(Circulation. 1997;95:885-891.)
© 1997 American Heart Association, Inc.
Articles |
Platelet Activation by Superoxide Anion and Hydroxyl Radicals Intrinsically Generated by Platelets That Had Undergone Anoxia and Then Reoxygenated
the Institute of Clinical Medicine I (R.L., L.I., A.R.C., F.V.) and the Department of Experimental Medicine and Pathology (F.M.P., P.P.), University La Sapienza, Rome, Italy; the Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia (D.P., G.A.F.); and the National Institute of Nutrition (A.G.), Rome, Italy.
Correspondence to Francesco Violi, MD, Institute of Clinical Medicine I, University La Sapienza, Policlinico Umberto I, 00185, Rome, Italy.
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Abstract |
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Background Platelet activation has been demonstrated in experimental and clinical models of ischemia-reperfusion, but the underlying mechanism is still unclear. We mimicked the ischemia-reperfusion model in vitro by exposing platelets to anoxia-reoxygenation (A-R) and evaluated the role of oxygen free radicals (OFRs), which are usually produced during the reperfusion phase, in inducing platelet activation.
Methods and Results Human platelets were exposed to 15 and 30 minutes of anoxia and then reoxygenated. Compared with control platelets kept in atmospheric conditions, platelets exposed to A-R showed spontaneous platelet aggregation (SPA), which was maximal after 30 minutes of anoxia. Superoxide dismutase (SOD) (–74%, P<.005), catalase (–67%, P<.005), SOD plus catalase (–82%, P<.005), and the hydroxyl radical (OH°) scavengers mannitol (–66%, P<.005) and deoxyribose (–55%, P<.005) inhibited SPA. Platelets that had undergone A-R released superoxide anion (O2–), as detected by lucigenin chemiluminescence. Also, platelets exposed to A-R and incubated with salicylic acid generated 2,3- and 2,5-dihydroxybenzoates, which derive from salicylic acid reaction with OH°. SPA was significantly inhibited by the cyclooxygenase enzyme inhibitors aspirin and indomethacin; by SQ29548, a thromboxane (Tx) A2 receptor antagonist; by diphenyliodonium, an inhibitor of flavoprotein-dependent enzymes; and by arachidonyl trifluoromethyl ketone, a selective inhibitor of cytosolic phospholipase A2. Platelets exposed to A-R markedly generated inositol 1,3,4-trisphosphate and TxA2, which were inhibited by incubation of platelets with SOD plus catalase.
Conclusions This study shows that platelets exposed to A-R intrinsically generated O2– and OH°, which in turn activate arachidonic acid metabolism via phospholipases A2 and C, and provides further support for the use of antioxidant agents as inhibitors of platelet function in ischemia-reperfusion models.
Key Words: platelets • free radicals • hypoxia
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Introduction |
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Experimental and clinical studies provide evidence that platelet activation occurs upon ischemia-reperfusion.1 In patients with myocardial infarction treated with tissue-type plasminogen activator, Kerins et al2 found an increased urinary excretion of 2,3-dinor-TxB2, a stable metabolite of TxA2, providing evidence that platelet activation occurs in vivo after thrombolytic treatment.2 Since platelet activation delays the time to reperfusion and induces vascular reocclusion,3 4 a knowledge of the mechanism leading to platelet activation during reperfusion would be useful to counteract its deleterious effects. Several hypotheses have been postulated for explaining platelet activation occurring upon thrombolysis. It has been proposed that platelets could be stimulated by plasmin, thrombin, or exposure to injured vascular bed.1 A burst of the OFRs O2– and OH° was reported in experimental models of ischemia-reperfusion.5 6 Studies from this laboratory have shown that OFRs favor PA, acting as second messengers.7 Therefore, it is conceivable that in models of ischemia-reperfusion, OFRs could have a role in stimulating platelet function. Support for this hypothesis was provided by experimental models of vascular occlusion-reperfusion in which OFRs mediated PA.8 9 However, it has never been examined whether intrinsic functional changes of platelets may account for their activation occurring after ischemia-reperfusion. In particular, it remains to be established whether platelets generate OFRs and how these compounds mediate PA.
A-R of tissues is an in vitro model that mimics in vivo experimental models of ischemia-reperfusion. Zweier et al10 demonstrated that human endothelial cells exposed to A-R intrinsically generate OFRs.
In the present study, we demonstrate that platelets exposed to A-R intrinsically generate O2– and OH°, which, in turn, mediate PA via AA metabolism.
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Methods |
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Blood samples were drawn without stasis from an antecubital vein with a 21-gauge needle. All venipunctures were performed between 8 and 9 AM. Blood mixed with 0.13 mol/L sodium citrate (ratio, 9:1) was collected from healthy volunteers (nonsmokers) who had not ingested any drugs known to interfere with platelet function for at least 15 days. All participants gave informed consent, and the study was approved by the appropriate hospital board.
Experimental Protocol
To keep platelets in anaerobic conditions, samples were purged with a gentle stream of pure nitrogen gas for 15 and 30 minutes. After that, platelets were reoxygenated and stirred in an LT aggregometer. Aggregation of washed platelets was studied in the presence of 1 mmol/L CaCl2 and 4.4 mmol/L fibrinogen after 1 minute of reoxygenation achieved by reexposure to atmospheric conditions. Platelets subjected to a gentle stream of room air were used as control.
In separate experiments, PA was measured in whole blood treated as described above.
Platelet Aggregation
Aggregation of washed platelets
PA (Born's method) was performed in washed platelets, which were prepared as previously described.7 Measurements were made at 37°C in plastic cuvettes with an LT aggregometer with constant stirring at 1000 rpm (Chrono-Log Corp). Aggregation was recorded for 3 minutes. Platelets were primed by addition of STCs of AA (0.1 to 0.3 µmol/L), ADP (0.1 µmol/L), and thrombin (20 mU/mL). STCs of agonist were defined as the lowest doses that induced <10% increase in LT.11 An increase of 
20% in LT was considered to indicate SPA.12 PA was also studied with threshold concentrations of agonists, defined as the lowest dose that induced >50% increase in LT. The effect of the following inhibitors on postanoxic PA was investigated: SOD (300 U/mL), which scavenges O2–; catalase (300 U/mL), which destroys H2O2; the cyclooxygenase inhibitors aspirin (100 µmol/L) and indomethacin (10 µmol/L); the TxA2 receptor antagonist SQ29548 (2 µmol/L); AACOCF3 (14 µmol/L), a selective inhibitor of the 85-kD cPLA213 ; the OH° scavengers mannitol (5 mmol/L) and deoxyribose (5 mmol/L); and the NADPH oxidase inhibitor DPI (50 µmol/L).14 As control, in all experiments washed platelets were added with the dilution medium.
Whole-blood PA
Whole-blood PA (impedance method) was studied with a Chronolog 540 whole-blood aggregometer (Chrono-Log Corp), as previously described.15 Platelets were primed by addition of an STC of AA (50 to 100 µmol/L). STCs of agonist were defined as the highest dose that induces a <5-
increase in electrical impedance. Experiments were always made within 60 minutes after blood sampling.
Biochemical Analysis
Platelet TxB2 and 8-epi-PGF2
production was measured in the supernatant with a gas chromatography/mass spectrometry assay, as previously described.16 The ions monitored were m/z 614 for TxB2, m/z 618 for internal standard [2H4]TxB2, m/z 695 for 8-epi-PGF2, and m/z 699 for the internal standard [2O18]8-epi-PGF2. To evaluate the source of AA utilized as substrate for TxB2 formation and the AA release from membranes during reoxygenation, washed platelets were loaded with 10 µg of [2H8]AA for 1 hour at 37°C, washed twice, resuspended, and treated as described above in the presence or absence of aspirin. The ions monitored were m/z 614 for TxB2, m/z 618 for [2H4]TxB2, m/z 622 for [2H8]TxB2, and m/z 303 for AA.
Measurement of LDH
As a parameter of cytoplasmic leakage, LDH activity was measured spectrophotometrically (Beckman, Dristat reagent). Three minutes after reoxygenation, samples were centrifuged 2 minutes at 12 000g in a Beckman Microfuge 11, and LDH activity measured in the supernatant was compared with the total enzyme activity of control platelets lysed by sonication.
Detection of O2–
The CL of lucigenin was detected with a Bio-Orbit 1251 luminometer. Disposable polystyrene cuvettes were used in all assays. The chemical specificity of this light-yielding reaction for O2– was reported previously.17 In this study, sensitivity and specificity of the assay were determined with xanthine (100 to 400 nmol/L) and xanthine oxidase (0.002 U) to generate O2– with and without SOD (0.5 U/mL). To assay for O2–, lucigenin (0.25 mmol/L) was dissolved to 1.0 mL final volume in Krebs-HEPES buffer containing (in mmol/L) NaCl 99.01, KCl 4.69, CaCl2 1.87, MgSO4 1.20, K2HPO4 1.03, NaHCO3 25.0, Na-HEPES 20.0; pH 7.4. CL in triplicate samples was measured in 1-minute integrate cycles for 10 minutes at 35°C and subtracted for blanks obtained from samples read just before the addition of xanthine oxidase. The amount of O2– produced by this system was assessed as previously described.17 18 Under the conditions of our assay, the yield of O2– produced was found to be equal to 27% of the total xanthine present. This value was used to calibrate the lucigenin signal obtained during reactions with xanthine–xanthine oxidase. The linear correlation coefficient between CL and O2– generated with a simple fit was r=.99. CL was detected in washed platelets at a fixed concentration of 3x108 cells/mL at 37°C. After 30 minutes of anoxia, each sample was added with 0.25 mmol/L lucigenin, CaCl2, and fibrinogen, and the CL obtained at intervals of 1 over 3 minutes was measured. Samples containing lucigenin plus components (with the exception of platelets) were counted, and these blank values were subtracted from the CL signals obtained from platelets. CL was expressed as nanomoles of O2–·mL-1·min-1. In some experiments, 300 U/mL SOD or 50 µmol/L DPI was added to the platelet suspension before treatment with nitrogen.
Measurement of OH°
The production of OH° by platelets was determined by incubation of washed platelets with 5 mmol/L salicylic acid and measurement of its hydroxylated byproducts 2,3- and 2,5-dihydroxybenzoates as previously described.19 Briefly, at the end of the aggregation studies, platelet samples (500 µL) were placed in prechilled 10-mL glass tubes containing 10 µL of 10 µmol/L 3,4-dihydroxibenzoate (internal standard) and 25 µL 1.0N HCl. Sample was twice extracted into 5.0 mL of HPLC-grade diethyl ether, and the organic layers were collected and evaporated to dryness under nitrogen. Samples were reconstituted with 1.0 mL mobile phase just before HPLC analysis.
PLC Activation
[32P]-labeled platelet suspension (1x109 cells/mL) was treated with 0.44N perchloric acid 1 minute after reexposure to air atmosphere. The platelet production of Ins-1,3,4-P3 was measured as previously described.20
Materials
EDTA, AA, mannitol, lucigenin, xanthine, xanthine oxidase (grade III), ferricytochrome c (type III), indomethacin, aspirin, salicylic acid, and all dihydroxybenzoate isomers were from Sigma Chemical Co; SOD and catalase from Calbiochem; deoxyribose and DPI from Aldrich; AACOCF3 from Cascade Biochem Ltd; SQ29548, [2H4]TxB2, [2O18]8-epi-PGF2, and [5,6,8,9,11,12,14,15-d8]-AA from Cayman Chemical Co; [2H4]orthophosphoric acid and [3H]oleic acid from Amersham; and [3H]Ins-1,3,4-P3 from NEN-DuPont de Nemours GmbH. All other chemicals not specified otherwise were of analytical grade and were obtained from Merck.
Statistical Analysis
Data are reported as mean±SD. The comparison between variables was analyzed by Student's t test for paired data. Significance was accepted at the P<.05 level.
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Results |
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PA Upon A-R
In comparison with control maintained at atmospheric conditions, platelets that had undergone A-R showed an SPA that was dependent on anoxic time. Thus, the highest aggregation rate was observed after 30 minutes of anoxia (Fig 1A
). PA was even more evident if platelets were primed with STCs of AA. Although primed platelets kept in atmospheric conditions did not aggregate, primed platelets that had undergone A-R showed remarkable aggregation, higher than that in nonprimed platelets (Fig 1A). Conversely, no increase of aggregation was observed with platelets primed with ADP or thrombin compared with nonprimed platelets (data not shown). Platelets exposed to 30 minutes of a gentle stream of room air did not show SPA (data not shown).
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Under our experimental conditions, nitrogen treatment did not produce any cytolytic effect, as demonstrated by measurement of LDH release in the medium; even with the highest anoxic time, LDH in the supernatant did not exceed 3% of the total platelet enzyme activity (data not shown). Platelets that had undergone A-R were also stimulated with threshold concentrations of other agonists to analyze whether PA was altered by A-R. This study showed that platelets exposed to A-R normally responded to ADP (2 µmol/L), AA (1 to 2 µmol/L), and thrombin (100 mU/mL) (data not shown). Similar findings were obtained when experiments were performed on whole blood. Thus, we found SPA in whole blood that had undergone A-R. This effect was dependent on anoxic time and was observed when platelets were primed with STCs of AA (Fig 1B
). Since the highest SPA was observed at 30 minutes of anoxia, all the following experiments were performed with platelets kept in anaerobic conditions for 30 minutes and then reoxygenated. To identify the species involved in SPA, we incubated washed platelets that had undergone A-R with different types of OFR scavengers. SPA was significantly inhibited by SOD (–74%, P<.005), catalase (–67%, P<.005), and the combination of these two enzymes (–82%, P<.005) (Fig 2), indicating a key role for O2–-derived H2O2 in the activation process. The effect of OH° scavengers was also investigated. Either mannitol or deoxyribose significantly reduced postanoxic SPA (Fig 2). Comparable effects were consistently observed in platelets primed with STCs of AA (Fig 2). The effect of OFR scavengers was also analyzed on washed platelets kept in atmospheric conditions and stimulated with low concentrations of AA (0.5 µmol/L). Platelets incubated with SOD plus catalase showed 33% reduction of aggregation compared with controls (29.6±1.8 versus 19.8±1.2; P<.005). Such inhibition was not observed with higher concentrations of AA.
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Measurement of OFRs in Reoxygenated Platelets
Because the above-reported experiments focused on the potential role of OFRs in SPA, we studied the formation of these species in washed platelets exposed to A-R. The release of O2– in platelets that had undergone A-R was measured by the CL probe lucigenin. Compared with platelets kept in atmospheric conditions, platelets reoxygenated after anoxia released O2– in a time-dependent manner (Fig 3A). Incubation of platelets with SOD reduced O2– release significantly (–68%, P<.005) (Fig 4). The incubation of platelets with DPI reduced O2– release significantly (–88%, P<.005) (Fig 4). DPI also consistently inhibited SPA (n=5; 29.18±6.18% versus 10.82±4.37%, P<.005). The release of O2– was also analyzed on washed platelets incubated with 100 µmol/L aspirin. We observed a significant reduction of platelet release of O2– (–53%, P<.005) compared with controls (Fig 4). Finally, we measured O2– release from platelets kept in atmospheric conditions and stimulated with threshold concentrations of AA (1 to 2 µmol/L) and thrombin (100 mU/mL). Whereas thrombin did not induce any change of O2– release, AA provoked a time-dependent release of O2– (Fig 3B). The release of OH° by platelets that had undergone A-R was assessed by measurement of salicylate hydroxylation. A hydrodynamic voltammogram, which represents the electrochemical characteristics of all hydroxylated salicylate byproducts, is shown in Fig 5. A potential of 350 mV was selected for analytic runs, because all standards showed sufficient response to that voltage and all compounds were fully oxidized. This allowed us to recycle the mobile phase avoiding all interferences. The ratios of peak height between 350 and 200 mV were 1.85±0.15 and 1.76±0.22 and the ratios for 200/100 mV were 4.43±0.51 and 4.48±0.39 for 2,3- and 2,5-dihydrobenzoates, respectively. Peak identity was determined by calculation of the ratios between the peaks obtained from analytic runs of platelet extracts compared with the ratios obtained with pure standard solutions. In addition to O2– production, and perhaps depending on it, platelets exposed to A-R also release OH°. This is shown in Fig 6, where the amount of OH° normally released by platelets maintained at atmospheric conditions and expressed as the sum of the two main hydroxylated salicylate byproducts becomes more than five times higher after A-R. This increase was present in nonprimed and, to a greater extent, in primed platelets. Fig 7 shows typical chromatographic runs obtained by two differently treated platelet samples. The runs reached completeness in 12 minutes, being 7.76±0.31, 8.24±0.42, and 10.3±0.76 times the retention times for 2,3-, 2,5-, and 3,4-dihydrobenzoates, respectively. No additional peaks were detectable after these times.
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indicates 2,3-dihydroxybenzoate; 
, 2,5-dihydroxybenzoate; and , 3,4-dihydroxybenzoate.
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Phospholipases A2 and C in OFRs Mediated SPA
The incubation of platelets exposed to A-R with AACOCF3, a potent and selective inhibitor of cPLA2, completely prevented SPA and priming-enhanced SPA (Table). Compared with platelets not exposed to A-R, SPA was associated with a consistent increase in Ins-1,3,4-P3 formation (879±253 versus 190±40 cpm, P<.005), which was prevented by the combination of SOD plus catalase (210±31 cpm) (Fig 8).
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AA Metabolism Activation in Platelets Exposed to A-R
SPA was inhibited by blocking either the cyclooxygenase enzyme with aspirin and indomethacin or the TxA2 receptor with SQ29548. TxB2 was below the detection level of its assay (0.1 ng/mL) in washed platelets maintained at atmospheric conditions. After A-R, a significant increase in TxB2 production was observed (Fig 9) (P<.005). The combination of SOD plus catalase prevented this increase. The rise in TxB2 was also observed in samples primed with STCs of AA; again, SOD plus catalase (Fig 9) and the cyclooxygenase inhibitors aspirin and indomethacin (data not shown) were able to block the production. Next, we decided to measure the level of 8-epi-PGF2 in the supernatant, because it has been reported that this isoprostane can be produced either by free radical attack of phospholipids containing AA or by cyclooxygenase activity.21 No 8-epi-PGF2 production was observed in platelets at air atmosphere; by contrast, in the setting of SPA after A-R, 8-epi-PGF2 levels reached 50±15 pg/mL (n=5). After A-R, higher levels of 8-epi-PGF2 were produced when platelets were primed with STCs of AA (110±20 pg/mL) (n=5). In both situations, aspirin or indomethacin completely abolished this production, suggesting a cyclooxygenase-dependent formation (data not shown). To evaluate the source of AA for TxB2 production and the AA release, platelets were loaded with 10 µg of [2H8]AA, washed twice, then treated as described above. The amount of [2H8]TxB2 produced when SPA was monitored after reoxygenation was 10±3 ng/mL (n=3); the AA released in the supernatant was 65±10 ng/mL (n=3). As predicted, aspirin or indomethacin was able to prevent the thromboxane formation; by contrast, no reduction in AA release was observed (78±18 ng/mL; n=3). This finding suggests that A-R results in AA release that in turn is metabolized by the cyclooxygenase enzyme.
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Discussion |
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SPA can be observed by the turbidimetric method usually after 10 minutes of stirring and may be used to study ex vivo platelet activation in humans.12 We found that platelets that had undergone A-R had remarkable activation, because after reoxygenation they showed a sharp aggregation, in contrast to platelets maintained at atmospheric conditions. Since previous studies using other cell lines provided evidence that A-R induces an intracellular burst of OFRs,10 we used this experimental model as a tool to investigate whether platelets release OFRs and which role they play in platelet function. There is evidence that platelets release OFRs, but this usually occurs upon platelet stimulation with agonists.22 However, small amounts of O2– may also be released by resting platelets, even if its biological value is unknown.23 In our experimental conditions, platelets produce two types of OFR, O2– and OH°. O2– was detected by means of lucigenin CL, which is a sensitive method of measuring O2–.24 Thus, previous studies demonstrated that lucigenin CL is produced by nanomolar concentrations of O2– and is insensitive to other oxidant species.25 26 27 Other studies have shown, however, we found that SOD did not completely suppress lucigenin CL, probably because of the impossibility of gaining access to the platelet cytoplasm.25 26 OH° was measured by the assay of two of its adducts with salicylic acid, which can be measured by electrochemical detection.19 After these two procedures, we demonstrated that upon A-R, platelets spontaneously release both O2– and OH°. The incubation of platelets with several types of OFR scavengers allowed us to demonstrate that these oxidant species are able to mediate PA. Inhibition of SPA was, in fact, achieved by SOD, by catalase, and by the combination of SOD plus catalase, indicating that both O2– and H2O2 are involved in SPA. Data concerning the role of H2O2 in platelet function are somewhat conflicting, because both stimulation and inhibition of PA have been elicited by H2O2.28 29 30 31 32 33 These studies, however, investigated the effect of exogenous H2O2 on platelet function, whereas we studied the role of oxidant species spontaneously released by platelets. The importance of H2O2 as a mediator of PA was reinforced by the finding that OH° scavengers inhibit SPA. OH° and O2–, in fact, are produced by H2O2 through a Fenton-type reaction.34 Our finding is consistent with that obtained by Khalid and Ashraf,35 who demonstrated that cardiomyocytes exposed to A-R produce both H2O2 and OH°.
To explore the mechanism leading to the generation of OFRs by platelets, we incubated platelets that had undergone A-R with DPI, which has been reported to inhibit NADPH oxidase but also other flavoprotein-dependent enzymes such as nitric oxide synthase.14 36 We demonstrated that DPI almost completely prevents platelet release of O2– and, according to a previous report,37 inhibits PA. This finding would suggest that NADPH oxidase could be the source of OFRs, but the unspecificity of DPI limits this interpretation. Another potential source of OFRs could be the activation of AA metabolism, which has already been shown to generate OFRs in other experimental models.38 39 Our data are in agreement with these studies, inasmuch as we found a significant increase of O2– in AA-stimulated platelets and a significant decrease of O2– in aspirin-treated platelets that had undergone A-R. Further study is necessary to elucidate the exact mechanism that generates OFRs from AA metabolism activation. Previous studies provided evidence that OFRs stimulated platelets by activating PLA2.40 In agreement with these studies, we demonstrated that a selective inhibitor of the cPLA213 prevented SPA. We found that in platelets that had undergone A-R, PLC was activated and that OFRs have a crucial role, inasmuch as the incubation of platelets with OFR scavengers significantly reduced the enzyme activation. Therefore, our experimental model suggests that OFRs stimulate both PLA2 and PLC, which in turn activate platelets via AA metabolism.41 In fact, we demonstrated that the mechanism that induces SPA is related to the activation of AA metabolism. Thus, SPA was dependent on intraplatelet AA release and its conversion to TxA2. The crucial role for AA metabolism activation was confirmed by SPA inhibition with either cyclooxygenase enzyme or TxA2 receptor blockers. To further investigate the role of AA metabolism via cyclooxygenase activation, we measured the level of 8-epi-PGF2. Previously, we have reported that this isoeicosanoid can be produced either by nonenzymatic or enzymatic metabolism of AA.21 In our system, 8-epi-PGF2 generation followed the kinetics of the thromboxane, and it was also completely prevented by cyclooxygenase inhibitors, suggesting that OFRs generated during A-R activate the enzymatic metabolism of AA via cyclooxygenase; this was further confirmed by the finding that aspirin or indomethacin, although able to prevent TxB2 synthesis, did not suppress the AA release from platelet membranes. This suggests that A-R results in AA release that in turn triggers cyclooxygenase activity. The biological importance of our data is demonstrated by experiments performed in whole blood, which contains both oxidant and antioxidant agents. For instance, red blood cells contain OFR scavengers, such as SOD and catalase, and release iron, which is a potent oxidant, whereas leukocytes are known to release OFRs upon stimulation. We found that PA also occurred in whole blood that had undergone A-R. In conclusion, this study demonstrated that platelets exposed to A-R generate two types of OFR, O2– and OH°, which in turn activate AA metabolism via phospholipase A2 and C pathways. This finding provides strong support to the hypothesis that OFRs contribute to platelet activation and offers a rationale to further explore antioxidant agents as modulators of PA in experimental models of ischemia-reperfusion.
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Selected Abbreviations and Acronyms |
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Received July 8, 1996; revision received November 18, 1996; accepted November 25, 1996.
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