Hoppe-Seyler's Z. Physiol. Chem. Bd. 360, S. 559-570, April 1979
Response of Platelets Exposed to Potassium Tetraperoxochromate, an Extracellular Source of Singlet Oxygen, Hydroxyl Radicals, Superoxide Anions and HydrogenPeroxide Peter WÖRNER, Heinrich PATSCHEKE and Wulf PASCHEN Institut für Biochemie II (Med.Fak.), Universität Heidelberg
(Received 8 January 1979)
Summary: When potassium tetraperoxochromate bituric acid-reactive material from platelets. Other (K3Cr08) is added to platelet suspension media scavengers of hydroxyl radicals such as mannitol, it decomposes to the oxygen species hydrogen dimethylsulfoxide, EDTA or histidine prevent the peroxide, Superoxide radicals, hydroxyl radicals, release and the formation of thiobarbituric acid and singlet oxygen. K3Cr08 induces a reversible chromogen. Interaction of hydroxyl radicals with shape change and aggregation of human platelets Tris or sucrose most likely results in the generaand, in the presence of Tris or sucrose, also the tion of short-lived intermediates which may act release of serotonin. Its effect on shape change on platelets to produce thiobarbituric acid and aggregation is due to the long-lived species chromogen and to promote serotonin release, hydrogen peroxide and is abolished by indo^^ effccts Qn latdets ^ mi ^.^ fc methacm and acetylsalicyhc acid^Superoxide acetylsaiicylic acid or indomethacin. Therefore radicals, which are formed from K 3 CrO 8 in ^ reactiye h d , radical and $. let fte HEPES-contammg media do not evoke a platelet oxygen> when generated extracellularly, do not response. mediate their effects via the enzyme-catalyzed The release of serotonin depends on an interacprostaglandin pathway, in contrast to those tion of hydroxyl radicals with Tris or sucrose and evoked by the less reactive hydrogen peroxide, is associated with excessive formation of thiobarReaktion von Thrombozyten auf Kaliumtetraperoxochromat, eine extrazelluläre Quelle der Sauerstoffspezies Singulet-Sauerstoff, Hydroxylradikal, Superoxidanion und Wasserstoffperoxid Zusammenfassung: Beim Zerfall von Kaliumtetraperoxochromat (K3Cr08) in Suspensionsmedien für Blutplättchen entstehen die Sauerstoffspezies Wasserstoffperoxid (H 2 0 2 ), Super-
oxid- (0|) und Hydroxylradikale (OH) und Singulet-Sauerstoff ( J Ag02). K3Cr08 bewirkt eine reversible Formveränderung von Blutplättchen und deren Aggregation. In Gegenwart von
Enzymes: Apyrase, ATP diphosphohydrolasc (EC 3.6.1.5); Catalase, hydrogen-peroxide:hydrogen-peroxide oxidoreductase (EC 1.11.1.6); Peroxidase (horse radish), donor:hydrogen-peroxide oxidoreductase (EC 1.11.1.7); Superoxide dismutase, Superoxide:Superoxide oxidoreductase (EC 1.15.1.1). Abbreviations: HEPES =W-2-hydroxycthylpipcrazincW-cthancsulfonic acid.
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P. Wörner, H. Patscheke and W. Paschen
Tris oder Saccharose setzen die ßlutplättchen auch Serotonin frei. Formveränderung und Aggregation der Thrombozyten beruhen auf der Wirkung des langlebigen Wasserstoffperoxids. Sie werden durch Acetylsalicylsäure oder Indometacin vollständig gehemmt. Superoxidradikale, die in Gegenwart von HEPES-Puffer aus K 3 Cr0 8 entstehen, haben keinen Einfluß auf die Plättchenfunktion. Die Serotoninfreisetzung ist Folge einer Wechselwirkung zwischen Hydroxylradikalen und Tris oder Saccharose. Dabei wird ThiobarbitursäureChromogen in den Plättchen gebildet. Hydroxylradikal-Reaktanten wie Mannit, Dimethylsulf-
Bd. 360 (1979)
oxid, Ethylendiamintetraessigsäure oder Histidin verhindern sowohl die Serotoninfreisetzung als auch die Bildung des Thiobarbitursäure-Chromogens. Für beide Reaktionen werden kurzlebige Zwischenprodukte verantwortlich gemacht, die bei der Einwirkung von Hydroxylradikalen auf Tris oder Saccharose entstehen. Lipidperoxidation und Freisetzung von Serotonin werden nicht durch Acetylsalicylsäure oder Indometacin gehemmt. Wenn die hochreaktiven Sauerstoffspezies. Hydroxylradikal oder Singulet-Sauerstoff, extrazellulär gebildet werden, beruht ihre Wirkung auf Plättchen nicht auf der enzymkatalysierten Synthese von Prostaglandinendoperoxiden. Dies gilt nicht für das reaktionsträgere Wasserstoffperoxid.
Key words: Platelets, potassium tetraperoxochromate, reactive oxygen species, hydrogen peroxide, prostuglandin synthesis.
Hydrogen peroxide (H 2 0 2 ) and Superoxide radicals (Of) have been reported to cause aggregation of blood platelets' 1 » 2 !. Platelet aggregation has been shown to be inhibited by a variety of antioxidants and by scavengers of hydroxyl radicals (OH) and of singlet oxygen (1Ag02)13-6 '. These findings suggest a role of reactive oxygen species in platelet activation. These species, which have a well-known harmful action on cells, are suggested to be generated in all aerobic cells during oxygen reduction and are believed to be involved in several enzyme-catalyzed oxidations (for review seeref.' 7 !).
media results in the formation of l Ag0 2 . OH. OJ, H 2 0 2 and OH" I16"19!. We used K 3 Cr0 8 as a source for the extracellular generation of these species and tried to identify those which are able to cause activation of platelets.
Methods and Materials
Preparation of washed discoid platelets. Blood was obtained from healthy donors who denied having taken untiinflammatory drugs. 6 ml of blood was mixed with 1 ml anticoagulant, N.I.H. formula A (0.8% citric acid, 2.2% sodium citrate, 2.45% hydrous glucose). Preparation of platelet-rich plasma, washing and labelThe oxygenation of arachidonate during prostaling with | 3 H]serotonin were performed as previously glandin biosynthesis has been assumed to involve 20 1 described' '. The washed discoid platelets were stored reactive oxygen species like Ag0 2> OH, Of or 8 11 as a concentrate (2 10 9 /m/) at room temperature in a H 2 O 2 /hydroperoxides (ROOH)l - '). Upon solution of 103mM NaCl, 36mM citric acid adjusted to stimulation, blood platelets produce prostaglandpH 6.5 with NaOH, 5mM glucose, 2mM CaCl 2 , I m M in endoperoxides and thromboxane A 2 , and these MgCl , 2 mg/m/ albumin and 200 pg apyrase/m/. Prior to 2 agents are potent inducers of platelet aggregation an experiment, 100 / of this concentrate was prewarmcd 12 13 and the release reaction' » '. The response of to 37 °C and the volume was made up to 1 ml to achieve platelets to some stimuli, e.g. added arachidonic final concentrations of 500 Mg albumin/m/, 50 g apyrase/m/, 96mM NaCl, 3.6mM citrate, 5mM glucose, 5mM acid' 12 ' 14 ' or H 2 0 2 completely depends on prostaglandin synthesis. Other stimuli, like throm- KG, 0.5mM CaCl2, O.lmM MgCl2 and 46.2mM of either bin, result in the synthesis of prostaglandins which Tris, HEPES or phosphate buffer, pH 7.4. (These solutions are heieafter referred to as Tris-, HEPES- or contribute to the overall response, but in addiphosphate-buffered medium.) The final platelet count tion cause aggregation and release by alternative was 2 108/m/, except for determination of 2-thiobar15 pathways' '. bituric-acid-reactive material (4 108/m/). The decomposition of potassium tetraperoxoWashed spheroid platelets were prepared from blood anticoagulated with EDTA. After differential centrifuchromate (K 3 Cr0 8 ) in slightly alkaline aqueous
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Bd. 360 (1979)
Platelet Response to Extracellular Reactive Oxygen Species
gation, labelling with | 3 H]serotonin and washing, platelets were stored as a concentrate (1010 m / ~ ] ) at 4 °C 1 21 1 and finally resuspended in saline containing 5mM KC1 buffered with either 46.2mM Tris; HEPES or phosphate, pH 7.4. In all experiments without platelets, solutions of similar composition were employed and hereafter referred to as Tris, HEPES or phosphate buffer. Aggregation was studied in platelet samples stirred at 250 rpm in the presence of fibrinogen (3 mg/m/) in a sixchannel aggregometer (modified model UDC 1A, Kontron/Labotron, Munich). Serotonin release and shape change were recorded as previously described' 20 ' 21 ' with the exception that the samples in shape change experiments did not contain EDTA. Absence or presence of aggregates was confirmed by phase contrast microscopy. The shape of platelets was estimated from oscillation amplitude and the decrease in transmittance after stopping the st irren 20 '. Scavengers were adjusted to pH 7.4. if required, and added as isotonic solutions. To account for dilution when large volumes of scavengers were added, the concentrations of apyrase. albumin and buffer were increased, the latter at the expense of saline, to achieve final concentrations as in controls without scavengers. Decomposition of potassium tetraperoxochromate (K3CrOs) in solutions buffered at pH 7.4 was followed at 37 °C by monitoring the increase in absorbance caused by chromate (CrO^6) formation* 16 1 The concentration of K CrOg was restricted to 0.1 mM because of the high absorbance of CrO^ at 375 nm. Hydrogen peroxide (1^02) was determined after complete decomposition of 0.5mM K^CrOg at 37 °C in 1 ml of either Tris, HEPES or phosphate buffer. 5-μ/ samples were diluted to 600 μ/ with buffered saline containing 8μΜ scopoletin. \\^2 was measured as the decrease in fluorescence intensity due to oxidation of scopoletin after addition of horse radish peroxidase (325 mU/m/) I 22 1. Excitation wavelength was 350 nm, emission was monitored at 46.8 nm. Calibration curves were obtained by addition of known amounts of H2 2. Addition of the scavengers employed did not interfere with the assay. 2-Thiobarbituric-acid-reactive material, which is supposed to result in the generation of malonaldchyde, was assayed by the thiobarbituric acid method^] jt s formation in 1-m/ portions of nonstirred platelet suspension (4 χ 10s m/~ l ) was terminated 5 min after addition of the stimulus by mixing with I ml of thiobarbituric acid reagent' 23 1 Absorbance was measured at 546 nm in an Eppendorf photometer in cuvettes of 5 cm path length. The values were corrected for the absorbance of blanks, i.e. complete reaction mixture with O.SmM KaCrOg without platelets. Calibration with 1,1,3,3-tetraethoxypropane, which yields stoichiomctric
561
amounts of malonaldehyde, was in good agreement with reported e-values. Thiobarbituric acid chromogcn generated from platelets was expressed as nmol of malonaldehyde/4 χ 108 platelets. Strong color formation from HEPES exposed to K^CrOg conflicted with the measurement of thiobarbituric acid chromogen generated from platelets in the presence of this buffer. Assay of Superoxide radicals (θ|). θ| causes reduction of nitro blue tetrazolium or ferricytochrome c and oxidation of epinephrine to adrenochrome. Inhibition of these reactions by Superoxide dismutase was used to identify O^. Superoxide dismutase was established to be active after exposure to K CrOg by its ability to completely prevent reduction of nitro blue tetrazolium by a known source of O^: phenazine methosulfate and NADH 2 1 24 1. The photometric assays were performed in a Beckman DB-GT spectrophotometer in cuvettes thcrmostated at 37 °C. The test medium consisted of saline buffered with Tris, HEPES or phosphate (46.2mM), pH 7.4, 5mM KC1 and O.SmM CaCl2. Reduction of nitro blue tetrazolium (245μΜ) to blue diformazan color was monitored at 546 nm' 2 5 '. K CrOg decomposition in phosphate buffer leads to the development of a brown color. Measurement of the reduction of nitro blue tetrazolium was therefore confined to Tris or HEPES buffer. Reduction of ferricytochrome c (50μΜ) was examined by measuring the increase in absorbance at 550 nml 26 ). Generation of adrenochrome by oxidation of epinephrine was followed by the increase in absorbance at 480 nml 26 1 In experiments with cytochrome c and epinephrine, catalase (325 U/m/) was included to prevent color decay. Solutions of nitro blue tetrazolium. cytochrome c, epinephrine and Superoxide dismutase were prepared immediately prior to experiments. MATERIALS Catalase from beef liver (65000 U/mg) and ferricytochrome c (horse heart, cristallinc) were products of Boehringer Mannheim GmbH, D-6800 Mannheim. A part of the experiments was performed with catalase dialyzed for 12 h against buffered saline to remove preservatives. Dialysis of catalase had no effect on the results. 1,1,3,3-tetraethoxypropane was obtained from K & K, Plainview, New York, U.S.A. Apyrase from potato (grade I, 227 5'-ATPunits/mg), Superoxide dismutase (bovine blood, 2700 U/mg), l-epinephrine and prostaglandin EI were obtained from Sigma Chemie, D-8021 Taufkirchen. Human fibrinogen was from Bchringwerke, D-3550 Marburg. 7-Hydroxy-6methoxycoumarin (scopoletin) \vas purchased from Fluka AG, Buchs, Switzerland. Nitro blue tetrazolium, bovine albumin and horse radish peroxidase (250 U/mg) were obtained from Biomol, D-6804 Ilvesheim. 1,4-Diazabicyclo[2.2.2loctane was obtained from Merck-
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P.W rner, H. Patscheke and W. Paschen
Schuchardt, D-8011 Hohenbrunn, hydrogen peroxide from Merck, D-6100 Darmstadt and cytochalasin B from Aldrich-Europe, Becrse, Belgium. Cristalline potassium tetraperoxochromate (K3CrO8) was prepared from alkaline potassium chromatc and icecold hydrogen peroxide according to Ricsenfeld et alJ 2 0 l K3CrO8 was added to 1 ml of a platelet suspension as 5 μ/ of a stable solution in 0.1 M NuOH. After addition of O.SmM CrO|°, the pH changed from 7.4 to 7.45 in HEPES-, to 7.47 in Tris- and to 7.49 in phosphate-buffered saline containing 46.2mM of buffer.
Results 1) Experiments on the decomposition ofK^CrOg in the absence of platelets K3Cr08 is stable when dissolved in 0.1 M NaOH but rapidly degrades to chromatel 16 ! at physiological pH values. The breakdown of 0.1 mM CrO|e was complete in less than l min in HEPESbuffered solution and occurred somewhat more slowly in Tris- or phosphate-buffered solution. In the presence of lOmM CaCl 2 , degradation was incomplete even after 10 min in either Tris or HEPES (Fig. 1).
Bd. 360 (1979)
It has to be emphasized that Trisi 19 ! or HEPES may interact with reactive products generated during K 3 Cr0 8 decomposition. These buffers may thereby alter the rate and mode of K 3 Cr0 8 decomposition. a) Generation ofO% during decomposition of K3CrO8 in HEPES buffer O| is able to oxidize epinephrine to adrenochrome and, on the other hand, to reduce nitro blue tetrazolium or ferricytochrome c. These reactions assay 0| when the formation of formazan, ferrocytochrome c or adrenochrome can be inhibited by Superoxide dismut se. This enzyme catalyzes the reaction 02 + Of -> 02 + H 2 0 2 i 2 6 J . Of was formed during decomposition of K 3 Cr0 8 in HEPES buffer. A small reduction of nitro blue tetrazolium or cytochrome c was observed which could partially be inhibited by prior addition of Superoxide dismut se. The formation of Of could be suppressed by prior addition of the OH scavengers mannitol, dimethylsulfoxide, histidine or EDTA. At a high concentration of CaCl 2 , which reduced the rate of K 3 Cr0 8 decomposition, more Of was formed (Fig. 2). b) Absence of Ο? during K3Cr08 decay in Tris and phosphate buffers Of could be detected neither in phosphate nor in Tris buffer. This was established with cytochrome c and nitro blue tetrazolium in the presence or absence of CaCl2. We also measured adrenochrome formation during K 3 Cr0 8 decomposition. Adrenochrome was formed from epinephrine, but the lack of inhibition by Superoxide dismut se indicates oxidation independent of Of.
0.2-
0
2
4 6 t [min] —^-
8
10
Fig. 1. Decomposition of K3CrO8. K3Cr08 (0. ImM) was added as 5 μ/ of a solution in 0.1 Μ NaOH to 1 m/ of saline containing 5mM KC1 buffered cither with 46.2mM HEPES (a, c) or Tris (b). lOmM CaCl2 was present in experiment c. Decomposition of K3CrC)8 at 37 °C was monitored by the increase of absorbance at 375 nm due to formation of chromate. Curves obtained in phosphate-buffered saline were identical with those in the presence of Tris.
The production of Of from K3Cr08 in the presence of Tris or phosphate could have been obscured due to the removal of Of by the reaction OH + Of -» ΟΗθ + 0 2 (£=1.01χ ΙΟ^Μ- 1 s'1 1 27 J). Furthermore, the oxidizing species 1 Ag0 2 l l8 l and OH may interfere with the reduction of nitro blue tetrazolium or cytochrome c or also may result in a formation of adrenochrome 128K Therefore we also included scavenging agents for *Ag02 and/or OH. The presence of ΙΟηΐΜ diazabicyclooctane, 20niM histidine, 20mM dimethylsulfoxide, 50mM mannitol or 50mM sucrose did not enable us to observe a reduction of cytochrome c or nitro blue tetrazolium. The
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Bd. 360 (1979)
Platelet Response to Extracellular Reactive Oxygen Species
563
Table 1. Estimation of hydrogen peroxide after decomposition of K3Cr08 0.5mM K3CrO8 was added to 1 ml of saline containing 5mM KC1 and 46.2mM of HEPES, Tris or phosphate buffer, pH 7.4, at 37 °C. H 2 O 2 was determined after complete decomposition of K3CrO8. Mean values of three determinations. For details see Methods.
Fig. 2. Reduction of nitro blue tctrazolium to diformazan color (546 nm) during decomposition of K3CrO8 in the presence of HEPES. Enhancement by calcium and inhibition by Superoxide dismutase. At 0 min 0.5mM K 3 Cr0 8 was added to 1 ml of 46.2mM HEPES-buffered saline containing 5mM KC1 and 245μΜ nitro blue tetrazolium at 37 °C. a) Control; b) plus lOmM CaC^: c) plus 108 U Superoxide dismutase/m/; d) plus lOmM CaCl2 and 108 U Superoxide dismutase/ ml. Inhibition by Superoxide dismutase (c, d) indicates that O| is formed during decomposition of K 3 CrO 8 in the presence of HEPES.
1
Δ§Ο2-scavenging agent diazabicyclooctane enhanced the formation of adrenochrome from epinephrine exposed to K 3 Cr0 8 , but generation of adrenochrome was not affected by the presence of Superoxide dismutase. Thus, 0| is unlikely to be formed from K 3 Cr0 8 in either Tris or phosphate buffer at 37 °C.
Buffer
Scavenging agent
H202 (mMl
Tris HEPES HEPES Phosphate Phosphate Phosphate Phosphate Phosphate
_ _ lOmMCa 2 * 50mM Dimcthylsulfoxide 50mM mannitol lOmM Diazabicyclooctane 50mM Sucrose
0.42 0.33 0.32 0.45 0.58 0.50 0.43 0.36
OH scavengers either augmented (dimethylsulfoxide) or reduced (sucrose) the yield of H 2 0 2 . Despite the generation of 0| which spontaneously dismutates to H 2 0 2 1 29 1, formation of H 2 0 2 in HEPES buffer was slightly reduced, even in the presence of lOmM CaCl 2 . Different proportions of the following reactions, depending on the compounds in die medium, may contribute to the overall yield of H 2 0 2 : 1) H 2 0 2 may be a direct product of K 3 Cr0 8 decomposition. 2) H 2 0 2 may result from the interaction of 20Hradicalsl 2 ? l. 3) H 2 0 2 may be formed by spontaneous dismutation of 02 which may be either generated directly or secondarily to an interaction of OH with a scavenger^30 L From the data obtained it cannot be concluded that there is one single mechanism of H 2 0 2 formation.
d) Production of thiobarbituric-acid-reactive material from HEPES exposed to K3Cr08 Decomposition of K 3 Cr0 8 in the presence of c) Formation ofHiOi during decomposition of HEPES resulted in the production of thiobarK3Cr08 bituric-acid-reactive material. However, this prodH 2 0 2 was generated during the decomposition of uct differed from malonaldehyde generated from tetraethoxypropane in that the absorption peak K 3 Cr0 8 in all solutions employed. In phosphate buffer, which is expected to interact weakly, if was at 505 nm and not at 546 nm. Since inclusion at all, with reactive decomposition products of of OH scavengers such as 50mM mannitol, 50mM dimethylsuifoxide or 5mM EDTA interfered with K 3 Cr0 8 , the molar ratio of H 2 0 2 formed from the generation of this material, OH may be the K 3 Cr0 8 was about 0.9 (Table 1). inclusion of
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P. W rner, H. Patscheke and W. Paschen
Bd. 360(1979)
species interacting with HEPES. Catalase a) Phosphate- or HEPES-buffered media (2600 U/m/) or Superoxide dismutase (108 U/m/) When K 3 Cr0 8 was added to platelets in phosphatedid not prevent generation of the reactive material. or HEPES-buffered media, discoid platelets were A high concentration of CaClo, which enhances initially transformed to spheroids with subsethe formation of 0|. inhibits the generation of quent partial recovery of their original shape this material from HEPES. No color developed (Fig. 3a). As expected from shape recovery and when HEPES was added after decomposition of from the absence of an increase in light transK 3 Cr0 8 . mission, which occurs concomitantly with a granule discharge^ 20 ', no serotonin release was detected. In stirred samples containing fibrino2) Platelet response to decomposingK3CrO$ gen, platelet aggregation was observed, followed The platelet response to K 3 Cr0 8 strongly depended on the composition of the suspending medium by a complete disaggregation (Fig. 3b). employed. Either a reversible disc-sphere transformation without release or an additional strong serotonin release was observed.
0
?
t
0
The shape change induced by K 3 CrO 8 was completely prevented by prior addition of 2μΜ prostaglandin Ε , , which increases platelet cAMP
2 1 . tfminj—^·
0
2
1
.
Fig. 3. Effects of K3CrO8 on washed discoid platelets suspended in phosphate- or Tris-buffered medium. Comparison with the effect of Η2θ2· a) Shape change experiments without fibrinogen. b) Aggregation is allowed to take place by the inclusion of 3 mg fibrinogcn/m/ in these experiments. 0.5mM K 3 CrO 8 (I, II) or O.SmM H2 2 (III) was applied at 0 min. Percentages indicate serotonin release. Prior to addition of K3CrO8 the stirrer was stopped for 20 s to evaluate the discoid shape of platelets' 20 '. 1: Phosphate-buffered medium: K 3 CrO 8 causes reversible shape change (= disc-sphere transformation) without serotonin release (a). Moderate serotonin release is found when aggregation takes place with fibrinogen (b). II: Tris-buffcred medium: Subsequent to the shape change, an increase in light transmission and concomitant strong serotonin release is observed (a). A minor part of the increase in light transmission is due to weak aggregation (followed by disaggregation) despite the absence of fibrinogen (a). Control experiments without stirring after addition of K3CrO8 established that the major part was due to granule discharge'20!. Ill: The response of platelets to \\2®2 was tne same whether the platelets were suspended in phosphate- or Trisbuffercd medium. The response was similar to that evoked by K 3 CrO 8 in phosphate-buffered medium.
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Bd.360 (1979)
Platelet Response to Extracellular Reactive Oxygen Species
by activation of adenylcyclasel 31 1, and by 0acetylsalicylic acid and indomethacin, which interfere with platelet prostaglandin endoperoxide synthesis'12'14!. Moreover, addition of 325 U catalase/m/prior to K 3 Cr0 8 completely inhibited this platelet response. Neither the scavengers for OH, 5OmM mannitol, 50mM dimethylsulfoxide and 20mM histidine, nor 108 U of the 02-scavenging enzyme Superoxide dismutase, nor the l Ag0 2 quenching agent diazabicyclooctane (10mM) inhibited the response. The platelet response was also observed when platelets were added after complete decomposition of K 3 Cr0 8 (15 min). Thus, a long-lived species generated during K 3 Cr0 8 decomposition was responsible for the shape change and aggregation. Addition of the decomposition product chromate (CrO|w) did not mimic the effect of K 3 Cr0 8 . 0.5mMH 2 0 2 (almost this amount had been generated during K 3 Cr0 8 decomposition) evoked the same effect as K 3 CrO 8 , i.e. reversible disc-sphere transformation, which could be completely prevented by acetylsalicylic acid, indomethacin, prostaglandin E{ or catalase, but not by Superoxide dismutase, diazabicyclooctane or OH scavengers. The small amount of serotonin release induced by H 2 0 2 or K 3 Cr0 8 in a phosphate-buffered medium (Fig. 3b) containing fibrinogen was a result of aggregation. H 2 O 2 or K 3 Cr0 8 did not evoke serotonin release, if the stirring mechanism was stopped 0.5 s after their addition.
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salicylic acid or indomethacin had only a weak inhibitory influence on the serotonin release induced by K 3 Cr0 8 . However, complete inhibition of release and a reduction to the level of reversible disc-sphere transformation was achieved by the addition of the OH scavenging agents histidine, dimethylsulfoxide, EDTA or HEPES (Fig. 4). Mannitol was somewhat less effective. The ^02quenching agent diazabicyclooctane enhanced the effect of K 3 Cr0 8 (Fig. 4). None of these scavengers interfered with shape change induced by 2μΜ ADP or 1.5mM H 2 0 2 ; however, EDTA and dimethylsulfoxide inhibited aggregation induced by these stimuli. High concentrations of catalase (5200 U/m/) suppressed the platelet response to K 3 Cr0 8 . It has to be emphasized that 325 U of catalase/m/ was enough to prevent shape change and aggregation induced by l.SniM H 2 0 2 , but catalase at this concentration did not interfere with serotonin release induced by K 3 Cr0 8 . When either Tris or sucrose was present, K 3 Cr0 8 also induced serotonin release in phosphate-buf-
b) Iris-buffered medium Shape change curves in the presence of Tris buffer differ from those obtained from platelets exposed to K 3 CrO 8 in HEPES or phosphate buffer in that after a short decrease in light transmission a strong increase occurred parallel to strong serotonin release (Fig. 3,11). In stirred samples oscilFig. 4. Platelet responses to K3CrO8 in Tris-buftcrcd medium at 37 °C. Inhibition by OH-scavenging agents lations ceased and platelets did not recover their original discoid shape. When fibrinogen was pres- and enhancement by diazabicyclooctane. The scavengers were added 2 min prior to O.SmM ent in stirred samples, K 3 Cr0 8 induced strong aggregation which was followed after about l min K 3 CrO 8 ; a) Control; b) 5mM EDTA; e) 7.5mM histidine;d) lOmM by disaggregation, despite release of serotonin diazabicyclooctane. The percentages given represent (Fig.3b). serotonin release. Inclusion of either 50mM dimethyl2μΜ Cytochalasin B, which strongly enhances the sulfoxide, 5mM HEPES or 50mM mannitol reduced the extrusion phase of the platelet release reaction platelet response to the level of a reversible disc-sphere I20'32!, hardly augmented serotonin release intransformation without serotonin release as shown for duced by K 3 Cr0 8 . Prostaglandin E 1 ? acetylEDTA and histidine (Curve b, c).
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P.Wörner, H. Patscheke and W. Paschen
fered medium. Other compounds containing hydroxyl groups such as mannitol or glucose could not be substituted for Tris or sucrose. The release of serotonin is effected by a shortlived species since it requires the simultaneous presence of Tris or sucrose and platelets during K 3 Cr0 8 decomposition. When either Tris or platelets were added 30s after K 3 Cr0 8 . a reversible shape change without release was observed. To exclude possible effects due to the interaction of reactive species generated from K 3 CrO 8 with compounds of the medium such as apyrase, albumin, citrate or glucose, the effect of K 3 Cr0 8 was additionally investigated on washed platelets resuspended in buffered saline supplemented only with 5mM KC1. Similar results were obtained: serotonin release and the concomitant increase in light transmission were restricted to those samples to which Tris buffer or sucrose had been added. c) Production of thiobarbitwic-acid-reactive material from platelets Addition of K 3 Cr0 8 to platelets in phosphatebuffered medium resulted in the formation of
Bd.360(1979)
thiobarbituric-acid-reactive material. In Tris-containing samples more chromogen was formed than in those buffered by phosphate and the development of this additional color correlated with the amount of serotonin release. The OH-scavenging agents dimethylsulfoxide and EDTA caused strong inhibition, while mannitol was less effective. Inhibition by indomethacin and acetylsalicylic acid was weak. Diazabicyclooctane enhanced the color development (Table 2).
Discussion Decomposition of K 3 Cr0 8 in slightly alkaline solution proved to be a very complex reaction in that not only the rate of decomposition but also the kind and the yield of products depended on the composition of the medium. H 2 0 2 was established as a decomposition product of K 3 Cr0 8 in all media employed. The yield of H 2 0 2 formed from K 3 Cr0 8 was increased in the presence of the OH scavenger dimethylsulfoxide and decreased in the presence of HEPES or sucrose.
Table 2. Formation of thioburbituric acid chromogen from platelets exposed to K3CrO8. O.SmM K3CrO8 was added to platelets (4 10 8 /m/) at 37°C. Inhibitory agents were added 2 min prior to K 3 CrO 8 . For details sec Methods. The numbers represent the mean values from 4 platelet preparations (.* = level of significance < 0.05; Wilcoxon's signed-rank test). Formation of a yellow color in the presence of sucrose conflicted with the assay. Chromogen equivalent to malonaldchydc |nmol/4x 108 platelets 1 Buffer Phosphate Phosphate Phosphate Tris Tris Tris Tris Tris Tris Tris
Compounds added
0.1 m M Indomethacin 0.1 mM Indomethacin 5mM EDTA 50mM Dimethylsulfoxide 50mM Mannitol 2400 U Catalase/m/ 1 OmM Diazabicyclooctane
K3Cr08 K 3 Cr0 8 K 3 Cr0 8 K3Cr08 K 3 Cr0 8 K3Cr08 K3Cr08 K3Cr08 K 3 Cr0 8
0.08 0.54 0.42 1.06 0.84* 0.26* 0.27* 0.41* 0.45* 1.26*
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Bd. 360 (1979)
Platelet Response to Extracellular Reactive Oxy£ in Species
In our experiments, the occurrence of another decomposition product, 0®, was restricted to HEPES-buffered solutions. Controversy exists in the literature as to whether or not Of is formed during K 3 Cr0 8 decomposition: no Of was detected in Tris-buffered medium^ 19 ', while it could be established in pyrophosphate-buffered medium in the presence of *AgO2 scavengersl18'. The divergence of results may be explained by differences in the composition of solutions, in temperatures, or in the assays for Of employed. Generation of OH from K 3 Cr0 8 has been reported^ 19 ' and is indicated by our experiments with OH scavengers. OH scavengers do not inhibit shape change of platelets promoted by other stimuli. They do, however, interfere with serotonin release and formation of thiobarbituric acid chromogen from platelets treated with K 3 Cr0 8 . OH scavengers also inhibited the formation of 0? in HEPES-buffered solutions and the generation of thiobarbituric-acid-reactive material from HEPES on application of K3Cr08.
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1) H 2 0 2 , in an amount which is formed during K 3 CrO 8 decomposition, evokes platelet effects similar to that of K 3 Cr0 8 after complete decomposition. 2) A low concentration of catalase completely prevents shape change, or aggregation in the presence of fibrinogen, induced by either K3Cr08 orH 2 0 2 . 3) Acetylsalicylic acid and indomethacin completely inhibit shape change promoted by K 3 CrO 8 and H 2 O 2 . Shape change and aggregation induced by the two agents depend on the prostaglandin endoperoxide pathway. Several results show that Of is not responsible for the effect of K 3 Cr0 8 on platelets.
1) K 3 Cr0 8 causes a similar platelet response regardless of whether HEPES or phosphate is employed as buffer. However, Of is detected in HEPES but not in phosphate buffer. 2) Superoxide dismutase effectively quenches Of but does not influence the shape change induced by K 3 Cr0 8 . K 3 Cr0 8 has been reported as a chemical source 117 19 331 3) Inclusion of OH scavengers, which prevent the of iAg02 - ' . If ^gO2 were responsible formation of Of in HEPES, does not inhibit shape for the platelet responses observed, inclusion of diazabicyclooctane, a ^g02-quenching agent^34' change induced by K 3 CrO 8 . 4) A high concentration of CaCl 2 , which incould be expected to inhibit them. However, it enhanced K 3 Cr0 8 -induced lipid peroxidation and creases the yield of Of formed in HEPES-bufserotonin release. It has been reported to prevent fered medium, does not augment the platelet relipid peroxidation in liver microsomes exposed to sponse. K 3 Cr0 8 l 3 3 L However, the concentration emPlatelet aggregation and release in the presence of ployed in these experiments (0.33M) is not apxanthine and xanthine oxidase have been attriplicable in platelet suspensions and it may have buted to Of I 2 '. However, the enzyme alone is acted by eliminating preformed lipid hydroalso able to induce platelet aggregation and the peroxides'35!. release reaction!361. The response of platelets to K 3 Cr0 8 depends on The involvement of a long-lived species argues the experimental conditions. A reversible discagainst a role of *Ag02 or OH in K 3 Cr0 8 -insphere transformation (= shape change) without duced shape change. !Ag02 has a life-time of serotonin release occurs when platelets are added about 2 MS in aqueous medial37! and OH also has after complete decomposition of K 3 Cr0 8 . Seroa very short life-time, as can be expected from its tonin release, however, is only brought about if high reactivity. Moreover, neither the *Ag02K 3 Cr0 8 is directly added to platelets. These find- quenching agent diazabicyclooctane nor high ings point to the involvement of a long-lived concentrations of the OH scavengers mannitol, species in the shape change, while additional re- EDTA, dimethylsulfoxide or histidine, which interact with OH at rates which are almost diffulease reaction requires short-lived decomposition sion controlled (fc OH > 10 9 M~ 1 s'1138-«°1), products. interfere with K 3 CrO 8 -induced shape change. The most likely candidate for the long-lived species which produces reversible shape change In contrast to shape change, K 3 Cr0 8 -promoted without release is H 2 0 2 : serotonin release involves a short-lived species. Brought to you by | Purdue University Libraries Authenticated Download Date | 5/31/15 1:11 PM
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P. W rner, H. Patscheke and W. Paschen
One of the species to be considered as a stimulus of this response is OH. However, serotonin release appears not to be the result of a direct action of OH on platelets. Serotonin release is not observed in phosphate-buffered medium: it requires the presence of either Tris or sucrose, which are known to act as OH scavengers^19»38'. The finding that other scavengers of OH such as EDTA, dimethylsulfoxide, histidine or mannitol inhibit the release of serotonin suggests a competition of these agents with Tris or sucrose for OH. Concentrations of catalase in excess of those required to inhibit the effect of H 2 O 2 on platelets may also inhibit due to the ability of catalase totrapOHl 4 1 !.
Bd. 360 (1979)
a possible trigger for release. Enhancement of serotonin release by the ! Ag0 2 -quenching agent diazabicyclooctane strongly argues against a requirement for J Ag0 2 . However, the OH-scavenging agents EDTA and histidinel 43 » 44 ', but not dimethylsulfoxide and mannitol, also have ^gOotrapping properties and they inhibit release at lower concentrations than do the latter. These results could be interpreted to mean that !Ag02 contributes to serotonin release. Thus, at present, we cannot definitely exclude a role of ]Ag02 in K 3 Cr0 8 -induced serotonin release.
A common reaction performed by free radicals is an abstraction of hydrogen atoms from target molecules and this effect is typical for OH^ 45 ·. Through their interaction with OH. OH-trapping Hydrogen abstraction from unsaturated lipids is agents are themselves transformed to radicals. presumably the initiating step for their peroxidaThese secondary radicals will either be eliminated tionl 46 !. OH and !Ag02 have been suggested to by dimerization or may represent the beginning cause lipid peroxidationl 35 ' 47 " 49 !. The occurrence of a radical chain. Scavenging of OH by dimethyl- of lipid peroxides can be detected by their desulfoxide may result in the generation of CH 3 I 3 °1 composition product, malonaldehyde, which rewhich may form dimers; from methanol ·ΟΗ 2 ΟΗ, acts with thiobarbituric acid to yield a red color •0 2 CH 2 OH and 0| may be formed^ 42 !. Since it [23,461 τ}^ chromogen is also formed during is not unequivocally known what does result from prostaglandin synthesis catalyzed by fatty acid the interaction of the different scavengers with cyclooxygenase. However, in contrast to non-enOH, any rationalization is hazardous. The most zymic lipid peroxidation, its generation is preventlogical assumption seems to be that intermediates ed by the inhibitors of cyclooxygenase, acetylgenerated by the interaction of Tris or sucrose salicylic acid and indomethacinl 50 ]. with OH induce serotonin release, while those H 2 0 2 -induced shape change and aggregation are generated from the other OH scavengers do not. The life-time of these intermediates is short since completely prevented by inhibitors of cyclooxygenase. These also interfere with shape change the release requires the simultaneous presence of and aggregation promoted by K 3 Cr0 8 in phosTris or sucrose and platelets when K 3 Cr0 8 is phate-buffered medium. However, the amount of added. thiobarbituric acid color from platelets exposed The results obtained in HEPES-containing media to K 3 Cr0 8 exceeds by far that from H 2 0 2 -treatdeserve further comment. HEPES may also act as ed platelets; and this color formation is inhibited an OH scavenger. Thiobarbituric-acid-reactive only to a minor extent by acetylsalicylic acid or material is formed from HEPES exposed to indomethacin. The latter is an indication for cyK 3 Cr0 8 even in the absence of platelets. Declooxygenase-indepcndent lipid peroxidation by velopment of this color is inhibited by high conspecies generated during K 3 Cr0 8 decomposition centrations of other OH scavengers. Moreover, other than H 2 0 2 . generation of Of in the presence of HEPES is prevented by OH scavengers. This indicates an inter- When K 3 Cr0 8 induces serotonin release, i.e. in action of OH with HEPES. HEPES may compete the presence of Tris buffer, even more thiobarwith Tris for OH and thereby inhibit serotonin re- bituric-acid-reactive material is formed than after K 3 Cr0 8 -promoted shape change (in phosphatelease even in the presence of Tris, as does mannibuffered medium). The small effect of indomethtol, for example. acin argues against an involvement of prostaglandin endoperoxides in serotonin release. Inhibition As the other short-lived intermediate generated by OH-scavenging agents indicates lipid peroxidafrom K 3 Cr0 8 , *Ag02 remains to be discussed as
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Bd. 360 (1979)
Platelet Response to Extracellular Reactive Oxygen Species
tion mediated by OH and secondary radicals generated by the interaction of OH with Tris. These radicals also promote serotonin release, which may be an event either independent of or secondary to membrane perturbation caused by the formation of lipid peroxides. The assumption that serotonin release induced by K 3 CrOg does not involve the physiological pathway is supported by the experiments with cytochalasin B. Cytochalasin B augments the release reaction from platelets activated by thrombin and collagen (20,321 ? b ut UOQS not enhance serotonin release by K 3 Cr0 8 .
569
branes. OH and possibly !Ag02 may be generated intracellularly from H 2 0 2 in the presence of a transition metal ion in a lower valence state [45,601
The authors thank Prof. R. Brossmer for his support of this study. The assistance of Misses/. Schlageter and C. Wabhel is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft within SFB 90 "Cardiovaskularcs System". Literature \
Hisashi,O., Kikuchi, Y. & Konno, K. (1972) Tohoku J.Exp.Med. 106,399-409. 2 Handin, R.I.. Karabin, R. & Boxer, G. J. (1977) / C///I. Invest. 59. 959-965. From the inhibitory action of extracellularly applied scavengers we conclude that only exogenous- 3 Steiner, M. & Anastasi,J. (1976)7. Clin. Invest. 59, 732-737. ly generated decomposition products of K 3 CrOg 4 Panganamala,R.V., Miller, J.S.. Gwebu,E.T., are responsible for its effect on platelets. ExtraSharma, H.M. & Cornwall, D.G. (1977) Prostacellularly «generated OH and 1Ag02 do not trigger glandins 13, 261-271. release by enzyme-catalyzed formation of prosta- 5 White, J.G., Rao, G.H.R. & Gerrard, J.M. (1977) Am. J. Pathol. 88, 387-402. glandin endoperoxides. However, this does not Greenberg,M.,Grady,R.W.& Peterson,CM. (1977) exclude a possible physiological role of highly re- 6 Br. J. Haematol. 37, 569-577. active radicals in prostaglandin endoperoxide syn- 7 Fridovich, I. (1976) in Free Radicals in Biology thesis within the cell. Prostaglandin endoperoxide (Pryor,\V.A., ed.) vol. 1, pp. 239-277, Academic Press, New York. synthesis by cyclooxygenase appears to require 8 Panganamala, R. V., Sharma, H. M., Sprecher, H., fatty acid peroxides for activity l 5 1 ' and these Geer, J.C. & Cornwall, D.G. (1974) Prostaglandins have been assumed to promote reactions usually 8,3-11. 52 attributed to ^AgC^ ', or they may undergo an 9 Vargaftig, B.B., Tränier, . & Chignard, . (1975) Eur. J. Pharmacol 33. 19-29. interaction yielding ^02'53'54'. On the other 10 Rahimtula, A. & O'Brien, P.J. (1976) Biochem. Biohand, fatty acid cyclooxygenase is deactivated phys. Res. Commun. 70, 893-899. during turnover by radicals^ 55 ' and ^gOo scav11 Panganamala, R.V., Sharma, H.M., Heikkila,R,E., engers protect the enzyme from self-destruction Gear, J.C & Cornwall, D.G. (1976) Prostaglandins 11,599-607. l l ° l . Chemiluminescence, which has commonly been attributed to ^02, 02 or excited carbonyl 12 Willis, A. L., Vane, F. M., Kühn, D.C, Scott, CG. & Petrin, M. (1974) Prostaglandins 8, 453-507. products generated during breakdown of dioxet13 Hamberg,M., Svensson, J. & Samuelsson, B. (1975) 56 57 anonest ' ', is observed during prostaglandin Proc. Nail. Acad. Sei. 72, 2994-2998. 14 Smith, J.B., Ingerman,C, Kocsis, J.J. & Silver, J.M. synthesis, and this chemiluminescence was in(1974)7: Clin. Invest. 53, 1468-1472. hibited by indomethacin or the l Ag0 2 -trapping 15 Kinlough-Rathbone,R.L., Packham,M.A. & Musagent carotene^ 58 '. Addition of arachidonic acid tard, J.F. (1977) Thromb. Res. 11,567-580. to platelets also leads to chemiluminescence 16 Riescnfcld,E.H.,\Vohlcrs,H.E. & Kutsch,W.A. which is prevented by acetylsalicylic acidl 59 '. (1905) Chem. Ber. 38, 1885-1898. 17 Peters, J.W., Pitts, J.N., Rosenthal, 1. & Fuhr, H. These results indicate that short-lived reactive (1972) J. Am. Chem. Soc. 94, 4348-4350. species may be involved in prostaglandin synthe18 Hodgson,E.K. & Fridovich,!. (1974) Biochemistry sis and/or in the self-deactivation of cyclooxy13,3811-3815. genase. When l Ag0 2 or OH plays this role, its 19 Paschen, W. & Wescr, U. (1975) Hoppe-Seyler's Z. Physiol. Chem. 356, 727-737. high and almost indiscriminating reactivity, which 20 Patscheke, H. & Worner, P. (1978) Thromb. Res. 12, limits diffusion, requires its generation in close 485-496. proximity to its targets. This presupposition is 21 Patscheke, H. & Wörner, P. (1977) Thromb. Res. 11, ! not realized when OH or Ag02 is provided by 391-402. an extracellular source. However, "lie relatively 22 Andreac,W.A. (1955) Nature (London) 175, stable species H 2 0 2 is able to penetrate mem859-860.
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23 Placer. Z. A., Cushman, L. L. & Johnson, B.C. (1966) Anal. Biochem. 16, 359-364. 24 Nishikimi,M., Rao,N.A. & Yagi, K. (1972) Biochem. Biophys. Res. Comtnun. 46, 849-854. 25 Beauchamp, C. & Fridovich, I. (l971) Anal Biochem. 44,276-287. 26 McCord, J.M. & Fridovich,!. (1969) / Biol Chem. 244, 6049-6055. 27 Schcstcd,K., Rasmusscn, O.L. & Fricke.H. (1968) J. Phys. Chem. 72,626-63i. 28 Bors, W., Michel, C, Saran,M. & Lengfclder, E. (1978) Biochim. Biophys. Ada 540, 162-1 72. 29 Czapski, G. (1971) Annu. Rev. Phys. dem. 22, 171-208. 30 Czapski, G. & Dorfman, L. M. (1964) /. Phys. Chem. 68, 1169-1177. 31 Ball, G., Brcreton, G. G., Fulwood, M., Ircland, D. M. & Yatcs,P. (1970) Biochem. J. 120, 709-718. 32 Haslam,R.J., Davidson, M.M. L. & McClenaghan, M.D. (1915) Nature (London) 273, 455-457. 33 Baird,M.B., Massie,H.R. & Pickiclniak, M.J. (1977) Chem.-Biol Interact. 16, 145-153. 34 Ouanncs,C. & Wilson, T. (1968)J. Am. Chem. Soc. 90,6527-6528. 35 Kcllog, E.W. & Fridovich, I. (1975) / Biol Chem. 250,8812-8817. 36 Patscheke, H., Paschen, W. & Wörner, P. (1978) Hoppe-Seyler's Z. Physiol. Chem. 359, 933-937. 37 Merkel, P. B. & Kearns,D.R. (1972) J. A m. Chem. Soc. 94, 7244-7253. 38 Dorfman, L. M. & Adams, G.E. (1973) Reactivity of the Hydroxyl Radical in Aqueous Solutions, National Bureau of Standards Publ. 46, Washington, D.C. 39 Bhattacharyya,S.N. & Kundu,K.P. (1972)7. Radial. Phys. Chem. 4, 31-41. 40 Chapman, J. D., Reuvcrs, A.P., Borsa, J. & Greenstock, C. L. (1973) Radiat.Res. 56, 291-306. 41 Michclson, A.M. & Durosay,P. (1977) Photochem. Photobiol. 25, 55-63. 42 Taniguchi,H.,Tagaki,H. & Hatano.H. (1972)/. Phys. Chem. 16, 136-138.
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43 Matheson,I.B.C, Etheridge, R.D., Kratowich, N. R. & Lce,J. (197 f 5) Photochem. Photobiol. 21, 165-171. 44 Lynch, R.E. & Fridovich, 1. (1978) / Biol Chem. 253, 1838-1845. 45 Hamilton, G. A. (1974) in Molecular Mechanisms of Oxygen Activation (Hayashi, O., cd.) pp. 405-451, Academic Press, New York-London. 46 Dahle, L.K., Hill, E.G. & Holman,R.T. (1962) Arch. Biochem. Biophys. 98, 253-261. 47 Fong,K.L., McCay,P.B. & Poycr, J.L. (1973) /. Biol. Chem. 248, 7792-7797. 48 Anderson, S.M., Krinsky, N.I., Stone, M. J. & Clagett, D.C. (1974) Photochem. Photobiol. 20, 65-69. 49 Kcllog, E. W. & Fridovich, 1. (1977) /. Biol. Chem. 252,6721-6728. 50 Smith, J.B.& Willis, A.L. (\9T\) Nature (London) New Biol. 231, 235-237. 51 Lands, W.E.M., Cook, H.W. & Rome, L.H. (1976) in Advances in Prostaglandin and Thromboxanc Research (Samuelsson, B. & Paoletti, R., eds.) vol. 1, pp. 7-17, Raven Press, New York. 52 Lai,E.K., Fong, K. & McCay.P.B. (1978) Biochim. Biophys. Acta 528, 497-506. 53 Nakano, M., Takayama, K., Shimizu, Y., Tsuji, Y., Inaba, H. & Migitä, T. (1976) /. Am. Chem. Soc. 98, 1974-1975. 54 King, M. M., Lai, E. K. & McCay, P. B. (1975) J. Biol. Chem. 250, 6496-6502. 55 Egan,R.\V., Paxton, J. & Kuchl.F.A. (1976)/. Biol. Chem. 251, 7329-7335. 56 Khan, A. V. & Kasha, M. (1963) Nature (London) 204,241-243. 57 Hastings, J.W.& Wilson, T. (1976) Photochem. Photobiol. 23, 461 -473. 58 Marnett, L. J., Wlodawer, P. & Samuelsson, B. (1974) Biochem. Biophys. Res. Commun. 60, 1286-1294. 59 Mills, E. L., Gerrard, J. M., Filipovich, D., White, J. D. &Quie,P.G. (1978) J. Clin. Invest. 61, 807-814. 60 Misra,H.P. & Fridovich,!. (1976) Arch. Biochem. Biophys. 176,577-581.
Dr. P. Wörner, Institut für Biochemie II (Med. Fak.), Universität Heidelberg, Im Neuenheimer Feld 328, D-6900 Heidelberg.
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Response of Platelets Exposed to Potassium Tetraperoxochromate, an Extracellular Source of Singlet Oxygen, Hydroxyl Radicals, Superoxide Anions and HydrogenPeroxide Peter WÖRNER, Heinrich PATSCHEKE and Wulf PASCHEN Institut für Biochemie II (Med.Fak.), Universität Heidelberg
(Received 8 January 1979)
Summary: When potassium tetraperoxochromate bituric acid-reactive material from platelets. Other (K3Cr08) is added to platelet suspension media scavengers of hydroxyl radicals such as mannitol, it decomposes to the oxygen species hydrogen dimethylsulfoxide, EDTA or histidine prevent the peroxide, Superoxide radicals, hydroxyl radicals, release and the formation of thiobarbituric acid and singlet oxygen. K3Cr08 induces a reversible chromogen. Interaction of hydroxyl radicals with shape change and aggregation of human platelets Tris or sucrose most likely results in the generaand, in the presence of Tris or sucrose, also the tion of short-lived intermediates which may act release of serotonin. Its effect on shape change on platelets to produce thiobarbituric acid and aggregation is due to the long-lived species chromogen and to promote serotonin release, hydrogen peroxide and is abolished by indo^^ effccts Qn latdets ^ mi ^.^ fc methacm and acetylsalicyhc acid^Superoxide acetylsaiicylic acid or indomethacin. Therefore radicals, which are formed from K 3 CrO 8 in ^ reactiye h d , radical and $. let fte HEPES-contammg media do not evoke a platelet oxygen> when generated extracellularly, do not response. mediate their effects via the enzyme-catalyzed The release of serotonin depends on an interacprostaglandin pathway, in contrast to those tion of hydroxyl radicals with Tris or sucrose and evoked by the less reactive hydrogen peroxide, is associated with excessive formation of thiobarReaktion von Thrombozyten auf Kaliumtetraperoxochromat, eine extrazelluläre Quelle der Sauerstoffspezies Singulet-Sauerstoff, Hydroxylradikal, Superoxidanion und Wasserstoffperoxid Zusammenfassung: Beim Zerfall von Kaliumtetraperoxochromat (K3Cr08) in Suspensionsmedien für Blutplättchen entstehen die Sauerstoffspezies Wasserstoffperoxid (H 2 0 2 ), Super-
oxid- (0|) und Hydroxylradikale (OH) und Singulet-Sauerstoff ( J Ag02). K3Cr08 bewirkt eine reversible Formveränderung von Blutplättchen und deren Aggregation. In Gegenwart von
Enzymes: Apyrase, ATP diphosphohydrolasc (EC 3.6.1.5); Catalase, hydrogen-peroxide:hydrogen-peroxide oxidoreductase (EC 1.11.1.6); Peroxidase (horse radish), donor:hydrogen-peroxide oxidoreductase (EC 1.11.1.7); Superoxide dismutase, Superoxide:Superoxide oxidoreductase (EC 1.15.1.1). Abbreviations: HEPES =W-2-hydroxycthylpipcrazincW-cthancsulfonic acid.
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P. Wörner, H. Patscheke and W. Paschen
Tris oder Saccharose setzen die ßlutplättchen auch Serotonin frei. Formveränderung und Aggregation der Thrombozyten beruhen auf der Wirkung des langlebigen Wasserstoffperoxids. Sie werden durch Acetylsalicylsäure oder Indometacin vollständig gehemmt. Superoxidradikale, die in Gegenwart von HEPES-Puffer aus K 3 Cr0 8 entstehen, haben keinen Einfluß auf die Plättchenfunktion. Die Serotoninfreisetzung ist Folge einer Wechselwirkung zwischen Hydroxylradikalen und Tris oder Saccharose. Dabei wird ThiobarbitursäureChromogen in den Plättchen gebildet. Hydroxylradikal-Reaktanten wie Mannit, Dimethylsulf-
Bd. 360 (1979)
oxid, Ethylendiamintetraessigsäure oder Histidin verhindern sowohl die Serotoninfreisetzung als auch die Bildung des Thiobarbitursäure-Chromogens. Für beide Reaktionen werden kurzlebige Zwischenprodukte verantwortlich gemacht, die bei der Einwirkung von Hydroxylradikalen auf Tris oder Saccharose entstehen. Lipidperoxidation und Freisetzung von Serotonin werden nicht durch Acetylsalicylsäure oder Indometacin gehemmt. Wenn die hochreaktiven Sauerstoffspezies. Hydroxylradikal oder Singulet-Sauerstoff, extrazellulär gebildet werden, beruht ihre Wirkung auf Plättchen nicht auf der enzymkatalysierten Synthese von Prostaglandinendoperoxiden. Dies gilt nicht für das reaktionsträgere Wasserstoffperoxid.
Key words: Platelets, potassium tetraperoxochromate, reactive oxygen species, hydrogen peroxide, prostuglandin synthesis.
Hydrogen peroxide (H 2 0 2 ) and Superoxide radicals (Of) have been reported to cause aggregation of blood platelets' 1 » 2 !. Platelet aggregation has been shown to be inhibited by a variety of antioxidants and by scavengers of hydroxyl radicals (OH) and of singlet oxygen (1Ag02)13-6 '. These findings suggest a role of reactive oxygen species in platelet activation. These species, which have a well-known harmful action on cells, are suggested to be generated in all aerobic cells during oxygen reduction and are believed to be involved in several enzyme-catalyzed oxidations (for review seeref.' 7 !).
media results in the formation of l Ag0 2 . OH. OJ, H 2 0 2 and OH" I16"19!. We used K 3 Cr0 8 as a source for the extracellular generation of these species and tried to identify those which are able to cause activation of platelets.
Methods and Materials
Preparation of washed discoid platelets. Blood was obtained from healthy donors who denied having taken untiinflammatory drugs. 6 ml of blood was mixed with 1 ml anticoagulant, N.I.H. formula A (0.8% citric acid, 2.2% sodium citrate, 2.45% hydrous glucose). Preparation of platelet-rich plasma, washing and labelThe oxygenation of arachidonate during prostaling with | 3 H]serotonin were performed as previously glandin biosynthesis has been assumed to involve 20 1 described' '. The washed discoid platelets were stored reactive oxygen species like Ag0 2> OH, Of or 8 11 as a concentrate (2 10 9 /m/) at room temperature in a H 2 O 2 /hydroperoxides (ROOH)l - '). Upon solution of 103mM NaCl, 36mM citric acid adjusted to stimulation, blood platelets produce prostaglandpH 6.5 with NaOH, 5mM glucose, 2mM CaCl 2 , I m M in endoperoxides and thromboxane A 2 , and these MgCl , 2 mg/m/ albumin and 200 pg apyrase/m/. Prior to 2 agents are potent inducers of platelet aggregation an experiment, 100 / of this concentrate was prewarmcd 12 13 and the release reaction' » '. The response of to 37 °C and the volume was made up to 1 ml to achieve platelets to some stimuli, e.g. added arachidonic final concentrations of 500 Mg albumin/m/, 50 g apyrase/m/, 96mM NaCl, 3.6mM citrate, 5mM glucose, 5mM acid' 12 ' 14 ' or H 2 0 2 completely depends on prostaglandin synthesis. Other stimuli, like throm- KG, 0.5mM CaCl2, O.lmM MgCl2 and 46.2mM of either bin, result in the synthesis of prostaglandins which Tris, HEPES or phosphate buffer, pH 7.4. (These solutions are heieafter referred to as Tris-, HEPES- or contribute to the overall response, but in addiphosphate-buffered medium.) The final platelet count tion cause aggregation and release by alternative was 2 108/m/, except for determination of 2-thiobar15 pathways' '. bituric-acid-reactive material (4 108/m/). The decomposition of potassium tetraperoxoWashed spheroid platelets were prepared from blood anticoagulated with EDTA. After differential centrifuchromate (K 3 Cr0 8 ) in slightly alkaline aqueous
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Bd. 360 (1979)
Platelet Response to Extracellular Reactive Oxygen Species
gation, labelling with | 3 H]serotonin and washing, platelets were stored as a concentrate (1010 m / ~ ] ) at 4 °C 1 21 1 and finally resuspended in saline containing 5mM KC1 buffered with either 46.2mM Tris; HEPES or phosphate, pH 7.4. In all experiments without platelets, solutions of similar composition were employed and hereafter referred to as Tris, HEPES or phosphate buffer. Aggregation was studied in platelet samples stirred at 250 rpm in the presence of fibrinogen (3 mg/m/) in a sixchannel aggregometer (modified model UDC 1A, Kontron/Labotron, Munich). Serotonin release and shape change were recorded as previously described' 20 ' 21 ' with the exception that the samples in shape change experiments did not contain EDTA. Absence or presence of aggregates was confirmed by phase contrast microscopy. The shape of platelets was estimated from oscillation amplitude and the decrease in transmittance after stopping the st irren 20 '. Scavengers were adjusted to pH 7.4. if required, and added as isotonic solutions. To account for dilution when large volumes of scavengers were added, the concentrations of apyrase. albumin and buffer were increased, the latter at the expense of saline, to achieve final concentrations as in controls without scavengers. Decomposition of potassium tetraperoxochromate (K3CrOs) in solutions buffered at pH 7.4 was followed at 37 °C by monitoring the increase in absorbance caused by chromate (CrO^6) formation* 16 1 The concentration of K CrOg was restricted to 0.1 mM because of the high absorbance of CrO^ at 375 nm. Hydrogen peroxide (1^02) was determined after complete decomposition of 0.5mM K^CrOg at 37 °C in 1 ml of either Tris, HEPES or phosphate buffer. 5-μ/ samples were diluted to 600 μ/ with buffered saline containing 8μΜ scopoletin. \\^2 was measured as the decrease in fluorescence intensity due to oxidation of scopoletin after addition of horse radish peroxidase (325 mU/m/) I 22 1. Excitation wavelength was 350 nm, emission was monitored at 46.8 nm. Calibration curves were obtained by addition of known amounts of H2 2. Addition of the scavengers employed did not interfere with the assay. 2-Thiobarbituric-acid-reactive material, which is supposed to result in the generation of malonaldchyde, was assayed by the thiobarbituric acid method^] jt s formation in 1-m/ portions of nonstirred platelet suspension (4 χ 10s m/~ l ) was terminated 5 min after addition of the stimulus by mixing with I ml of thiobarbituric acid reagent' 23 1 Absorbance was measured at 546 nm in an Eppendorf photometer in cuvettes of 5 cm path length. The values were corrected for the absorbance of blanks, i.e. complete reaction mixture with O.SmM KaCrOg without platelets. Calibration with 1,1,3,3-tetraethoxypropane, which yields stoichiomctric
561
amounts of malonaldehyde, was in good agreement with reported e-values. Thiobarbituric acid chromogcn generated from platelets was expressed as nmol of malonaldehyde/4 χ 108 platelets. Strong color formation from HEPES exposed to K^CrOg conflicted with the measurement of thiobarbituric acid chromogen generated from platelets in the presence of this buffer. Assay of Superoxide radicals (θ|). θ| causes reduction of nitro blue tetrazolium or ferricytochrome c and oxidation of epinephrine to adrenochrome. Inhibition of these reactions by Superoxide dismutase was used to identify O^. Superoxide dismutase was established to be active after exposure to K CrOg by its ability to completely prevent reduction of nitro blue tetrazolium by a known source of O^: phenazine methosulfate and NADH 2 1 24 1. The photometric assays were performed in a Beckman DB-GT spectrophotometer in cuvettes thcrmostated at 37 °C. The test medium consisted of saline buffered with Tris, HEPES or phosphate (46.2mM), pH 7.4, 5mM KC1 and O.SmM CaCl2. Reduction of nitro blue tetrazolium (245μΜ) to blue diformazan color was monitored at 546 nm' 2 5 '. K CrOg decomposition in phosphate buffer leads to the development of a brown color. Measurement of the reduction of nitro blue tetrazolium was therefore confined to Tris or HEPES buffer. Reduction of ferricytochrome c (50μΜ) was examined by measuring the increase in absorbance at 550 nml 26 ). Generation of adrenochrome by oxidation of epinephrine was followed by the increase in absorbance at 480 nml 26 1 In experiments with cytochrome c and epinephrine, catalase (325 U/m/) was included to prevent color decay. Solutions of nitro blue tetrazolium. cytochrome c, epinephrine and Superoxide dismutase were prepared immediately prior to experiments. MATERIALS Catalase from beef liver (65000 U/mg) and ferricytochrome c (horse heart, cristallinc) were products of Boehringer Mannheim GmbH, D-6800 Mannheim. A part of the experiments was performed with catalase dialyzed for 12 h against buffered saline to remove preservatives. Dialysis of catalase had no effect on the results. 1,1,3,3-tetraethoxypropane was obtained from K & K, Plainview, New York, U.S.A. Apyrase from potato (grade I, 227 5'-ATPunits/mg), Superoxide dismutase (bovine blood, 2700 U/mg), l-epinephrine and prostaglandin EI were obtained from Sigma Chemie, D-8021 Taufkirchen. Human fibrinogen was from Bchringwerke, D-3550 Marburg. 7-Hydroxy-6methoxycoumarin (scopoletin) \vas purchased from Fluka AG, Buchs, Switzerland. Nitro blue tetrazolium, bovine albumin and horse radish peroxidase (250 U/mg) were obtained from Biomol, D-6804 Ilvesheim. 1,4-Diazabicyclo[2.2.2loctane was obtained from Merck-
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562
P.W rner, H. Patscheke and W. Paschen
Schuchardt, D-8011 Hohenbrunn, hydrogen peroxide from Merck, D-6100 Darmstadt and cytochalasin B from Aldrich-Europe, Becrse, Belgium. Cristalline potassium tetraperoxochromate (K3CrO8) was prepared from alkaline potassium chromatc and icecold hydrogen peroxide according to Ricsenfeld et alJ 2 0 l K3CrO8 was added to 1 ml of a platelet suspension as 5 μ/ of a stable solution in 0.1 M NuOH. After addition of O.SmM CrO|°, the pH changed from 7.4 to 7.45 in HEPES-, to 7.47 in Tris- and to 7.49 in phosphate-buffered saline containing 46.2mM of buffer.
Results 1) Experiments on the decomposition ofK^CrOg in the absence of platelets K3Cr08 is stable when dissolved in 0.1 M NaOH but rapidly degrades to chromatel 16 ! at physiological pH values. The breakdown of 0.1 mM CrO|e was complete in less than l min in HEPESbuffered solution and occurred somewhat more slowly in Tris- or phosphate-buffered solution. In the presence of lOmM CaCl 2 , degradation was incomplete even after 10 min in either Tris or HEPES (Fig. 1).
Bd. 360 (1979)
It has to be emphasized that Trisi 19 ! or HEPES may interact with reactive products generated during K 3 Cr0 8 decomposition. These buffers may thereby alter the rate and mode of K 3 Cr0 8 decomposition. a) Generation ofO% during decomposition of K3CrO8 in HEPES buffer O| is able to oxidize epinephrine to adrenochrome and, on the other hand, to reduce nitro blue tetrazolium or ferricytochrome c. These reactions assay 0| when the formation of formazan, ferrocytochrome c or adrenochrome can be inhibited by Superoxide dismut se. This enzyme catalyzes the reaction 02 + Of -> 02 + H 2 0 2 i 2 6 J . Of was formed during decomposition of K 3 Cr0 8 in HEPES buffer. A small reduction of nitro blue tetrazolium or cytochrome c was observed which could partially be inhibited by prior addition of Superoxide dismut se. The formation of Of could be suppressed by prior addition of the OH scavengers mannitol, dimethylsulfoxide, histidine or EDTA. At a high concentration of CaCl 2 , which reduced the rate of K 3 Cr0 8 decomposition, more Of was formed (Fig. 2). b) Absence of Ο? during K3Cr08 decay in Tris and phosphate buffers Of could be detected neither in phosphate nor in Tris buffer. This was established with cytochrome c and nitro blue tetrazolium in the presence or absence of CaCl2. We also measured adrenochrome formation during K 3 Cr0 8 decomposition. Adrenochrome was formed from epinephrine, but the lack of inhibition by Superoxide dismut se indicates oxidation independent of Of.
0.2-
0
2
4 6 t [min] —^-
8
10
Fig. 1. Decomposition of K3CrO8. K3Cr08 (0. ImM) was added as 5 μ/ of a solution in 0.1 Μ NaOH to 1 m/ of saline containing 5mM KC1 buffered cither with 46.2mM HEPES (a, c) or Tris (b). lOmM CaCl2 was present in experiment c. Decomposition of K3CrC)8 at 37 °C was monitored by the increase of absorbance at 375 nm due to formation of chromate. Curves obtained in phosphate-buffered saline were identical with those in the presence of Tris.
The production of Of from K3Cr08 in the presence of Tris or phosphate could have been obscured due to the removal of Of by the reaction OH + Of -» ΟΗθ + 0 2 (£=1.01χ ΙΟ^Μ- 1 s'1 1 27 J). Furthermore, the oxidizing species 1 Ag0 2 l l8 l and OH may interfere with the reduction of nitro blue tetrazolium or cytochrome c or also may result in a formation of adrenochrome 128K Therefore we also included scavenging agents for *Ag02 and/or OH. The presence of ΙΟηΐΜ diazabicyclooctane, 20niM histidine, 20mM dimethylsulfoxide, 50mM mannitol or 50mM sucrose did not enable us to observe a reduction of cytochrome c or nitro blue tetrazolium. The
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Bd. 360 (1979)
Platelet Response to Extracellular Reactive Oxygen Species
563
Table 1. Estimation of hydrogen peroxide after decomposition of K3Cr08 0.5mM K3CrO8 was added to 1 ml of saline containing 5mM KC1 and 46.2mM of HEPES, Tris or phosphate buffer, pH 7.4, at 37 °C. H 2 O 2 was determined after complete decomposition of K3CrO8. Mean values of three determinations. For details see Methods.
Fig. 2. Reduction of nitro blue tctrazolium to diformazan color (546 nm) during decomposition of K3CrO8 in the presence of HEPES. Enhancement by calcium and inhibition by Superoxide dismutase. At 0 min 0.5mM K 3 Cr0 8 was added to 1 ml of 46.2mM HEPES-buffered saline containing 5mM KC1 and 245μΜ nitro blue tetrazolium at 37 °C. a) Control; b) plus lOmM CaC^: c) plus 108 U Superoxide dismutase/m/; d) plus lOmM CaCl2 and 108 U Superoxide dismutase/ ml. Inhibition by Superoxide dismutase (c, d) indicates that O| is formed during decomposition of K 3 CrO 8 in the presence of HEPES.
1
Δ§Ο2-scavenging agent diazabicyclooctane enhanced the formation of adrenochrome from epinephrine exposed to K 3 Cr0 8 , but generation of adrenochrome was not affected by the presence of Superoxide dismutase. Thus, 0| is unlikely to be formed from K 3 Cr0 8 in either Tris or phosphate buffer at 37 °C.
Buffer
Scavenging agent
H202 (mMl
Tris HEPES HEPES Phosphate Phosphate Phosphate Phosphate Phosphate
_ _ lOmMCa 2 * 50mM Dimcthylsulfoxide 50mM mannitol lOmM Diazabicyclooctane 50mM Sucrose
0.42 0.33 0.32 0.45 0.58 0.50 0.43 0.36
OH scavengers either augmented (dimethylsulfoxide) or reduced (sucrose) the yield of H 2 0 2 . Despite the generation of 0| which spontaneously dismutates to H 2 0 2 1 29 1, formation of H 2 0 2 in HEPES buffer was slightly reduced, even in the presence of lOmM CaCl 2 . Different proportions of the following reactions, depending on the compounds in die medium, may contribute to the overall yield of H 2 0 2 : 1) H 2 0 2 may be a direct product of K 3 Cr0 8 decomposition. 2) H 2 0 2 may result from the interaction of 20Hradicalsl 2 ? l. 3) H 2 0 2 may be formed by spontaneous dismutation of 02 which may be either generated directly or secondarily to an interaction of OH with a scavenger^30 L From the data obtained it cannot be concluded that there is one single mechanism of H 2 0 2 formation.
d) Production of thiobarbituric-acid-reactive material from HEPES exposed to K3Cr08 Decomposition of K 3 Cr0 8 in the presence of c) Formation ofHiOi during decomposition of HEPES resulted in the production of thiobarK3Cr08 bituric-acid-reactive material. However, this prodH 2 0 2 was generated during the decomposition of uct differed from malonaldehyde generated from tetraethoxypropane in that the absorption peak K 3 Cr0 8 in all solutions employed. In phosphate buffer, which is expected to interact weakly, if was at 505 nm and not at 546 nm. Since inclusion at all, with reactive decomposition products of of OH scavengers such as 50mM mannitol, 50mM dimethylsuifoxide or 5mM EDTA interfered with K 3 Cr0 8 , the molar ratio of H 2 0 2 formed from the generation of this material, OH may be the K 3 Cr0 8 was about 0.9 (Table 1). inclusion of
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564
P. W rner, H. Patscheke and W. Paschen
Bd. 360(1979)
species interacting with HEPES. Catalase a) Phosphate- or HEPES-buffered media (2600 U/m/) or Superoxide dismutase (108 U/m/) When K 3 Cr0 8 was added to platelets in phosphatedid not prevent generation of the reactive material. or HEPES-buffered media, discoid platelets were A high concentration of CaClo, which enhances initially transformed to spheroids with subsethe formation of 0|. inhibits the generation of quent partial recovery of their original shape this material from HEPES. No color developed (Fig. 3a). As expected from shape recovery and when HEPES was added after decomposition of from the absence of an increase in light transK 3 Cr0 8 . mission, which occurs concomitantly with a granule discharge^ 20 ', no serotonin release was detected. In stirred samples containing fibrino2) Platelet response to decomposingK3CrO$ gen, platelet aggregation was observed, followed The platelet response to K 3 Cr0 8 strongly depended on the composition of the suspending medium by a complete disaggregation (Fig. 3b). employed. Either a reversible disc-sphere transformation without release or an additional strong serotonin release was observed.
0
?
t
0
The shape change induced by K 3 CrO 8 was completely prevented by prior addition of 2μΜ prostaglandin Ε , , which increases platelet cAMP
2 1 . tfminj—^·
0
2
1
.
Fig. 3. Effects of K3CrO8 on washed discoid platelets suspended in phosphate- or Tris-buffered medium. Comparison with the effect of Η2θ2· a) Shape change experiments without fibrinogen. b) Aggregation is allowed to take place by the inclusion of 3 mg fibrinogcn/m/ in these experiments. 0.5mM K 3 CrO 8 (I, II) or O.SmM H2 2 (III) was applied at 0 min. Percentages indicate serotonin release. Prior to addition of K3CrO8 the stirrer was stopped for 20 s to evaluate the discoid shape of platelets' 20 '. 1: Phosphate-buffered medium: K 3 CrO 8 causes reversible shape change (= disc-sphere transformation) without serotonin release (a). Moderate serotonin release is found when aggregation takes place with fibrinogen (b). II: Tris-buffcred medium: Subsequent to the shape change, an increase in light transmission and concomitant strong serotonin release is observed (a). A minor part of the increase in light transmission is due to weak aggregation (followed by disaggregation) despite the absence of fibrinogen (a). Control experiments without stirring after addition of K3CrO8 established that the major part was due to granule discharge'20!. Ill: The response of platelets to \\2®2 was tne same whether the platelets were suspended in phosphate- or Trisbuffercd medium. The response was similar to that evoked by K 3 CrO 8 in phosphate-buffered medium.
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Bd.360 (1979)
Platelet Response to Extracellular Reactive Oxygen Species
by activation of adenylcyclasel 31 1, and by 0acetylsalicylic acid and indomethacin, which interfere with platelet prostaglandin endoperoxide synthesis'12'14!. Moreover, addition of 325 U catalase/m/prior to K 3 Cr0 8 completely inhibited this platelet response. Neither the scavengers for OH, 5OmM mannitol, 50mM dimethylsulfoxide and 20mM histidine, nor 108 U of the 02-scavenging enzyme Superoxide dismutase, nor the l Ag0 2 quenching agent diazabicyclooctane (10mM) inhibited the response. The platelet response was also observed when platelets were added after complete decomposition of K 3 Cr0 8 (15 min). Thus, a long-lived species generated during K 3 Cr0 8 decomposition was responsible for the shape change and aggregation. Addition of the decomposition product chromate (CrO|w) did not mimic the effect of K 3 Cr0 8 . 0.5mMH 2 0 2 (almost this amount had been generated during K 3 Cr0 8 decomposition) evoked the same effect as K 3 CrO 8 , i.e. reversible disc-sphere transformation, which could be completely prevented by acetylsalicylic acid, indomethacin, prostaglandin E{ or catalase, but not by Superoxide dismutase, diazabicyclooctane or OH scavengers. The small amount of serotonin release induced by H 2 0 2 or K 3 Cr0 8 in a phosphate-buffered medium (Fig. 3b) containing fibrinogen was a result of aggregation. H 2 O 2 or K 3 Cr0 8 did not evoke serotonin release, if the stirring mechanism was stopped 0.5 s after their addition.
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salicylic acid or indomethacin had only a weak inhibitory influence on the serotonin release induced by K 3 Cr0 8 . However, complete inhibition of release and a reduction to the level of reversible disc-sphere transformation was achieved by the addition of the OH scavenging agents histidine, dimethylsulfoxide, EDTA or HEPES (Fig. 4). Mannitol was somewhat less effective. The ^02quenching agent diazabicyclooctane enhanced the effect of K 3 Cr0 8 (Fig. 4). None of these scavengers interfered with shape change induced by 2μΜ ADP or 1.5mM H 2 0 2 ; however, EDTA and dimethylsulfoxide inhibited aggregation induced by these stimuli. High concentrations of catalase (5200 U/m/) suppressed the platelet response to K 3 Cr0 8 . It has to be emphasized that 325 U of catalase/m/ was enough to prevent shape change and aggregation induced by l.SniM H 2 0 2 , but catalase at this concentration did not interfere with serotonin release induced by K 3 Cr0 8 . When either Tris or sucrose was present, K 3 Cr0 8 also induced serotonin release in phosphate-buf-
b) Iris-buffered medium Shape change curves in the presence of Tris buffer differ from those obtained from platelets exposed to K 3 CrO 8 in HEPES or phosphate buffer in that after a short decrease in light transmission a strong increase occurred parallel to strong serotonin release (Fig. 3,11). In stirred samples oscilFig. 4. Platelet responses to K3CrO8 in Tris-buftcrcd medium at 37 °C. Inhibition by OH-scavenging agents lations ceased and platelets did not recover their original discoid shape. When fibrinogen was pres- and enhancement by diazabicyclooctane. The scavengers were added 2 min prior to O.SmM ent in stirred samples, K 3 Cr0 8 induced strong aggregation which was followed after about l min K 3 CrO 8 ; a) Control; b) 5mM EDTA; e) 7.5mM histidine;d) lOmM by disaggregation, despite release of serotonin diazabicyclooctane. The percentages given represent (Fig.3b). serotonin release. Inclusion of either 50mM dimethyl2μΜ Cytochalasin B, which strongly enhances the sulfoxide, 5mM HEPES or 50mM mannitol reduced the extrusion phase of the platelet release reaction platelet response to the level of a reversible disc-sphere I20'32!, hardly augmented serotonin release intransformation without serotonin release as shown for duced by K 3 Cr0 8 . Prostaglandin E 1 ? acetylEDTA and histidine (Curve b, c).
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P.Wörner, H. Patscheke and W. Paschen
fered medium. Other compounds containing hydroxyl groups such as mannitol or glucose could not be substituted for Tris or sucrose. The release of serotonin is effected by a shortlived species since it requires the simultaneous presence of Tris or sucrose and platelets during K 3 Cr0 8 decomposition. When either Tris or platelets were added 30s after K 3 Cr0 8 . a reversible shape change without release was observed. To exclude possible effects due to the interaction of reactive species generated from K 3 CrO 8 with compounds of the medium such as apyrase, albumin, citrate or glucose, the effect of K 3 Cr0 8 was additionally investigated on washed platelets resuspended in buffered saline supplemented only with 5mM KC1. Similar results were obtained: serotonin release and the concomitant increase in light transmission were restricted to those samples to which Tris buffer or sucrose had been added. c) Production of thiobarbitwic-acid-reactive material from platelets Addition of K 3 Cr0 8 to platelets in phosphatebuffered medium resulted in the formation of
Bd.360(1979)
thiobarbituric-acid-reactive material. In Tris-containing samples more chromogen was formed than in those buffered by phosphate and the development of this additional color correlated with the amount of serotonin release. The OH-scavenging agents dimethylsulfoxide and EDTA caused strong inhibition, while mannitol was less effective. Inhibition by indomethacin and acetylsalicylic acid was weak. Diazabicyclooctane enhanced the color development (Table 2).
Discussion Decomposition of K 3 Cr0 8 in slightly alkaline solution proved to be a very complex reaction in that not only the rate of decomposition but also the kind and the yield of products depended on the composition of the medium. H 2 0 2 was established as a decomposition product of K 3 Cr0 8 in all media employed. The yield of H 2 0 2 formed from K 3 Cr0 8 was increased in the presence of the OH scavenger dimethylsulfoxide and decreased in the presence of HEPES or sucrose.
Table 2. Formation of thioburbituric acid chromogen from platelets exposed to K3CrO8. O.SmM K3CrO8 was added to platelets (4 10 8 /m/) at 37°C. Inhibitory agents were added 2 min prior to K 3 CrO 8 . For details sec Methods. The numbers represent the mean values from 4 platelet preparations (.* = level of significance < 0.05; Wilcoxon's signed-rank test). Formation of a yellow color in the presence of sucrose conflicted with the assay. Chromogen equivalent to malonaldchydc |nmol/4x 108 platelets 1 Buffer Phosphate Phosphate Phosphate Tris Tris Tris Tris Tris Tris Tris
Compounds added
0.1 m M Indomethacin 0.1 mM Indomethacin 5mM EDTA 50mM Dimethylsulfoxide 50mM Mannitol 2400 U Catalase/m/ 1 OmM Diazabicyclooctane
K3Cr08 K 3 Cr0 8 K 3 Cr0 8 K3Cr08 K 3 Cr0 8 K3Cr08 K3Cr08 K3Cr08 K 3 Cr0 8
0.08 0.54 0.42 1.06 0.84* 0.26* 0.27* 0.41* 0.45* 1.26*
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Bd. 360 (1979)
Platelet Response to Extracellular Reactive Oxy£ in Species
In our experiments, the occurrence of another decomposition product, 0®, was restricted to HEPES-buffered solutions. Controversy exists in the literature as to whether or not Of is formed during K 3 Cr0 8 decomposition: no Of was detected in Tris-buffered medium^ 19 ', while it could be established in pyrophosphate-buffered medium in the presence of *AgO2 scavengersl18'. The divergence of results may be explained by differences in the composition of solutions, in temperatures, or in the assays for Of employed. Generation of OH from K 3 Cr0 8 has been reported^ 19 ' and is indicated by our experiments with OH scavengers. OH scavengers do not inhibit shape change of platelets promoted by other stimuli. They do, however, interfere with serotonin release and formation of thiobarbituric acid chromogen from platelets treated with K 3 Cr0 8 . OH scavengers also inhibited the formation of 0? in HEPES-buffered solutions and the generation of thiobarbituric-acid-reactive material from HEPES on application of K3Cr08.
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1) H 2 0 2 , in an amount which is formed during K 3 CrO 8 decomposition, evokes platelet effects similar to that of K 3 Cr0 8 after complete decomposition. 2) A low concentration of catalase completely prevents shape change, or aggregation in the presence of fibrinogen, induced by either K3Cr08 orH 2 0 2 . 3) Acetylsalicylic acid and indomethacin completely inhibit shape change promoted by K 3 CrO 8 and H 2 O 2 . Shape change and aggregation induced by the two agents depend on the prostaglandin endoperoxide pathway. Several results show that Of is not responsible for the effect of K 3 Cr0 8 on platelets.
1) K 3 Cr0 8 causes a similar platelet response regardless of whether HEPES or phosphate is employed as buffer. However, Of is detected in HEPES but not in phosphate buffer. 2) Superoxide dismutase effectively quenches Of but does not influence the shape change induced by K 3 Cr0 8 . K 3 Cr0 8 has been reported as a chemical source 117 19 331 3) Inclusion of OH scavengers, which prevent the of iAg02 - ' . If ^gO2 were responsible formation of Of in HEPES, does not inhibit shape for the platelet responses observed, inclusion of diazabicyclooctane, a ^g02-quenching agent^34' change induced by K 3 CrO 8 . 4) A high concentration of CaCl 2 , which incould be expected to inhibit them. However, it enhanced K 3 Cr0 8 -induced lipid peroxidation and creases the yield of Of formed in HEPES-bufserotonin release. It has been reported to prevent fered medium, does not augment the platelet relipid peroxidation in liver microsomes exposed to sponse. K 3 Cr0 8 l 3 3 L However, the concentration emPlatelet aggregation and release in the presence of ployed in these experiments (0.33M) is not apxanthine and xanthine oxidase have been attriplicable in platelet suspensions and it may have buted to Of I 2 '. However, the enzyme alone is acted by eliminating preformed lipid hydroalso able to induce platelet aggregation and the peroxides'35!. release reaction!361. The response of platelets to K 3 Cr0 8 depends on The involvement of a long-lived species argues the experimental conditions. A reversible discagainst a role of *Ag02 or OH in K 3 Cr0 8 -insphere transformation (= shape change) without duced shape change. !Ag02 has a life-time of serotonin release occurs when platelets are added about 2 MS in aqueous medial37! and OH also has after complete decomposition of K 3 Cr0 8 . Seroa very short life-time, as can be expected from its tonin release, however, is only brought about if high reactivity. Moreover, neither the *Ag02K 3 Cr0 8 is directly added to platelets. These find- quenching agent diazabicyclooctane nor high ings point to the involvement of a long-lived concentrations of the OH scavengers mannitol, species in the shape change, while additional re- EDTA, dimethylsulfoxide or histidine, which interact with OH at rates which are almost diffulease reaction requires short-lived decomposition sion controlled (fc OH > 10 9 M~ 1 s'1138-«°1), products. interfere with K 3 CrO 8 -induced shape change. The most likely candidate for the long-lived species which produces reversible shape change In contrast to shape change, K 3 Cr0 8 -promoted without release is H 2 0 2 : serotonin release involves a short-lived species. Brought to you by | Purdue University Libraries Authenticated Download Date | 5/31/15 1:11 PM
568
P. W rner, H. Patscheke and W. Paschen
One of the species to be considered as a stimulus of this response is OH. However, serotonin release appears not to be the result of a direct action of OH on platelets. Serotonin release is not observed in phosphate-buffered medium: it requires the presence of either Tris or sucrose, which are known to act as OH scavengers^19»38'. The finding that other scavengers of OH such as EDTA, dimethylsulfoxide, histidine or mannitol inhibit the release of serotonin suggests a competition of these agents with Tris or sucrose for OH. Concentrations of catalase in excess of those required to inhibit the effect of H 2 O 2 on platelets may also inhibit due to the ability of catalase totrapOHl 4 1 !.
Bd. 360 (1979)
a possible trigger for release. Enhancement of serotonin release by the ! Ag0 2 -quenching agent diazabicyclooctane strongly argues against a requirement for J Ag0 2 . However, the OH-scavenging agents EDTA and histidinel 43 » 44 ', but not dimethylsulfoxide and mannitol, also have ^gOotrapping properties and they inhibit release at lower concentrations than do the latter. These results could be interpreted to mean that !Ag02 contributes to serotonin release. Thus, at present, we cannot definitely exclude a role of ]Ag02 in K 3 Cr0 8 -induced serotonin release.
A common reaction performed by free radicals is an abstraction of hydrogen atoms from target molecules and this effect is typical for OH^ 45 ·. Through their interaction with OH. OH-trapping Hydrogen abstraction from unsaturated lipids is agents are themselves transformed to radicals. presumably the initiating step for their peroxidaThese secondary radicals will either be eliminated tionl 46 !. OH and !Ag02 have been suggested to by dimerization or may represent the beginning cause lipid peroxidationl 35 ' 47 " 49 !. The occurrence of a radical chain. Scavenging of OH by dimethyl- of lipid peroxides can be detected by their desulfoxide may result in the generation of CH 3 I 3 °1 composition product, malonaldehyde, which rewhich may form dimers; from methanol ·ΟΗ 2 ΟΗ, acts with thiobarbituric acid to yield a red color •0 2 CH 2 OH and 0| may be formed^ 42 !. Since it [23,461 τ}^ chromogen is also formed during is not unequivocally known what does result from prostaglandin synthesis catalyzed by fatty acid the interaction of the different scavengers with cyclooxygenase. However, in contrast to non-enOH, any rationalization is hazardous. The most zymic lipid peroxidation, its generation is preventlogical assumption seems to be that intermediates ed by the inhibitors of cyclooxygenase, acetylgenerated by the interaction of Tris or sucrose salicylic acid and indomethacinl 50 ]. with OH induce serotonin release, while those H 2 0 2 -induced shape change and aggregation are generated from the other OH scavengers do not. The life-time of these intermediates is short since completely prevented by inhibitors of cyclooxygenase. These also interfere with shape change the release requires the simultaneous presence of and aggregation promoted by K 3 Cr0 8 in phosTris or sucrose and platelets when K 3 Cr0 8 is phate-buffered medium. However, the amount of added. thiobarbituric acid color from platelets exposed The results obtained in HEPES-containing media to K 3 Cr0 8 exceeds by far that from H 2 0 2 -treatdeserve further comment. HEPES may also act as ed platelets; and this color formation is inhibited an OH scavenger. Thiobarbituric-acid-reactive only to a minor extent by acetylsalicylic acid or material is formed from HEPES exposed to indomethacin. The latter is an indication for cyK 3 Cr0 8 even in the absence of platelets. Declooxygenase-indepcndent lipid peroxidation by velopment of this color is inhibited by high conspecies generated during K 3 Cr0 8 decomposition centrations of other OH scavengers. Moreover, other than H 2 0 2 . generation of Of in the presence of HEPES is prevented by OH scavengers. This indicates an inter- When K 3 Cr0 8 induces serotonin release, i.e. in action of OH with HEPES. HEPES may compete the presence of Tris buffer, even more thiobarwith Tris for OH and thereby inhibit serotonin re- bituric-acid-reactive material is formed than after K 3 Cr0 8 -promoted shape change (in phosphatelease even in the presence of Tris, as does mannibuffered medium). The small effect of indomethtol, for example. acin argues against an involvement of prostaglandin endoperoxides in serotonin release. Inhibition As the other short-lived intermediate generated by OH-scavenging agents indicates lipid peroxidafrom K 3 Cr0 8 , *Ag02 remains to be discussed as
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Bd. 360 (1979)
Platelet Response to Extracellular Reactive Oxygen Species
tion mediated by OH and secondary radicals generated by the interaction of OH with Tris. These radicals also promote serotonin release, which may be an event either independent of or secondary to membrane perturbation caused by the formation of lipid peroxides. The assumption that serotonin release induced by K 3 CrOg does not involve the physiological pathway is supported by the experiments with cytochalasin B. Cytochalasin B augments the release reaction from platelets activated by thrombin and collagen (20,321 ? b ut UOQS not enhance serotonin release by K 3 Cr0 8 .
569
branes. OH and possibly !Ag02 may be generated intracellularly from H 2 0 2 in the presence of a transition metal ion in a lower valence state [45,601
The authors thank Prof. R. Brossmer for his support of this study. The assistance of Misses/. Schlageter and C. Wabhel is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft within SFB 90 "Cardiovaskularcs System". Literature \
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Dr. P. Wörner, Institut für Biochemie II (Med. Fak.), Universität Heidelberg, Im Neuenheimer Feld 328, D-6900 Heidelberg.
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