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Superoxide dismutase triggers activation of “primed” platelets
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 289, No. 1, August 15, pp. 180-183, 1991
Superoxide Dismutase Triggers Activation of “Primed” Platelets’ L. Iuliano, Institute
D. Pratic6, A. Ghiselli,
of 1st Clinical Medicine,
Received February
University
M. S. Bonavita,
and F. Violi’
of Rome ‘La Sapienza, ” Rome, Italy
25, 1991, and in revised form April 29, 1993
Superoxide dismutase (SOD) triggers activation of human platelets exposed to subthreshold concentrations of arachidonic acid and collagen. The subthreshold concentrations used are not able to activate platelets but “prime” platelets to be activated by SOD. The addition of SOD to arachidonic acid- or collagen-primed platelets induced aggregation, thromboxane AZ production, and release of [‘Hlserotonin. Superoxide dismutase does not have any effect on resting platelets and ADP-, thrombin-, calcium ionophore A23187-, PAF-, or U46619-stimulated platelets. Furthermore, superoxide dismutase-dependent platelet activation is fully prevented by catalase and/or aspirin, suggesting a role for HzOz and the involvement of the cyclooxygenase pathway of arachidonic acid in such activation. o 1991 Academic press, IN.
There is a growing body of evidence that oxygen free radicals are involved in several forms of human diseases, including acute respiratory distress syndrome, atherosclerosis, and autoimmune diseases (1). Recent studies have focused on the pathophysiologic role of OFRs3 in coronary heart disease, in particular in biological and clinical abnormalities following the ischemia-reperfusion process (2,3). Indeed, the use of electron spin resonance spectroscopy, either alone or in combination with spin-traps, has demonstrated that reperfusion of an ischemic or hypoxic myocardium is associated with a burst of free radicals production (4-7). OFRs are responsible for increased lipid peroxidation, myocardial ’ This research was supported by Andrea Cesalpino Foundation. ’ To whom correspondence should be addressed. a Abbreviations used: OFRs, oxygen free radicals; SOD, Cu, Zn-superoxide dismutase; dSOD, denaturated Cu, Zn-superoxide-dismutase; hr-SOD, human recombinant superoxide dismutase; CAT, catalase; dCAT, denatured catalase; TxA2, thromboxane A?; OF, superoxide anion; 5HT, serotonin; PAF, platelet activating factor; U46619, 9,11-dideoxylla,9ol-epoxymethanoprostaglandin F,,.
damage, and arrhythmias. Reperfusion is also accompanied by an intracoronary platelet activation as suggested by histological and functional studies (8,9), and enhanced production of TxAz, a potent vasoconstrictor and platelet aggregating substance, has been demonstrated (10). Increased TxA, production and OFRs production could be two related phenomena, assuming that OFRs may activate platelet function. This problem has been investigated in previous studies with conflicting findings (ll17). It has been reported that exogenous SOD has no effect upon ADP-, epinephrine-, collagen-, and arachidonateinduced platelet aggregation (12-15). However, Salvemini et al. reported that SOD inhibits platelet aggregation induced by low concentrations of thrombin, but has no effect when high concentrations of thrombin are used (17). The functional relation between SOD and platelets could be important for several reasons. If platelets are activated by OFRs, in particular by 02, then the administration of SOD during reperfusion may be useful both to scavenge 0; and to reduce platelet activation. In addition, platelets are a potential source of 0; (ll), thus the presence of SOD in the medium may represent an important modulator of platelet function. Taking into account that platelets are activated during reperfusion and that they play an important role in the reocclusion phenomenon (8-lo), the investigation of the role of SOD on platelet function is relevant. This study reports on the effect of SOD on platelets stimulated in vitro by several agonists. MATERIALS
AND
METHODS
Platelet isolation and aggregation. Blood mixed with 3.8% Na-citrate (ratio 9:l) was collected from healthy volunteers (11 males age 28-40, nonsmokers) who had not taken any drugs known to interfere with platelet function for at least 15 days before the beginning of the study. Platelets were isolated as previously described (16) and suspended in Hank’s balanced salt solution (calcium and magnesium free) containing 10% autologous platelet poor plasma. The platelet count was adjusted at 2 X 10s ml-‘. No neutrophils or red blood cells were present in the platelet suspension when examined by light microscopy. Platelet aggregation, by Born’s method (18), was measured 3 min after adding a subthreshold concentration of agonist (final concentrations: arachidonic
180
0003.g&361/91
$3.00
Copyright 0 1991 by Academic Press, Inc. All
rights
of reproduction
in any
form
reserved.
PLATELET
ACTIVATION
BY SUPEROXIDE
acid, lo-20 pM; collagen, 0.1-0.2 pg X ml-‘; ADP, 0.1-0.2 pM, A23187, 0.6-0.8 pM, thrombin, 0.02-0.04 units X ml-‘, PAF, l-3 nM, U46619, SO-120 nM). Subthreshold concentrations of agonist were defined as the lowest doses which induced less than a 10% increase in light transmission. Platelet aggregation was also studied using threshold concentrations of agonists defined as the lowest dose which induced ~50% increase in light transmission. As control in all experiments, washed platelets were added with the vehicles used to dissolve the substances studied. In order to explore the involvement of the cyclooxygenase pathway, platelets were incubated 5 min at 37’C with 100 pM aspirin before the washing procedure. TxA, measurement. Platelet TxAZ formation was measured as its stable metabolite thromboxane B? by radioimmunoassay using kits purchased from New England Nuclear (Du Pont de Nemours, Florence, Italy), as previously described (198). [3H]Serotonin release. Before being washed platelets were incubated 30 min at 22’C with 0.2 HIM Ci X ml-’ [3H]serotonin. The platelets were washed twice and finally resuspended in Hank’s balanced salt solution containing 5 pM imipramine to :inhibit the reuptake. Three minutes after the addition of stimuli 700 ~1 of the sample was immediately transferred to an Eppendorf microfuge vial containing 70 ~1 of the following solution: EDTA, 5 mM; theophylline, 5 mM; aspirin, 500 PM, and PGEi, 0.2 pg X ml-‘. After centrifugation, 2 min at 12,OOOg, 100 ~1 of the supernatant was transferred to a scintillation vial and 2 ml of aqueous scintillation fluid was added. The amount of 3H was determined using a Beckman scintillation counter. The [3H] was always expressed as a percentage of the total amount osf [3H]serotonin that had been accumulated by the platelets. Each experiment was performed in duplicate. SOD and CAT actiuity. Superoxide dismutases (EC 1.15.1.1) were dialyzed by ultrafiltration in a Spectrum apparatus (Spectrum, Haverton, USA). Enzyme activity was measured by a spectrophotometer using the xanthine/xanthine oxidase/cytochrome c system according to McCord and Fridovich (20). CAT (EC 1.11.1.6) was dialyzed to remove thymol and activity was measured by a spectrophotometer, by following the disappearance rate of H202 (21). SOD and CAT were denaturated by boiling the enzymes for 10 min. This procedure could give >95% inactivation. Statistical analysis. Data are reported as means + standard deviation. The comparison between variables was analyzed by Student’s t test for paired data. Significance was accepted at the P < 0.05 level. The following materials were obtained from Sigma Materials. Chemical Co. (Sigma Chimica, M&no, Italy): arachidonic acid, U46619, PAF, ADP, A23187, EDTA, theophiline, PGE,, MnSOD. [HHydroxytriptamine binoxilate (sp act 15-30 Ci X mmol-‘) was purchased
TABLE
I
Effect of SOD (300 U X ml--‘) on Platelet Aggregation Induced by Different Types of Agonists at Threshold Concentrations (the Lowest Dose Which Induces at Least a 50% Increase in Light Transmission [LT%]). LT%’
Thrombin A23187 ADP Collagen Arachidonate ’ Mean + standard deviation b P < 0.005.
-SOD
+SOD
60 57 53 57 54
58 f 4 55 f 3 54 + 4 72 f 3* 71+5*
f 6 ?I 5 f 6 + 4 -t 5
of five separate experiments.
181
DISMUTASE TABLE
II
Effect of SOD on Human Platelet “Primed” with Subthreshold Concentrations of Different Agonists
SOD Thrombin Thrombin + SOD A23187 A23187 + SOD ADP ADP + SOD Collagen Collagen + SOD Collagen + SOD + CAT Collagen + H202 Collagen + dSOD Collagen + SOD + dCAT Collagen + SOD + Aspirin Collagen + hr-SOD Collagen + Mn-SOD Arachidonate Arachidonate + SOD Archidonate + SOD + CAT Arachidonate + HzOz Arachidonate + dSOD Arachidonate + SOD + dCAT Arachidonate + SOD + Aspirin Arachidonate + hr-SOD Arachidonate + Mn-SOD
LT
TxA,
3 4 3 5 5 4 5 3 2 4
[3H]5HT
4+3 55 f 4 5+2 48 + 3
522 61 f 7 4+2 52 + 3
Note. Values of percentage of light transmission (LT), TxAz (ngxmll’), and [sH]5HT release (W), taken 3 min after adding the agonists, represent the mean f standard deviation of five separate experiments. Final concentrations: thrombin, 0.02-0.04 units X ml-‘; A23187, 0.6-0.8 pM; ADP, 0.1-0.2 jiM; collagen, 0.1-0.2 pg X ml-‘; arachidonic acid, lo-20 pM; superoxide-dismutases and CAT, 300 units X ml-‘; H,Oz, 0.5 FM.
from New England Nuclear (Du Pont de Nemours, Florence, Italy). Thrombin, CuZn-SOD, and hr-SOD were obtained from Calbiochem (San Diego, CA). Hydrogen peroxide was from Merck (Darmstadt, Germany). Collagen (bovine tendon) was purchased from Menarini (Florence, Italy). Aspirin as its soluble lysin salt was from Maggioni (Milan, Italy). Arachidonic acid, 15 mM stck solution, was dissolved in absolute ethanol under Nz atmosphere and stored at -20°C. Calcium ionophore A23187 was dissolved in dimethyl sulfoxide. HzOz was titrated with KMnO,. All the other reagents were dissolved in double-distilled water and diluted in physiologic saline to the appropriate concentrations. RESULTS
Table I shows platelet aggregation observed using different types of platelet stimulation in the presence or absence of SOD. No significant changes were observed in samples stimulated with threshold concentrations of ADP, thrombin, and the calcium ionophore A23187; a significant increase in platelet aggregation by SOD was observed when collagen or arachidonic acid was used as platelet stimulus (P < 0.005). Table II reports on platelet changes observed using subthreshold concentrations of several platelet stimuli in
182
IULIANO
the presence or absence of SOD; neither platelet aggregation nor TxAz production was induced by the various platelet agonists at subthreshold concentrations or by SOD alone. Platelet stimulation by ADP, thrombin, or calcium ionophore A23187 was not modified in the presence of SOD. Platelet stimulation by PAF and the stable endoperoxide analogue U46619 was not influenced in the presence of SOD, either at threshold or subthreshold concentrations of these platelet stimuli (data not shown). On the contrary, when subthreshold concentrations of arachidonic acid or collagen were tested on platelets with SOD added, a large increase in aggregation, TxAz production, and [3H]serotonin release was seen (a typical tracing of the aggregatory response is depicted in Fig. 1). The effects of SOD on primed platelets were concentration dependent up to a level of 300 units; the dose-response curves of platelet aggregation to increasing amounts of SOD are depicted in Fig. 2. The amplification of platelet response by SOD was dependent on the catalytic activity of the enzyme as indicated by: (i) dSOD not activating “primed” platelets; (ii) active CAT, but not dCAT, fully preventing platelet activation by SOD; (iii) H202 mimicking the effect of SOD on “primed” platelets. Additional evidence was given by experiments in which other forms of the enzyme were used: Mn-SOD and hr-SOD produced effect on platelets similar to those of CuZn-SOD (Table II). The detection of TxAz in SOD-activated platelets suggested the involvement of the cyclooxygenase pathway of arachidonic acid. This was further corroborated by experiments with aspirin pre-treated platelets: blockade of cyclooxygenase activity prevented both TxAz production and aggregation of platelets stimulated with SOD and
0
60
120 seconds
180
FIG. 1. Representative platelet aggregation tracings showing effect of SOD on platelets stimulated with subthreshold concentrations of arachidonic acid. A, arachidonic acid (15 PM); B, A + SOD (300 U X mll’); C, A + dSOD; D, B + CAT (300 U X ml-‘); E, A + 0.5 pM H,O,.
ET AL.
-0
loo
200 SOD uni ts x ml-’
FIG. 2. Aggregation of primed platelets tration. The aggregation was measured 3 subthreshold concentration of arachidonic lagen (0.16 ag X ml-‘; triangles) along with tions.
sub-threshold concentrations lagen (Table II).
as function of SOD concenmin after the addition of a acid (22 PM; circles) or colincreasing SOD concentra-
of arachidonic
acid or col-
DISCUSSION
This study indicates that the enzyme SOD per se does not influence platelet function but can induce an irreversible aggregation, TxA2 production, and [3H]serotonin release when added to primed platelets. Such an amplification was observed using agonists which act through a cyclooxygenase-dependent mechanism. This is shown by the amplification of platelet response observed when we have used subthreshold concentrations of collagen or arachidonic acid, which activate platelets through the cyclooxygenase pathway. The same was not observed when other agonists such as ADP, thrombin, A23187, PAF, or U46619 were employed. The absence of the amplification response by SOD could be due to different pathways of platelet activation induced by these agonists. In fact, even though the latter agonists may enhance arachidonic acid release and oxygenation, this is not their primary mechanism of platelet activation (22). Taken together, these findings indicate that SOD could be considered as a trigger of platelet aggregation, which acts by amplifying the response to other agonists, only when stimuli acting mainly through the arachidonic acid cascade are employed. This suggestion is supported by the finding of increased amounts of TxAz in samples substimulated with arachidonic acid or collagen in the presence of SOD and by the disappearance of both platelet aggregation and TxA2 formation in platelets pretreated with aspirin.
PLATELET
Furthermore, reaction
ACTIVATION
BY SUPEROXIDE
HzOz generated by SOD according to the
20; + 2H+ + HzOz + O2 was directly involved in the activation of platelets because the SOD-dependent aggregation, TxAZ generation, and [3H]serotonin release were fully prevented by catalase, which eliminates HzOz. Tlhis observation indicates that the amplification of platellet response by SOD is due to the catalytic activity and is mediated by H202. In fact denaturated SOD and denaturated catalase did not produce any effect. Therefore our findings could be explained by assuming that the platelet-produced 0; (11) is converted, by the addition of SOD, to Ha02, which in turn facilitates the activation of arachidonic acid metabolism. This suggestion is demonstrated by adding exogenous H,O, instead of SOD: Hz02, at nanomolar concentrations, which mimicks the effect of active SOD on primed platelets. Furthermore, the role for HzOz is supported by previous findings showing that Hz02 enhances platelet aggregation (23, 24) and modulates the mobilization of arachidonate from platelet membrane (25). The demonstration that SOD stimulates the cyclooxygenase pathway of platelet arachidonic acid metabolism might help to explain some recent findings observed in human and experimental model systems. In experiments of ischemic reperfusion, an elevated formation of oxygen free radicals has been detected (4-7). The metabolism of arachidonic acid has recently been studied in this system, and, in fact, an increased formation of TxAz has been detected during reperfusion (10). This finding may be important because platelet activation and, in particular, the increased TxA, formation, can contribute to vascular reocclusion (8, 9). Our results suggest that this increased TxA, formation observed during reperfusion could be mediated by OFRs. The amplification of platelet response by SOD should also be taken into account in experimental models of ischemia-reperfusion where this enzyme is used to prevent the myocardial damage induced by OFRs (26-29). ACKNOWLEDGMENTS We are grateful to Prof. C. Patron0 and Prof. G. Rotilio for their helpful criticism and suggestions .and to Mrs. C. Piccheri for technical assistance.
REFERENCES 1. Halliwell, B., and Gutteridge, J. M. C. (Eds.) (1989) Free Radicals in Biology and Medicine, 2nd ed., Clarendon Press, Oxford.
DISMUTASE
183
2. Simpson, P. J., and Lucchesi, B. R. (1987) J. Lab. Clin. Med. 110, 13-30. 3. Kloner, R. A., Przyklenk, K., and Whittaker, P. (1989) Circulation 80, 1115-1127. 4. Zweir, J. L. (1988) J. Biol. Chem. 263, 1353-1357. 5. Bolli, R., Patel, B. S., Jeroudi, M. O., Lai, E. K., and McCay, P. B. (1988) J. Clin. Incest. 82, 476485. 6. Bolli, R., Jeroudi, M. O., Patel, B. S., DuBose, C. M., Lai, E. K., Roberts, R., and McCay, P. B. (1989) Proc. Natl. Acad. Sci. 1JSA 86,4695&4699. 7. Kuzuya, T., Hoshida, S., Kim, Y., Nishida, M., Fuji, H., Kitabatake, A., Tada, M., and Kamada, T. (1990) Circ. Res. 66, 1160-1165. 8. Yasuda, T., Gold, H. K., Fallon, J. T., Leinbach, R. C., Guerrero, J. L., Scudder, L. E., Kanke, M., Shealy, D., Ross, M. J., Collen, D., and Caller, B. S. (1988) J. Clin. Znuest. 81, 1284-1291. 9. Yasuda, T., Gold, H. K., Fallon, J. T., Leinbach, R. C., Garabedian, H. D., Guerrero, J. L., and Cohen, D. (1989) J. Am. Coil. Cardiol. 13, 1409-1414. 10. Fitzgerald, D. J., Wright, F., and FitzGerald, G. A. (1989) Circ. Res. 65,83%94. 11. Marcus, A. J., Silk, S. T., Safier, L. B., and Ullman, H. L. (1977) J. Clin. Invest. 59, 149-158. 12. Bressler, N. M., Broekman, M. J., and Marcus, A. J. (1979) Blood 53,167-178. 13. Iatridis, S. G., Iatridis, P. G., Kyrkou, K. A. et al. (1979) Thromb. Res. 15,733%741. 14. Krinsky, N. I., Sladdin, D. G., Levine, P. H., Taub, L. A., and Simic, H. G. (1981) Thromb. Haemostasis 42, 116-120. 15. Clemmons, R. M., Dorsey Lee, M. R., Bliss, E. L., Asbury, A. C., Cook, D., and Brown, V. (1985) Am. J. Vet. Res. 46, 110441106. 16. Violi, F., Ghiselli, A., Iuliano, L., Alessandri, C., Cordova, C., and Balsano, F. (1988) Haemostasis 18, 91-98. 17. Salvemini, D., De Nucci, G., Sneddon, J. M., and Vane, J. R. (1989) Brit. J. Pharmacol. 97, 1145-1150. 18. Born, G. V. R., and Cross, M. J. (1963) J. Physiol. 168, 178-195. 19. Violi, F., Ghiselli, G., Iuliano, L., Pratico, D., Alessandri, A., and Balsano, F. (1988) Eur. J. Clin. Pharmacol. 33, 599-602. 20. McCord, J. M., and Fridovich, I. (1969) J. Biol. Chem. 244, 60496055. 21. Beers, R. F., and Sizer, I. W. (1952) J. Biol. Chem. 195, 133-140. 22. Seiss, W. (1989) Physiol. Rev. 69, 58-174. 23. Canoso, R. T., Rodvien, R., Scoon, K., and Levine, P. H. (1974) Blood 43, 645-656. 24. Del Principe, D., Menichelli, A., De Matteis, W., Di Corpo, M. L., Di Giulio, S., and Finazzi-Agro, A. (1985) FEBS Lett. 185, 142146. 25. Hornberger, W., and Patscheke, H. (1990) Eur. J. Biochem. 187, 1755181. 26. Engler, R., and Gilpin, E. (1989) Circulation 79, 1137-1142. 27. Patel, B. S., Jeroudi, M. O., O’Neill, P. G., Roberts, R., and Bolli, R. (1990) Am. J. Physiol. 258, H369-H380. 28. Omar, B. A., Gad, N. M., Jordan, M. C., Striplin, S. P., Russell, W. J., Downey, J. M., and McCord, J. M. (1990) Free Rad. Biol. Med. 9,465-471. 29. Omar, B. A., and McCord, J. M. (1990) Free Rad. Biol. Med. 9,473478.
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 289, No. 1, August 15, pp. 180-183, 1991
Superoxide Dismutase Triggers Activation of “Primed” Platelets’ L. Iuliano, Institute
D. Pratic6, A. Ghiselli,
of 1st Clinical Medicine,
Received February
University
M. S. Bonavita,
and F. Violi’
of Rome ‘La Sapienza, ” Rome, Italy
25, 1991, and in revised form April 29, 1993
Superoxide dismutase (SOD) triggers activation of human platelets exposed to subthreshold concentrations of arachidonic acid and collagen. The subthreshold concentrations used are not able to activate platelets but “prime” platelets to be activated by SOD. The addition of SOD to arachidonic acid- or collagen-primed platelets induced aggregation, thromboxane AZ production, and release of [‘Hlserotonin. Superoxide dismutase does not have any effect on resting platelets and ADP-, thrombin-, calcium ionophore A23187-, PAF-, or U46619-stimulated platelets. Furthermore, superoxide dismutase-dependent platelet activation is fully prevented by catalase and/or aspirin, suggesting a role for HzOz and the involvement of the cyclooxygenase pathway of arachidonic acid in such activation. o 1991 Academic press, IN.
There is a growing body of evidence that oxygen free radicals are involved in several forms of human diseases, including acute respiratory distress syndrome, atherosclerosis, and autoimmune diseases (1). Recent studies have focused on the pathophysiologic role of OFRs3 in coronary heart disease, in particular in biological and clinical abnormalities following the ischemia-reperfusion process (2,3). Indeed, the use of electron spin resonance spectroscopy, either alone or in combination with spin-traps, has demonstrated that reperfusion of an ischemic or hypoxic myocardium is associated with a burst of free radicals production (4-7). OFRs are responsible for increased lipid peroxidation, myocardial ’ This research was supported by Andrea Cesalpino Foundation. ’ To whom correspondence should be addressed. a Abbreviations used: OFRs, oxygen free radicals; SOD, Cu, Zn-superoxide dismutase; dSOD, denaturated Cu, Zn-superoxide-dismutase; hr-SOD, human recombinant superoxide dismutase; CAT, catalase; dCAT, denatured catalase; TxA2, thromboxane A?; OF, superoxide anion; 5HT, serotonin; PAF, platelet activating factor; U46619, 9,11-dideoxylla,9ol-epoxymethanoprostaglandin F,,.
damage, and arrhythmias. Reperfusion is also accompanied by an intracoronary platelet activation as suggested by histological and functional studies (8,9), and enhanced production of TxAz, a potent vasoconstrictor and platelet aggregating substance, has been demonstrated (10). Increased TxA, production and OFRs production could be two related phenomena, assuming that OFRs may activate platelet function. This problem has been investigated in previous studies with conflicting findings (ll17). It has been reported that exogenous SOD has no effect upon ADP-, epinephrine-, collagen-, and arachidonateinduced platelet aggregation (12-15). However, Salvemini et al. reported that SOD inhibits platelet aggregation induced by low concentrations of thrombin, but has no effect when high concentrations of thrombin are used (17). The functional relation between SOD and platelets could be important for several reasons. If platelets are activated by OFRs, in particular by 02, then the administration of SOD during reperfusion may be useful both to scavenge 0; and to reduce platelet activation. In addition, platelets are a potential source of 0; (ll), thus the presence of SOD in the medium may represent an important modulator of platelet function. Taking into account that platelets are activated during reperfusion and that they play an important role in the reocclusion phenomenon (8-lo), the investigation of the role of SOD on platelet function is relevant. This study reports on the effect of SOD on platelets stimulated in vitro by several agonists. MATERIALS
AND
METHODS
Platelet isolation and aggregation. Blood mixed with 3.8% Na-citrate (ratio 9:l) was collected from healthy volunteers (11 males age 28-40, nonsmokers) who had not taken any drugs known to interfere with platelet function for at least 15 days before the beginning of the study. Platelets were isolated as previously described (16) and suspended in Hank’s balanced salt solution (calcium and magnesium free) containing 10% autologous platelet poor plasma. The platelet count was adjusted at 2 X 10s ml-‘. No neutrophils or red blood cells were present in the platelet suspension when examined by light microscopy. Platelet aggregation, by Born’s method (18), was measured 3 min after adding a subthreshold concentration of agonist (final concentrations: arachidonic
180
0003.g&361/91
$3.00
Copyright 0 1991 by Academic Press, Inc. All
rights
of reproduction
in any
form
reserved.
PLATELET
ACTIVATION
BY SUPEROXIDE
acid, lo-20 pM; collagen, 0.1-0.2 pg X ml-‘; ADP, 0.1-0.2 pM, A23187, 0.6-0.8 pM, thrombin, 0.02-0.04 units X ml-‘, PAF, l-3 nM, U46619, SO-120 nM). Subthreshold concentrations of agonist were defined as the lowest doses which induced less than a 10% increase in light transmission. Platelet aggregation was also studied using threshold concentrations of agonists defined as the lowest dose which induced ~50% increase in light transmission. As control in all experiments, washed platelets were added with the vehicles used to dissolve the substances studied. In order to explore the involvement of the cyclooxygenase pathway, platelets were incubated 5 min at 37’C with 100 pM aspirin before the washing procedure. TxA, measurement. Platelet TxAZ formation was measured as its stable metabolite thromboxane B? by radioimmunoassay using kits purchased from New England Nuclear (Du Pont de Nemours, Florence, Italy), as previously described (198). [3H]Serotonin release. Before being washed platelets were incubated 30 min at 22’C with 0.2 HIM Ci X ml-’ [3H]serotonin. The platelets were washed twice and finally resuspended in Hank’s balanced salt solution containing 5 pM imipramine to :inhibit the reuptake. Three minutes after the addition of stimuli 700 ~1 of the sample was immediately transferred to an Eppendorf microfuge vial containing 70 ~1 of the following solution: EDTA, 5 mM; theophylline, 5 mM; aspirin, 500 PM, and PGEi, 0.2 pg X ml-‘. After centrifugation, 2 min at 12,OOOg, 100 ~1 of the supernatant was transferred to a scintillation vial and 2 ml of aqueous scintillation fluid was added. The amount of 3H was determined using a Beckman scintillation counter. The [3H] was always expressed as a percentage of the total amount osf [3H]serotonin that had been accumulated by the platelets. Each experiment was performed in duplicate. SOD and CAT actiuity. Superoxide dismutases (EC 1.15.1.1) were dialyzed by ultrafiltration in a Spectrum apparatus (Spectrum, Haverton, USA). Enzyme activity was measured by a spectrophotometer using the xanthine/xanthine oxidase/cytochrome c system according to McCord and Fridovich (20). CAT (EC 1.11.1.6) was dialyzed to remove thymol and activity was measured by a spectrophotometer, by following the disappearance rate of H202 (21). SOD and CAT were denaturated by boiling the enzymes for 10 min. This procedure could give >95% inactivation. Statistical analysis. Data are reported as means + standard deviation. The comparison between variables was analyzed by Student’s t test for paired data. Significance was accepted at the P < 0.05 level. The following materials were obtained from Sigma Materials. Chemical Co. (Sigma Chimica, M&no, Italy): arachidonic acid, U46619, PAF, ADP, A23187, EDTA, theophiline, PGE,, MnSOD. [HHydroxytriptamine binoxilate (sp act 15-30 Ci X mmol-‘) was purchased
TABLE
I
Effect of SOD (300 U X ml--‘) on Platelet Aggregation Induced by Different Types of Agonists at Threshold Concentrations (the Lowest Dose Which Induces at Least a 50% Increase in Light Transmission [LT%]). LT%’
Thrombin A23187 ADP Collagen Arachidonate ’ Mean + standard deviation b P < 0.005.
-SOD
+SOD
60 57 53 57 54
58 f 4 55 f 3 54 + 4 72 f 3* 71+5*
f 6 ?I 5 f 6 + 4 -t 5
of five separate experiments.
181
DISMUTASE TABLE
II
Effect of SOD on Human Platelet “Primed” with Subthreshold Concentrations of Different Agonists
SOD Thrombin Thrombin + SOD A23187 A23187 + SOD ADP ADP + SOD Collagen Collagen + SOD Collagen + SOD + CAT Collagen + H202 Collagen + dSOD Collagen + SOD + dCAT Collagen + SOD + Aspirin Collagen + hr-SOD Collagen + Mn-SOD Arachidonate Arachidonate + SOD Archidonate + SOD + CAT Arachidonate + HzOz Arachidonate + dSOD Arachidonate + SOD + dCAT Arachidonate + SOD + Aspirin Arachidonate + hr-SOD Arachidonate + Mn-SOD
LT
TxA,
3 4 3 5 5 4 5 3 2 4
[3H]5HT
4+3 55 f 4 5+2 48 + 3
522 61 f 7 4+2 52 + 3
Note. Values of percentage of light transmission (LT), TxAz (ngxmll’), and [sH]5HT release (W), taken 3 min after adding the agonists, represent the mean f standard deviation of five separate experiments. Final concentrations: thrombin, 0.02-0.04 units X ml-‘; A23187, 0.6-0.8 pM; ADP, 0.1-0.2 jiM; collagen, 0.1-0.2 pg X ml-‘; arachidonic acid, lo-20 pM; superoxide-dismutases and CAT, 300 units X ml-‘; H,Oz, 0.5 FM.
from New England Nuclear (Du Pont de Nemours, Florence, Italy). Thrombin, CuZn-SOD, and hr-SOD were obtained from Calbiochem (San Diego, CA). Hydrogen peroxide was from Merck (Darmstadt, Germany). Collagen (bovine tendon) was purchased from Menarini (Florence, Italy). Aspirin as its soluble lysin salt was from Maggioni (Milan, Italy). Arachidonic acid, 15 mM stck solution, was dissolved in absolute ethanol under Nz atmosphere and stored at -20°C. Calcium ionophore A23187 was dissolved in dimethyl sulfoxide. HzOz was titrated with KMnO,. All the other reagents were dissolved in double-distilled water and diluted in physiologic saline to the appropriate concentrations. RESULTS
Table I shows platelet aggregation observed using different types of platelet stimulation in the presence or absence of SOD. No significant changes were observed in samples stimulated with threshold concentrations of ADP, thrombin, and the calcium ionophore A23187; a significant increase in platelet aggregation by SOD was observed when collagen or arachidonic acid was used as platelet stimulus (P < 0.005). Table II reports on platelet changes observed using subthreshold concentrations of several platelet stimuli in
182
IULIANO
the presence or absence of SOD; neither platelet aggregation nor TxAz production was induced by the various platelet agonists at subthreshold concentrations or by SOD alone. Platelet stimulation by ADP, thrombin, or calcium ionophore A23187 was not modified in the presence of SOD. Platelet stimulation by PAF and the stable endoperoxide analogue U46619 was not influenced in the presence of SOD, either at threshold or subthreshold concentrations of these platelet stimuli (data not shown). On the contrary, when subthreshold concentrations of arachidonic acid or collagen were tested on platelets with SOD added, a large increase in aggregation, TxAz production, and [3H]serotonin release was seen (a typical tracing of the aggregatory response is depicted in Fig. 1). The effects of SOD on primed platelets were concentration dependent up to a level of 300 units; the dose-response curves of platelet aggregation to increasing amounts of SOD are depicted in Fig. 2. The amplification of platelet response by SOD was dependent on the catalytic activity of the enzyme as indicated by: (i) dSOD not activating “primed” platelets; (ii) active CAT, but not dCAT, fully preventing platelet activation by SOD; (iii) H202 mimicking the effect of SOD on “primed” platelets. Additional evidence was given by experiments in which other forms of the enzyme were used: Mn-SOD and hr-SOD produced effect on platelets similar to those of CuZn-SOD (Table II). The detection of TxAz in SOD-activated platelets suggested the involvement of the cyclooxygenase pathway of arachidonic acid. This was further corroborated by experiments with aspirin pre-treated platelets: blockade of cyclooxygenase activity prevented both TxAz production and aggregation of platelets stimulated with SOD and
0
60
120 seconds
180
FIG. 1. Representative platelet aggregation tracings showing effect of SOD on platelets stimulated with subthreshold concentrations of arachidonic acid. A, arachidonic acid (15 PM); B, A + SOD (300 U X mll’); C, A + dSOD; D, B + CAT (300 U X ml-‘); E, A + 0.5 pM H,O,.
ET AL.
-0
loo
200 SOD uni ts x ml-’
FIG. 2. Aggregation of primed platelets tration. The aggregation was measured 3 subthreshold concentration of arachidonic lagen (0.16 ag X ml-‘; triangles) along with tions.
sub-threshold concentrations lagen (Table II).
as function of SOD concenmin after the addition of a acid (22 PM; circles) or colincreasing SOD concentra-
of arachidonic
acid or col-
DISCUSSION
This study indicates that the enzyme SOD per se does not influence platelet function but can induce an irreversible aggregation, TxA2 production, and [3H]serotonin release when added to primed platelets. Such an amplification was observed using agonists which act through a cyclooxygenase-dependent mechanism. This is shown by the amplification of platelet response observed when we have used subthreshold concentrations of collagen or arachidonic acid, which activate platelets through the cyclooxygenase pathway. The same was not observed when other agonists such as ADP, thrombin, A23187, PAF, or U46619 were employed. The absence of the amplification response by SOD could be due to different pathways of platelet activation induced by these agonists. In fact, even though the latter agonists may enhance arachidonic acid release and oxygenation, this is not their primary mechanism of platelet activation (22). Taken together, these findings indicate that SOD could be considered as a trigger of platelet aggregation, which acts by amplifying the response to other agonists, only when stimuli acting mainly through the arachidonic acid cascade are employed. This suggestion is supported by the finding of increased amounts of TxAz in samples substimulated with arachidonic acid or collagen in the presence of SOD and by the disappearance of both platelet aggregation and TxA2 formation in platelets pretreated with aspirin.
PLATELET
Furthermore, reaction
ACTIVATION
BY SUPEROXIDE
HzOz generated by SOD according to the
20; + 2H+ + HzOz + O2 was directly involved in the activation of platelets because the SOD-dependent aggregation, TxAZ generation, and [3H]serotonin release were fully prevented by catalase, which eliminates HzOz. Tlhis observation indicates that the amplification of platellet response by SOD is due to the catalytic activity and is mediated by H202. In fact denaturated SOD and denaturated catalase did not produce any effect. Therefore our findings could be explained by assuming that the platelet-produced 0; (11) is converted, by the addition of SOD, to Ha02, which in turn facilitates the activation of arachidonic acid metabolism. This suggestion is demonstrated by adding exogenous H,O, instead of SOD: Hz02, at nanomolar concentrations, which mimicks the effect of active SOD on primed platelets. Furthermore, the role for HzOz is supported by previous findings showing that Hz02 enhances platelet aggregation (23, 24) and modulates the mobilization of arachidonate from platelet membrane (25). The demonstration that SOD stimulates the cyclooxygenase pathway of platelet arachidonic acid metabolism might help to explain some recent findings observed in human and experimental model systems. In experiments of ischemic reperfusion, an elevated formation of oxygen free radicals has been detected (4-7). The metabolism of arachidonic acid has recently been studied in this system, and, in fact, an increased formation of TxAz has been detected during reperfusion (10). This finding may be important because platelet activation and, in particular, the increased TxA, formation, can contribute to vascular reocclusion (8, 9). Our results suggest that this increased TxA, formation observed during reperfusion could be mediated by OFRs. The amplification of platelet response by SOD should also be taken into account in experimental models of ischemia-reperfusion where this enzyme is used to prevent the myocardial damage induced by OFRs (26-29). ACKNOWLEDGMENTS We are grateful to Prof. C. Patron0 and Prof. G. Rotilio for their helpful criticism and suggestions .and to Mrs. C. Piccheri for technical assistance.
REFERENCES 1. Halliwell, B., and Gutteridge, J. M. C. (Eds.) (1989) Free Radicals in Biology and Medicine, 2nd ed., Clarendon Press, Oxford.
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2. Simpson, P. J., and Lucchesi, B. R. (1987) J. Lab. Clin. Med. 110, 13-30. 3. Kloner, R. A., Przyklenk, K., and Whittaker, P. (1989) Circulation 80, 1115-1127. 4. Zweir, J. L. (1988) J. Biol. Chem. 263, 1353-1357. 5. Bolli, R., Patel, B. S., Jeroudi, M. O., Lai, E. K., and McCay, P. B. (1988) J. Clin. Incest. 82, 476485. 6. Bolli, R., Jeroudi, M. O., Patel, B. S., DuBose, C. M., Lai, E. K., Roberts, R., and McCay, P. B. (1989) Proc. Natl. Acad. Sci. 1JSA 86,4695&4699. 7. Kuzuya, T., Hoshida, S., Kim, Y., Nishida, M., Fuji, H., Kitabatake, A., Tada, M., and Kamada, T. (1990) Circ. Res. 66, 1160-1165. 8. Yasuda, T., Gold, H. K., Fallon, J. T., Leinbach, R. C., Guerrero, J. L., Scudder, L. E., Kanke, M., Shealy, D., Ross, M. J., Collen, D., and Caller, B. S. (1988) J. Clin. Znuest. 81, 1284-1291. 9. Yasuda, T., Gold, H. K., Fallon, J. T., Leinbach, R. C., Garabedian, H. D., Guerrero, J. L., and Cohen, D. (1989) J. Am. Coil. Cardiol. 13, 1409-1414. 10. Fitzgerald, D. J., Wright, F., and FitzGerald, G. A. (1989) Circ. Res. 65,83%94. 11. Marcus, A. J., Silk, S. T., Safier, L. B., and Ullman, H. L. (1977) J. Clin. Invest. 59, 149-158. 12. Bressler, N. M., Broekman, M. J., and Marcus, A. J. (1979) Blood 53,167-178. 13. Iatridis, S. G., Iatridis, P. G., Kyrkou, K. A. et al. (1979) Thromb. Res. 15,733%741. 14. Krinsky, N. I., Sladdin, D. G., Levine, P. H., Taub, L. A., and Simic, H. G. (1981) Thromb. Haemostasis 42, 116-120. 15. Clemmons, R. M., Dorsey Lee, M. R., Bliss, E. L., Asbury, A. C., Cook, D., and Brown, V. (1985) Am. J. Vet. Res. 46, 110441106. 16. Violi, F., Ghiselli, A., Iuliano, L., Alessandri, C., Cordova, C., and Balsano, F. (1988) Haemostasis 18, 91-98. 17. Salvemini, D., De Nucci, G., Sneddon, J. M., and Vane, J. R. (1989) Brit. J. Pharmacol. 97, 1145-1150. 18. Born, G. V. R., and Cross, M. J. (1963) J. Physiol. 168, 178-195. 19. Violi, F., Ghiselli, G., Iuliano, L., Pratico, D., Alessandri, A., and Balsano, F. (1988) Eur. J. Clin. Pharmacol. 33, 599-602. 20. McCord, J. M., and Fridovich, I. (1969) J. Biol. Chem. 244, 60496055. 21. Beers, R. F., and Sizer, I. W. (1952) J. Biol. Chem. 195, 133-140. 22. Seiss, W. (1989) Physiol. Rev. 69, 58-174. 23. Canoso, R. T., Rodvien, R., Scoon, K., and Levine, P. H. (1974) Blood 43, 645-656. 24. Del Principe, D., Menichelli, A., De Matteis, W., Di Corpo, M. L., Di Giulio, S., and Finazzi-Agro, A. (1985) FEBS Lett. 185, 142146. 25. Hornberger, W., and Patscheke, H. (1990) Eur. J. Biochem. 187, 1755181. 26. Engler, R., and Gilpin, E. (1989) Circulation 79, 1137-1142. 27. Patel, B. S., Jeroudi, M. O., O’Neill, P. G., Roberts, R., and Bolli, R. (1990) Am. J. Physiol. 258, H369-H380. 28. Omar, B. A., Gad, N. M., Jordan, M. C., Striplin, S. P., Russell, W. J., Downey, J. M., and McCord, J. M. (1990) Free Rad. Biol. Med. 9,465-471. 29. Omar, B. A., and McCord, J. M. (1990) Free Rad. Biol. Med. 9,473478.