Hoppe-Seyler's Z. Physiol. Chem. Bd. 359, S. 933-937, August 1978
Superoxide-Independent Platelet Response to Xanthine Oxidase Heinrich PATSCHEKE, Wulf PASCHEN and Peter WÖRNER Institut für Biochemie II, Universität Heidelberg (Med. Fak.)
(Received 21 March 1978)
Summary: Xanthine oxidase (1—5 Mg/m/) from cow's milk induces shape change, aggregation, and the release reaction of human washed platelets. Xanthine oxidase plus xanthine produce superoxide radicals, which reduce nitro blue tetrazolium. Superoxide dismutase, allopurinol, or ommission of xanthine inhibits the reduction of nitro blue tetrazolium but has no influence on the platelet response to xanthine oxidase. In con-
trast, small amounts of plasma or apyrase from potatoes abolish the effect on platelets, but not the enzyme activity of xanthine oxidase. Comparison of two xanthine oxidase preparations shows that higher specific enzyme activity corresponds to a lesser effect on platelets. The results suggest that platelet and enzyme activities reside in different components of xanthine oxidase preparations.
Superoxid-unabhängige Reaktion von Thrombozyten auf Xanthin-Oxidase Zusammenfassung: Xanthin-Oxidase (1-5 Mg/m/) aus Kuhmilch induziert Formänderung, Aggregation und Freisetzungsreaktion von gewaschenen Thrombozyten vom Menschen. Xanthin-Oxidase plus Xanthin produzieren Superoxidradikale, die Nitroblautetrazolium reduzieren. SuperoxidDismutase, Allopurinol oder Weglassen von Xanthin hemmen die Reduktion von Nitroblautetrazolium, haben aber keinen Einfluß auf die Thrombozytenreaktion auf Xanthin-Oxidase. Im Gegensatz dazu hemmen geringe Mengen von
Plasma oder Kartoffel-Apyrase den Effekt auf die Thrombozyten, aber nicht die Enzymaktivität von Xanthin-Oxidase. Ein Vergleich von zwei Präparationen von Xanthin-Oxidase zeigt, daß eine höhere spezifische Enzymaktivität mit einem schwächeren Effekt auf die Thrombozyten korrespondiert. Die Ergebnisse deuten darauf hin, daß verschiedene Komponenten in XanthinOxidase-Präprarationen für Thrombozytenstimulierung und Enzymaktivität verantwortlich sind.
Key words: Xanthine oxidase, Superoxide radicals, blood platelets Enzymes: Apyrase, ATP diphosphohydrolase (EC 3.6.1.5); Superoxide dismutase, stiperoxideisuperoxide oxidoreductase (EC 1.15.1.1); Thrombin (EC 3.4.21.5); Xanthine oxidase, aldehyde:oxygen oxidoreductase (EC 1.2.3.1). 0018-4888/78/0359-0933 $02.00 © by Walter de Gruyter & CoLibraries · Berlin · New York Brought to you by | Purdue University Authenticated Download Date | 5/31/15 5:13 AM
934
H. Patscheke, W. Paschen and P. W rner
Xanthine oxidase has recently been shown to induce aggregation and release of washed platelets'1 1. The platelet-stimulating effect of xanthine oxidase has been ascribed to Superoxide anions which are generated during aerobic conversion of xanthine to uric acid'1'. We report here that xanthine oxidase from cow's milk stimulates platelets without its enzymic action being involved. Methods and Materials Suspensions of discoid and spheroid washed platelets were prepared as has been described^ 2 ). Spheroid platelets resulted from washing with EDTA at 22 °C. The discoid platelet shape was preserved during a washing procedure with acid citrate dextrose, pH 6.5 at 22 °C. Nanomolar concentrations of prostaglandin EI and the presence of apyrase, Ca2®, and Mg2® helped preserve the shape' 21. Platelet-poor plasma was obtained from citrated platelet-rich plasma (0.31% sodium citrate) by centrifugation (Eppendorf 3 200) for 30 s at 8 000 χ g and 4 °C. Platelet serotonin was labelled with [ 3 HJserotonin (Amersham-Buchler TRA 223), and the [3H]serotonin release was measured as has previously been describedl2'3!. Shape change and aggregation were followed as the changes in light transmission in the aggregometer'2'3'. Platelet concentration was 2 χ 108 m/"1. Shape change of discoid platelets was recorded with continuous stirring of the platelet suspension, which was required to demonstrate the disc-sphere transformation = shape change Π 2 '. With spheroid platelets, the stirring mechanism was switched on for less than l s on addition of xanthine oxidasel 2 » 3 '. The shape change curves then showed shape change II and the extrusion phase of the release reaction^ 2 1.
Bd. 359(1978)
indicated tinder Results. A xanthine oxidase preparation (0.5 U/mg) from Sigma had been used by the authors referred to in the introduction. The xanthine oxidase preparations were supplied as suspensions in 3.2M (Bochringcr) or 2.3M (Sigma) ammonium sulfate. After dialysis at 4 °C against Tris/HCl-buffered saline, pH 7.4, containing 0.02mM sodium EDTA, the xanthine oxidase from Boehringer had lost some of its platelet activity. This reduction exceeded the inhibition by ammonium sulfate of the platelet response when non-dialyzed xanthine oxidase was used. Therefore, dialysis was omitted in experiments with xanthine oxidase from Boehringer. Ammonium sulfate applied together with 10 Mg xanthine oxidase/m/ (highest concentration used) showed weak inhibition of platelet activation by 0.09 U thrombin/m/. Since xanthine oxidase from Sigma required much higher concentrations to activate platelets, this preparation had to be dialyzed prior to its application to platelets. Apyrase from potatoes (Grade I), Superoxide dismutase (3 000 U/mg) and phenazine methosulfate were from Sigma. Allopurinol was from Boehringer Mannheim GmbH, cytochalasin B was from Aldrich-Europe, Beerse, Belgium, and nitro blue tetrazolium chloride was from Biomol, E. Barth, D-6804 Ilvesheim. Prostaglandin El was kindly supplied by Dr. Pike, Upjohn Co., Kalamazoo, Michigan, U.S.A. Potassium Superoxide was a gift of Prof. Dr. U. Weser, Biochemical Institute, University T bingen.
Results.
The enzymic activity of xanthine oxidase was determined as the reduction of nitro blue tctrazolium chloride to formazane. These tests were performed in the platelet suspension medium composed of 5mM glucose, 500 Mg albumin/m/, 5mM KC1, ImM CaCl2, or ImM sodium EDTA, 115mM NaCl, 31mM Tris/HCl buffer, pH 7.4. The test samples contained an additional 0.15mM xanthine and 0.15mM nitro blue tetrazolium and 3 g gelatin//. The blue colour which developed on addition of xanthine oxidase was followed in an Eppendorf Photometer at 546nm. Materials. Xanthine oxidase from cow's milk (0.4 U/mg) was from Boehringer Mannheim GmbH. Xanthine oxidase (Grade I, 0.64 U/mg, from buttermilk) obtained from Sigma was used for comparative experiments where
Washed platelets treated with 2 μg xanthine oxidase/m/strongly aggregate and release [3H]serotonin. The effectiveness of xanthine oxidase is destroyed by boiling it for 5 min prior to its addition to platelets. The effects of the enzyme are independent of addition of xanthine or hypoxanthine. They are inhibited neither by 0.2niM allopurinol nor Superoxide dismutase, up to a concentration of 200 jug/m/, nor by 125 μg nitro blue tetrazolium/m/. Xanthine oxidase leads to a reduction of nitro blue tetrazolium only if xanthine or hypoxanthine has been added. Hypoxanthine is the end product of the platelet adenine nucleotide metabolism and may permeate the plasma membrane of platelets^ H The amount of hypoxanthine (or xanthine) from platelets is, however, too little to serve as sufficient substrate for reduction by xanthine oxidase of nitro blue tetrazolium. This was established in experiments in which a platelet suspension from which platelets had been separated
Brought to you by | Purdue University Libraries Authenticated Download Date | 5/31/15 5:13 AM
Bd. 359(1978)
Superoxide-Independent Platelet Response to Xanthine Oxidase
by centrifugation was used for the nitro blue tetrazolium test without xanthine addition. The reduction of nitro blue tetrazolium with xanthine oxidase and xanthine is suppressed by 20μΜ allopurinol or 20 ^g Superoxide dismutase/m/ (Table). These experiments were performed in the same medium in which platelets were suspended in aggregation or shape-change experiments.
935
Apyrase, which is required in the suspension medium for washed discoid plateltsl 2 ' 5 !, completely inhibits the platelet response to 2 Mg xanthine oxidase/m/ at an apyrase concentration of only 10^g/m/. Higher concentrations of xanthine oxidase are, therefore, necessary to stimulate washed discoid platelets suspended with apyrase (Figure).
10 pg apyrase/m/ does not, however, impair the enzymic action of xanthine oxidase in the nitro blue tetrazolium test (Table). 0.2% (υ/υ) plateletTable. Reduction of nitro blue tetrazolium by 5 μ§ xanpoor plasma abolishes the effects of 5 g xanthine thine oxidase/m/ plus xanthine. oxidase/m/ on platelets but strongly enhances Change in absorbance per min (Δ A^/min) is calculated nitro blue tetrazolium reduction by the enzyme from linear change in absorbance between 2 and 7 min (Table). The increase in nitro blue tetrazolium after addition of xanthine oxidase. Experiments were reduction was completely suppressed by 20 μg performed in duplicate, which gave identical values. Slight Superoxide dismutase/m/(Table). changes in stimulation by plasma from three blood donors were observed. Platelet stimulation by xanthine oxidase is accompanied by shape change I = disc-sphere transAdditive Amount Δ A 546/min formation, shape change II and serotonin extrusionl2! for which extracellular Ca 2 ® is not reControl 0.019 quired (Figure), in contrast to aggregation by Superoxide 20 ^g/m/ 0.003 xanthine oxidase. Aggregation in the presence of dismutase Ca 2 ®, however, enhances the release. The platelet 20μΜ Allopurinol 0.000 response to xanthine oxidase is inhibited by Apyrase 20 Mg/m/ 0.018 prostaglandin Ej (Figure). Serotonin release and Platelet-poor 0.2% (υ/υ) 0.031 the concomitant change in light transmission!31 plasma are enhanced by 2μΜ cytochalasin Β (Figure). Platelet-poor, 0.2% (υ/υ) plasma plus Comparative experiments revealed weak platelet 0.003 Superoxide activity of a xanthine oxidase preparation ob20 Mg/m/ dismutase tained from Sigma. Shape change and serotonin
A)
stirrer: on—|off|on-
20
0.9
22
B) off
•|off
on *of f
20
22
t
XO 10pg/ml
I
PGE1 1pM
XO 10jjg/ml
30.7
t [min \
Figure. Shape change and serotonin release of discoid (A) and spheroid (B) washed platelets stimulated by xanthine oxidase (XO). Numbers at ends of curves show percentage of serotonin release measured 5 min after addition of xanthine oxidase. A) Xanthine-oxidase-induced discsphere transformation (decrease in light transmission, stirred). Inhibition by prostaglandin E j (PGEj). B) Xanthine-oxidase-induced shape change II (decrease in light transmission, non-stirred) and subsequent extrusion (increase in light transmission). Stimulation by cytochalasin B (CB) of extrusion and of concomitant serotonin release.
Brought to you by | Purdue University Libraries Authenticated Download Date | 5/31/15 5:13 AM
936
H. Patscheke, W. Paschen and P. Wörner
release remained weak and required high xanthine oxidase concentrations above 80 Mg/m/. This effect was not enhanced by 0.15mM xanthine, and was not inhibited by 100 Mg Superoxide dismutase/m/. The specific enzyme activity measured with the nitro blue tetrazolium test was, according to the producer specifications, 1.6-fold higher with xanthine oxidase from Sigma than with the enzyme from Boehringer. Two batches of the preparation from Boehringer (Control No. 1027154 and Control No. 1227356) gave about the same results, which are described above.
Bd. 359 (1978)
platelet function. Since the enzymic action of xanthine oxidase continues in the presence of apyrase, or even increases with plasma, the platelet and the enzyme activities of xanthine oxidase may reside in different components of xanthine oxidase.
A typical platelet activation by xanthine oxidase is demonstrated by the platelet shape changes, the inhibition by prostaglandin E{ and the stimulation of the extrusion by cytochalasin B. Prostagiandin synthesis by platelets is obviously not involved. Our results!91 with indomethacin and the measurements of malonic dialdehyde production confirmed earlier observations of other authors^ 1 1. Discussion The protein concentration of xanthine oxidase The enzymic oxidation of xanthine or hypoxansufficient to elicit maximal platelet activation, thine with xanthine oxidase is associated with 5 Mg/m/ is low compared to that of other highthe formation of Superoxide radicals^ 6 1. Reducmolecular-weight platelet stimuli, e.g. certain tion of nitro blue tetrazolium is proof of the pro- lectins'3!. Highly purified xanthine oxidase is obduction of Superoxide radicals, if the reaction can tained from cow's milk by comparatively simple be abolished by Superoxide dismutasel7L Xanmethods'] °~ 12 1 Preparations of xanthine oxidase, thine-oxidase-induced nitro blue tetrazolium rehowever, contain an impurity which consists of duction was suppressed by addition of Superoxide polymerized apo-xanthine-oxidase protein with dismutase, allopurinol, or by the absence of the low or no enzyme activity' 14 L The dissociation substrates, xanthine or hypoxanthine; but the of platelet and enzyme activities shown here, pareffect of xanthine oxidase on platelets was not ticularly the experiments with allopurinol, suginhibited under any of these conditions. This sug- gests the independence of platelet activity from gests independence of the platelet response from the enzymic site of xanthine oxidase. The enan enzymic action of xanthine oxidase or an atzymically inactive xanthine oxidase polymers tack of Superoxide radicals on platelets. Super(molecular weight 2 500000 or morel 131) may, oxide-generating agents, e.g. potassium supertherefore, be considered as the platelet-activating oxide = K02 or phenazine methosulfate plus components of xanthine oxidase. Our finding NADH, also could not simulate the platelet rethat a high specific enzyme activity (0.64 U/mg, sponse to xanthine oxidase (to be published). The Sigma) corresponds to low platelet activity, and inhibitory effect of Superoxide dismutase shown that a lower specific enzyme activity is found in by other authors^ 11 with the high Superoxide dis- the highly platelet-reactive preparation (0.4 U/mg, mutase concentration of 200 Mg/m/ was probably Boehringer Mannheim GmbH) confirm the sugnot due to an enzymic effect of Superoxide disgestion that the two activities reside in different mutase. Only 20 g Superoxide dismutase/m/ was components of xanthine oxidase preparations. enough to abolish the nitro blue tetrazolium reThe assistance of Miss Rita Klein is,gratefully acknowduction by xanthine oxidase, while even 200 g dismutase/m/ did not impair the platelet response ledged. The authors thank Prof. R. Brossmer for his support of this study. This work was supported by the in our experiments. Deutsche Forschungsgemeinschaft within SFB 90 "CarApyrase or minute amounts of human plasma in- diovasculäres System". hibited the effects of xanthine oxidase on platelets, but did not impair its enzyme activity. Apyrase Literature and plasma factors maintain platelet reactivity to 1 Handin, R. I., Karabin, R. & Boxer, G. J. (1977) ADP and the aggregation response to certain in/. Clin. Invest. 59, 959-965. 5 8 duced » '. Therefore, the inhibition is due to in- 2 Patscheke, H. & Wörner, P. (1978) Thromb. Res. 12, terference with xanthine oxidase rather than with 485-496.
Brought to you by | Purdue University Libraries Authenticated Download Date | 5/31/15 5:13 AM
Bd. 359 (1978)
Superoxide-Independent Platelet Response to Xanthine Oxidase
Patscheke, H. & Wörner, P. (1977) Thromb. Res. 11, 391-402. Holmsen, H. & Day, H. J. (1971) Ser. Haeniatol. 4,28-58. Mustard, J. F., Perry, D. W., Ardlie, N. G. & Packham, M.A. (\9Tl)Br.J.HaematoL 22, 193-202. Knowles, P. F., Gibson, J. F., Pick, F. M. & Bray,R. C. (1969) Biochem. J. 111,53-58. Fridovich, \.(\912) Acc.Chem.Res. 5, 321-326. Kinlough-Rathbone, R. L., Mustard, J. F., Packham, . ., Perry, D.W., Reimers, H.-J. & Cazenave, J.-P. (1977) Thromb.Haemostas. 37,291-308.
937
9 Patscheke, H. & Wörner, P. (1977) Hoppe-Seyler's Z. Physiol.Chem. 358,1256. 10 Nelson, C. A. & Handler, P. (1968) /. Biol Chem. 243, 5368-5373. 11 Massey, V., Brumby, P. E., Komai, H. & Palmer, G. (1969) J. Biol Chem. 244, 1682-1691. 12 Hart, L. I., McGartoU, . ., Chapman, H. R. & Bray, R.C. (1910) Biochem. J. 116,851-864. 13 Bray, R. C., Chisholm, A. J., Hart, L. I., Meriwether, L. S. & Watts, D. C. (1966) Flavins and Flavoproteins (Slater, E.G., ed.) pp. 117-129, Elsevier Publ. Comp., Amsterdam.
Dr. H. Patscheke, Dr. W. Paschen und Dr. P. Wörner, Institut für Biochemie II der Universität Heidelberg, Im Neuenheimer Feld 328, D-6900 Heidelberg 1.
Brought to you by | Purdue University Libraries Authenticated Download Date | 5/31/15 5:13 AM
Brought to you by | Purdue University Libraries Authenticated Download Date | 5/31/15 5:13 AM
Superoxide-Independent Platelet Response to Xanthine Oxidase Heinrich PATSCHEKE, Wulf PASCHEN and Peter WÖRNER Institut für Biochemie II, Universität Heidelberg (Med. Fak.)
(Received 21 March 1978)
Summary: Xanthine oxidase (1—5 Mg/m/) from cow's milk induces shape change, aggregation, and the release reaction of human washed platelets. Xanthine oxidase plus xanthine produce superoxide radicals, which reduce nitro blue tetrazolium. Superoxide dismutase, allopurinol, or ommission of xanthine inhibits the reduction of nitro blue tetrazolium but has no influence on the platelet response to xanthine oxidase. In con-
trast, small amounts of plasma or apyrase from potatoes abolish the effect on platelets, but not the enzyme activity of xanthine oxidase. Comparison of two xanthine oxidase preparations shows that higher specific enzyme activity corresponds to a lesser effect on platelets. The results suggest that platelet and enzyme activities reside in different components of xanthine oxidase preparations.
Superoxid-unabhängige Reaktion von Thrombozyten auf Xanthin-Oxidase Zusammenfassung: Xanthin-Oxidase (1-5 Mg/m/) aus Kuhmilch induziert Formänderung, Aggregation und Freisetzungsreaktion von gewaschenen Thrombozyten vom Menschen. Xanthin-Oxidase plus Xanthin produzieren Superoxidradikale, die Nitroblautetrazolium reduzieren. SuperoxidDismutase, Allopurinol oder Weglassen von Xanthin hemmen die Reduktion von Nitroblautetrazolium, haben aber keinen Einfluß auf die Thrombozytenreaktion auf Xanthin-Oxidase. Im Gegensatz dazu hemmen geringe Mengen von
Plasma oder Kartoffel-Apyrase den Effekt auf die Thrombozyten, aber nicht die Enzymaktivität von Xanthin-Oxidase. Ein Vergleich von zwei Präparationen von Xanthin-Oxidase zeigt, daß eine höhere spezifische Enzymaktivität mit einem schwächeren Effekt auf die Thrombozyten korrespondiert. Die Ergebnisse deuten darauf hin, daß verschiedene Komponenten in XanthinOxidase-Präprarationen für Thrombozytenstimulierung und Enzymaktivität verantwortlich sind.
Key words: Xanthine oxidase, Superoxide radicals, blood platelets Enzymes: Apyrase, ATP diphosphohydrolase (EC 3.6.1.5); Superoxide dismutase, stiperoxideisuperoxide oxidoreductase (EC 1.15.1.1); Thrombin (EC 3.4.21.5); Xanthine oxidase, aldehyde:oxygen oxidoreductase (EC 1.2.3.1). 0018-4888/78/0359-0933 $02.00 © by Walter de Gruyter & CoLibraries · Berlin · New York Brought to you by | Purdue University Authenticated Download Date | 5/31/15 5:13 AM
934
H. Patscheke, W. Paschen and P. W rner
Xanthine oxidase has recently been shown to induce aggregation and release of washed platelets'1 1. The platelet-stimulating effect of xanthine oxidase has been ascribed to Superoxide anions which are generated during aerobic conversion of xanthine to uric acid'1'. We report here that xanthine oxidase from cow's milk stimulates platelets without its enzymic action being involved. Methods and Materials Suspensions of discoid and spheroid washed platelets were prepared as has been described^ 2 ). Spheroid platelets resulted from washing with EDTA at 22 °C. The discoid platelet shape was preserved during a washing procedure with acid citrate dextrose, pH 6.5 at 22 °C. Nanomolar concentrations of prostaglandin EI and the presence of apyrase, Ca2®, and Mg2® helped preserve the shape' 21. Platelet-poor plasma was obtained from citrated platelet-rich plasma (0.31% sodium citrate) by centrifugation (Eppendorf 3 200) for 30 s at 8 000 χ g and 4 °C. Platelet serotonin was labelled with [ 3 HJserotonin (Amersham-Buchler TRA 223), and the [3H]serotonin release was measured as has previously been describedl2'3!. Shape change and aggregation were followed as the changes in light transmission in the aggregometer'2'3'. Platelet concentration was 2 χ 108 m/"1. Shape change of discoid platelets was recorded with continuous stirring of the platelet suspension, which was required to demonstrate the disc-sphere transformation = shape change Π 2 '. With spheroid platelets, the stirring mechanism was switched on for less than l s on addition of xanthine oxidasel 2 » 3 '. The shape change curves then showed shape change II and the extrusion phase of the release reaction^ 2 1.
Bd. 359(1978)
indicated tinder Results. A xanthine oxidase preparation (0.5 U/mg) from Sigma had been used by the authors referred to in the introduction. The xanthine oxidase preparations were supplied as suspensions in 3.2M (Bochringcr) or 2.3M (Sigma) ammonium sulfate. After dialysis at 4 °C against Tris/HCl-buffered saline, pH 7.4, containing 0.02mM sodium EDTA, the xanthine oxidase from Boehringer had lost some of its platelet activity. This reduction exceeded the inhibition by ammonium sulfate of the platelet response when non-dialyzed xanthine oxidase was used. Therefore, dialysis was omitted in experiments with xanthine oxidase from Boehringer. Ammonium sulfate applied together with 10 Mg xanthine oxidase/m/ (highest concentration used) showed weak inhibition of platelet activation by 0.09 U thrombin/m/. Since xanthine oxidase from Sigma required much higher concentrations to activate platelets, this preparation had to be dialyzed prior to its application to platelets. Apyrase from potatoes (Grade I), Superoxide dismutase (3 000 U/mg) and phenazine methosulfate were from Sigma. Allopurinol was from Boehringer Mannheim GmbH, cytochalasin B was from Aldrich-Europe, Beerse, Belgium, and nitro blue tetrazolium chloride was from Biomol, E. Barth, D-6804 Ilvesheim. Prostaglandin El was kindly supplied by Dr. Pike, Upjohn Co., Kalamazoo, Michigan, U.S.A. Potassium Superoxide was a gift of Prof. Dr. U. Weser, Biochemical Institute, University T bingen.
Results.
The enzymic activity of xanthine oxidase was determined as the reduction of nitro blue tctrazolium chloride to formazane. These tests were performed in the platelet suspension medium composed of 5mM glucose, 500 Mg albumin/m/, 5mM KC1, ImM CaCl2, or ImM sodium EDTA, 115mM NaCl, 31mM Tris/HCl buffer, pH 7.4. The test samples contained an additional 0.15mM xanthine and 0.15mM nitro blue tetrazolium and 3 g gelatin//. The blue colour which developed on addition of xanthine oxidase was followed in an Eppendorf Photometer at 546nm. Materials. Xanthine oxidase from cow's milk (0.4 U/mg) was from Boehringer Mannheim GmbH. Xanthine oxidase (Grade I, 0.64 U/mg, from buttermilk) obtained from Sigma was used for comparative experiments where
Washed platelets treated with 2 μg xanthine oxidase/m/strongly aggregate and release [3H]serotonin. The effectiveness of xanthine oxidase is destroyed by boiling it for 5 min prior to its addition to platelets. The effects of the enzyme are independent of addition of xanthine or hypoxanthine. They are inhibited neither by 0.2niM allopurinol nor Superoxide dismutase, up to a concentration of 200 jug/m/, nor by 125 μg nitro blue tetrazolium/m/. Xanthine oxidase leads to a reduction of nitro blue tetrazolium only if xanthine or hypoxanthine has been added. Hypoxanthine is the end product of the platelet adenine nucleotide metabolism and may permeate the plasma membrane of platelets^ H The amount of hypoxanthine (or xanthine) from platelets is, however, too little to serve as sufficient substrate for reduction by xanthine oxidase of nitro blue tetrazolium. This was established in experiments in which a platelet suspension from which platelets had been separated
Brought to you by | Purdue University Libraries Authenticated Download Date | 5/31/15 5:13 AM
Bd. 359(1978)
Superoxide-Independent Platelet Response to Xanthine Oxidase
by centrifugation was used for the nitro blue tetrazolium test without xanthine addition. The reduction of nitro blue tetrazolium with xanthine oxidase and xanthine is suppressed by 20μΜ allopurinol or 20 ^g Superoxide dismutase/m/ (Table). These experiments were performed in the same medium in which platelets were suspended in aggregation or shape-change experiments.
935
Apyrase, which is required in the suspension medium for washed discoid plateltsl 2 ' 5 !, completely inhibits the platelet response to 2 Mg xanthine oxidase/m/ at an apyrase concentration of only 10^g/m/. Higher concentrations of xanthine oxidase are, therefore, necessary to stimulate washed discoid platelets suspended with apyrase (Figure).
10 pg apyrase/m/ does not, however, impair the enzymic action of xanthine oxidase in the nitro blue tetrazolium test (Table). 0.2% (υ/υ) plateletTable. Reduction of nitro blue tetrazolium by 5 μ§ xanpoor plasma abolishes the effects of 5 g xanthine thine oxidase/m/ plus xanthine. oxidase/m/ on platelets but strongly enhances Change in absorbance per min (Δ A^/min) is calculated nitro blue tetrazolium reduction by the enzyme from linear change in absorbance between 2 and 7 min (Table). The increase in nitro blue tetrazolium after addition of xanthine oxidase. Experiments were reduction was completely suppressed by 20 μg performed in duplicate, which gave identical values. Slight Superoxide dismutase/m/(Table). changes in stimulation by plasma from three blood donors were observed. Platelet stimulation by xanthine oxidase is accompanied by shape change I = disc-sphere transAdditive Amount Δ A 546/min formation, shape change II and serotonin extrusionl2! for which extracellular Ca 2 ® is not reControl 0.019 quired (Figure), in contrast to aggregation by Superoxide 20 ^g/m/ 0.003 xanthine oxidase. Aggregation in the presence of dismutase Ca 2 ®, however, enhances the release. The platelet 20μΜ Allopurinol 0.000 response to xanthine oxidase is inhibited by Apyrase 20 Mg/m/ 0.018 prostaglandin Ej (Figure). Serotonin release and Platelet-poor 0.2% (υ/υ) 0.031 the concomitant change in light transmission!31 plasma are enhanced by 2μΜ cytochalasin Β (Figure). Platelet-poor, 0.2% (υ/υ) plasma plus Comparative experiments revealed weak platelet 0.003 Superoxide activity of a xanthine oxidase preparation ob20 Mg/m/ dismutase tained from Sigma. Shape change and serotonin
A)
stirrer: on—|off|on-
20
0.9
22
B) off
•|off
on *of f
20
22
t
XO 10pg/ml
I
PGE1 1pM
XO 10jjg/ml
30.7
t [min \
Figure. Shape change and serotonin release of discoid (A) and spheroid (B) washed platelets stimulated by xanthine oxidase (XO). Numbers at ends of curves show percentage of serotonin release measured 5 min after addition of xanthine oxidase. A) Xanthine-oxidase-induced discsphere transformation (decrease in light transmission, stirred). Inhibition by prostaglandin E j (PGEj). B) Xanthine-oxidase-induced shape change II (decrease in light transmission, non-stirred) and subsequent extrusion (increase in light transmission). Stimulation by cytochalasin B (CB) of extrusion and of concomitant serotonin release.
Brought to you by | Purdue University Libraries Authenticated Download Date | 5/31/15 5:13 AM
936
H. Patscheke, W. Paschen and P. Wörner
release remained weak and required high xanthine oxidase concentrations above 80 Mg/m/. This effect was not enhanced by 0.15mM xanthine, and was not inhibited by 100 Mg Superoxide dismutase/m/. The specific enzyme activity measured with the nitro blue tetrazolium test was, according to the producer specifications, 1.6-fold higher with xanthine oxidase from Sigma than with the enzyme from Boehringer. Two batches of the preparation from Boehringer (Control No. 1027154 and Control No. 1227356) gave about the same results, which are described above.
Bd. 359 (1978)
platelet function. Since the enzymic action of xanthine oxidase continues in the presence of apyrase, or even increases with plasma, the platelet and the enzyme activities of xanthine oxidase may reside in different components of xanthine oxidase.
A typical platelet activation by xanthine oxidase is demonstrated by the platelet shape changes, the inhibition by prostaglandin E{ and the stimulation of the extrusion by cytochalasin B. Prostagiandin synthesis by platelets is obviously not involved. Our results!91 with indomethacin and the measurements of malonic dialdehyde production confirmed earlier observations of other authors^ 1 1. Discussion The protein concentration of xanthine oxidase The enzymic oxidation of xanthine or hypoxansufficient to elicit maximal platelet activation, thine with xanthine oxidase is associated with 5 Mg/m/ is low compared to that of other highthe formation of Superoxide radicals^ 6 1. Reducmolecular-weight platelet stimuli, e.g. certain tion of nitro blue tetrazolium is proof of the pro- lectins'3!. Highly purified xanthine oxidase is obduction of Superoxide radicals, if the reaction can tained from cow's milk by comparatively simple be abolished by Superoxide dismutasel7L Xanmethods'] °~ 12 1 Preparations of xanthine oxidase, thine-oxidase-induced nitro blue tetrazolium rehowever, contain an impurity which consists of duction was suppressed by addition of Superoxide polymerized apo-xanthine-oxidase protein with dismutase, allopurinol, or by the absence of the low or no enzyme activity' 14 L The dissociation substrates, xanthine or hypoxanthine; but the of platelet and enzyme activities shown here, pareffect of xanthine oxidase on platelets was not ticularly the experiments with allopurinol, suginhibited under any of these conditions. This sug- gests the independence of platelet activity from gests independence of the platelet response from the enzymic site of xanthine oxidase. The enan enzymic action of xanthine oxidase or an atzymically inactive xanthine oxidase polymers tack of Superoxide radicals on platelets. Super(molecular weight 2 500000 or morel 131) may, oxide-generating agents, e.g. potassium supertherefore, be considered as the platelet-activating oxide = K02 or phenazine methosulfate plus components of xanthine oxidase. Our finding NADH, also could not simulate the platelet rethat a high specific enzyme activity (0.64 U/mg, sponse to xanthine oxidase (to be published). The Sigma) corresponds to low platelet activity, and inhibitory effect of Superoxide dismutase shown that a lower specific enzyme activity is found in by other authors^ 11 with the high Superoxide dis- the highly platelet-reactive preparation (0.4 U/mg, mutase concentration of 200 Mg/m/ was probably Boehringer Mannheim GmbH) confirm the sugnot due to an enzymic effect of Superoxide disgestion that the two activities reside in different mutase. Only 20 g Superoxide dismutase/m/ was components of xanthine oxidase preparations. enough to abolish the nitro blue tetrazolium reThe assistance of Miss Rita Klein is,gratefully acknowduction by xanthine oxidase, while even 200 g dismutase/m/ did not impair the platelet response ledged. The authors thank Prof. R. Brossmer for his support of this study. This work was supported by the in our experiments. Deutsche Forschungsgemeinschaft within SFB 90 "CarApyrase or minute amounts of human plasma in- diovasculäres System". hibited the effects of xanthine oxidase on platelets, but did not impair its enzyme activity. Apyrase Literature and plasma factors maintain platelet reactivity to 1 Handin, R. I., Karabin, R. & Boxer, G. J. (1977) ADP and the aggregation response to certain in/. Clin. Invest. 59, 959-965. 5 8 duced » '. Therefore, the inhibition is due to in- 2 Patscheke, H. & Wörner, P. (1978) Thromb. Res. 12, terference with xanthine oxidase rather than with 485-496.
Brought to you by | Purdue University Libraries Authenticated Download Date | 5/31/15 5:13 AM
Bd. 359 (1978)
Superoxide-Independent Platelet Response to Xanthine Oxidase
Patscheke, H. & Wörner, P. (1977) Thromb. Res. 11, 391-402. Holmsen, H. & Day, H. J. (1971) Ser. Haeniatol. 4,28-58. Mustard, J. F., Perry, D. W., Ardlie, N. G. & Packham, M.A. (\9Tl)Br.J.HaematoL 22, 193-202. Knowles, P. F., Gibson, J. F., Pick, F. M. & Bray,R. C. (1969) Biochem. J. 111,53-58. Fridovich, \.(\912) Acc.Chem.Res. 5, 321-326. Kinlough-Rathbone, R. L., Mustard, J. F., Packham, . ., Perry, D.W., Reimers, H.-J. & Cazenave, J.-P. (1977) Thromb.Haemostas. 37,291-308.
937
9 Patscheke, H. & Wörner, P. (1977) Hoppe-Seyler's Z. Physiol.Chem. 358,1256. 10 Nelson, C. A. & Handler, P. (1968) /. Biol Chem. 243, 5368-5373. 11 Massey, V., Brumby, P. E., Komai, H. & Palmer, G. (1969) J. Biol Chem. 244, 1682-1691. 12 Hart, L. I., McGartoU, . ., Chapman, H. R. & Bray, R.C. (1910) Biochem. J. 116,851-864. 13 Bray, R. C., Chisholm, A. J., Hart, L. I., Meriwether, L. S. & Watts, D. C. (1966) Flavins and Flavoproteins (Slater, E.G., ed.) pp. 117-129, Elsevier Publ. Comp., Amsterdam.
Dr. H. Patscheke, Dr. W. Paschen und Dr. P. Wörner, Institut für Biochemie II der Universität Heidelberg, Im Neuenheimer Feld 328, D-6900 Heidelberg 1.
Brought to you by | Purdue University Libraries Authenticated Download Date | 5/31/15 5:13 AM
Brought to you by | Purdue University Libraries Authenticated Download Date | 5/31/15 5:13 AM
