. Nutritional status of all the subjects included were normal (measured by skin fold thickness). None of the subjects had taken antioxidants, vitamins or nutritional supplements for at least 4 weeks prior to enrollment for the study. Blood was taken from the cubital vein in sodium citrate (3.8% w/v; 9:1 v/v). Blood was also collected from the femoral vein of Rhesus monkeys in sodium citrate (3.8% w/v; 9:1 v/v). While, rat (male, Sprague –Dawley, 200– 225 g) blood was collected by cardiac puncture under ether anaesthesia in sodium citrate (3.8%, 9:1). PMNs were isolated from the blood as reported earlier [13,18]. The PMNs were suspended in calcium and magnesium free Hank’s balanced salt solution (HBSS, pH 7.4). Cell viability was tested by Trypan blue exclusion test, which was never less than 95%. 2.3. Platelet aggregation Blood from human volunteers, monkeys or rat was collected in sodium citrate (3.8% w/v for humans and monkeys and 1.9% w/v for rats, 9:1 v/v) and centrifuged at 200 –250 g for 15 min to obtain the platelet-rich plasma (PRP). Platelet counts were adjusted to 2 108 cells/ml with platelet-poor plasma, which was obtained on further centrifugation of the blood at 2000 g for 20 min at 20 jC. Aggregation was monitored on a dual channel Lumi aggregometer (Chronolog, Haverton, CA, USA) with constant stirring at 1000 rpm at 37 jC.
2. Materials and methods
2.4. Effect of PMNs on ADP-induced platelet aggregation
2.1. Materials
To investigate the effect of PMNs on platelet aggregation, PRP was suspended with PMNs (106 cells) for 5, 15, 30 min and subsequently aggregation was monitored with ADP (5 AM). In these experiments, autologus PMNs were used to assess the effect of PMNs on ADP-induced platelet aggregation. All the interventions used in the study were also evaluated for their per se effect on ADP-induced platelet aggregation. PRP was incubated with PMNs (106 cells to 5 106 cells) for 5, 15 or 30 min prior to aggregation with ADP (5 AM). Control experiments were performed by incubating PRP with vehicle (HBSS) for the same incubation period prior to ADP-induced aggregation. Autologus PMNs were used for evaluating the effect on ADP-induced platelet aggregation.
Ascorbic acid, adenosine 5V-diphosphate (ADP), catalase, dehydroascorbic acid, 4,5-diaminofluorescein diacetate, Dglucose, 1H-[1,2,4]-oxadiazolo[4,3]quinoxalin-1-one (ODQ), hemoglobin (Hb), methylene blue, 2-phenyl4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), sodium citrate, superoxide dismutase were obtained from Sigma (St. Louis, USA). All other chemicals were obtained from E. Merck, Mumbai, India. Cyclic GMP enzyme immunoassay kit was obtained from Cayman Chemical (USA). Animals were obtained from the animal colony of CDRI, Lucknow (India) and the animal experiments conducted were in accordance of ‘‘Principles of laboratory animal care’’ (NIH publication No. 85 – 23,
S.A.V. Raghavan et al. / Thrombosis Research 110 (2003) 117–126
2.5. Effect of ascorbate or dehydroascorbate on the PMNs response Autologus PMNs were used through out for evaluating the effect of ascorbate/dehydroascorbate on ADP-induced platelet aggregation. PMNs was treated with ascorbate (100 or 300 AM) or dehydroascorbate (100 or 300 AM) for 5, 15 or 30 min prior to ADP-induced aggregation in rat, human or monkey platelets. Control experiments were performed by incubating PRP with ascorbate or dehydroascorbate. To characterise the mechanism of ascorbate-induced modulation of PMNs response on platelet aggregation, PMNs was pretreated with D-glucose (10 mM), an inhibitor of dehydroascorbate uptake prior to addition of ascorbate (300 AM) and subsequently ADP-induced aggregation was evaluated in the presence of these PMNs. PMNs and ascorbate effect was also investigated in the presence of reactive oxygen scavengers (ROS) scavengers, SOD (100 U/ml) and catalase (500 U/ml) for 30 min prior to addition of ascorbate (300 AM) and subsequently ADPinduced aggregation was induced. To investigate the involvement of NO/cGMP, rat or human PRP was incubated with NO scavenger, PTIO (100 AM) or inhibitor of soluble guanylyl cyclase (ODQ, 10 AM) for 15 min prior to the addition of PMNs treated with ascorbate or dehydroascorbate and subsequently ADP-induced aggregation was evaluated. While, monkey PRP was incubated with haemoglobin (10 AM), a NO scavenger or with methylene blue (10 AM), an inhibitor of soluble guanylyl cyclase for 15 min prior to addition of PMNs treated with ascorbate (300 AM) and subsequently ADPinduced aggregation was evaluated. Aggregation to ADP (5 AM) was considered as 100% and aggregation to ADP in the presence of various interventions are represented as percentage of the ADP-induced aggregation [18].
119
action was stopped with 6% (final concentration) ice-cold trichloroacetic acid (TCA). Precipitated proteins were removed by centrifugation at 5000 g for 10 min at 4 jC. The supernatant was separated and TCA was extracted with 4 volumes of water saturated diethylether (five times). The ethereal layer was discarded and aqueous layer was heated at 55 – 60 jC for 5 min to remove residual ether. The samples were then lyophilised and cGMP was estimated on acetylation by enzyme immunoassay according to the manufacturer’s instructions (Cayman Chemical). Platelet cGMP values are expressed as nM/106 platelets. 2.8. Statistical analysis All results are expressed as mean F S.E.M. of at least five experiments. The statistical significance between two columns was calculated by using unpaired Student’s t-test and multiple columns were compared by one-way ANOVA followed by Student – Newman– Keuls test. The difference between two groups was considered significant at p < 0.05.
2.6. Estimation of NO generation by flow cytometry PMNs from rats and humans were used for assessing the release of NO by flow cytometry. PMNs (2 106 cells/ml) were incubated for 5 min with 4,5-diaminofluorescein diacetate (DAF-2 DA, 10 AM) [19] and were subsequently treated with ascorbate or DHA for 30 min at 37 jC. Each sample was evaluated on FACS Calibur (Becton Dickinson, USA). The increase in fluorescence were analyzed by Cell Quest program (Becton Dickinson) and results are represented as shifts in the histogram corresponding to the increased NO generation [20,21]. 2.7. Estimation of cyclic GMP levels As the trend in the ascorbate/dehydroascorbate-mediated response on platelets was similar in PMNs, mechanism of inhibition of ADP-induced aggregation was elucidated in rat PMNs. At the end of platelet aggregation, the platelet re-
Fig. 1. (A) Inhibition of ADP (5 AM)-induced platelet aggregation by PMNs (106 to 5 106 cells) in rat PRP. PMNs-mediated inhibition were evaluated from the same rat PRP (n = 5 in each set). *P < 0.001 in the presence of PMNs compared to vehicle treatment. #P < 0.05 compared to 106 PMNs. (B) Time-dependent inhibition of ADP (5 AM)-induced aggregation in PRP from human (n = 5), rat (n = 10) and monkey (n = 5). PMNs and PRP were obtained from the same animal/human. *P < 0.05, **P < 0.01, ***P < 0.001 in PMNs (106 cells) at different time intervals compared to their respective group at 5 min of incubation.
120
S.A.V. Raghavan et al. / Thrombosis Research 110 (2003) 117–126
Data were analyzed using Graph Pad Prism 3.0 statistical package (San Diego, CA, USA).
3. Results 3.1. Effect of PMNs on ADP-induced platelet aggregation ADP (5 AM) produced an irreversible aggregation ( f 60%) over a duration of 5 –7 min in all the three species under study. Addition of rat PMNs to platelet-rich plasma produced a cell number (1 106 to 5 106 PMNs) dependent inhibition of ADP-induced aggregation. Significant inhibition of aggregation was observed with 106 PMNs after 5 min of incubation with PRP (Fig. 1A) and the same cell number was used for the subsequent experiments for different time intervals in all the species under study. ADP-
Fig. 3. Time-dependent effects of dehydroascorbate (100 AM and 300 AM) on the PMNs (106 cells)-induced inhibition of ADP (5 AM) in rat PRP. *P < 0.05, **P < 0.01, ***P < 0.001 at 15 and 30 min of incubation compared to their respective treatments at 5 min of incubation. #P < 0.05 in the presence of dehydroascorbate (300 AM) compared to their absence at 30 min of incubation (n = 10 in each group). @P < 0.05 in the presence of 300 AM compared to 100 AM dehydroascorbate at 30 min of incubation (n = 10 in each group).
induced aggregation by rat, human or monkey PMNs (Fig. 1B) was also inhibited in a time-dependent manner. The maximal inhibition of aggregation was observed at the end of 30 min and further incubation did not produce any significant enhancement of the inhibitory response. 3.2. Effect of ascorbate on the PMNs-mediated response
Fig. 2. (A) Effect of ascorbate (100 AM and 300 AM) on PMNs (106 cells)mediated inhibition of ADP (5 AM)-induced aggregation in rat PRP at 5, 15 and 30 min of incubation. *P < 0.01, **P < 0.001 in different groups at 15 and 30 min compared to their respective treatments at 5 min of incubation (n = 10 in each group). (B) Effect of scavengers of ROS, SOD (100 U/ ml) + Catalase (500 U/ml) on ascorbate (300 AM)-induced augmentation of PMNs (106 cells)-mediated inhibition of ADP (5 AM)-induced aggregation in rat PRP. *P < 0.001 at 15 and 30 min of incubation compared to their respective treatments at 5 min of incubation (n = 10 in each group). # P < 0.05 in the presence of scavengers of ROS compared to their absence at 30 min of incubation.
Incubation of rat PRP with different concentrations of ascorbate (1 AM – 1 mM) did not produce significant changes in the ADP-induced aggregations (data not shown). However, pretreatment of rat PMNs (106 cells) with ascorbate augmented the inhibition of ADP induced aggregation in a concentration- and time-dependent manner. Significant attenuation of ADP-induced aggregation was observed only after 30 min of incubation of 100 AM ascorbate with PRP, whereas 300 AM ascorbate showed significant inhibition at 15 min of incubation (Fig. 2A). Treatment of PMNs with concentrations greater than 300 AM ascorbate produced significant inhibitions of ADP-induced aggregation even at lesser incubation intervals (maximal aggregation at 1 mM ascorbate being, 28 F 2.5% (n = 3) and 14 F 3.8% (n = 3) after 15 and 30 min of incubation, respectively). Pretreatment of the rat PMNs with scavengers of ROS, i.e., combination of superoxide dismutase (SOD) and catalase, augmented the inhibitory responses of ascorbate (Fig. 2B). The inhibitory response was potentiated at 30 min of incubation with SOD and catalase (Fig. 2B). 3.3. Effect of dehydroascorbate on the PMNs-mediated response Effect of dehydroascorbate was evaluated on the cosuspension of PMNs and PRP in rats. The inhibition of
S.A.V. Raghavan et al. / Thrombosis Research 110 (2003) 117–126
aggregation of PMNs treated with dehydroascorbate was more than that observed on PMNs treated with ascorbate, and DHA (300 AM)-mediated effects was comparable to that of ascorbate (1 – 2 mM). Similar to ascorbate, dehydroascorbate at 300 AM showed significant inhibition at lesser incubation intervals compared to 100 AM dehydroascorbate (Fig. 3). In all the species studied, dehydroascorbate (300 AM) produced significant potentiation of PMNs-mediated inhibition at 30 min of incubation compared to ascorbate (300 AM) (Fig. 4A and B). Ascorbate by itself possesses minimal capacity to enter PMNs. Ascorbate enters the cell as its oxidized product, dehydroascorbic acid, through the GLUT1 transporter, and is inhibited by high D-glucose [22]. Hence, we performed experiments to ascertain the hypothesis that the effects of ascorbic acid are indeed mediated by dehydroascorbic acid. Rat PRP was preincubated with glucose (10 mM), which inhibited the uptake of dehydroascorbic acid into the PMNs. Pretreatment with glucose significantly inhibited the atten-
121
Fig. 5. Effect of glucose (10 mM) on the ascorbate (100 AM and 300 AM)induced augmentation of PMNs (106 cells)-mediated inhibition of ADP (5 AM)-induced aggregation in rat PRP. *P < 0.05, **P < 0.01 in the presence of glucose compared to their respective treatments (n = 10 and n = 5 in the absence and presence of glucose, respectively).
uation in aggregation responses by PMNs alone or PMNs incubated with ascorbate (Fig. 5), confirming that ascorbic acid-induced effects on platelet aggregation was indeed mediated by its oxidized product, dehydroascorbic acid. Ascorbate in the suspension medium was spontaneously oxidized to dehydroascorbate by molecular oxygen and hence entered the PMNs to elicit the inhibitory effects. Subsequently, experiments were performed to characterize the mechanism behind the ascorbate/DHA-mediated augmentation of inhibition of aggregation. 3.4. Involvement of NO/cGMP in ascorbate- or dehydroascorbate-mediated response
Fig. 4. (A) Time-dependent effects of ascorbate (300 AM) and DHA (300 AM) on the PMNs (106 cells)-induced inhibition of ADP (5 AM)-induced aggregation in human PRP. *P < 0.05, **P < 0.01 in the respective treatments at 15 and 30 min of incubation compared to the same treatment at 5 min of incubation. #P < 0.05 in the ascorbate- and DHA-treated group in comparison to PMNs alone at 30 min of incubation. (B) Timedependent effects of ascorbate (300 AM) and DHA (300 AM) on the PMNs (106 cells)-induced inhibition of ADP (5 AM)-induced aggregation in human PRP. *P < 0.05, **P < 0.01 in the respective treatments at 30 min of incubation compared to the same treatment at 5 min of incubation. # P < 0.05 in the DHA-treated group in comparison to PMNs alone at 30 min of incubation.
Dehydroascorbate like ascorbate did not inhibit ADPinduced aggregations similar to ascorbate. Since no significant alterations in the aggregation responses was observed on treatment of PRP with varying concentrations of ascorbate/ dehydroascorbate, it seemed likely that the effects of ascorbate were due to the release/production of some intermediates from PMNs, which exerted antiaggregatory effects on the platelets. Previous work from our lab has indicated towards the involvement of the potent antiaggregatory factor, NO. To confirm the involvement of NO, PRP was incubated with NO scavenger, PTIO or hemoglobin, and NO-sensitive guanylyl cyclase inhibitor, ODQ or methylene blue, before treatment with ascorbate- or dehydroascorbate-treated PMNs. Pretreatment of PRP with PTIO (to scavenge NO) and ODQ (to inhibit soluble guanylyl cyclase) significantly inhibited the potentiation of inhibition by PMNs at all time intervals in rats. Human PRP incubated with PTIO or ODQ also showed similar trend at 30 min of incubation with either ascorbate or dehydroascorbate. Monkey PRP incubated with hemoglobin (another scavenger of NO) or methylene blue (guanylyl cyclase inhibitor), also reversed the potentiation of inhibition
122
S.A.V. Raghavan et al. / Thrombosis Research 110 (2003) 117–126
by ascorbate (300 AM; 30 min) or dehydroascorbate (300 AM; 30 min). NO scavengers and guanylyl cyclase inhibitors reversed the inhibition of aggregation, confirming the involvement of NO-mediated activation of soluble guanylyl cyclase (Fig. 6A – C). 3.5. NO generation in ascorbate-treated PMNs In the present study, it seemed that NO released from the PMNs was responsible for the observed effects with ascor-
Fig. 6. (A) Effect of NO scavenger, PTIO (100 AM) and guanylyl cyclase inhibitor, ODQ (10 AM) on the ascorbate (300 AM)- and dehydroascorbate (300 AM)-induced effects on PMNs (106 cells)-mediated inhibition of ADP (5 AM)-induced aggregation in rat PRP. **P < 0.01 in the PTIO and ODQ pretreated group in comparison to their respective controls. #P < 0.05 at 30 min compared to the same treatment at 15 min of incubation. (B) Effect of NO scavenger, PTIO (100 AM) and guanylyl cyclase inhibitor, ODQ (10 AM) on the ascorbate (300 AM)- and dehydroascorbate (DHA, 300 AM)induced augmentation of PMNs (106 cells)-induced inhibition of ADP (5 AM)-induced aggregation in human PRP. *P < 0.01 in the PTIO and ODQ pretreated group in comparison to their respective controls. (C) Effect of NO scavenger, hemoglobin (10 AM) and guanylyl cyclase inhibitor, methylene blue (10 AM) on the ascorbate (300 AM)-induced augmentation of PMNs (106 cells)-mediated inhibition of ADP (5 AM)-induced aggregation in monkey PRP. *P < 0.01 in the PTIO and ODQ pretreated group in comparison to their respective controls.
Fig. 7. (A) Representative histogram indicating increased fluorescence to 4V,5V-diaminofluorescein diacetate from rat PMNs (2 106 cells/ml) treated with ascorbate 300 AM after 30 min of incubation. Shifts indicated in figure are representations of one out of five independent experiments. (B) Representative histogram indicating increased fluorescence to 4V,5Vdiaminofluorescein diacetate from human PMNs (2 106 cells/ml) treated with ascorbate 300 AM after 30 min of incubation. Shifts indicated in figure are representations of one out of three independent experiments.
S.A.V. Raghavan et al. / Thrombosis Research 110 (2003) 117–126
Fig. 8. Bar diagram depicting increased platelet cGMP levels estimated by enzyme immunoassay from PMNs (106 cells) alone or from PMNs treated with ascorbate (300 AM; 30 min)/dehydroascorbate (DHA, 300 AM; 30 min) on the ADP (5 AM)-induced aggregation in rat PRP. *P < 0.05 in the DHA-treated group compared to PMNs receiving no treatment.
bate and dehydroascorbate. PMNs loaded with 4,5-diaminofluorescein diacetate, a dye specific for nitric oxide measurement, prior to treatment with ascorbate also exhibited formation of NO. Mean fluorescence was significantly increased in both the species after 30 min of incubation, indicating that PMNs treated with ascorbate released significant amounts of NO (Fig. 7A and B). 3.6. Effect of ascorbate/dehydroascorbate on platelet cGMP levels To further ascertain the activation of guanylyl cyclase by NO, platelet cGMP levels were estimated in presence of PMNs treated with either ascorbate or dehydroascorbate. Incubation of PRP with PMNs (106 cells) elevated the cGMP levels significantly (Fig. 8). Platelet cGMP levels were further increased in the presence of PMNs incubated with ascorbate/dehydroascorbate, suggesting that incubation of PMNs with ascorbate or dehydroascorbate potentiated the inhibitory responses to ADP-induced aggregation by elevating cGMP levels.
4. Discussion L-Ascorbic acid has been described to exert multiple beneficial effects in cardiovascular disorders associated with impaired nitric oxide/cGMP signalling. Thus, endothelial dysfunction in the course of essential hypertension was improved by supplementation of vitamin C [23]. Others described the preventive effect on the development of tolerance during long-term administration of organic nitrates [24]. Recently, stimulation of NO biosynthesis by L-ascorbic acid has been observed in human endothelial cells [17] and
123
the antioxidant was shown to sensitize isolated coronary arteries towards NO-induced vasodilation [25]. Oral administration of vitamin C, reaching up to a plasma concentration of 100 AM, has been reported to reduce arterial stiffness and platelet aggregation [26]. However, the mechanism involving protection of NO from inactivation in this study by vitamin C was not confirmed. Moreover, the mechanism behind this antiaggregatory effect has not been explained to date. Vitamin C has been reported to exert a pro-oxidant effect in addition to its antioxidant effect [27,28] and particularly increases the nitrosative stress in cells [29,30]. Hence, it is unlikely that the antiaggregatory effects of this vitamin could be mainly due to the antioxidant effect, associated with this vitamin. In the present study, we did not observe any significant inhibition in vitro of ADP-induced aggregation with ascorbate (1 AM to 1 mM). The present study was a part of the study which was aimed to evaluate the role of ascorbic acid in regulating PMNs function. We initially tested the idea in rat cells, and the outcome of the study prompted us to confirm it in primates and humans. PMNs, the co-habitant also synthesize NO in substantial amounts [8,18,31] and like endothelial cells, also store high concentrations of ascorbate [14]. Role of ascorbate in PMNs is not so well defined. Previous work from our lab has already demonstrated inhibition of ADP-induced aggregation by the factors released from PMNs [13]. One of the most potent inhibitory factor released by PMNs is NO [8,18,31]. Moreover, PMNs also play a significant role in hemostasis and thrombosis [3,4,18]. Hence, we found it worthwhile to investigate the role of ascorbate in PMNs and platelet interactions. Co-suspension of PMNs with platelets showed augmented inhibition of aggregation on treatment with ascorbate. As ascorbate by itself did not elicit any effect on platelet aggregation, it seemed likely that ascorbate-mediated inhibition of aggregation were due to its effect on PMNs. Ascorbate potentiated the PMNs-mediated inhibition of aggregation in a time- and concentration-dependent manner (Fig. 2A). Pretreatment of PRP with scavengers of ROS indicated that the effects of ascorbate were not mediated due to its antioxidant action (Fig. 2B). Had ascorbate exerted its antioxidant effect, such high concentration of ascorbate used in this study, should have been sufficient to scavenge superoxide radicals, and further addition of scavengers would not have any additional effect. Vitamin C exists primarily as ascorbic acid (reduced form) in human plasma and enters the cells slowly by a Na-dependent transport. Transiently produced dehydroascorbic acid from the activated PMNs is transported into cells readily by the GLUT1 transporter. No measurable dehydroascorbic acid is detected in human blood [32,33]. Oxidants in circulation have the capacity to oxidize extracellular ascorbate to dehydroascorbic acid. Dehydroascorbic acid is more rapidly transported than ascorbate and is immediately reduced intracellularly to ascorbate [14]. This observation
124
S.A.V. Raghavan et al. / Thrombosis Research 110 (2003) 117–126
was confirmed in our studies as pretreatment of PRP with Dglucose (10 mM) inhibited the anti aggregatory effects observed with PMNs treated with either ascorbate/dehydroascorbate. Moreover, extracellular administration of dehydroascorbate elicited higher inhibition in comparison to ascorbate indicating the ready transport and immediate effect (Figs. 3 and 4A,B). Also, Takajo et al. [34] observed that following oral administration of vitamin C, concentrations of vitamin C were higher in the platelets than plasma suggesting the transport of vitamin C into the cells. Treatment of the co-suspension of platelets and PMNs with ascorbate, though reduces the oxidative stress, however, seems to increase the nitrosative stress. The nitrosative stress is of particular interest since this can generate NO donors, based upon modification of polyhydroxylated compounds and thiols [35 –38]. The chemical identity of the NO donor(s) generated is likely to involve the formation of either S-nitrosothiols or organic nitrites [35,36]. Synthesis of NO from PMNs has been well reported and the NO modulates release of other free radicals from PMNs [11,20,39]. Recently, Sharma et al. [21] have observed that increased oxidative stress in the PMNs seem to favor release of other free radicals over NO. Results from the present study also suggest to this conclusion, as ascorbate augmented the release of NO. The mechanism of release of NO from PMNs was not explored in detail in the present study, but, could probably involve one or more of the mechanism suggested for endothelial cells which is presently under investigation in our lab. Ascorbate acts intracellularly, [40] and could improve the availability for NOS of tetrahydrobiopterin [17], or sparing intracellular thiols [41,42]. To investigate the involvement of NO, we used DAF, specific probe for NO, in the flow cytometry experiments. Increase in the DAF fluorescence in the PMNs treated with ascorbate indicated the involvement of NO (Fig. 7A and B). This result was confirmed on preincubation of PRP with scavengers of NO (Fig. 6A –C). NO subsequently activates soluble guanylyl cyclase elevating platelet cGMP levels. The reversal of inhibition with guanylyl cyclase inhibitors (Fig. 6A –C) and elevated cGMP levels from rat platelets (Fig. 8) confirmed the involvement of NO – sGC system in the potentiation of PMNs induced inhibition of aggregation by both ascorbate and dehydroascorbate. The NO-mediated antiaggregatory effect on platelet has been reported to be mediated by an increase in cGMP levels [31,43,44]. However, cGMP-independent mechanisms of inhibition of platelet aggregation by NO and related species have been documented recently [45 – 47]. A possible explanation of the role of cGMP in the inhibition of platelet activation could be due to the nitration of platelet proteins by the formation of NO and NO donors, subsequently forming S-nitrosothiols and consequent inhibition of platelet aggregation [47]. An increase in cAMP in the platelets cannot be dismissed in the inhibitory effects observed in the present study. However, NO-mediated increase of intraplatelet levels of cAMP have been observed in many studies
where elevation of cGMP [48 – 51] is explained due to the reduction of cGMP-inhibited cAMP phosphodiesterase [52,53]. We however did not investigate involvement of cGMP-independent effect in the ascorbate-mediated effects. In hyperglycemia, where there is a decreased cellular content of L-arginine [54,55], supplementation of ascorbate could enhance the synthesis of NO by increasing the intracellular tetrahydrobiopterin levels [56,57]. High glucose concentration might also interfere with ascorbate/dehydroascorbate uptake and thereby down regulating NO bioavailability. A prothrombotic state is very common in diabetic patients. Ascorbate could also enhance the release of NO in circulation and could control the renin – angiotensin activation [58], preventing the progression of diabetic nephropathy. Though the role of ascorbate in platelet function has been enumerated, the results of the present study give a better understanding of ascorbate in hemostasis. In circulation, ascorbate prevents the activation of platelets not only by acting as an antioxidant, but also by potentiating the release of NO from the PMNs. The NO released from the PMNs subsequently activates the guanylate cyclase, elevating cGMP levels in the platelets and inhibits the aggregation of platelets. The present study is yet another confirmation of the regulatory role of the potent relaxing and antiaggregatory factor, nitric oxide. Acknowledgements Financial support in form of grant-in-aid to MD from the Department of Biotechnology, New Delhi, India is gratefully acknowledged. SAVR and PS acknowledge CSIR, New Delhi for the Senior Research Fellowship. Technical assistance of Mr. A.L. Viswakarma during the flow cytometry experiments is acknowledged. References [1] Marcus AJ. Recent progress in the role of platelets in occlusive vascular disease. Stroke 1983;14:475 – 9. [2] Dikshit M, Kumari R. Modulation of platelet aggregation response by factors released from polymorphonuclear leukocytes. Hematology 1997;2:39 – 53. [3] Marcus AJ. Thrombosis and inflammation as multicellular process: pathophysiologic significance of transcellular metabolism. Blood 1990;66:1093 – 7. [4] Cerletti C, Evangelista V, de Gaetono G. Polymorphonuclear leukocyte-dependent modulation of platelet function: relevance to the pathogenesis of thrombosis. Pharmacol Res 1992;26:168 – 261. [5] Bazzoni G, Dejana E, Delmaschio A. Platelet – neutrophil interactions—possible relevance in the pathogenesis of thrombosis and inflammation. Haematologica 1991;76:491 – 9. [6] Faint RW. Platelet neutrophil interactions: their significance. Blood Rev 1992;6:83 – 91. [7] Stewart BJ. Neutrophils and deep venous thrombosis. Haemostasis 1993;23:127 – 40. [8] Salvemini D, de Nucci G, Sneddon JM, Vane JR. Human neutrophils and mononuclear cells inhibit platelet aggregation by releasing a nitric oxide-like factor. Proc Natl Acad Sci U S A 1989;86:6328 – 32.
S.A.V. Raghavan et al. / Thrombosis Research 110 (2003) 117–126 [9] Yoshimoto H, Suchiro A, Kakishita E. Exogenous nitric oxide inhibits platelet activation in whole blood. J Cardiovasc Pharmacol 1999;33: 109 – 15. [10] Rubanyi GM, Ho EH, Cantor EH, et al. Cytoprotective function of nitric oxide: Inactivation of superoxide radicals produced by human leukocytes. Biochem Biophys Res Commun 1991;181:1392 – 7. [11] Seth P, Kumari R, Dikshit M, Srimal RC. Modulation of rat peripheral polymorphonuclear leukocyte response by nitric oxide and arginine. Blood 1994;84:2741 – 8. [12] Sethi S, Dikshit M. Modulation of polymorphonuclear leukocytes function by nitric oxide. Thromb Res 2000;100:223 – 47. [13] Kumari R, Singh MP, Seth P, Dikshit M. Inhibition of platelet aggregation by a protein factor present in rat neutrophil supernatant. Thromb Res 1998;91:75 – 82. [14] Welch RW, Wang Y, Crossman A, et al. Accumulation of vitamin C (ascorbate) and its oxidized metabolite dehydroascorbic acid occurs by separate mechanisms. J Biol Chem 1995;21:12584 – 92. [15] Park JB, Levine M. Purification, cloning and expression of dehydroascorbic acid reducing activity from human neutrophils: identification as glutaredoxin. Biochem J 1996;315:931 – 8. [16] Gotoh N, Niki E. Rates of interactions of superoxide with vitamin E, vitamin C and related compounds as measured by chemiluminescence. Biochim Biophys Acta 1992;1115:201 – 7. [17] Heller R, Munscher-Paulig F, Grabner R, Till U. L-ascorbic acid potentiates nitric oxide synthesis in endothelial cells. J Biol Chem 1999;274:8254 – 60. [18] Dikshit M, Kumari R, Srimal RC. Pulmonary thromboembolism induced alterations in nitric oxide release from rat circulating neutrophils. J Pharmacol Exp Ther 1993;265:1369 – 72. [19] Qiu W, Kass DA, Hu Q, Ziegelstein RC. Determination of shear stress-stimulated endothelial NO production assessed in real time by 4,5-diaminofluorescein fluorescence. Biochem Biophys Res Commun 2001;286:328 – 35. [20] Sethi S, Sharma P, Dikshit M. Nitric oxide and oxygen derived free radical generation from control and lipopolysaccharide treated rat polymorphonuclear leukocyte. Nitric Oxide 2001;5:482 – 93. [21] Sharma P, Barthwal MK, Dikshit M. NO synthesis and its regulation in the arachidonic acid stimulated polymorphonuclear leukocytes. Nitric Oxide 2002;7:119 – 26. [22] Washko P, Levine M. Inhibition of ascorbic acid transport in human neutrophils by glucose. J Biol Chem 1992;267:23568 – 74. [23] Taddei S, Virdis A, Ghiadoni L, et al. Vitamin C improves endothelium-dependent vasodilation by restoring nitric oxide activity in essential hypertension. Circulation 1998;97:2222 – 9. [24] Hinz B, Schroder H. Vitamin C attenuates nitrate tolerance independently of its antioxidant effect. FEBS Lett 1998;428:97 – 9. [25] Murphy ME. Ascorbate and dehydroascorbate modulate nitric oxideinduced vasodilations of rat coronary arteries. J Cardiovasc Pharmacol 1999;34:295 – 303. [26] Wilkinson IB, Megson IL, MacCallum H, et al. Oral vitamin C reduces arterial stiffness and platelet aggregation in humans. J Cardiovasc Pharmacol 1999;34:690 – 3. [27] Halliwell B, Gutteridge JMC. Free radicals in biology and medicine. New York: Clarendon Press; Oxford, UK: Oxford Univ. Press; 1999. [28] Roginsky VA, Stegmann HB. Ascorbyl radical as natural indicator of oxidative stress: quantitative regularities. Free Radic Biol Med 1994; 17:93 – 103. [29] Childs A, Jacobs C, Kaminski T, et al. Supplementation with vitamin C and N-acetylcysteine increases oxidative stress in humans after an acute muscle injury induced by eccentric exercise. Free Radic Biol Med 2001;31:745 – 53. [30] Brown AS, Moro MA, Masse JM, et al. Nitric oxide-dependent and independent effects on human platelets treated with peroxynitrite. Cardiovasc Res 1998;40:380 – 8. [31] Faint RW, Mackie IJ, Machin SJ. Platelet aggregation is inhibited by a nitric oxide like factor released from human neutrophils in vitro. Br J Hematol 1991;77:539 – 45.
125
[32] Koshiishi I, Mamura Y, Liu J, Lmanari T. Evaluation of an acidic deproteinization for the measurement of ascorbate and dehydroascorbate in plasma samples. Clin Chem 1998;44:863 – 8. [33] Levine M, Dhariwal KR, Wang Y, et al. In: Frei B, editor. Natural antioxidants in human health and disease. San Diego, CA: Academic Press; 1994. p. 469 – 84. [34] Takajo Y, Ikeda H, Haramaki N, et al. Augmented oxidative stress of platelets in oxidative stress in chronic smokers. J Am Coll Cardiol 2001;38:1320 – 7. [35] Moro MA, Darley-Usmar VM, Goodwin DA, et al. Paradoxical and biological action of peroxynitrite on human platelets. Proc Natl Acad Sci U S A 1994;91:6702 – 6. [36] Moro MA, Darley-Usmar VM, Lizasoain J, et al. The formation of nitric oxide donors from peroxynitrite. Br J Pharmacol 1995;116: 1999 – 2004. [37] White CR, Moellering D, Patel R, et al. Formation of the NO donors glyceryl mononitrate and glyceryl mononitrite from the reaction of peroxynitrite with glycerol. Biochem J 1997;328:517 – 24. [38] Mayer B, Schrammel A, Klatt P, et al. Peroxynitrite-induced accumulation of cyclic GMP in endothelial cells and stimulation of purified soluble guanylyl cyclase. Dependence on glutathione and possible role of S-nitrosation. J Biol Chem 1995;270:17355 – 60. [39] Sethi S, Singh MP, Dikshit M. Nitric oxide mediated augmentation of polymorphonuclear leukocyte free radical generation after hypoxiareoxygenation. Blood 1999;9:333 – 40. [40] Martin A, Frei B. Both intracellular and extracellular vitamin C inhibit atherogenic modification of LDL by human vascular endothelial cells. Arterioscler Thromb Vasc Biol 1997;17:1583 – 90. [41] Carr AC, Frei B. Toward a new recommended dietary allowance for vitamin C based on antioxidant and health effects in humans. Am J Clin Nutr 1999;69:1086 – 107. [42] Carr A, Frei B. The role of natural antioxidants in preserving the biological activity of endothelium-derived nitric oxide. Free Radic Biol Med 2000;28:1806 – 14. [43] Gerzer R, Karrenbrock B, Siess W, Heim J-M. Direct comparison of the effects of nitroprusside, SIN-1, and various nitrates on platelet aggregation and soluble guanylyl cyclase activity. Thromb Res 1988;52: 11 – 21. [44] Ivanova K, Schaefer M, Drummer C, Gerzer R. Effects of nitric oxidecontaining compounds on increases in cytosolic ionized Ca2 + and on aggregation of human platelets. Eur J Pharmacol 1993;244:37 – 47. [45] Sogo N, Magid KS, Shaw CA, et al. Inhibition of human platelet aggregation by nitric oxide donor drugs: Relative contribution of cGMP-independent mechanisms. Biochem Biophys Res Commun 2000;279:412 – 9. [46] Tsikas D, Ikic M, Tewes KS, et al. Inhibition of platelet aggregation by S-nitrosocysteine via cGMP-independent mechanisms: evidence of inhibition of thromboxane A2 synthesis in human blood platelets. FEBS Lett 1999;442:162 – 6. [47] Low SY, Sabetkar M, Bruckdorfer KR, Naseem KM. The role of protein nitration in the inhibition of platelet activation by peroxynitrite. FEBS Lett 2002;511:59 – 64. [48] Macdonald PS, Read MA, Dusting GJ. Synergistic inhibition of platelet aggregation by endothelium-derived relaxing factor and prostacyclin. Thromb Res 1988;49:437 – 49. [49] Anfossi G, Massucco P, Mularoni E, et al. Organic nitrates and compounds that increase intraplatelet guanosine monophosphate (cGMP) levels enhance the antiaggregating effects of the stable prostacyclin analogue iloprost. Prostaglandins Leukot Essent Fatty Acids 1993;49: 439 – 45. [50] Anfossi G, Massucco P, Mularoni E, et al. Effects of forskolin and organic nitrate on aggregation and intracellular cyclic nucleotide content in human platelets. Gen Pharmacol 1994;25:1093 – 100. [51] Cavallini L, Coassin MG, Borean A, Alexandre A. Prostacyclin and sodium nitroprusside inhibit the activity of the platelet inositol-1,4,5triphosphate receptor and promote its phosphorylation. J Biol Chem 1996;8:5545 – 51.
126
S.A.V. Raghavan et al. / Thrombosis Research 110 (2003) 117–126
[52] Maruice DH, Haslam RJ. Molecular basis of the synergistic inhibition of platelet function by nitrovasodilators and activators of adenylate cyclase: inhibition of cyclic AMP breakdown by cyclic GMP. Mol Pharmacol 1990;37:671 – 81. [53] Anfossi G, Russo I, Massucco P, et al. Studies on inhibition of human platelet function by sodium nitroprusside. Kinetic evaluation of the effect on aggregation and cyclic nucleotide content. Thromb Res 1999;102:319 – 20. [54] Haneda M, Kikkawa R, Horide N, et al. Glucose enhances type IV collagen production in cultured rat glomerular mesangial cells. Diabetologia 1991;34:198 – 200. [55] Prabhakar SS. Tetrahydrobiopterin reverses the inhibition of nitric
oxide by high glucose in cultured endothelial cells. Am J Physiol Renal Physiol 2001;281:F179 – 88. [56] Ting HH, Timimi FK, Boles KS, et al. Vitamin C improves endothelial dependent vasodilatation in non insulin dependent diabetes mellitus. J Clin Invest 1996;97:22 – 8. [57] Huang A, Vita JA, Venema RC, Keany JF. Ascorbic acid enhances endothelial nitric oxide synthase activity by increasing intracellular tetrahydrobiopterin. J Biol Chem 2000;275:17399 – 406. [58] Lewis EJ, Hunsicker LG, Brain RP, Rohde RD. The effect of angiotensin converting enzyme inhibition on diabetic nephropathy. N Engl J Med 1993;329:1456 – 62.