Superoxide production and reducing activity in human platelets. A J Marcus, … , L B Safier, H L Ullman J Clin Invest. 1977;59(1):149-158. https://doi.org/10.1172/JCI108613. Research Article Human platelets contain the cuprozinc (cytoplasmic) and manganese (mitochondrial) forms of superoxide dismutase. Nevertheless, superoxide radicals were detectable in the surrounding medium of metabolically viable platelet suspensions by using two assay systems: cytochrome c and nitroblue tetrazolium. The quantity of superoxide generated by platelets (5 X 10(5) superoxide radicals/platelet per 10 min) was constant and did not increase after aggregation by agents such as collagen and thrombin. The superoxide-generating system was present in the supernate of both aggregated and resting platelets and therefore was not platelet-bound. Platelet superoxide production was unaffected by prior ingestion of aspirin, indicating that the prostaglandin and thromboxane pathways were not involved. Both resting and aggregated platelets exhibited a reductive capacity toward cytochrome c and nitroblue tetrazolium which was unrelated to superoxide production. Furthermore, the aggregation process always resulted in a marked increase in this reduction. The nonsuperoxide reduction associated with aggregation was found to be membrane bound and to decrease with an apparent first order reaction rate (k1 = 0.067 min-1). In addition, accumulative, time-dependent nonsuperoxide-related cytochrome c reduction was also detected. Since there is no superoxide dismutase in plasma, the presence of superoxide radicals in the surrounding medium of platelets may have in vitro significance for platelet and leukocyte concentration and storage and in vivo significance for hemostasis, coagulation, and thrombosis. The nonsuperoxide-related reducing […] Find the latest version: https://jci.me/108613/pdf Superoxide Production and Reducing Activity in Human Platelets AARON J. MARCUS, SUSAN T. SILK, LENORE B. SAFIER, and HARRIS L. ULLMAN From the Hematology Section, New York Veterans Administration Hospital, New York, New York 10010, and the Department of Medicine, Division of Hematology-Oncology, New York Hospital- Cornell Medical Center, New York, New York 10021 A B S T R A C T Human platelets contain the cupro- INTRODUCTION zinc (cytoplasmic) and manganese (mitochondrial) forms of superoxide dismutase. Nevertheless, super- Aerobic cells contain the enzyme superoxide dis- oxide radicals were detectable in the surrounding mutase (SOD),1 which serves as protection from de- medium of metabolically viable platelet suspensions structive effects of superoxide radicals (1, 2). These by using two assay systems: cytochrome c and nitro- free radicals form as toxic intermediates in oxidative blue tetrazolium. The quantity of superoxide gener- metabolic processes because the spin state of oxygen ated by platelets (5 x 105 superoxide radicals/platelet favors univalent pathways of reduction (1-3). SOD per 10 min) was constant and did not increase after catalyzes the reaction: O + O2+ 2H->+ 02 + H202. aggregation by agents such as collagen and thrombin. Huiman polymorphontuclear leuikocytes uitilize suiper- The superoxide-generating system was present in the oxide radicals for bacterial killing (4-6), while in the supernate of both aggregated and resting platelets disorder chronic granuloinatous disease, generation of and therefore was not platelet-bound. Platelet super- superoxide radicals is defective (6, 7). Leukocytes also oxide production was unaffected by prior ingestion of generate °2 in response to cell surface perturbation aspirin, indicating that the prostaglandin and throm- in the absence of phagocytosis (8, 9). Superoxide boxane pathways were not involved. Both resting and production in lung tissue has been implicated in the aggregated platelets exhibited a reductive capacity pathogenesis of poisoning by the herbicide, paraquat toward cytochrome c and nitroblue tetrazolium which (10), and in the pulmonary fibrosis resulting from was unrelated to superoxide production. Further- long-term ingestion of nitrofurantoin (11). Thus, clin- more, the aggregation process always resulted in a ical consequences of exposure of cells and tissues to marked increase in this reduction. The nonsuper- superoxide radicals are becoming apparent. oxide reduction associated with aggregation was found In the course of an unrelated study involving a lipid- to be membrane bound and to decrease with an ap- protein fraction from human platelets (12) SOD was parent first order reaction rate (k1 = 0.067 min-'). identified and an investigation of the role of this en- In addition, accumulative, time-dependent nonsuper- zyme in platelet function was undertaken. Since oxide-related cytochrome c reduction was also de- superoxide radicals are capable of damaging cell tected. Since there is no superoxide dismutase in membranes and macromolecules (1-3), and since there plasma, the presence of superoxide radicals in the sur- is essentially no SOD in plasma (13), the presence rounding medium of platelets may have in vitro of superoxide radicals in the external milieu of significance for platelet and leukocyte concentration stimulated or unstimulated platelets may have sig- and storage and in vivo significance for hemostasis, nificant implications for hemostasis, coagulation, and coagulation, and thrombosis. The nonsuperoxide- occlusive vascular disease. Furthermore, the genera- related reducing activities may represent a biochemical tion of °2 could be an important determinant of basis for platelet-blood vessel interactions, with platelet viability during processing, concentration, particular reference to blood vessel integrity. and storage for clinical transfusion purposes. Received for publication 19 July 1976 and in revised form 'Abbreviations used in this paper: NBT, nitroblue 20 September 1976. tetrazolium; SOD, superoxide dismutase. TheJournal of Clinical Investigation Volume 59 January 1977-149-158 149 ethylene dropper) in 5 ml of washing fluid (370C) and the suspensions were pooled into two tubes, each diluted to a total volume of 35 ml. Two washes were carried out with centrifugation at 370C for 15 min (1,100 g). Before each centrifugation the platelets were allowed to incubate for 15 min (37°C). The platelets were finally pooled and counts adjusted to an average of 2.8 x 109/ml (4.78 mg protein/ml) utilizing the microhematocrit technique of Tollefsen and as- sociates (16). Contaminating erythrocytes and leukocytes were removed by 'trapping' in the cone of the centrifuge tubes. Examination of stained smears prepared from final platelet suspensions revealed that total leukocyte contamina- tion was less than 0.1%, of which 99.8% were lymphocytes. All experiments reported were carried out with samples re- moved from the 'stock' suspension, which was maintained at 37°C. The processing time from blood collection to final suspension was 3-4 h. Radioactive serotonin uptake was used as an indicator of metabolic viability of the platelets (17). In addition, oxygen consumption, fluctuations in pH, UNAGGREGATED and alterations in aggregation responses to collagen were (NO Collogen) Cytocirome (NO Collagen)CytochromeC or monitored. C or N BT, Platelets, SOD NBT, Platelets, NO SOD Assays for SOD. The method of McCord and Fridovich (18) was employed for quantification of platelet SOD. Washed, FIGURE 1 Diagram ofprocedure utilized for platelet aggrega- frozen, and thawed platelets, sonified in a cup-horn instru- tion and simultaneous assay of cytochrome c or NBT reduc- ment (model L Sonifier Converter, Heat Systems-Ultra- tion in the presence or absence of SOD. Platelets were sonics, Inc., Plainview, N. Y.) (four 15-s intervals, output transferred from stock suspension to four assay tubes (con- setting at '8,' 15°C), as well as supernatant fractions from taining appropriate reagents) in a sequential manner at 15-s ultracentrifuged platelet homogenates, (19) were used for as- intervals. Successive sets of experiments were spaced say. To inhibit cytochrome c oxidase (19), a potential source of approximately 30 min apart. In the cytochrome c assay it was error (20), KCN (10 ,um) was included in each assay. necessary to note the exact time of platelet addition since Manganese SOD was measured by inhibiting the copper-zinc values increased with the age of platelets in the stock suspen- form with 1 mM cyanide (21, 22). Triton X-100 (0.033%, sion. This was due to cytochrome c reduction unrelated to final concentration) was included in the assays for solubiliza- superoxide generation. Thus the cytochrome c data were tion of any SOD that may have been particle-bound (23).2 plotted on a time scale with the venipuncture taken as zero Measurements were made in a Cary 16 spectrophotometer time (Figs. 4-7). with full scale expanded to 0.2 absorbance units. A calibra- tion curve was constructed with beef erythrocyte SOD. (Truett Laboratories, Dallas, Tex. Preparations used in aggregation This report describes the qualitative and quantita- studies were dialyzed and lyophilized before use). To avoid tive distribution of SOD in human platelets. The ap- reduction or oxidation of cytochrome c by endogenous plate- pearance of O2 in the surrounding medium of washed let substances and at the same time remain in the linear platelets incubated alone and in the presence of ag- portion of the standard curve, it was necessary to limit the range of platelet protein assayed to 45-200 ,g. Sample gregating agents will be documented. Finally, data on blanks