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Basic aggregation properties of washed rat platelets: Correlation between aggregation, phospholipid degradation, malondialdehyde, and thromboxane formation
Basic Aggregation Properties of Washed Rat Platelets: Correlation between Aggregation, Phospholipid Degradation, Mafondialdehyde, and Thromboxane Formation
TAKAKO TOMITA, KEIZO UMEGAKI, AND EIICHI HAYASHI
The basic characteristics of washed rat platelets in aggregation and biochemical responses to thrombin and collagen were investigated. Maximum aggregation response to thrombin and collagen was observed in the presence of l-3 mM extracellular CA’+. Breakdown of endogenous phospholipids was followed by HPTLC. Phosphatidylinositol degraded most rapidly and greatly, followed by ph~sphatidyiethanolamine, and phosphatidylchoiine. Sphingomyelin did not change on stimulation. Formation of malondialdehyde was closely associated with thrombin- and collagen-induced aggregation within the range of the concentration employed. In addition, it was correlated with TXBz formation and phosphatidylinositol degradation. These results indicated that, despite known species variation, thrombin-induced aggregation is mediated by TXA2 and malondialdehyde formation reflects both cycle-oxygenase and thromboxane synthaseactivities, and phospholipid degradation in rat platelets as well as in human platelets.
Key Words:
Platelet aggregation; Washed rat platelets; Malondialdehyde; pholipid degradation; Thromboxane
Phos-
INTRODUCTION Considerable information is available about platelet aggregation and the accompanying biochemical events. The information pertaining to platelet aggregation, however, has been gained mostly from human platelets, and such information is lacking for other species of animals, particularly for widely used laboratory animals such as the rat. The rat is a very useful animal species for studying the changes of platelet function due to various pathological conditions and also drug treatment. It has been pointed out that there are great differences in platelet morphology and function in species. Platelet size, circulating numbers, and volume vary enormously between species Won Behrens, 1972). The platelet size is smaller, and the circulating platelet counts (8-12 x 108/ml) are higher in the rat than in humans (1.54.0 x lOa/mI). Rat platelets behave differently, in general, with aggregating agents and aggregation-inhibiting agents. Adrenalin, a proaggregator in human platelets, is inhibitor to thrombin-induced aggregation in the rat (Vu and Latour, 1977). AdenFrom the Department of Pharmacology, Shizuoka College of Pharmaceutical Sciences, Shizuoka, Japan Address requests for reprints to: Takako Tomita, Department of Pharmacology, Shizuoka College of Pharmaceutical Sciences, Ohika, Shizuoka, Japan 422. Received January 25,1983; revised and accepted April l&1983.
31 Journal of Pharmacological Q, 1983 Elsevier
Science
Methods Publishing
IO, 31-44 Co.,
Inc.,
(1983) 52 Vanderbilt
Avenue,
New
York,
NY 10017
32
T. Tomita et al.
osine (Philp, 1970), PGD, (Hayaishi et al., 1982) and dipryidamole (Philp, 1970), known inhibitors of aggretation, do not exert inhibition on the rat platelet aggregation. Dipyridamole is reported to potentiate rat platelet aggregation. The mechanism of platelet aggregation has been extensively studied and a large body of evidence now indicates that TXA, plays a central role in platelet aggregation. Thromboxane synthetase catalyzes the concomitant isomerization and dismutation of PGHp to give TXA2, MDA, and HHT (12-L-hydroxyheptadecatrienoic acid). Thus platelet MDA can be regarded as an indicator of the activities of both cyclo-oxygenase and thromboxane synthetase, and of the release of arachidionic acid from platelet membrane phospholipids (Smith et al., 1976). In the case of rat platelets, however, there is some evidence that questions the role of TXA2 in aggregation. Hwang (1980) reported that preincubation of rat platelet-rich plasma with imidazole, a known inhibitor of thromboxane synthetase, did not attenuate the aggretation induced by ADP or collagen, whereas the synthesis of TXB, was inhibited by more than 90%. In human platelets, imidazole was shown to inhibit thromboxane synthesis and accompanying aggregation induced by PGH, in human platelet rich plasma (Fitzpatrick and Gorman, 1977). Dutilh (Dutilh et al., 1979) demonstrated that prostaglandin endoperoxide, which was rapidly converted into thromboxane AZ, did not cause aggregation in rat platelets, and also that collagen-induced aggregation of rat platelets was completely inhibited by eicosatetrayenoic acid at the concentration at which lipoxygenase activity was completely blocked with an apparent stimulation of cyclooxygenase. Therefore, this study was designed to investigate the basic aggregation characteristics of washed rat platelets, and particularly to assess the significance of MDA assay in aggregation reaction. METHODS Preparation
of Washed
Platelets
Washed platelets were prepared principally according to Baenziger and Majerus (1974). Blood was collected into a siliconized centrifuging tube containing one portion of acid-citrate-dextrose anticoagulant (0.085 M trisodium citrate, 0.065 M citric acid, 2% dextrose) to five portions of blood, and the mixture was consecutively centrifuged for 15 min at 230 x g (1200 rpm) and for 3 min at 800 x g (220 rpm) at room temperature. This two-step centrifugation was employed to prevent a loss of platelets of larger sizes. PRP (platelet-rich plasma) separated from the two centrifugations were mixed, and centrifuged for 7 min at 120 x g (800 rpm) to sediment red and white cells. The supernatant was then centrifuged for 15 min at 1700 x g (3200 rpm). The sedimented platelets were resuspended in the same volume of a washing buffer (0.113 M NaCI, 4.3 mM K2HP04, 4.3 mM Na2HP04, 24.4 mM NaH2P04, 5.5 mM glucose, 1 mM EDTA, pH 6.5) as the whole blood and centrifuged for 15 min, at 1500 x g (3000 rpm) at 4°C. The washed platelets were resuspended in a resuspending buffer (0.14 M NaCI, 15 mM Tris-HCI, 5 mM glucose, pH 7.4) to make a suspension
of about 4 x IO8 cells/ml.
The washed
platelet
suspensions
were
kept at 4°C until used, within 4 hr. The intactness of platelets was ensured throughout all the preparatory stages by measuring the released of lactic acid dehydrogenase activity in the supernatant.
Aggregation Properties of Washed Rat Platelets Measurement
of Platelet Aggregation
The degree of platelet aggregation was measured by the turbidometric method using 4 channeled NKK Hematracer 1 (Niko Bioscience, Tokyo). To 200 ~1 of platelet suspension (adjusted to contain about 4 x IO8 cells/ml), IO ~1 of CaCI, was added to give a final concentration indicated in the legends. 20 ~1 of various concentrations of thrombin or collagen were added after 1 min incubation at 37°C with constant stirring at 1000 rpm, and changes in turbidity were recorded. A maximum aggregation was measured 3 min and 5 min after the addition of thrombin and collagen, respectively. Human plasma thrombin (Midori Cross Co., Osaka, Japan) was diluted with the resuspending buffer. The solution of collagen (Type I, insoluble, Sigma, St. Louis, MO) was prepared according to lnoshita et al. (1976), and collagen in the solution was determined by the method of ltzhaki et al. (1964). Incubation
of Platelets
A suspension of washed rat platelets (4 x IO8 cells/ml) was incubated either with collagen or with thrombin at 37°C in the presence of 1.5 mM Ca’+. Incubation conditions are described in detail in the legend for each figure. Extraction of Platelet Phospholipids To 0.5 ml of platelet suspension, 2.2 ml of a mixture of CHC&: MeOH (1 : 2) was added to stop the incubation. The mixture was shaken vigorously for 1 min with 0.71 ml of CHC& and 0.71 ml of 2 M KCI solution containing 5 mM EDTA, and centrifuged for 30 min at 3000 rpm. The lower layer was withdrawn and the solvent was removed under a vacuum. Analysis of Platelet Phospholipids The phospholipid extract was dissolved in 50 ~1 of a mixture of CHC& : MeOH : HZ0 (75 : 25 : 2), and a portion of the extract was applied on a HPTLC plate (Kieselgel 60, art No. 5641, 10 x 20 cm, Merch, Darmstadt, Germany.), which was rinsed with a mixture of CHC13 : MeOH (2 : 1) overnight and activated by heating for 1 hr at 110°C. The plate was developed at 4°C in a mixture of CHC&: MeOH: CH3NH2 (40%) (63 : 35 : 10). After the solvents were completely removed under a hot air stream, the plate was heated for 10 hr at 150°C with ammonium bicarbonate in a sealed chamber to develop fluorescence (Kupke, 1978). The intensity of fluorescence in each phospholipids was measured by a CAMAG TLC Scanner (Muttenz, Swiss). Analysis of MDA To 0.25 ml of platelet suspension, 0.1 ml of 3.1 M trichloroacetic acid (final concentration, 0.8 M) was added to stop the incubation. Malondialdehyde formed by platelet reaction was measured principally according to Ohkawa et al. (1978). A portion of the mixture (0.4 ml) was mixed with 0.4 ml of 3% sodium dodecylsulfate (Wako Chemicals, Tokyo), and then heated at 70°C for 45 min with 1.6 ml of 0.1 N HCI, 0.8 ml of 10% phosphotungstic acid, and 0.8 ml of 0.7% thiobarbituric acid (Nakarai Chemicals, Tokyo). The reaction was terminated by chilling, and the flu-
33
34
T. Tomita et al.
orescent products were extracted with 4 ml of n-butanol. The intensity of fluorescence was measured as percent fluorescence of Rhodamin B (20 ng/ml) by a Hitachi fluorescence Photmeter (Hitachi 204, Hitachi Co. LTD, Tokyo) (excitation 515 nm, emission 553 nm). For a calibration curve, 50-860 p mol of 1, 1, 3, 3-tetraethoxypropane (a gift from Eizai Pharmaceuticals, Tokyo, Japan) in 0.4 ml of 0.8 M trichloroacetic acid, was submitted to the reaction as described above, and the fluorescence was measured. Radioimmunoassay
of TXB2
To 0.5 ml of platelet suspension, 2 M citric acid was added to adjust pH to 3.0 and to stop the reaction. The mixture was chilled in liquid nitrogen and kept at -80°C till assayed. The thawed samples were neutralized with 1 M NaOH, and thromboxane B2 in the diluted samples was assayed by using thromboxane B, t3Hl RIA Kit (NEN, Boston, MA).
RESULTS Aggregation
Response
in Rat-Washed
Platelets
Figure 1 shows typical aggregation response to thrombin and collagen in washed platelets from Wistar Kyoto rats in the presence of 1.5 mM Ca*‘. Aggregation reached a maximum at 3 min with thrombin and at 5 min with collagen. As Figure 2A shows, aggregation was dose-dependent in the range of 0.1-0.3 U of thrombin/ml
83.7 )rg/ml 0.44 U/ml
16.7 pg/ml 0.17 U/ml 8.4 pg/ml
20
0.13 U/ml
tr 0-
12
3
'123456
4 Time
(
min
)
FIGURE 1. Typical aggregation response to three concentrations of a) thrombin and b) collagen in washed rat (Wistar Kyoto) platelets in the presence of 1.5 mM Ca2+.
100.
35 _-_--A ___--/___-----;--
80'
l
/-&---
"
/
/'/'
60 .
/
,' E .c +I z?
i
:
;40. 4"
:'
I a
f
0'
0.1
0.2
0
50
100
0.3 150
0.4
Thranbin
U/ml
200
Collagen
pg/ml
FIGURE 2A. Platelet aggregation induced by various concentrations of thrombin (-•-_) and collagen (-A-). Washed rat (WKY) platelets were stimulated either with thrombin (0.087, 0.11, 0.13, 0.15, 0.17, 0.22, 0.26, and 0.44 U/ml) or with collagen (6.2, 7.5, 9.3, 12.5, 18.7, 46.7, 93.4, and 187 pg/ml) in the presence of 1.5 mM Ca*+.
FIGURE 2B. Effects of Ca*’ concentration on platelet aggregation. Washed rat (WKY) platelets were stimulated either with thrombin (0.22 U/ml) in the presence of Ca’+ (0.125, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, 10.0, and 20.0 mM) or with collagen (lo.7 pg/ml) in the presence of Ca2+ (0.5, 1.0, 2.0, 9.0, and 23.0 mM). Each point represents mean for triplicates.
343 T. Tomita et al. and IO-100 pg collagen/ml. The thrombin concentration at which 50% aggregation was induced varies even within strains of rats. It was between 0.15-0.2 U/ml in Wistar Kyoto rats, but between 0.6-0.7 U/ml in Wistar Mishima. lndomethacin (42 pgiml) inhibited thrombin (0.42 U/ml)-induced aggregation by 69% in the presence of 1.5 mM Ca*‘. Fig 2 B shows the strong dependency of rat platelet aggregation on Ca” concentration. Thrombin (0.22 U/ml) or collagen (10.7 F./ml) scarcely induced aggregation with washed rat platelets in the absence of Ca2+. The presence of l-3 mM Ca*‘, however, induced a maximum aggregation response to thrombin and collagen, while at the higher concentration aggregation was inversely reduced with the concentration. Thrombin-induced aggregation was completely inhibited at 20 mM of Ca*‘. The cell density of rat platelet preparations did not affect the aggregability within the range of 2-6 x IO’ cells/ml (Figure 3). Analysis of Platelet Phospholipids Phospholipids extracted from platelets before and after stimulation with thrombin were separated by HPTLC and fluorescence was developed by heating the plate with ammoniumbicarbonate for IO hr at 150°C. Figure 4 shows the profile of rat platelet phospholipids. A linear correlation was observed between the intensity of fluorescence developed on each phospholipid and the concentration of phospholipids (Figure 5). Contents of PC, PE, and PI in washed rat platelets (unstimulated) 100
I
-0 .------_-__-______ A
0
1 2.0 Cell density
4.0
( x l@
6.0 Cells
8.0 / ml
)
FIGURE 3. The dependency of aggregation on the cell density of rat platelet suspension. Washed rat (WKY) platelets were stimulated with thrombin (-•-) (0.44 U/ml) and with collagen (---A---) (46.7 @ml) in the presence of 1.5 mM Ca*+.
Control
(thrombln
Stimulated 12.5
U/ml,
3 min
)
PE
PC SM
PI
i
FIGURE 4. A profile of rat platelet phospholipids before and after stimulation with thrombin (12.5 U/ml) for 3 min. Platelet phospholipids were extracted by CHCI,: methanol (1 :2), and separated by HPTLC using CHC&: menthanol:methylamine (63:35: 10) as a developing solvent. Intensity of fluorescence in each phospholipid was measured by CAMAG HPTLC scanner. PE
0
/ 0
PI
PC
1
0
0.5
1 .o
1.5
2.0
( p9
I
0
0.5
1.0
1.5
2.0
PE ( pg
)
0
1 .o
2.0
3.0
4.0
PC ( p3
1
FIGURE 5. Standard curves of phospholipids. determination.
PI
Each point represents the mean of triplicate
38
-----______ --------______ -----*
SM
m PC .\A
PE
l
-o--------.
0‘
2.0
4.0
PI
8.0
6.0
Thrombin
10.0 (
U 1 ml
12.0
14.0
16.0
)
FIGURE 6A. Effects of thrombin concentration on phospholipid degradation in platelets. Washed rat platelets were incubated at 37°C for 3 min with thrombin (0.88, 2.63, 4.39, 7.02, 8.77, and 17.5 U/ml) in the presence of 3.5 mM Ca*+. Each point represents the mean of duplicates.
01
1
2 3 Incubation time ( min )
lncubaffo”
time
( min
1
o-
Incubation time ( min
,
Incubation time ( min )
FIGURE 6B. Time course of changes in platelet phospholipid composition following thrombin stimulation. Washed rat platelets were incubated for 5, 30 set, 1, 2, and 3 min at 37°C with thrombin (0.88 U/ml) in the presence of 2.9 mM Ca”. Each points represents the mean and S. E. of triplicates.
0
10
*Clca*+( liw
)
FIGURE 7A. The effect of Ca2* on the formation of malandialdehyde in rat platelets stimulated either with thrombin (-O--)(1.78 U/ml) for 3 min in the presence of 0.54,f.09,2.17, 4.35 10.3, and 20 .0 mM Ca2* I or with collagen f---A---) f25.0 t.kgfml) for 5 min in the presence of Ok~4~1.09, 2.17, $35, 8.09, 13.4, and 21.7 mM Ca”,
Time after stimulation
( min )
FIGURE 7&. Time course of the formation of mafondialdehyde in rat platekts &mutation either with thtombin (-a--_) (8.8 U/ml) or with collagen (---A---) (100 tt,g/mlI in the presence of25* m&t Caz’ .
r = 0.967
80 -
(A)
60 -
40.
20 *
A
OLb.
0.6
0.4 MDA
01
(p mol/pg
1.0
0.8 protein)
I
L
1.0
2.0
I
3.0
MDA (p mol/ug protein)
Correlation between aggregation and the formation of malondialdehyde in FIGURE 8A,B. rat platelets stimulated A) with thrombin (0.097-0.87 U/ml) for 3 min, and B) with collagen (0, 9.34, 18.7, 46.7, 93.4 and 186 @ml) for 5 min in the presence of 1.5 mM Ca*+.
Aggregation
0'
0.5
Properties of Washed Rat Platelets
1.0
TXB2 ( p mol / pg protein
1.5
2.0
)
FIGURE 8C. Correlation between the formation of thromboxane B2 and malondialdehyde in rat platelets stimulated with thrombin (0, 0.087, 0.11, 0.13, 0.15, 0.17, 0.22, 0.26, 0.44, 0.87, 1.74, and 8.71 U/ml) for 3 min in the presence of 1.5 mM Ca’+.
were 73 t 8.3 (8), 47 t 5.7 (8), and 8.7 IL 0.85 (6) kg/IO’ cells, respectively, measured by this micromethod. Phospholipid
Degradation
by Thrombin
when
Stimulation
The microanalysis of phospholipids described above made it possible to follow the stimulation-induced changes in the small amounts of phospholipids in platelet membranes without previous labeling that may result in unequal distribution of labeled substances in membrane phospholipids. Figure 6A shows percent changes of phospholipids when platelets were stimulated with thrombin at various concentrations. The degradation of PI reached a maximum at 7 U of thrombin/mI, while that of PC and PE reached a plateau at 3 U of thrombin/ml. The rate of degradation was greatest in Pi, followed by PE and PC, while the amount of sphingomyelin was unchanged by thrombin-stimulation. Figure 6B) shows the time course of phospholipid degradation when platelets were stimulated with 8.8 U of thrombin/ml. Degradation of PI occurred rapidly (21%, 30%, 39%, 44% at 30 set, 1, 2, and 3 min after being stimulated with thrombin). Formation
of MDA
Formation of MDA in platelets following stimulation was absolutely Ca*+-dependent. A maximum formation was observed at l-3 mM Ca2’, and the formation was gradually reduced with increasing concentrations over 3 mM. The Ca*’ dependency
41
42
T. Tomita et al.
(Figure
7A) and the time
with those formation
of platelet induced
Correlation
course
of MDA
aggregation.
by thrombin
between
production
lndomethacin
(0.42 U/ml)
(Figure
7B) were
(42 tJ,g/ml) inhibited
in the presence
consistent
by 76% MDA
of 1.5 mM Ca*+.
Aggregation,
MDA,
and TXBz Formation,
and MDA
formation
were measured
and PI
Degradation Aggregation
response
concomitantly
3 min
after the platelets were stimulated with various amounts of thrombin (0.057-0.87 U/ml) in the presence of 1.5 mM Ca*’ and the result is shown in Figure 8A which demonstrates 88 shows amounts
a strong
a similar of collagen
simultaneous
correlation
result
between aggregation
obtained
(9.34-186
assay of TXB2
when
platelets
and MDA formation.
were
stimulated
Kg/ml) for 5 min in the presence
and MDA
in platelets
stimulated
with
Figure various
of 1.5 mM Ca’+. A
with thrombin
(0.087-
8.71 U/ml) for 3 min in the presence of 1.5 mM Ca*+, shows a good correlation between the formation of the two substances in rat platelets (Figure 80. As shown in Figure
9, MDA
formation
phospholipid which shows dicate that MDA formation genase and thromboxane
also was correlated
with
the degradation
of PI, the
the greatest change after stimulation. These results inreflects phospholipid degradation, and also cyclo-oxysynthetase
activities
in rat platelets.
0.987 40 -
,’
@/ l
o
0
H .z 30 . z 2 0P =
20 .
r
lo: 0
l
l
/
.
/ 100
200
MDA ( p mol / pg protein
300
)
FIGURE 9. Correlation between malondialdehyde formation and the degradation of phosphatodylinositol (percent) in rat platelets stimulated with thrombin in the presence of 2.9 mM Ca’+. Each point represents the mean of triplicate determination.
Aggregation
Properties of Washed Rat Platelets
DISCUSSION Although aggregation response with platelet-rich plasma reflects platelet aggregability, and thus, platelet-rich plasma is widely used, various humoral factors influencing platelet aggregation are present in such preparations. Rat plasma contains four times more TXA2 than human plasma (Hwang et al., 19801, and no prostaglandin D2 (Moncada and Vene, 19781, which is inhibitory to human platelet aggregation. Production of PG12, a potent inhibitor of aggregation, also is found to be much higher in rat aorta than in the rabbit, the porcine, and the bovine (Morita et al., 1983). In addition, the concentration of these factors may fluctuate according to pathological conditions. For these reasons, washed platelets were used in this investigation to study rat platelet function. The presence of extracellular Ca2+ was required for the aggregation in washed rat platelets. The optimum concentration is about l-3 mM and concentrations greater than 3 mM reduced aggregation. Malondialdehyde formation was proposed by Smith et al. (1976) as an indicator of cycle-oxygenase and thromboxane synthetase activities and also the release of arachidonate in human platelets. Since there exists wide species variation in platelet responses to proaggregators and inhibitors (Dodds, 1978), and also in the role of TXA2 in aggregation, a simultaneous study of aggregation, MDA, and TXB, formation, and phospholipid degradation was undertaken in washed rat platelets. At the concentrations of thrombin and collagen indicated in the legends of the figures, a good correlation between aggregation and formation of MDA was observed. Furthermore, MDA formation was well correlated with TXB2 formation and PI degradation. Thrombin-induced aggregation and MDA formation were inhibited by indomethacin. These results strongly indicate that TXA, is an important endogenous proaggregator, and MDA formation reflects cycle-oxygenase and thromboxane synthetase activities, and phospholipid degradation in rat platelets as well as in human platelets. The authors are grateful to MS C. Nanjo for her technical assistance and to MS Y. Serizawa for typing the manuscript.
REFERENCES Baenziger NL, Majerus PW (1974) Isolation of human platelets and platelet surface membranes. Methods Enzyme/31:149-155. Dodds WJ (1978) Platelet function in animals: species specificities. In: Platelets: A Multidisciplinary Approach. Eds., C De Gaetano and S Garattini. New York: Raven Press, pp. 45-59. Dutilh CE, Haddeman E, Jouvenaz GH, Ten Hoor F, Nugteren DH (1979) Study of the two pathways for arachidonate oxygenation in blood platelets. Lipids 14:241-246.
Fitzpatrick F, Gorman RR (1977) Platelet rich plasma transforms exogenous prostaglandin endoperoxide Hz into thromboxane AZ. Prostaglandins 14:881-889.
Hayaishi 0, Watanabe T, Yamashita A, Ogorochi T, Narumiya S, Shimizu T (1982) Antithrombotic action of prostaglandins: Different roles of postacyclin and PGDZ. Adv Pharmacol Therap II 4:251260.
Hwang DH (1980) Aggregation and inhibition of rat platelets and the formation of endoperoxide me-
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T. Tomita et al. tabolites.
Prostaglandms
and
Medicine
5:163-
Hwang
DH,
variation their
Codke
RA,
in serum
precursor
lnoshita
K, lmaoka
K, Kosaki
Rings
levels
RW
(1980)
of prostaglandins
test.
Blood
suspension & Vessel
proteins.
IR, Zeugner
formance plasma plication
of 0.5 ~1 samples
I Chromatogr
after
tidisciplinary S Garattini. Morita fects
for
high-perin
direct
ap-
to the silica-gel
Eds.,
Approach.
I, Takahashi
In:
layer.
A Mu/-
G De Gaetano and
R, Saito Y, Murota
of eicosapentaenoic
platelet ag-
Platelets:
Raven Press,
H, Ohishi
pp. 239-258. S (1983) Ef-
acid on arachidonic
cells
Thromb
and
Res,
in
Philp
RB
Anal.
Biochem
(1970)
JB, lngerman
landin production Med
Behrens
WE
Silver
the
MJ (1976)
as an indicator
rat.
Malon-
of prostag-
by human platelets.
Latour
hibition
(1972) Evidence
of the circulating
man. Thromb
Diath
Haemorrh
JG (1977)
by B-adrenergic
let aggregation. and
in
23:129-139.
/. Lab C/in
881167-172.
canalisation Yu SK,
CM,
formation
of
and dipyri-
potentiation
Haemorrh
acid
inhibition
by adenosine
Paradoxical Diath
dialdehyde
by thiobarbituric 95:351-358.
Species-dependent
aggregation
damole: Smith
N, Yagi K (1978) Assay for lipid
in animal tissues
reaction.
Von
and thrombosis. New York:
Ohkawa
Thromb
of lipids
vascular
difference.
press.
platelet
146:261-271.
Moncada S, Vane JR (1978) Prostacyclin, gregation,
method
chromatography homogenates
for plate-
9:401-410.
S (1978) Quantitative
thin-layer
and liver
Anal Biochem
H, lshi
7:719-726.
RF, Gill DM (1964) A micro-biuret
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Species
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S, Ogawa M, Sasakuma
G (1976) Collagen
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Potentiation stimulations
A comparative platelets.
of phylogenetic platelet
by cy and inof rat plate-
study with human
Thrombos
Haemostas
TAKAKO TOMITA, KEIZO UMEGAKI, AND EIICHI HAYASHI
The basic characteristics of washed rat platelets in aggregation and biochemical responses to thrombin and collagen were investigated. Maximum aggregation response to thrombin and collagen was observed in the presence of l-3 mM extracellular CA’+. Breakdown of endogenous phospholipids was followed by HPTLC. Phosphatidylinositol degraded most rapidly and greatly, followed by ph~sphatidyiethanolamine, and phosphatidylchoiine. Sphingomyelin did not change on stimulation. Formation of malondialdehyde was closely associated with thrombin- and collagen-induced aggregation within the range of the concentration employed. In addition, it was correlated with TXBz formation and phosphatidylinositol degradation. These results indicated that, despite known species variation, thrombin-induced aggregation is mediated by TXA2 and malondialdehyde formation reflects both cycle-oxygenase and thromboxane synthaseactivities, and phospholipid degradation in rat platelets as well as in human platelets.
Key Words:
Platelet aggregation; Washed rat platelets; Malondialdehyde; pholipid degradation; Thromboxane
Phos-
INTRODUCTION Considerable information is available about platelet aggregation and the accompanying biochemical events. The information pertaining to platelet aggregation, however, has been gained mostly from human platelets, and such information is lacking for other species of animals, particularly for widely used laboratory animals such as the rat. The rat is a very useful animal species for studying the changes of platelet function due to various pathological conditions and also drug treatment. It has been pointed out that there are great differences in platelet morphology and function in species. Platelet size, circulating numbers, and volume vary enormously between species Won Behrens, 1972). The platelet size is smaller, and the circulating platelet counts (8-12 x 108/ml) are higher in the rat than in humans (1.54.0 x lOa/mI). Rat platelets behave differently, in general, with aggregating agents and aggregation-inhibiting agents. Adrenalin, a proaggregator in human platelets, is inhibitor to thrombin-induced aggregation in the rat (Vu and Latour, 1977). AdenFrom the Department of Pharmacology, Shizuoka College of Pharmaceutical Sciences, Shizuoka, Japan Address requests for reprints to: Takako Tomita, Department of Pharmacology, Shizuoka College of Pharmaceutical Sciences, Ohika, Shizuoka, Japan 422. Received January 25,1983; revised and accepted April l&1983.
31 Journal of Pharmacological Q, 1983 Elsevier
Science
Methods Publishing
IO, 31-44 Co.,
Inc.,
(1983) 52 Vanderbilt
Avenue,
New
York,
NY 10017
32
T. Tomita et al.
osine (Philp, 1970), PGD, (Hayaishi et al., 1982) and dipryidamole (Philp, 1970), known inhibitors of aggretation, do not exert inhibition on the rat platelet aggregation. Dipyridamole is reported to potentiate rat platelet aggregation. The mechanism of platelet aggregation has been extensively studied and a large body of evidence now indicates that TXA, plays a central role in platelet aggregation. Thromboxane synthetase catalyzes the concomitant isomerization and dismutation of PGHp to give TXA2, MDA, and HHT (12-L-hydroxyheptadecatrienoic acid). Thus platelet MDA can be regarded as an indicator of the activities of both cyclo-oxygenase and thromboxane synthetase, and of the release of arachidionic acid from platelet membrane phospholipids (Smith et al., 1976). In the case of rat platelets, however, there is some evidence that questions the role of TXA2 in aggregation. Hwang (1980) reported that preincubation of rat platelet-rich plasma with imidazole, a known inhibitor of thromboxane synthetase, did not attenuate the aggretation induced by ADP or collagen, whereas the synthesis of TXB, was inhibited by more than 90%. In human platelets, imidazole was shown to inhibit thromboxane synthesis and accompanying aggregation induced by PGH, in human platelet rich plasma (Fitzpatrick and Gorman, 1977). Dutilh (Dutilh et al., 1979) demonstrated that prostaglandin endoperoxide, which was rapidly converted into thromboxane AZ, did not cause aggregation in rat platelets, and also that collagen-induced aggregation of rat platelets was completely inhibited by eicosatetrayenoic acid at the concentration at which lipoxygenase activity was completely blocked with an apparent stimulation of cyclooxygenase. Therefore, this study was designed to investigate the basic aggregation characteristics of washed rat platelets, and particularly to assess the significance of MDA assay in aggregation reaction. METHODS Preparation
of Washed
Platelets
Washed platelets were prepared principally according to Baenziger and Majerus (1974). Blood was collected into a siliconized centrifuging tube containing one portion of acid-citrate-dextrose anticoagulant (0.085 M trisodium citrate, 0.065 M citric acid, 2% dextrose) to five portions of blood, and the mixture was consecutively centrifuged for 15 min at 230 x g (1200 rpm) and for 3 min at 800 x g (220 rpm) at room temperature. This two-step centrifugation was employed to prevent a loss of platelets of larger sizes. PRP (platelet-rich plasma) separated from the two centrifugations were mixed, and centrifuged for 7 min at 120 x g (800 rpm) to sediment red and white cells. The supernatant was then centrifuged for 15 min at 1700 x g (3200 rpm). The sedimented platelets were resuspended in the same volume of a washing buffer (0.113 M NaCI, 4.3 mM K2HP04, 4.3 mM Na2HP04, 24.4 mM NaH2P04, 5.5 mM glucose, 1 mM EDTA, pH 6.5) as the whole blood and centrifuged for 15 min, at 1500 x g (3000 rpm) at 4°C. The washed platelets were resuspended in a resuspending buffer (0.14 M NaCI, 15 mM Tris-HCI, 5 mM glucose, pH 7.4) to make a suspension
of about 4 x IO8 cells/ml.
The washed
platelet
suspensions
were
kept at 4°C until used, within 4 hr. The intactness of platelets was ensured throughout all the preparatory stages by measuring the released of lactic acid dehydrogenase activity in the supernatant.
Aggregation Properties of Washed Rat Platelets Measurement
of Platelet Aggregation
The degree of platelet aggregation was measured by the turbidometric method using 4 channeled NKK Hematracer 1 (Niko Bioscience, Tokyo). To 200 ~1 of platelet suspension (adjusted to contain about 4 x IO8 cells/ml), IO ~1 of CaCI, was added to give a final concentration indicated in the legends. 20 ~1 of various concentrations of thrombin or collagen were added after 1 min incubation at 37°C with constant stirring at 1000 rpm, and changes in turbidity were recorded. A maximum aggregation was measured 3 min and 5 min after the addition of thrombin and collagen, respectively. Human plasma thrombin (Midori Cross Co., Osaka, Japan) was diluted with the resuspending buffer. The solution of collagen (Type I, insoluble, Sigma, St. Louis, MO) was prepared according to lnoshita et al. (1976), and collagen in the solution was determined by the method of ltzhaki et al. (1964). Incubation
of Platelets
A suspension of washed rat platelets (4 x IO8 cells/ml) was incubated either with collagen or with thrombin at 37°C in the presence of 1.5 mM Ca’+. Incubation conditions are described in detail in the legend for each figure. Extraction of Platelet Phospholipids To 0.5 ml of platelet suspension, 2.2 ml of a mixture of CHC&: MeOH (1 : 2) was added to stop the incubation. The mixture was shaken vigorously for 1 min with 0.71 ml of CHC& and 0.71 ml of 2 M KCI solution containing 5 mM EDTA, and centrifuged for 30 min at 3000 rpm. The lower layer was withdrawn and the solvent was removed under a vacuum. Analysis of Platelet Phospholipids The phospholipid extract was dissolved in 50 ~1 of a mixture of CHC& : MeOH : HZ0 (75 : 25 : 2), and a portion of the extract was applied on a HPTLC plate (Kieselgel 60, art No. 5641, 10 x 20 cm, Merch, Darmstadt, Germany.), which was rinsed with a mixture of CHC13 : MeOH (2 : 1) overnight and activated by heating for 1 hr at 110°C. The plate was developed at 4°C in a mixture of CHC&: MeOH: CH3NH2 (40%) (63 : 35 : 10). After the solvents were completely removed under a hot air stream, the plate was heated for 10 hr at 150°C with ammonium bicarbonate in a sealed chamber to develop fluorescence (Kupke, 1978). The intensity of fluorescence in each phospholipids was measured by a CAMAG TLC Scanner (Muttenz, Swiss). Analysis of MDA To 0.25 ml of platelet suspension, 0.1 ml of 3.1 M trichloroacetic acid (final concentration, 0.8 M) was added to stop the incubation. Malondialdehyde formed by platelet reaction was measured principally according to Ohkawa et al. (1978). A portion of the mixture (0.4 ml) was mixed with 0.4 ml of 3% sodium dodecylsulfate (Wako Chemicals, Tokyo), and then heated at 70°C for 45 min with 1.6 ml of 0.1 N HCI, 0.8 ml of 10% phosphotungstic acid, and 0.8 ml of 0.7% thiobarbituric acid (Nakarai Chemicals, Tokyo). The reaction was terminated by chilling, and the flu-
33
34
T. Tomita et al.
orescent products were extracted with 4 ml of n-butanol. The intensity of fluorescence was measured as percent fluorescence of Rhodamin B (20 ng/ml) by a Hitachi fluorescence Photmeter (Hitachi 204, Hitachi Co. LTD, Tokyo) (excitation 515 nm, emission 553 nm). For a calibration curve, 50-860 p mol of 1, 1, 3, 3-tetraethoxypropane (a gift from Eizai Pharmaceuticals, Tokyo, Japan) in 0.4 ml of 0.8 M trichloroacetic acid, was submitted to the reaction as described above, and the fluorescence was measured. Radioimmunoassay
of TXB2
To 0.5 ml of platelet suspension, 2 M citric acid was added to adjust pH to 3.0 and to stop the reaction. The mixture was chilled in liquid nitrogen and kept at -80°C till assayed. The thawed samples were neutralized with 1 M NaOH, and thromboxane B2 in the diluted samples was assayed by using thromboxane B, t3Hl RIA Kit (NEN, Boston, MA).
RESULTS Aggregation
Response
in Rat-Washed
Platelets
Figure 1 shows typical aggregation response to thrombin and collagen in washed platelets from Wistar Kyoto rats in the presence of 1.5 mM Ca*‘. Aggregation reached a maximum at 3 min with thrombin and at 5 min with collagen. As Figure 2A shows, aggregation was dose-dependent in the range of 0.1-0.3 U of thrombin/ml
83.7 )rg/ml 0.44 U/ml
16.7 pg/ml 0.17 U/ml 8.4 pg/ml
20
0.13 U/ml
tr 0-
12
3
'123456
4 Time
(
min
)
FIGURE 1. Typical aggregation response to three concentrations of a) thrombin and b) collagen in washed rat (Wistar Kyoto) platelets in the presence of 1.5 mM Ca2+.
100.
35 _-_--A ___--/___-----;--
80'
l
/-&---
"
/
/'/'
60 .
/
,' E .c +I z?
i
:
;40. 4"
:'
I a
f
0'
0.1
0.2
0
50
100
0.3 150
0.4
Thranbin
U/ml
200
Collagen
pg/ml
FIGURE 2A. Platelet aggregation induced by various concentrations of thrombin (-•-_) and collagen (-A-). Washed rat (WKY) platelets were stimulated either with thrombin (0.087, 0.11, 0.13, 0.15, 0.17, 0.22, 0.26, and 0.44 U/ml) or with collagen (6.2, 7.5, 9.3, 12.5, 18.7, 46.7, 93.4, and 187 pg/ml) in the presence of 1.5 mM Ca*+.
FIGURE 2B. Effects of Ca*’ concentration on platelet aggregation. Washed rat (WKY) platelets were stimulated either with thrombin (0.22 U/ml) in the presence of Ca’+ (0.125, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, 10.0, and 20.0 mM) or with collagen (lo.7 pg/ml) in the presence of Ca2+ (0.5, 1.0, 2.0, 9.0, and 23.0 mM). Each point represents mean for triplicates.
343 T. Tomita et al. and IO-100 pg collagen/ml. The thrombin concentration at which 50% aggregation was induced varies even within strains of rats. It was between 0.15-0.2 U/ml in Wistar Kyoto rats, but between 0.6-0.7 U/ml in Wistar Mishima. lndomethacin (42 pgiml) inhibited thrombin (0.42 U/ml)-induced aggregation by 69% in the presence of 1.5 mM Ca*‘. Fig 2 B shows the strong dependency of rat platelet aggregation on Ca” concentration. Thrombin (0.22 U/ml) or collagen (10.7 F./ml) scarcely induced aggregation with washed rat platelets in the absence of Ca2+. The presence of l-3 mM Ca*‘, however, induced a maximum aggregation response to thrombin and collagen, while at the higher concentration aggregation was inversely reduced with the concentration. Thrombin-induced aggregation was completely inhibited at 20 mM of Ca*‘. The cell density of rat platelet preparations did not affect the aggregability within the range of 2-6 x IO’ cells/ml (Figure 3). Analysis of Platelet Phospholipids Phospholipids extracted from platelets before and after stimulation with thrombin were separated by HPTLC and fluorescence was developed by heating the plate with ammoniumbicarbonate for IO hr at 150°C. Figure 4 shows the profile of rat platelet phospholipids. A linear correlation was observed between the intensity of fluorescence developed on each phospholipid and the concentration of phospholipids (Figure 5). Contents of PC, PE, and PI in washed rat platelets (unstimulated) 100
I
-0 .------_-__-______ A
0
1 2.0 Cell density
4.0
( x l@
6.0 Cells
8.0 / ml
)
FIGURE 3. The dependency of aggregation on the cell density of rat platelet suspension. Washed rat (WKY) platelets were stimulated with thrombin (-•-) (0.44 U/ml) and with collagen (---A---) (46.7 @ml) in the presence of 1.5 mM Ca*+.
Control
(thrombln
Stimulated 12.5
U/ml,
3 min
)
PE
PC SM
PI
i
FIGURE 4. A profile of rat platelet phospholipids before and after stimulation with thrombin (12.5 U/ml) for 3 min. Platelet phospholipids were extracted by CHCI,: methanol (1 :2), and separated by HPTLC using CHC&: menthanol:methylamine (63:35: 10) as a developing solvent. Intensity of fluorescence in each phospholipid was measured by CAMAG HPTLC scanner. PE
0
/ 0
PI
PC
1
0
0.5
1 .o
1.5
2.0
( p9
I
0
0.5
1.0
1.5
2.0
PE ( pg
)
0
1 .o
2.0
3.0
4.0
PC ( p3
1
FIGURE 5. Standard curves of phospholipids. determination.
PI
Each point represents the mean of triplicate
38
-----______ --------______ -----*
SM
m PC .\A
PE
l
-o--------.
0‘
2.0
4.0
PI
8.0
6.0
Thrombin
10.0 (
U 1 ml
12.0
14.0
16.0
)
FIGURE 6A. Effects of thrombin concentration on phospholipid degradation in platelets. Washed rat platelets were incubated at 37°C for 3 min with thrombin (0.88, 2.63, 4.39, 7.02, 8.77, and 17.5 U/ml) in the presence of 3.5 mM Ca*+. Each point represents the mean of duplicates.
01
1
2 3 Incubation time ( min )
lncubaffo”
time
( min
1
o-
Incubation time ( min
,
Incubation time ( min )
FIGURE 6B. Time course of changes in platelet phospholipid composition following thrombin stimulation. Washed rat platelets were incubated for 5, 30 set, 1, 2, and 3 min at 37°C with thrombin (0.88 U/ml) in the presence of 2.9 mM Ca”. Each points represents the mean and S. E. of triplicates.
0
10
*Clca*+( liw
)
FIGURE 7A. The effect of Ca2* on the formation of malandialdehyde in rat platelets stimulated either with thrombin (-O--)(1.78 U/ml) for 3 min in the presence of 0.54,f.09,2.17, 4.35 10.3, and 20 .0 mM Ca2* I or with collagen f---A---) f25.0 t.kgfml) for 5 min in the presence of Ok~4~1.09, 2.17, $35, 8.09, 13.4, and 21.7 mM Ca”,
Time after stimulation
( min )
FIGURE 7&. Time course of the formation of mafondialdehyde in rat platekts &mutation either with thtombin (-a--_) (8.8 U/ml) or with collagen (---A---) (100 tt,g/mlI in the presence of25* m&t Caz’ .
r = 0.967
80 -
(A)
60 -
40.
20 *
A
OLb.
0.6
0.4 MDA
01
(p mol/pg
1.0
0.8 protein)
I
L
1.0
2.0
I
3.0
MDA (p mol/ug protein)
Correlation between aggregation and the formation of malondialdehyde in FIGURE 8A,B. rat platelets stimulated A) with thrombin (0.097-0.87 U/ml) for 3 min, and B) with collagen (0, 9.34, 18.7, 46.7, 93.4 and 186 @ml) for 5 min in the presence of 1.5 mM Ca*+.
Aggregation
0'
0.5
Properties of Washed Rat Platelets
1.0
TXB2 ( p mol / pg protein
1.5
2.0
)
FIGURE 8C. Correlation between the formation of thromboxane B2 and malondialdehyde in rat platelets stimulated with thrombin (0, 0.087, 0.11, 0.13, 0.15, 0.17, 0.22, 0.26, 0.44, 0.87, 1.74, and 8.71 U/ml) for 3 min in the presence of 1.5 mM Ca’+.
were 73 t 8.3 (8), 47 t 5.7 (8), and 8.7 IL 0.85 (6) kg/IO’ cells, respectively, measured by this micromethod. Phospholipid
Degradation
by Thrombin
when
Stimulation
The microanalysis of phospholipids described above made it possible to follow the stimulation-induced changes in the small amounts of phospholipids in platelet membranes without previous labeling that may result in unequal distribution of labeled substances in membrane phospholipids. Figure 6A shows percent changes of phospholipids when platelets were stimulated with thrombin at various concentrations. The degradation of PI reached a maximum at 7 U of thrombin/mI, while that of PC and PE reached a plateau at 3 U of thrombin/ml. The rate of degradation was greatest in Pi, followed by PE and PC, while the amount of sphingomyelin was unchanged by thrombin-stimulation. Figure 6B) shows the time course of phospholipid degradation when platelets were stimulated with 8.8 U of thrombin/ml. Degradation of PI occurred rapidly (21%, 30%, 39%, 44% at 30 set, 1, 2, and 3 min after being stimulated with thrombin). Formation
of MDA
Formation of MDA in platelets following stimulation was absolutely Ca*+-dependent. A maximum formation was observed at l-3 mM Ca2’, and the formation was gradually reduced with increasing concentrations over 3 mM. The Ca*’ dependency
41
42
T. Tomita et al.
(Figure
7A) and the time
with those formation
of platelet induced
Correlation
course
of MDA
aggregation.
by thrombin
between
production
lndomethacin
(0.42 U/ml)
(Figure
7B) were
(42 tJ,g/ml) inhibited
in the presence
consistent
by 76% MDA
of 1.5 mM Ca*+.
Aggregation,
MDA,
and TXBz Formation,
and MDA
formation
were measured
and PI
Degradation Aggregation
response
concomitantly
3 min
after the platelets were stimulated with various amounts of thrombin (0.057-0.87 U/ml) in the presence of 1.5 mM Ca*’ and the result is shown in Figure 8A which demonstrates 88 shows amounts
a strong
a similar of collagen
simultaneous
correlation
result
between aggregation
obtained
(9.34-186
assay of TXB2
when
platelets
and MDA formation.
were
stimulated
Kg/ml) for 5 min in the presence
and MDA
in platelets
stimulated
with
Figure various
of 1.5 mM Ca’+. A
with thrombin
(0.087-
8.71 U/ml) for 3 min in the presence of 1.5 mM Ca*+, shows a good correlation between the formation of the two substances in rat platelets (Figure 80. As shown in Figure
9, MDA
formation
phospholipid which shows dicate that MDA formation genase and thromboxane
also was correlated
with
the degradation
of PI, the
the greatest change after stimulation. These results inreflects phospholipid degradation, and also cyclo-oxysynthetase
activities
in rat platelets.
0.987 40 -
,’
@/ l
o
0
H .z 30 . z 2 0P =
20 .
r
lo: 0
l
l
/
.
/ 100
200
MDA ( p mol / pg protein
300
)
FIGURE 9. Correlation between malondialdehyde formation and the degradation of phosphatodylinositol (percent) in rat platelets stimulated with thrombin in the presence of 2.9 mM Ca’+. Each point represents the mean of triplicate determination.
Aggregation
Properties of Washed Rat Platelets
DISCUSSION Although aggregation response with platelet-rich plasma reflects platelet aggregability, and thus, platelet-rich plasma is widely used, various humoral factors influencing platelet aggregation are present in such preparations. Rat plasma contains four times more TXA2 than human plasma (Hwang et al., 19801, and no prostaglandin D2 (Moncada and Vene, 19781, which is inhibitory to human platelet aggregation. Production of PG12, a potent inhibitor of aggregation, also is found to be much higher in rat aorta than in the rabbit, the porcine, and the bovine (Morita et al., 1983). In addition, the concentration of these factors may fluctuate according to pathological conditions. For these reasons, washed platelets were used in this investigation to study rat platelet function. The presence of extracellular Ca2+ was required for the aggregation in washed rat platelets. The optimum concentration is about l-3 mM and concentrations greater than 3 mM reduced aggregation. Malondialdehyde formation was proposed by Smith et al. (1976) as an indicator of cycle-oxygenase and thromboxane synthetase activities and also the release of arachidonate in human platelets. Since there exists wide species variation in platelet responses to proaggregators and inhibitors (Dodds, 1978), and also in the role of TXA2 in aggregation, a simultaneous study of aggregation, MDA, and TXB, formation, and phospholipid degradation was undertaken in washed rat platelets. At the concentrations of thrombin and collagen indicated in the legends of the figures, a good correlation between aggregation and formation of MDA was observed. Furthermore, MDA formation was well correlated with TXB2 formation and PI degradation. Thrombin-induced aggregation and MDA formation were inhibited by indomethacin. These results strongly indicate that TXA, is an important endogenous proaggregator, and MDA formation reflects cycle-oxygenase and thromboxane synthetase activities, and phospholipid degradation in rat platelets as well as in human platelets. The authors are grateful to MS C. Nanjo for her technical assistance and to MS Y. Serizawa for typing the manuscript.
REFERENCES Baenziger NL, Majerus PW (1974) Isolation of human platelets and platelet surface membranes. Methods Enzyme/31:149-155. Dodds WJ (1978) Platelet function in animals: species specificities. In: Platelets: A Multidisciplinary Approach. Eds., C De Gaetano and S Garattini. New York: Raven Press, pp. 45-59. Dutilh CE, Haddeman E, Jouvenaz GH, Ten Hoor F, Nugteren DH (1979) Study of the two pathways for arachidonate oxygenation in blood platelets. Lipids 14:241-246.
Fitzpatrick F, Gorman RR (1977) Platelet rich plasma transforms exogenous prostaglandin endoperoxide Hz into thromboxane AZ. Prostaglandins 14:881-889.
Hayaishi 0, Watanabe T, Yamashita A, Ogorochi T, Narumiya S, Shimizu T (1982) Antithrombotic action of prostaglandins: Different roles of postacyclin and PGDZ. Adv Pharmacol Therap II 4:251260.
Hwang DH (1980) Aggregation and inhibition of rat platelets and the formation of endoperoxide me-
43
44
T. Tomita et al. tabolites.
Prostaglandms
and
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5:163-
Hwang
DH,
variation their
Codke
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precursor
lnoshita
K, lmaoka
K, Kosaki
Rings
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test.
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tidisciplinary S Garattini. Morita fects
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Approach.
I, Takahashi
In:
layer.
A Mu/-
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acid on arachidonic
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landin production Med
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Silver
the
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and dipyri-
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N, Yagi K (1978) Assay for lipid
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Moncada S, Vane JR (1978) Prostacyclin, gregation,
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RF, Gill DM (1964) A micro-biuret
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G (1976) Collagen
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A comparative platelets.
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