ARCHIVES

OF BIOCHEMISTRY

AND BIOPHYSICS

Vol. 289, No. 1, August 15, pp. 47-52, 1991

Lipid Peroxide Makes Rabbit Platelet Hyperaggregable to Agonists through Phospholipase A2 Activation Tsutomu Hashizume, Hiroyoshi Yamaguchi, Akihiko Akira Tamura,* Takashi Sato,l and Tatsuzo Fujii

Kawamoto,

Department of Biochemistry, Kyoto Pharmaceutical University, Kyoto 607; and *Department Faculty of Home Economics, Chukyo Women’s University, Aichi 474, Japan

Received January

of Food and Nutrition,

22, 1991, and in revised form May 1, 1991

Treatment of rabbit platelets with tert-butyl hydroperoxide and Fe’+ caused increasing arachidonic acid release, lysophosphatidylcholine formation, and aggregation with increasing concentrations of Fe’+. A combination of tert-butyl hydroperoxide and a low concentration of Fe’+, which by itself causes slight or no such activation, elicited synergistic release of arachidonic acid and aggregation under stimulation with a suboptimal concentration of collagen or arachidonic acid as an agonist. These responses were inhibited by pretreatment of the platelets with vitamin E or mepacrine in a concentration-dependent manner, but not by uric acid. The arachidonic acid release was dependent on the presence of Ca2+ in the medium. Synergistic formation of lysophosphatidylcholine, but not diacylglycerol, was also observed under this condition. The aggregation was also inhibited by indomethacin, a cyclooxygenase inhibitor. Cyclooxygenase activity was not affected by the oxidative treatment. These results suggest that lipid peroxide formed in membranes causes phospholipase A2 to become hypersusceptible to the agonist used, making the platelets hyperaggregable. o 1991 Academic PEES, IIW.

Formation of phospholipids is lipase AZ (PLA2)” acids (l-6) which

peroxidized fatty acids in membrane known to activate endogenous phosphoto liberate preferentially the peroxidized are then reduced by the catalytic action

1 To whom correspondence should be addressed at Department of Biochemistry, Kyoto Pharmaceutical University, Misasagi, Yamashinaku, Kyoto 607, Japan. ’ Abbreviations used: t-BHP, tert-butyl hydroperoxide; PLAz, phospholipase A,; BSA, bovine serum albumin; PRP, platelet-rich plasma; EGTA, ethylene glycol bis(&aminoethyl ether) N,N’-tetraacetic acid.

0003-9861/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form

of glutathione peroxidase (7, 8). This event is widely accepted as a detoxification process to protect membrane phospholipids against peroxidative damage (9). Although hydrogen peroxide (10-12) or pyrogaroll(13), which generates active oxygen species, is reported to enhance platelet aggregation, the precise mechanism underlying these effects has not been elucidated. However, it is recently reported that an oxidant, tert-butyl hydroperoxide (t-BHP), can increase PLAz activity and stimulate the release of arachidonic acid in the pulmonary arterial endothelial cells (6). This observation may explain the mechanism by which the oxidants stimulate platelet activation, since thromboxane A2 formed subsequently to the release of arachidonic acid via PLA2 activation is a powerful agonist to induce platelet aggregation. Therefore, we examined in the present work whether or not the PLA2 in rabbit platelets exposed to slight oxidative stress become hypersensitive to stimuli, thereby being involved in the mechanism by which platelet activation is accelerated. MATERIALS

AND

METHODS

Materials. Collagen (type I), arachidonic acid, t-BHP, mepacrine, and indomethacin were obtained from Sigma Chemical Co. Vitamin E (dl-ol-tocopherol) and uric acid were from Wako Pure Chemical Industries Ltd. (Japan). [3H]Arachidonic acid (100 Ci/mmol) and [3H]glycerol (200 mCi/mmol) were from New England Nuclear. Washed rabbitplatelets. Platelet-rich plasma (PRP), obtained from rabbit blood anticoagulated with 0.1% EDTA, was centrifuged and the platelets obtained were washed with Cazf-free Hepes buffer (142 mM NaCl, 2.7 mM KCl, 1 mM MgCl,, 5.6 mM glucose, 3.8 mM Hepes, pH 6.5) containing 0.35% bovine serum albumin (BSA) and 1 mM EGTA, as described previously (14). Finally, the platelets were resuspended in the same buffer without BSA and EGTA (pH 7.4) at a concentration of 5 X 10’ cells/ml. Platelet aggregation. Platelet aggregation was determined in the presence of 1 mM CaCl, by the increase in light transmission in an aggregometer (NKK Hema Tracer I, Niko Bioscience Co., Japan).

47 Inc. reserved.

48

HASHIZUME

ET AL.

Assay for phospholipase activity. [3H]Arachidonic acid- or [%glycerol-labeled platelets, which were prepared by incubation of PRP with [3H]arachidonic acid (2 @i/ml) or [3H]glycerol (80 pCi/ml) at 37°C for 1 or 3 h, respectively, and then washed, were pretreated with 50 pM BW755C (3-amino-l-[m-(trifluoromethyl)phenyl]-2-pyrazoline, a cyclooxygenase and lipoxygenase inhibitor) at 37°C for 2 min. The platelets were treated with 30 pM t-BHP plus 30 pM FeSO, for 1 min, in the presence of 1 mM CaCl,, and then stimulated with various concentrations of collagen or arachidonic acid for an additional 5 min. After lipid extraction with CHClz/CHBOH/HC1 (200/200/l, v/v/v), each lipid fraction was separated by thin-layer chromatography on Silica Gel G plates with a developing solvent as follows: for the detection of arachidonic acid and diacylglycerol derived from [3H]arachidonic acid-labeled platelets, petroleum ether/diethyl ether/acetic acid (60/45/l, v/v/v); for lysophosphatidylcholine from [3H]glycerol-labeled platelets, CHC13/ CH,OH/7 M NH,OH @O/54/11, v/v/v) in the first dimension and CHC13/ CH,OH/acetic acid/Hz0 (30/15/4/2, v/v/v/v) in the second dimension. The area corresponding to each lipid fraction was scraped off and the radioactivity was determined. Oxidatiue change in membrane lipids. [3H]Arachidonic acid-labeled platelets were pretreated with 20 pM mepacrine at 37°C for 2 min. The platelets were treated with or without 20 pM vitamin E for 2 min, and then exposed to 30 pM t-BHP plus 30 FM FeSO, for various periods of time. After lipid extraction with CHC13/CH30H/HC1 (200/200/l, v/v/v), the radioactivity in the water-soluble phase separated from organic extracts was determined. Determination of thromboxane B2 production. [3H]Arachidonic acidlabeled platelets were treated with 30 pM t-BHP and 30 pM FeSO, at 37°C for 1 min in the presence of 1 mM CaCl,, and then stimulated with 2 pg/ml collagen or 200 nM arachidonic acid at 37°C for 5 min. In the experiment of assay for cyclooxygenase activity, washed platelets were treated with oxidant as above, and then 60 pM [3H]arachidonic acid was added and incubated for 5 min in the presence of 1 mM CaCl,. These reactions were terminated by adding 5 mM EGTA (pH 3.0) and the lipids were extracted with ethyl acetate. The endoperoxide metabolites were separated by thin-layer chromatography developed with the upper phase of ethyl acetate/2,2,4-trimethylpentane/acetic acid/water (45/25/10/50, v/v/v/v), according to the method of Walenga et al. (15). Thromboxane Bz fraction which was identified by comigration with authentic standard was scraped off and the radioactivity was determined.

RESULTS

Effect of F2’

on t-BHP-Induced

Platelet Activation

As shown in Fig. 1, when [3H]arachidonic acid-labeled platelets were treated with 30 I.LM t-BHP and various concentrations of FeS04, the release of [3H]arachidonic acid was increased in an Fe’+ concentration-dependent manner, except at 100 PM FeS04. On the other hand, [3H]lysophosphatidylcholine formation from [3H]glycerol-labeled platelets and the aggregation of the platelets increased as Fe’+ concentration increased, even at high concentrations. Therefore, the decline in arachidonic acid release at 100 PM FeSO, may be due to oxidation of the arachidonic acid released. Each agent alone did not induce any aggregation. These results suggest that lipid peroxides produced in platelet membranes under the actions of t-BHP and Fe’+ could stimulate endogenous PLA2 activation as shown in other systems (l-6, 9), and ultimately induce aggregation of the platelets.

FeS04

(PM)

1. Effect of Fe’+ on t-BHP-induced platelet aggregation (O), arachidonic acid release (A), and lysophosphatidylcholine formation (B). Washed or [3H]arachidonic acid- or [3H]glycerol-labeled platelets were treated with FeSO, at 37’C for 5 min in the presence of 30 pM t-BHP and 1 mM CaCl,. Aggregation is expressed as the percentage of the maximal response. The radioactivity of [3H]arachidonic acid released and [3H]lysophosphatidylcholine formed was determined as described under Materials and Methods. Values represent the mean of two separate experiments. FIG.

Lipid Peroxidation-Induced to Agonist

Hyperaggregability

It is of interest to investigate the possibility that a slight formation of lipid peroxides in membranes renders platelets hypersensitive to agonists. Therefore, we examined whether or not t-BHP and a low concentration of Fe’+ act synergistically with suboptimal concentrations of agonist to enhance aggregation. The treatment of platelets with 30 PM t-BHP and 30 /*M Fe’+ significantly enhanced the aggregation under stimulation with collagen of low concentration ranges that induced only slight aggregation (Fig. 2A). With arachidonic acid as a stimulus, further representative enhancement was seen in the concentration ranges that induced no aggregation (Fig. 2B). The treatment with each or both t-BHP and Fe’+ in the absence of agonist did not exert any effect on aggregation under the concentration used here. Aggregation was inhibited by pretreatment with vitamin E or mepacrine in a concentration-dependent manner up to 20 pM. The concentrations of vitamin E or mepacrine required to inhibit the aggregation by 50% in response to oxidizing agents and an agonist (collagen or arachidonic acid), are as follows: with collagen 15 and 13 PM; and with arachidonic acid 14 or 13 PM, respectively. Indomethacin, a cyclooxygenase inhibitor, at 10 /*M also inhibited almost completely the aggregation in response to either combinations of t-BHP and collagen or arachidonic acid (data not shown). Uric acid, known as a water-soluble radical scavenger, did not affect aggregation even at concentra-

LIPID 75 -

PEROXIDE-INDUCED

PLATELET

HYPERAGGREGABILITY

49

TO AGONIST

JL (A)

(B) 0

ii! .a 50 .-z E e $ 25 E Ij $ 0’Li!Yil 012

3

Collagen

4

(vg/ml)

50 -G-T

Arachidonic

6

5

acid (-IogM)

Time (min)

FIG. 2. Synergistic effect of t-BHP and Fe’+ with collagen (A) or arachidonic acid (B) on platelet aggregation. Washed platelets were treated with (0) or without (0) 30 pM t-BHP plus 30 pM FeSO, at 37°C for 1 min in the presence of 1 mM CaCl, and then exposed to each agonist for 5 min. Aggregation is expressed as the percentage of the maximal response. Values represent the mean of two separate experiments.

FIG. 3. Time-dependent increase in the water-soluble radioactivity as an indicator of the oxidative change in membrane lipids, and the inhibition by vitamin E. [3H]Arachidonic acid-labeled platelets were pretreated with 20 ELM mepacrine at 37’C for 2 min. For experimental details see Materials and Methods. (0) with vitamin E, (0) without vitamin E. Values represent the mean k SD of four separate experiments performed in duplicate.

tions as high as 1 mM. Vitamin E at the highest concentration used here (20 pM) did not affect the aggregation induced by high concentrations of collagen (5 pg/ml) or arachidonic acid (10 PM) with no oxidant. Effect of the oxidant on thrombin-induced aggregation could not be evaluated because of the inactivation of thrombin in the presence of the oxidant.

Lipid Peroxidation-Induced of PLAz to Agonist

Determination

of Membrane

Phospholipid

Peroxidation

To confirm the oxidative changes in the fatty acyl chain of membrane phospholipids, we tried to determine thiobarbiturate-reactive substances in the platelets treated with t-BHP and Fe’+. However, no data to indicate the generation of such substances in the platelets were obtained. This seems to be due to a low sensitivity of the reaction. Therefore, we tried to detect water-soluble substances liberated from [3H]arachidonic acid-labeled platelets by treatment with t-BHP and Fe’+, in the presence of mepacrine to eliminate the PLAz-derived substances. The results in Fig. 3 show that liberation of significant amounts of radioactive substance into a watersoluble fraction ended after chloroform-methanol extraction was detected, and that this was completely inhibited by pretreatment with vitamin E. The radioactive substance could not be free arachidonic acid itself or subsequently formed cyclooxygenase-product, because the experiment was performed in the presence of mepacrine, a PLAz inhibitor. Hence, it is supposed to be certain peroxidative substance(s) liberated from radiolabeled membrane phospholipids. The inhibition by vitamin E supports this idea.

Hypersensitivity

Effect of membrane lipid peroxidation induced by t-BHP and Fe’+, and a weak stimulation with agonist, on PLAl activation in [3H]arachidonic acid-labeled platelets was studied. As shown in Fig. 4A, a significant amount of [3H]arachidonic acid was liberated synergistically by the stimulation with both the oxidant and the low concentrations of collagen which by itself could cause only a slight arachidonic acid liberation. Upon arachidonic acid

012

34

Collagen

(pg/ml)

50

6

Arachidonic

7

6

5

acid (-logbl)

FIG. 4. Synergistic effect of t-BHP and Fe*+ with collagen (A) or arachidonic acid (B) on [3H]arachidonic acid release (0, 0) and [sH]diacylglycerol formation (A, A). The radioactivity of each lipid from [3H]arachidonic acid-labeled platelets after stimulation with (closed symbol) or without (open symbol) t-BHP and Fez+ and exposure to each agonist for 5 min was determined as described under Materials and Methods. Values represent the mean of two separate experiments.

50

HASHIZUME

ET AL. TABLE

I

Suppressive Effect of Vitamin E (20 PM) or Mepacrine (20 PM) on Synergistic Release of Arachidonic Acid” [3H]Arachidonic

acid (dpm) t-BHP

Treatment None Vitamin E Mepacrine

None ‘-•HP Fe%

con

AA

t-BHP FG+

t-BHP Fe*+

con AA FIG. 5. Synergistic effect of t-BHP and Fe’+ with collagen (Coll, 2 fig/ml) or arachidonic acid (AA, 200 nM) on [3H]thromboxane B2 formation. The radioactivity of thromboxane B, formed from [Harachidonic acid-labeled platelets for 5 min after stimulation was determined as described under Materials and Methods. Values represent the mean of two separate experiments.

stimulation, almost no liberation was observed in the absence of the oxidant in any concentration ranges used, whereas considerable liberation was induced by pretreatment with the oxidant (Fig. 4B). The oxidant itself without the stimulant induced only a small amount of arachidonic acid liberation (l.l- to 1.3-fold above the intact level). In addition, arachidonic acid liberation in the absence of Ca2+ did not occur (data not shown). Under stimulation with the combination of the oxidant and the stimulant, production of thromboxane Bz was markedly enhanced (Fig. 5), whereas no increase in diacylglycerol ,formation was observed (Fig. 4). To confirm that the arachidonic acid release is due to the enhanced activation of PLA2 subsequent to the peroxidation of lipid, the effect of vitamin E and mepacrine on the release was examined. The formation of lysophosphatidylcholine, another hydrolysis product by PLAz action, was also examined. As shown in Table I, pretreatment of platelets with vitamin E or mepacrine markedly inhibited the release of arachidonic acid. Furthermore, formation of lysophosphatidylcholine in [3H]glycerol-labeled platelets was significantly increased, in comparison with that by each stimulus alone (Fig. 6). These facts indicate that arachidonic acid release results from the action of PLAz activated synergistically by both the oxidant and a weak stimulant. Effect of Lipid

Peroxidation

on Cyclooxygenase

Activity

It is reported that hydrogen peroxides which cause platelets to be hypersensitive to agonist enhance cyclooxygenase activity (12, 16). Indeed, the activity of this

+ Fe’+ Arachidonic

None

Collagen

1048 k 81

2517 -+ 131 1472 f 90 1378 f 115

acid

2593 f 135 1226 f 162 1288 + 189

’ [3H]Arachidonic acid-labeled platelets were pretreated with each compound at 37°C for 2 min, and exposed to t-BHP in the presence of 2 pg/ml collagen or 200 nM arachidonic acid for 5 min. The radioactivity of arachidonic acid released was determined as described under Materials and Methods. Values represent the mean f SD of three separate experiments performed in duplicate.

enzyme is known to be augmented continuously by a peroxidized compound such as prostaglandin GZ under physiological conditions, and hence to facilitate the conversion of arachidonic acid to prostaglandin H2 (17). Since there is a possibility in the present study that generated peroxides in platelet may cause direct stimulation of cyclooxygenase activity, thus making the platelets hyperaggregable, we studied the effect of t-BHP and Fe*+ on the activity. However, no significant difference between the untreated platelets and those treated with t-BHP plus Fe*+ or each alone was apparent in the rate of thrombox-

T T1’

None I-BHP F&

cow

AA

I-BHP Fe% Cdl

I-•HP Fe* AA

FIG. 6. Synergistic effect of t-BHP and Fe*+ with collagen (Coil, 2 pg/ml) or arachidonic acid (AA, 200 nM) on [3H]lysophosphatidylcholine formation. The radioactivity of lysophosphatidylcholine formed from [3H]glycerol-labeled platelets for 5 min after stimulation was determined as described under Materials and Methods. Values represent the mean f SD of four separate experiments performed in duplicate.

LIPID

PEROXIDE-INDUCED

PLATELET

ane BZ production from exogenously added arachidonic acid under the conditions described under Materials and Methods; the production of thromboxane Bz was within the range 0.305 + 0.06 nmol/lO’ cells. DISCUSSION

Some oxidizing agents to generate hydroperoxide of unsaturated fatty acids are reported to facilitate platelet aggregation (10-13). Although the mechanism underlying the effect is far from clear, we studied in the present work whether or not PLAz is involved, because this enzyme is known to be activated by oxidized fatty acids in membranes (l-6,9). We first demonstrated in the present work that t-BHP and Fe2+ induced platelet aggregation, arachidonic acid liberation, and lysophosphatidylcholine formation as a function of Fe2+ concentration (Fig. l), suggesting that peroxides formed in the platelet membranes could cause endogenous PLA2 activation, as shown in other systems (l-6, 9), and subsequent aggregation. However, it seems unlikely that platelets are exposed to such severe oxidative stress under physiological conditions during circulation in vivo. Therefore, we investigated whether a lower degree of peroxidative damage in the membranes makes platelet hypersensitive to agonists, using the combination of t-BHP plus a low concentration of Fe2+, which by itself exerts a little effect on platelet activation, and suboptimal concentrations of agonist. It was determined that such a combination of t-BHP and Fe2+ actually produced certain peroxidation product(s) from the membrane phospholipids of the treated platelets, by measuring 3H-labeled compound(s) in the water-soluble fraction of the [3H]arachidonic acid-labeled platelets after treatment with the oxidant. Production of peroxidized lipids was inhibited by vitamin E during the oxidative treatment (Fig. 3). The combination of oxidizing agents and suboptimal concentrations of stimuli synergistically enhanced arachidonic acid release without causing any increase in diacylglycerol formation (Fig. 4). This indicates that the release was caused by hydrolytic action of PLA2, not by combined actions of phospholipase C and diacylglycerol lipase, as evidenced by the lysophosphatidylcholine formation (Fig. 6) and the inhibition by mepacrine (a PLAz inhibitor) (Table I). Although it is reported that mepacrine is not necessarily an inhibitor specific for PLAz (18), the concentrations used here (up to 20 pM) are lower than those that exhibit its nonspecific action on cell membranes (greater than 200 yM). Therefore, the result obtained appears to be attributable to a specific inhibition of PLA2. Furthermore, the arachidonic acid release was also inhibited by a pretreatment of the platelets with vitamin E (an antioxidant) (Table I). This result suggests that a slight formation of peroxides in platelet membranes renders the PLA2 hypersusceptible to agonist.

HYPERAGGREGABILITY

TO AGONIST

51

Such a combination of an oxidizing agent and suboptimal levels of agonist also enhanced thromboxane B2 formation from [3H]arachidonic acid incorporated into the membrane phospholipids (Fig. 5). The direct formation of the eicosanoid from exogenously added [3H]arachidonic acid was not affected by the oxidant (shown in the results section), indicating that the peroxides produced have no effect on cyclooxygenase activity. Since enhanced aggregation by the same combination was inhibited by vitamin E, mepacrine, or indomethacin (a cyclooxygenase inhibitor) (see Fig. 2), we conclude that in the case of the combination with collagen, both the lipid peroxides generated in the membranes and the receptormediated weak stimulation by collagen synergistically enhanced PLA2 activity and subsequently released arachidonic acid to induce platelet aggregation after the conversion to thromboxane A2. With arachidonic acid as an agonist, a significant amount of peroxides from both endogenous fatty acids of membrane phospholipids and the exogenously added arachidonic acid could exert significant effects on the PLAz activation and lead to hyperaggregation. The mechanism underlying PLA2 activation in platelets has not been elucidated in the present paper. However, we obtained evidence that in the absence of added Ca2+, the combination of the oxidizing agents and a weak agonist failed to release arachidonic acid (Results section of Fig. 4), suggesting that increased Ca2+ influx may be involved in enhancing PLA2 activation. An increase in membrane permeability associated with lipid peroxidation has been reported for glucose and some pigments (19,20). Although our evidence is derived from results obtained in an in vitro oxidative system, it is known that active oxygen radicals are generated at sites of ischemia-reperfusion or inflammation. Furthermore, it is reported that oxidized low-density lipoprotein can facilitate platelet aggregation (21), and that platelets could release iron from transferrin, which is a promoting factor in oxygen radical formation (22). Therefore, it seems likely that the platelets exposed to even a slight oxidative stress become hyperaggregable to agonists and hence contribute to the etiology of thrombotic disease in vivo. REFERENCES 1. Yasuda, M., and Fujita, T. (1977) Jpn. J. Pharmacol. 27, 429-435. 2. Sevanian, A., Stein, R. A., and Mead, J. F. (1981) Lipids 16, 781789. 3. Weglicki, W. B., Dickens, B. F., and Mak, I. T. (1984) Biochem. Biophys. Res. Commun. 124. 229-235. 4. Beckman, J. K., Borowitz, Chem. 262, 1479-1481.

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5. Borowitz, S. M., and Montgomery, Commun. 158, 1021-1028.

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6. Chakraborti, S., Gurtner, G. H., and Michael, Physiol. 257, L430-L437.

J. R. (1989) Am. J.

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7. Grossmann, A., and Wendel, A. (1983) Eur. J. Biochem. 135,549552. 8. Tan, K. H., Meyer, D. J., Belin, J., and Ketterer, B. (1984) Biochem. J. 220, 243-252. 9. van Kuijk, F. J. G. M., Sevanian, A., Handelman, G. J., and Dram, E. A. (1987) Trends Biochem. Sci. 12,31-34. 10. Iatridis, S. G., Iatridis, P. G., Kyrkou, K. A., Markidou, S. G., and Iatridi, I. S. (1979) Thromb. Res. 15, 733-741. 11. Del Principe, D., Menichelli, A., De Matteis, W., Di Corpo, M. L., Di Giulio, S., and Finazzi-Agro, A. (1985) FEBS Lett. 185, 142146. 12. Hill, T. D., White, J. G., and Rao, G. H. R. (1989) T/womb. Res. 53,447-455. 13. Salvemini, D., de Nucci, G., Sneddon, J. M., and Vane, J. R. (1989) Br. J. Pharmacol. 97, 11451150. 14. Akiba, S., Sato, T., and Fujii, T. (1990) Biochim. Biophys. Actu 1044, 291-296.

ET AL. 15. Walenga, R. W., Opas, E. E., and Feinstein, Chem. 256, 12,523-12,528.

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16. Hemler, M. E., Cook, H. W., and Lands, W. E. M. (1979) Arch. Biochem. Biophys. 193, 340-345. 17. Hemler, M. E., Lands, W. E. M., and Graff, G. (1978) Biochem. Biophys. Res. Commun. 85, 1325-1331. 18. Dise, C. A., Burch, J. W., and Goodman, D. B. P. (1982) J. Biol. Chem. 257,4701-4704. 19. Kunimoto, M., Inoue, K., and Nojima, S. (1981) Biochim. Biophys. Acta 646, 169-178. 20. Tanfani, F., and Bertoli, E. (1989) Biochem. Biophys. Res. Commun. 163, 241-246. 21. Ardlie, N. G., Selley, M. L., and Simons, L. A. (1989) Atherosclerosis 76, 117-124. 22. Brieland, J. K., Vissers, M. C. M., Phan, S. H., and Fantone, J. C. (1989) Biochim. Biophys. Acta 978, 191-196.

Lipid peroxide makes rabbit platelet hyperaggregable to agonists through phospholipase A2 activation.

Treatment of rabbit platelets with tert-butyl hydroperoxide and Fe2+ caused increasing arachidonic acid release, lysophosphatidylcholine formation, an...
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