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


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



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




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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 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 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



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.



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.




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



Superoxide dismutase triggers activation of "primed" platelets.

Superoxide dismutase (SOD) triggers activation of human platelets exposed to subthreshold concentrations of arachidonic acid and collagen. The subthre...
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