ARCHIVES
OF BIOCHEMISTKY
Vol. 288, No. 1, July,
AND
pp. 282-286,
BIOPHYSICS
1991
Oxygen-Radical-Mediated Inhibition of ADP-Induced Takao
Ohyashiki,’
Department
Received
Masayuki
of Biochemistry,
October
Lipid Peroxidation and Platelet Aggregation
Kobayashi,
and Katsuhiko
School of Pharmacy,
29, 1990, and in revised
form
March
Hokuriku
University,
Kanagawa-machi,
Kanazawa,
Ishikawa
920-l 1, Japan
4, 1991
The effects of lipid peroxidation on ADP-induced aggregation of washed rat platelets were examined using a oxygen-radical-generating system consisting of HzOz and ferrous ion. Lipid peroxidation was assessed by measurement of thiobarbituric acid-reactive substances (TBARS). Incubation of the platelets with various concentrations of HzOz (2-10 mM) in the presence of 10 PM Fe” resulted in a decrease of the aggregating capacity and an increase of TBARS value, depending on the concentrations of HzO,. Addition of catalase (0.1 mg/ml) to the incubation medium containing 10 PM Fe2+ and 10 mM H202 effectively protected the aggregating capacity, hut superoxide dismutase (0.1 mg/ml) did not protect H202/ Fe2’-induced inhibition of the platelet aggregation. The results of kinetic studies on the platelet aggregation with varying ADP and Ca2+ concentrations suggested that treatment of the platelets with Hz02/Fe2+ causes decreases in the binding affinities of ADP and Ca2+ for the platelets. On the basis of these results, change in the aggregating capacity of the platelets by treatment with H20,/Fe2.+ is discussed in relation to lipid peroxidation. se) IwI Academic Press,
Matsui
Inc.
One of important roles of platelets in uiuo is thrombus formation. As is well known (l-3), platelets are activated by several physiological agonists such as thrombin, collagen, and ADP, resulting in platelet shape change, aggregation, and secretion of intracellular materials. It is generally accepted (4-6) that the expression of these functional responses of platelets is preceded by mobilization of free Ca2+ from intracellular Ca”+ storage sites. On the other hand, recently, Wang et al. (7) have demonstrated that change in the cell-surface Ca” binding capacity is also related to modulation of platelet functions, ’ To whom correspondence should be addressed. ’ Abbreviations used: TBARS, thiobarbituric acid-reactive MDA, malondialdehyde; BHA, 3(2)-k-t-butyl-4-hydroxyanisole.
substances;
although several investigators have reported that extracellular Cazf is not directly involved in platelet functions (f&9). Recently, lipid peroxidation has been suggested to be associated with a variety of pathological events such as postischemic injury of the heart (10, 11) and pulmonary oxygen toxicity (12), including the aging of cells (13). It involves a rearrangement and destruction of the double bonds in the unsaturated fatty acids of membrane phospholipids by a free-radical-mediated chain reaction (14-16). The formation of lipid hydroperoxides in biological membranes would result in damage of the membrane structures and functions (17). In fact, it has been reported by several investigators that lipid peroxidation of biological membranes causes a decrease of their lipid fluidity (18, 19), modification of protein conformation (20, al), and changes in membrane-bound enzyme activities (2224). In addition, it has been also reported that the damage of proteins resulting from oxidative stress may be due to fragmentation, cross-linking formation, and amino acid modification in the protein molecules (25). Recently, Mirabelli et al. (26) have reported that treatment of human platelets with menadione results in an elevation of cytosolic Ca2+ concentration and cytoskeletal alterations, i.e. formation of high-molecular-weight aggregation of actin, cr-actinin, and actin-binding proteins. This finding suggests that menadione-induced lipid peroxidation modifies cytoskeletal organization and intracellular Ca’~’ homeostasis of the platelets. However, most studies on lipid peroxidation and/or oxidative stress of blood cells have been done on erythrocytes and erythrocyte membranes (27-30) and there are very few reports concerning lipid peroxidation of platelets. Therefore, it will be important to obtain further information about the effects of lipid peroxidation on the functions of platelets in order to understand the mechanisms of lipid peroxidationand/or oxidative stress-induced injury in platelets.
282 All
Copyright (0 1991 by Academic Press, Inc. rights of reproduction in any form reserved.
LIPID
PEROXIDATION
283
OF PLATELETS
For this purpose, in the present study, we examined the effects of HzOa/Fe2+ treatment on ADP-induced aggregation of washed rat platelets in relation to lipid peroxidation. MATERIALS
AND
METHODS
Materials. ADP (dipotassium salt), superoxide dismutase (3000 units/mg protein), and cat&se (3100 units/mg protein) were purchased from Sigma Chemical Co. 2-Thiobarbituric acid and 3(2)-tert-butyl-4hydroxyanisole (BHA) were obtained from Wako Pure Chemical Co. All other chemicals used were of the purest grade commercially obtainable. Preparation o/ washedplatelets. Whole blood was collected from male Wister rats weighing 250 g with 0.1 vol of 3.8% (v/v) trisodium citrate solution. Platelet-rich plasma was obtained by centrifugation at 170g for 10 min at room temperature. The platelets were then sedimented by centrifugation at 145Og for 10 min at room temperature. The sedimented platelets were washed twice with 10 mM Tris-HCl buffer (pH 7.4) containing 145 mM NaCl, 5 mM KCl, and 1 mM MgSO,. Finally, the sedimented platelets were suspended in the same buffer and adjusted to 1.8-2.0 X 10s cells/ml. The washed platelets were used within 3 h after preparation in the present study. The protein concentration was assayed by the procedure of Lowry et al. (31) using bovine serum albumin as the standard. Lipid per-oxidation of washed platelets. The washed platelets (about 2 X lo” cells/ml) were incubated with 10 PM FeS04 and 10 mM HeOz in 10 mM Tris-HCl buffer (pH 7.4) containing 145 mM NaCl, 5 mM KCl, and 1 mM MgSO, for 30 min at 37’C, unless otherwise specified. After termination of the reaction by the addition of 1 mM BHA (as a final concentration), ADP-induced aggregation of the platelets was measured. The control platelets were treated in a similar manner without H,O,/ Fe”. The measurement of TBARS formation was performed as described in our previous paper (24). The washed platelets (0.3 mg protein/ml) were incubated with H202/Fe2+ in a reaction medium of 1 ml under the same conditions described above. The reaction was terminated by the addition of 5 mM BHA (as a final concentration) and then the reaction mixture was heated at 90°C for 30 min with 1 ml of 10% trichloroacetic acid and 1 ml of 0.67% 2-thiobarbituric acid (50% CH,COOH). After cooling to room temperature, the reaction mixture was centrifuged at 5200g for 10 min and then the absorbance at 530 nm of the supernatant was measured. The amount of TBARS formed was calculated using the molar extinction coefficient of 1.53 X lo” Mm’ cm-’ and expressed as equivalents of malondialdehyde (MDA). Assay of platelet aggregation. Platelet aggregation was followed by recording light transmission through a stirred platelet suspension in an aggregometer cuvette at 37°C in a final volume of 0.31 ml using a Sysmex aggregation analyzer AA-100 (Toa Medical Electronics Co., Tokyo). The aggregation was initiated by the addition of 0.1 mM ADP (as a final concentration) to the washed platelet suspension (about 2 X lo5 cells/ ml) in 10 mM Tris-HCl buffer (pH 7.4) containing 145 mM NaCl, 5 mM KCl, 1 mM MgSO,, and 2 mM CaCl*, unless otherwise specified, and measured as the percentage change in light transmission. In the kinetic studies on platelet aggregation with varying concentrations of ADP or Ca’+, the platelet aggregation was measured after 30 s of the addition of ADP, because the turbidity change of the platelet suspension linearly proceeded until 1 min after the addition of ADP.
RESULTS Aggregation
AND
DISCUSSION
and TBARS
Formation
of Washed Platelets
Figure 1 shows the time-course of ADP-induced aggregation of control and 10 mM Hz02/10 yM Fe2+-treated platelets at 37°C.
50
0 0
2
4 Time
6
8
10
min
FIG. 1. Time-course of ADP-induced platelet aggregation. The procedure and conditions of platelet aggregation were described under Materials and Methods. Symbols: 0, control platelets; 0, 10 mM H,OJlO PM Fe’+-treated platelets. Values are expressed as means of triplicate determinations of the same platelet preparation.
In the control platelets, the turbidity of the platelet suspension markedly increased by the addition of 0.1 mM ADP and reached almost a constant level after 7 min of the addition of the inducer. On the other hand, the rate of the turbidity change and the extent of the maximal aggregation were markedly suppressed by treatment of the platelets with H202/Fe2+. Next, we examined the relationship between changes in the maximal aggregation and TBARS formation induced by treatment of the platelets with various concentrations of H202 in the presence of 10 /*M Fe2+. As can be seen in Fig. 2, the amount of TBARS formed during the reaction increased linearly with increasing concentrations of H202 in the reaction medium. On the other hand, the extent of H,02/Fe2+-induced inhibition of the maximal aggregation was enhanced with increasing concentrations of HzOz. In this case, treatment of the platelets with 10 PM Fe2+ alone did not affect on their aggregating capacity (data not shown). Although the TBARS value is a qualitative measure of lipid peroxidation, this result suggests that the inhibition of platelet aggregation by treatment with Hz02/Fe2+ is involved in lipid peroxidation of their membrane lipids. Next, we further examined the effects of antioxidants on ADP-induced platelet aggregation and TBARS formation in order to examine this interpretation. As shown in Table I, the addition of catalase to the platelet suspension effectively inhibited H,0z/Fe2+-induced TBARS formation and protected ADP-induced platelet aggregation. On the other hand, superoxide dismutase did not prevent the inhibition of the platelet aggregation and gave no effect on TBARS formation even at high concentration of the enzyme. In this experiment,
284
OHYASHIKI,
KOBAYASHI,
AND
MATSUI TABLE
I
Effects of Antioxidants on ADP-Induced Platelet Aggregation and TBARS Formation Addition No addition SOD (0.1 mg/ml) Catalase (0.1 mg/ml)
0
2
4
6
10
8
H202 Concn.
Aggregation (So)
Amount of TBARS formed (nmol MDA/mg protein/30 min)
29.8 f 1.0 28.6 f
4.60 rt 0.30 4.75 zk 0.25
1.5
91.0 + 2.0
1.45 f 0.20
Note. The platelets were preincubated for 5 min at 37°C with SOD or catalase before treatment with 10 mM H,O.JlO pM Fe’+. The platelet aggregation was measured after 10 min of the addition of 0.1 mM ADP and expressed as relative to that of the control ones without antioxidants (92.3 f 3%). Values are expressed as means (n = 9) + SD for three different platelet preparations.
mbl
FIG. 2. Concentration dependence of HZ02 on ADP-induced platelet aggregation and TBARS formation in the presence of 10 yM Fe’+. The concentration of H20s was varied from 2 to 10 mM. The platelet aggregation was measured after 10 min of the addition of 0.1 mM ADP. Other experimental conditions were the same as those described in the legend to Fig. 1. The extent of platelet aggregation was expressed as relative to that in the absence of H20,/Fe2+. Symbols: 0, aggregation; 0, TBARS value. Values are expressed as means (n = 9) f SD for three different platelet preparations.
the addition of superoxide dismutase or catalase alone to the control platelets did not affect on the maximal extent of ADP-induced aggregation of the platelets (92 + 2 and 93 + 1% for the platelets with superoxide dismutase and catalase, respectively). From these results, it is clear that the inhibition of ADP-induced platelet aggregation by treatment with H,O,/Fe’+ is due to lipid peroxidation and that superoxide anions are not directly involved in this phenomenon. As is well known (32), the reaction of HaOz and ferrous ion easily generates hydroxyl radicals by the Fenton reaction as follows,
kinetic studies on platelet aggregation were performed with varying ADP concentrations. The double reciprocal plot of the extent of platelet aggregation after 30 s of the addition of ADP versus the ADP concentration added gave a linear relationship over the concentration range of ADP (lo-400 PM; data not shown) and the apparent dissociation constant (Kd) of the complex between ADP and the control platelets was estimated to be 11.0 & 1.5 PM (n = 9; SD) (Fig. 4). By treatment of the platelets with Hz02/Fe2+, this value increased depending on the H202 concentration in the incubation medium, and it was found that there is a linear relationship between the Kd values and the extents of platelet aggregation (Fig. 5). These results suggest that the decrease of ADP-binding
H202 + Fe2+ + ‘OH + OH- + Fe3’ Therefore, it seems likely that H202/Fezt-induced inhibition of platelet aggregation may be due to the formation of hydroxyl radicals during the reaction. Kinetic Study of Platelet Aggregation In order to investigate what kind of factor is related to H202/Fe2+-induced inhibition of platelet aggregation, the effects of ADP and Ca2+ concentrations were examined. As shown in Fig. 3, the extent of aggregation of the control platelets increased as a function of ADP concentration and reached a constant level above 150 PM ADP. On the other hand, the extent of the maximal aggregation of H,O,/Fe’+-treated platelets at each concentration of ADP was lower than that of the control ones. Next, the
0
100
200 ADP Concn,
300
400 PM
FIG. 3. ADP concentration dependence of platelet aggregation. The concentration of ADP was varied from 10 to 400 jiM. Other experimental conditions were the same as those described in the legend to Fig. 2. Symbols: 0, control platelets; l ,4 mM H102/10 pM Fe*+-treated platelets; 0.6 mM HZ02/10 pM Fe’+-treated platelets; a, 10 mM H202/10 pM Fe*+treated platelets. Values are expressed as means of triplicate determinations of the same platelet preparation.
LIPID PEROXIDATION OF PLATELETS
0
I
I
I
1
I
2
II
6
8
10
H202 Concn.
285
J I
0
mM
10
I
I
20
30
Kd(AJJP)
FIG. 4. Effects of H202 concentration on the Kd value for ADP. The Kd values were determined from the reciprocal plots of the extent of platelet aggregation against l/[ADP]. The ADP concentration was varied from 10 to 400 pM. The platelet aggregation was measured 30 s after the addition of ADP. Other experimental conditions were the same as those described in the legend to Fig. 2. Values are expressed as means (n = 9) + SD for three different platelet preparations.
affinity to the agonist-binding sites on the platelet membrane surface is one of the factors concerning HzOz/Fe2+induced inhibition of platelet aggregation. On the other hand, it has been known (7,33, 34) that Ca2+ is also an important factor affecting platelet functions. In fact, it has been reported (35-37) that extracellular Ca”+ is a cofactor for platelet aggregation and the binding of fibrinogen to the platelet surface. In addition, recently, Brass and Shattil (38) have demonstrated that activation of platelets by ADP or epinephrin induces an increase of the amount of bound Ca’+ to the platelet surface membrane. Therefore, we examined the effects of H,O,/Fe’+ treatment on Ca2+ requirement of ADP-induced platelet aggregation. As can be seen in Fig. 6, the reciprocal plots of the extent of platelet aggregation against l/[CaCl,] in all systems showed a linear relation over the concentration range of CaCl, tested (0.1-2 mM). From these plots, the Ca”+ concentrations which are required to induce half-maximal aggregation of the control, 6 mM HzOz/10 yM Fe’+-treated and 10 mM H202/10 PM Fe’+-treated platelets were estimated to be 138.9 +- 3.8, 158.7 * 7.4, and 178.6 + 3.2 PM, respectively. This result indicates that the binding affinity of Ca2+ on the platelet membrane surface is also sensitively modified by treatment of the platelets with H,0,/Fe2’. Taken together, our findings suggest that the inhibition of ADP-induced aggregation of washed rat platelets by treatment with H202/Fe2+ is related to decreases in the binding affinities of ADP and Ca2+ on their membrane surfaces. Although the observations of in vitro experi-
P
5. Relationship between the extents of platelet aggregation and the Kd values for ADP. The Kd values were obtained from Fig. 4. The extent of the aggregation described on the ordinate represents the value which was measured 10 min after the addition of 0.1 mM ADP and expressed relative to that in the absence of H20,/Fe”‘. Other experimental conditions were the same as those described in the legend to Fig. 2. Values are expressed as means (n = 9) + SD for three different platelet preparations. FIG.
ments cannot be directly related to in uivo conditions, it seems that the results obtained in the present study may give a clue in the analysis of mechanisms of lipid peroxidation-induced cellular injury in platelets.
0
5 1/Icoc121
10 mM
FIG. 6. Double reciprocal plots of the extent of ADP-induced aggregation and CaCl, concentration. The platelet aggregation was measured 30 s after the addition of 0.1 mM ADP. Other experimental conditions were the same as those described in the legend to Fig. 2. Symbols: 0, control platelets; 0, 6 mM H,OJlO pM Fe”-treated platelets; 0, 10 mM H202/10 pM Fe*+-treated platelets. Values are expressed as means (n = 9) + SD for three different platelet preparations.
286
OHYASHIKI,
KOBAYASHI,
Recently, Tsai et al. (34) and Brass and Shattil (38) have reported that the binding capacity of Ca2+ on the membrane surface plays an important role in several membrane reactions involved in platelet activation. In addition, it seems that the Ca2+-binding sites might be located on membrane proteins or glycoproteins which are located within or near platelet surface membrane, as well as phospholipids. As is well known (17-21), lipid peroxidation of biological membranes induces perturbation in the dynamic properties of the membranes, i.e., lipid fluidity and protein conformation. Therefore, it could be considered that modification of the membrane components of the platelets may be partly related to HzOz/Fe2+-induced changes in the binding affinities of ADP and Ca2+ on the platelet membrane surface. Further detailed analysis about the effects of lipid peroxidation on the dynamic Properties of the membrane components in the platelets could shed additional light on this problem. REFERENCES 1. Gordon, J. L. (1981) in Platelets in Biology and Pathology (Gordon, J. L., Ed.), Vol. 2, pp. l-17, Elsevier/North-Holland Biochemical Press, Amsterdam. 2. Gerrard, J. M., Peterson, D. A., and White, J. G. (1981) in Platelets in Biology and Pathology (Gordon, J. L., Ed.), Vol. 2, pp. 407-436, Elsevier/North-Holland Biochemical Press, Amsterdam. 3. Frojmovic, M. M., and Milton, J. (1982) Physiol. Reu. 62, 1855261. 4. Feinstein, M. B., Zavoico, G. B., and Halenda, S. P. (1985) in The Platelets, Physiology and Pharmacology (Longenecker, G. L., Ed.), pp. 237-269, Academic Press, San Diego. 5. Johnson, P., Ware, A., Cliveden, P., Smith, M., Dvorek, A., and Salzman, E. (1985) J. Biol. Chem. 260, 2069-2076. 6. Hallam, T. J., and Rink, T. J. (1985) FEBS Lett. 186, 175-179. 7. Wang, C. T., Shiao, Y. T., Chen, T. C., Tsai, W. J., and Young, C. C. (1986) Biochim. Biophys. Acta 856, 244-258. 8. Hovig, T. (1964) Thromb. Diath. Haemorrh. 12, 179-200. 9. Zuker, M. B., and Grant, R. A. (1978) Blood 52,505-514. 10. Stewart,, J. R., Blackwell, W. H., Crute, S. L., Loughlin, V., and Greenfield, L. J. (1984) Thorac. Cardiouasc. Surg. 86, 262-272. 11. Peterson, D. A., Asinger, R. W., Elsperger, K. J., Homans, D. C., andEaton, J. W. (1985) Biochem. Biophys. Res. Commun. 127,8793. 12. Crapo, J. D., Freeman, B. A., Barry, B. E., Turrens, J. F., and Young, S. L. (1983) Physiologist 26, 170-176.
AND
MATSUI
13. Mead, J. F. (1982) in Free Radicals in Biology (Pryor, W. A., Ed.), Vol. 1, pp. 51-68, Academic Press, New York. 14. Tappel, A. L. (1973) Fed. Proc. 32, 1870-1874. 15. Buege, J. A., and Aust, S. D. (1978) in Methods in Enzymology (Fleisher, S. and Packer, L., Eds.), Vol. 52, pp. 302-351, Academic Press, New York. 16. Svingen, B. A., Buege, J. A., O’Neal, F. O., and Aust, S. D. (1979) J. Biol. Chem. 254, 5892-5899. 17. Kagan, V. E. (1988) in Lipid Peroxidation in Biomembranes, CRC Press, Boca Raton, FL. 18. Rice-Evans, C., and Hochstein, P. (1981) Biochem. Biophys. Res. Commun. 100, 1537-1542. 19. Ohyashiki, T., Ohtsuka, T., and Mohri, T. (1986) Biochim. Biophys. Acta 861,311-318. 20. Ohyashiki, T., Ohtsuka, T., and Mohri, T. (1988) Biochim. Biophys. Acta 939, 383-392. 21. Ohyashiki, T., Yamamoto, T., and Mohri, T. (1989) Biochim. Biophys. Acta 981, 235-242. 22. Nicotera, P., Moore, M., and Orrenius, s. (1986) FEBS Lett. 161, 1499153. 23. Kukreja, R., Okabe, E., Schrier, G. M., and Hess, M. L. (1988) Arch. Biochem. Biophys. 261,447-457. 24. Ohta, A., Mohri, T., and Ohyashiki, T. (1989) Biochim. Biophys. Acta 984, 151-157. 25. Dean, R. T., Thomas, S. M., and Garner, A. (1986) Biochem. J. 240,489-494. 26. Mirabelli, F., Salis, A., Vairetti, M., Bellomo, G., Thor, H., and Orrenius, S. (1989) Arch. Biochem. Biophys. 270, 478-488. 27. Stern, A. (1985) in Oxidative Stress (Sies, H., Ed.), pp. 331-345, Academic Press, New York. 28. Arduini, A., Stern, A., Storto, S., Belfiglio, M., Mancinelli, G., Scurti, R., and Federici, G. (1989) Arch. Biochem. Biophys. 273,112-120. 29. Sheerin, H., Snyder, L. M., and Fairbanks, G. (1989) Biochim. Biophys. Acta 983, 65-76. 30. Moore, R. B., Brummitt, M. L., and Mankad, V. N. (1989) Arch. Biochem. Biophys. 273, 527-534. 31. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 32. Aust, S. D., Morehouse, L. A., and Thomas, C. E. (1985) J. Free Radicals Biol. Med. 1, 3-25. 33. Murer, E. M. (1972) Biochim. Biophys. Acta 26 1,435-443. 34. Tsai, W. J., Chen, J. C., and Wang, C. T. (1988) Biochim. Biophys. Acta 940, 105-120. 35. Bennett, J. S., and Vilaire, G. (1979) J. Clin. Invest. 64,1393-1401. 36. Lages, B., and Weiss, H. J. (1981) Thromb. Haemost. 45, 173-179. 37. Heptinstall, S. (1976) Thromb. Haemost. 36, 208-220. 38. Brass, L. F., and Shattil, S. T. (1982) J. Biol. Chem. 257, 14,00014,005.
OF BIOCHEMISTKY
Vol. 288, No. 1, July,
AND
pp. 282-286,
BIOPHYSICS
1991
Oxygen-Radical-Mediated Inhibition of ADP-Induced Takao
Ohyashiki,’
Department
Received
Masayuki
of Biochemistry,
October
Lipid Peroxidation and Platelet Aggregation
Kobayashi,
and Katsuhiko
School of Pharmacy,
29, 1990, and in revised
form
March
Hokuriku
University,
Kanagawa-machi,
Kanazawa,
Ishikawa
920-l 1, Japan
4, 1991
The effects of lipid peroxidation on ADP-induced aggregation of washed rat platelets were examined using a oxygen-radical-generating system consisting of HzOz and ferrous ion. Lipid peroxidation was assessed by measurement of thiobarbituric acid-reactive substances (TBARS). Incubation of the platelets with various concentrations of HzOz (2-10 mM) in the presence of 10 PM Fe” resulted in a decrease of the aggregating capacity and an increase of TBARS value, depending on the concentrations of HzO,. Addition of catalase (0.1 mg/ml) to the incubation medium containing 10 PM Fe2+ and 10 mM H202 effectively protected the aggregating capacity, hut superoxide dismutase (0.1 mg/ml) did not protect H202/ Fe2’-induced inhibition of the platelet aggregation. The results of kinetic studies on the platelet aggregation with varying ADP and Ca2+ concentrations suggested that treatment of the platelets with Hz02/Fe2+ causes decreases in the binding affinities of ADP and Ca2+ for the platelets. On the basis of these results, change in the aggregating capacity of the platelets by treatment with H20,/Fe2.+ is discussed in relation to lipid peroxidation. se) IwI Academic Press,
Matsui
Inc.
One of important roles of platelets in uiuo is thrombus formation. As is well known (l-3), platelets are activated by several physiological agonists such as thrombin, collagen, and ADP, resulting in platelet shape change, aggregation, and secretion of intracellular materials. It is generally accepted (4-6) that the expression of these functional responses of platelets is preceded by mobilization of free Ca2+ from intracellular Ca”+ storage sites. On the other hand, recently, Wang et al. (7) have demonstrated that change in the cell-surface Ca” binding capacity is also related to modulation of platelet functions, ’ To whom correspondence should be addressed. ’ Abbreviations used: TBARS, thiobarbituric acid-reactive MDA, malondialdehyde; BHA, 3(2)-k-t-butyl-4-hydroxyanisole.
substances;
although several investigators have reported that extracellular Cazf is not directly involved in platelet functions (f&9). Recently, lipid peroxidation has been suggested to be associated with a variety of pathological events such as postischemic injury of the heart (10, 11) and pulmonary oxygen toxicity (12), including the aging of cells (13). It involves a rearrangement and destruction of the double bonds in the unsaturated fatty acids of membrane phospholipids by a free-radical-mediated chain reaction (14-16). The formation of lipid hydroperoxides in biological membranes would result in damage of the membrane structures and functions (17). In fact, it has been reported by several investigators that lipid peroxidation of biological membranes causes a decrease of their lipid fluidity (18, 19), modification of protein conformation (20, al), and changes in membrane-bound enzyme activities (2224). In addition, it has been also reported that the damage of proteins resulting from oxidative stress may be due to fragmentation, cross-linking formation, and amino acid modification in the protein molecules (25). Recently, Mirabelli et al. (26) have reported that treatment of human platelets with menadione results in an elevation of cytosolic Ca2+ concentration and cytoskeletal alterations, i.e. formation of high-molecular-weight aggregation of actin, cr-actinin, and actin-binding proteins. This finding suggests that menadione-induced lipid peroxidation modifies cytoskeletal organization and intracellular Ca’~’ homeostasis of the platelets. However, most studies on lipid peroxidation and/or oxidative stress of blood cells have been done on erythrocytes and erythrocyte membranes (27-30) and there are very few reports concerning lipid peroxidation of platelets. Therefore, it will be important to obtain further information about the effects of lipid peroxidation on the functions of platelets in order to understand the mechanisms of lipid peroxidationand/or oxidative stress-induced injury in platelets.
282 All
Copyright (0 1991 by Academic Press, Inc. rights of reproduction in any form reserved.
LIPID
PEROXIDATION
283
OF PLATELETS
For this purpose, in the present study, we examined the effects of HzOa/Fe2+ treatment on ADP-induced aggregation of washed rat platelets in relation to lipid peroxidation. MATERIALS
AND
METHODS
Materials. ADP (dipotassium salt), superoxide dismutase (3000 units/mg protein), and cat&se (3100 units/mg protein) were purchased from Sigma Chemical Co. 2-Thiobarbituric acid and 3(2)-tert-butyl-4hydroxyanisole (BHA) were obtained from Wako Pure Chemical Co. All other chemicals used were of the purest grade commercially obtainable. Preparation o/ washedplatelets. Whole blood was collected from male Wister rats weighing 250 g with 0.1 vol of 3.8% (v/v) trisodium citrate solution. Platelet-rich plasma was obtained by centrifugation at 170g for 10 min at room temperature. The platelets were then sedimented by centrifugation at 145Og for 10 min at room temperature. The sedimented platelets were washed twice with 10 mM Tris-HCl buffer (pH 7.4) containing 145 mM NaCl, 5 mM KCl, and 1 mM MgSO,. Finally, the sedimented platelets were suspended in the same buffer and adjusted to 1.8-2.0 X 10s cells/ml. The washed platelets were used within 3 h after preparation in the present study. The protein concentration was assayed by the procedure of Lowry et al. (31) using bovine serum albumin as the standard. Lipid per-oxidation of washed platelets. The washed platelets (about 2 X lo” cells/ml) were incubated with 10 PM FeS04 and 10 mM HeOz in 10 mM Tris-HCl buffer (pH 7.4) containing 145 mM NaCl, 5 mM KCl, and 1 mM MgSO, for 30 min at 37’C, unless otherwise specified. After termination of the reaction by the addition of 1 mM BHA (as a final concentration), ADP-induced aggregation of the platelets was measured. The control platelets were treated in a similar manner without H,O,/ Fe”. The measurement of TBARS formation was performed as described in our previous paper (24). The washed platelets (0.3 mg protein/ml) were incubated with H202/Fe2+ in a reaction medium of 1 ml under the same conditions described above. The reaction was terminated by the addition of 5 mM BHA (as a final concentration) and then the reaction mixture was heated at 90°C for 30 min with 1 ml of 10% trichloroacetic acid and 1 ml of 0.67% 2-thiobarbituric acid (50% CH,COOH). After cooling to room temperature, the reaction mixture was centrifuged at 5200g for 10 min and then the absorbance at 530 nm of the supernatant was measured. The amount of TBARS formed was calculated using the molar extinction coefficient of 1.53 X lo” Mm’ cm-’ and expressed as equivalents of malondialdehyde (MDA). Assay of platelet aggregation. Platelet aggregation was followed by recording light transmission through a stirred platelet suspension in an aggregometer cuvette at 37°C in a final volume of 0.31 ml using a Sysmex aggregation analyzer AA-100 (Toa Medical Electronics Co., Tokyo). The aggregation was initiated by the addition of 0.1 mM ADP (as a final concentration) to the washed platelet suspension (about 2 X lo5 cells/ ml) in 10 mM Tris-HCl buffer (pH 7.4) containing 145 mM NaCl, 5 mM KCl, 1 mM MgSO,, and 2 mM CaCl*, unless otherwise specified, and measured as the percentage change in light transmission. In the kinetic studies on platelet aggregation with varying concentrations of ADP or Ca’+, the platelet aggregation was measured after 30 s of the addition of ADP, because the turbidity change of the platelet suspension linearly proceeded until 1 min after the addition of ADP.
RESULTS Aggregation
AND
DISCUSSION
and TBARS
Formation
of Washed Platelets
Figure 1 shows the time-course of ADP-induced aggregation of control and 10 mM Hz02/10 yM Fe2+-treated platelets at 37°C.
50
0 0
2
4 Time
6
8
10
min
FIG. 1. Time-course of ADP-induced platelet aggregation. The procedure and conditions of platelet aggregation were described under Materials and Methods. Symbols: 0, control platelets; 0, 10 mM H,OJlO PM Fe’+-treated platelets. Values are expressed as means of triplicate determinations of the same platelet preparation.
In the control platelets, the turbidity of the platelet suspension markedly increased by the addition of 0.1 mM ADP and reached almost a constant level after 7 min of the addition of the inducer. On the other hand, the rate of the turbidity change and the extent of the maximal aggregation were markedly suppressed by treatment of the platelets with H202/Fe2+. Next, we examined the relationship between changes in the maximal aggregation and TBARS formation induced by treatment of the platelets with various concentrations of H202 in the presence of 10 /*M Fe2+. As can be seen in Fig. 2, the amount of TBARS formed during the reaction increased linearly with increasing concentrations of H202 in the reaction medium. On the other hand, the extent of H,02/Fe2+-induced inhibition of the maximal aggregation was enhanced with increasing concentrations of HzOz. In this case, treatment of the platelets with 10 PM Fe2+ alone did not affect on their aggregating capacity (data not shown). Although the TBARS value is a qualitative measure of lipid peroxidation, this result suggests that the inhibition of platelet aggregation by treatment with Hz02/Fe2+ is involved in lipid peroxidation of their membrane lipids. Next, we further examined the effects of antioxidants on ADP-induced platelet aggregation and TBARS formation in order to examine this interpretation. As shown in Table I, the addition of catalase to the platelet suspension effectively inhibited H,0z/Fe2+-induced TBARS formation and protected ADP-induced platelet aggregation. On the other hand, superoxide dismutase did not prevent the inhibition of the platelet aggregation and gave no effect on TBARS formation even at high concentration of the enzyme. In this experiment,
284
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AND
MATSUI TABLE
I
Effects of Antioxidants on ADP-Induced Platelet Aggregation and TBARS Formation Addition No addition SOD (0.1 mg/ml) Catalase (0.1 mg/ml)
0
2
4
6
10
8
H202 Concn.
Aggregation (So)
Amount of TBARS formed (nmol MDA/mg protein/30 min)
29.8 f 1.0 28.6 f
4.60 rt 0.30 4.75 zk 0.25
1.5
91.0 + 2.0
1.45 f 0.20
Note. The platelets were preincubated for 5 min at 37°C with SOD or catalase before treatment with 10 mM H,O.JlO pM Fe’+. The platelet aggregation was measured after 10 min of the addition of 0.1 mM ADP and expressed as relative to that of the control ones without antioxidants (92.3 f 3%). Values are expressed as means (n = 9) + SD for three different platelet preparations.
mbl
FIG. 2. Concentration dependence of HZ02 on ADP-induced platelet aggregation and TBARS formation in the presence of 10 yM Fe’+. The concentration of H20s was varied from 2 to 10 mM. The platelet aggregation was measured after 10 min of the addition of 0.1 mM ADP. Other experimental conditions were the same as those described in the legend to Fig. 1. The extent of platelet aggregation was expressed as relative to that in the absence of H20,/Fe2+. Symbols: 0, aggregation; 0, TBARS value. Values are expressed as means (n = 9) f SD for three different platelet preparations.
the addition of superoxide dismutase or catalase alone to the control platelets did not affect on the maximal extent of ADP-induced aggregation of the platelets (92 + 2 and 93 + 1% for the platelets with superoxide dismutase and catalase, respectively). From these results, it is clear that the inhibition of ADP-induced platelet aggregation by treatment with H,O,/Fe’+ is due to lipid peroxidation and that superoxide anions are not directly involved in this phenomenon. As is well known (32), the reaction of HaOz and ferrous ion easily generates hydroxyl radicals by the Fenton reaction as follows,
kinetic studies on platelet aggregation were performed with varying ADP concentrations. The double reciprocal plot of the extent of platelet aggregation after 30 s of the addition of ADP versus the ADP concentration added gave a linear relationship over the concentration range of ADP (lo-400 PM; data not shown) and the apparent dissociation constant (Kd) of the complex between ADP and the control platelets was estimated to be 11.0 & 1.5 PM (n = 9; SD) (Fig. 4). By treatment of the platelets with Hz02/Fe2+, this value increased depending on the H202 concentration in the incubation medium, and it was found that there is a linear relationship between the Kd values and the extents of platelet aggregation (Fig. 5). These results suggest that the decrease of ADP-binding
H202 + Fe2+ + ‘OH + OH- + Fe3’ Therefore, it seems likely that H202/Fezt-induced inhibition of platelet aggregation may be due to the formation of hydroxyl radicals during the reaction. Kinetic Study of Platelet Aggregation In order to investigate what kind of factor is related to H202/Fe2+-induced inhibition of platelet aggregation, the effects of ADP and Ca2+ concentrations were examined. As shown in Fig. 3, the extent of aggregation of the control platelets increased as a function of ADP concentration and reached a constant level above 150 PM ADP. On the other hand, the extent of the maximal aggregation of H,O,/Fe’+-treated platelets at each concentration of ADP was lower than that of the control ones. Next, the
0
100
200 ADP Concn,
300
400 PM
FIG. 3. ADP concentration dependence of platelet aggregation. The concentration of ADP was varied from 10 to 400 jiM. Other experimental conditions were the same as those described in the legend to Fig. 2. Symbols: 0, control platelets; l ,4 mM H102/10 pM Fe*+-treated platelets; 0.6 mM HZ02/10 pM Fe’+-treated platelets; a, 10 mM H202/10 pM Fe*+treated platelets. Values are expressed as means of triplicate determinations of the same platelet preparation.
LIPID PEROXIDATION OF PLATELETS
0
I
I
I
1
I
2
II
6
8
10
H202 Concn.
285
J I
0
mM
10
I
I
20
30
Kd(AJJP)
FIG. 4. Effects of H202 concentration on the Kd value for ADP. The Kd values were determined from the reciprocal plots of the extent of platelet aggregation against l/[ADP]. The ADP concentration was varied from 10 to 400 pM. The platelet aggregation was measured 30 s after the addition of ADP. Other experimental conditions were the same as those described in the legend to Fig. 2. Values are expressed as means (n = 9) + SD for three different platelet preparations.
affinity to the agonist-binding sites on the platelet membrane surface is one of the factors concerning HzOz/Fe2+induced inhibition of platelet aggregation. On the other hand, it has been known (7,33, 34) that Ca2+ is also an important factor affecting platelet functions. In fact, it has been reported (35-37) that extracellular Ca”+ is a cofactor for platelet aggregation and the binding of fibrinogen to the platelet surface. In addition, recently, Brass and Shattil (38) have demonstrated that activation of platelets by ADP or epinephrin induces an increase of the amount of bound Ca’+ to the platelet surface membrane. Therefore, we examined the effects of H,O,/Fe’+ treatment on Ca2+ requirement of ADP-induced platelet aggregation. As can be seen in Fig. 6, the reciprocal plots of the extent of platelet aggregation against l/[CaCl,] in all systems showed a linear relation over the concentration range of CaCl, tested (0.1-2 mM). From these plots, the Ca”+ concentrations which are required to induce half-maximal aggregation of the control, 6 mM HzOz/10 yM Fe’+-treated and 10 mM H202/10 PM Fe’+-treated platelets were estimated to be 138.9 +- 3.8, 158.7 * 7.4, and 178.6 + 3.2 PM, respectively. This result indicates that the binding affinity of Ca2+ on the platelet membrane surface is also sensitively modified by treatment of the platelets with H,0,/Fe2’. Taken together, our findings suggest that the inhibition of ADP-induced aggregation of washed rat platelets by treatment with H202/Fe2+ is related to decreases in the binding affinities of ADP and Ca2+ on their membrane surfaces. Although the observations of in vitro experi-
P
5. Relationship between the extents of platelet aggregation and the Kd values for ADP. The Kd values were obtained from Fig. 4. The extent of the aggregation described on the ordinate represents the value which was measured 10 min after the addition of 0.1 mM ADP and expressed relative to that in the absence of H20,/Fe”‘. Other experimental conditions were the same as those described in the legend to Fig. 2. Values are expressed as means (n = 9) + SD for three different platelet preparations. FIG.
ments cannot be directly related to in uivo conditions, it seems that the results obtained in the present study may give a clue in the analysis of mechanisms of lipid peroxidation-induced cellular injury in platelets.
0
5 1/Icoc121
10 mM
FIG. 6. Double reciprocal plots of the extent of ADP-induced aggregation and CaCl, concentration. The platelet aggregation was measured 30 s after the addition of 0.1 mM ADP. Other experimental conditions were the same as those described in the legend to Fig. 2. Symbols: 0, control platelets; 0, 6 mM H,OJlO pM Fe”-treated platelets; 0, 10 mM H202/10 pM Fe*+-treated platelets. Values are expressed as means (n = 9) + SD for three different platelet preparations.
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Recently, Tsai et al. (34) and Brass and Shattil (38) have reported that the binding capacity of Ca2+ on the membrane surface plays an important role in several membrane reactions involved in platelet activation. In addition, it seems that the Ca2+-binding sites might be located on membrane proteins or glycoproteins which are located within or near platelet surface membrane, as well as phospholipids. As is well known (17-21), lipid peroxidation of biological membranes induces perturbation in the dynamic properties of the membranes, i.e., lipid fluidity and protein conformation. Therefore, it could be considered that modification of the membrane components of the platelets may be partly related to HzOz/Fe2+-induced changes in the binding affinities of ADP and Ca2+ on the platelet membrane surface. Further detailed analysis about the effects of lipid peroxidation on the dynamic Properties of the membrane components in the platelets could shed additional light on this problem. REFERENCES 1. Gordon, J. L. (1981) in Platelets in Biology and Pathology (Gordon, J. L., Ed.), Vol. 2, pp. l-17, Elsevier/North-Holland Biochemical Press, Amsterdam. 2. Gerrard, J. M., Peterson, D. A., and White, J. G. (1981) in Platelets in Biology and Pathology (Gordon, J. L., Ed.), Vol. 2, pp. 407-436, Elsevier/North-Holland Biochemical Press, Amsterdam. 3. Frojmovic, M. M., and Milton, J. (1982) Physiol. Reu. 62, 1855261. 4. Feinstein, M. B., Zavoico, G. B., and Halenda, S. P. (1985) in The Platelets, Physiology and Pharmacology (Longenecker, G. L., Ed.), pp. 237-269, Academic Press, San Diego. 5. Johnson, P., Ware, A., Cliveden, P., Smith, M., Dvorek, A., and Salzman, E. (1985) J. Biol. Chem. 260, 2069-2076. 6. Hallam, T. J., and Rink, T. J. (1985) FEBS Lett. 186, 175-179. 7. Wang, C. T., Shiao, Y. T., Chen, T. C., Tsai, W. J., and Young, C. C. (1986) Biochim. Biophys. Acta 856, 244-258. 8. Hovig, T. (1964) Thromb. Diath. Haemorrh. 12, 179-200. 9. Zuker, M. B., and Grant, R. A. (1978) Blood 52,505-514. 10. Stewart,, J. R., Blackwell, W. H., Crute, S. L., Loughlin, V., and Greenfield, L. J. (1984) Thorac. Cardiouasc. Surg. 86, 262-272. 11. Peterson, D. A., Asinger, R. W., Elsperger, K. J., Homans, D. C., andEaton, J. W. (1985) Biochem. Biophys. Res. Commun. 127,8793. 12. Crapo, J. D., Freeman, B. A., Barry, B. E., Turrens, J. F., and Young, S. L. (1983) Physiologist 26, 170-176.
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