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Some properties of rat platelet aggregation and effects of butylated hydroxytoluene, warfarin and aspirin
Fd Chem. Toxic. Vol. 29, No. 3, pp. 173-183, 1991
0278-6915/91 $3.00+ 0.00 Copyright© 1991 PergamonPress plc
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SOME PROPERTIES OF RAT PLATELET AGGREGATION A N D EFFECTS OF BUTYLATED HYDROXYTOLUENE, W A R F A R I N A N D ASPIRIN O. TAKAHASHI Department of Toxicology, Tokyo Metropolitan Research Laboratory of Public Health, 24-1 Hyakunincho 3-chome, Shinjuku-ku, Tokyo 169, Japan (Received 20 July 1990; revisions received 29 November 1990)
Abstract--The platelet aggregation characteristics of male Sprague-Dawley (JcI:SD) rats were investigated. Epinephrine, ristocetin, serotonin and platelet-activating factor were ineffective in rat platelets. Heparinized platelet-rich plasma (PRP) was more sensitive than citrated PRP to three aggregating agents, ADP, collagen and arachidonic acid. Butylated hydroxytoluene (BHT) and BHT quinone methide (2,6-di.tert-butyl-4-methylene-2,5-cyclohexadienone) inhibited ADP- and collagen-induced aggregation at concentrations over 10-3M in vitro. The ADP-, collagen- and arachidonic acid (0.5-2.0mM)-induced aggregations of PRP obtained from rats given 1.20% BHT in the diet for 7 days were normal, while arachidonic acid (3.9 mM)-induced aggregation of PRP from BHT-fed rats was significantly lower than control. PRP from rats given aspirin and warfarin also aggregated normally with ADP or collagen addition. These results suggest that heparinized PRP may be preferable in platelet aggregation analyses in rats and reaffirmed that effects on platelet aggregation may not play a key role in BHT-induced bleeding. Platelet aggregation capacity also does not necessarily reduce in haemorrhages induced by aspirin or warfarin. INTRODUCTION It is well known that the food antioxidant butylated hydroxytoluene (BHT) can cause haemorrhagic death in rats (Takahashi and Hiraga, 1978). This effect is both strain- and sex-dependent. Compared with other strains, Jcl:SD male rats had the highest death rate when exposed to BHT, and females were less sensitive to BHT than males (Takahashi et al., 1980). We have reported that a central event in BHT-induced bleeding may be defective blood coagulation due to a deficiency in coagulation factors II, VII, IX and X following the inhibition of the biosynthesis of those four vitamin-K-dependent factors by 2,6-di-tertbutyl-4-methylene-2,5-cyclohexadienone (BHT quinone methide), an active metabolite of BHT, in the liver (Takahashi, 1986, 1987 and 1988a,b). We have also examined the effects of BHT on some properties and functions of platelets: particle distribution, fatty-acid composition, factor-3 availability and aggregation activity, and have concluded that those factors probably do not play such a critical role as inadequate coagulation in the early stages of this bleeding (Takahashi, 1985 and 1986; Takahashi and Hiraga, 1981 and 1984). However, the method used to assay platelet aggregation capacity described in our previous reports was not necessarily a standardized one because we had no platelet aggregation analyser. Since we have now obtained this instrument, we now report our re-examination of the effects of BHT on platelet aggregation. It has been established that the aggregation properties of rat platelets differ greatly from those of man Abbreviations: BHT = butylated hydroxytoluene; DMSO =
dimethylsulpboxide; PAF = platelet activating factor; PPP = platelet-poorplasma; PRP = platelet-rich plasma.
and other species; rat platelets do not show the secondary aggregation that human platelets do (MacMillan and Sim, 1970). The doses of A D P and collagen required to induce aggregation in rat platelets are much higher than in human platelets (MacMillan and Sim, 1970). The relationship between the aggregation of rat platelets and drugs is yet more complicated, the reactions have different features from those of human platelets (Tanimoto, 1982). Furthermore, although it is unequivocal that platelets are a prerequisite for human haemostasis and a haemostatic model made from the study of human platelets is tentatively used for an interpretation of the rat haemostatic mechanism, the role of rat platelets in stopping bleeding is extremely obscure. It is questionable whether the human model is entirely applicable to rats. It is believed that platelet thrombi play a key role in stopping bleeding. The formation of platelet thrombi can actually be observed at an injured site of a vessel in a human model. Thilo and Bfhm (1973), however, did not find this formation in a rat model. There is, in fact, one report of the possible formation of white thrombi at injured tissues, which may be a crucial stage in human haemostasis (Thilo and Bohm, 1973). To shed further light on the behaviour of rat platelets, we have also examined the effects of warfarin and aspirin on the aggregation of rat platelets, since these drugs are known oral anticoagulants and antiaggregants for human platelets. MATERIALS AND METHODS
Chemicals. Platelet aggregation reagents, adenosine diphosphate (ADP) and calf-tendon collagen were obtained from Niko Bioscience Inc. (Tokyo, Japan), and arachidonic acid from porcine liver
173
174
O. TAKAHASHI
(purity c. 90%), fl-acetyl-7-O-(octadec-9-cis-enyl)L-~t-phosphatidylcholine (platelet activating factor: PAF; purity c. 99%), ristocetin, epinephrine and calf-skin collagen were from Sigma Chemical Co. or Sigma Diagnostics (St Louis, MO, USA). Sodium salts of citric acid, oxalic acid and heparin were purchased from Wako Pure Chemical Industries (Osaka, Japan), and 0.02 M-calcium chloride solution was from International Reagent Corp. (Kobe, Japan). BHT, 3-(~t-acetonylbenzyl)-4-hydroxycoumarin (warfarin) and acetylsalicylic acid (aspirin; Japan Pharmacopoeia) were from Tokyo Kasei Kogyo Co. (Tokyo, Japan), Sigma Chemical Co. (St Louis, MO, USA) and Tsukishima Yakuhin K. K. (Tokyo, Japan), respectively. BHT quinone methide was synthesized as described previously (Takahashi and Hiraga, 1979a). Animals and treatments. Male Sprague-Dawley (Jcl:SD) rats, purchased when 4 wk old, were used for all experiments. For experiments 1, 2 and 3, rats (10-20wk old) raised on a laboratory ration were killed to collect platelets after starvation for 18 hr. In experiments 4 and 5, rats (5-6 wk old) were given BHT, warfarin or aspirin in a purified diet for 4-7 days. The composition of the diet was as described previously (Takahashi and Hiraga, 1978 and 1979b). Preparation ofplatelets. Whole blood was collected (under ether anaesthesia) from the inferior vena cava into plastic syringes containing 0.124M-sodium citrate solution, 0.124M-sodium oxalate solution or 1000 units/ml sodium heparin (in saline) solution (anticoagulant solution-blood, 1:9, v/v), carefully avoiding formation of a bubble. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared by centrifugation of blood at 60-80g for 10 min and 1550 g for 25 min, respectively, at room temperature. Platelet aggregation analysis. Platelet aggregation of PRP was determined by the optical method described originally by Born (1962) using an NBS eight-channel platelet aggregometer (NBS Hema Tracer, Niko Bioscience Inc., Tokyo, Japan). PRP (200/zl) was placed in a siliconized cuvette, containing a siliconized stirring bar, placed in an aggregometer at 37°C and stirred at 1000 rpm. After the light-transmittance of PRP was adjusted at 0% against PPP of 100%, aggregation was observed as the change in turbidity.
Effects of aggregating agents on the aggregation of platelets from normal rats (experiment I). Following the addition of ADP, collagen, arachidonic acid, PAF, ristocetin or epinephrine to PRP, platelet aggregation was recorded. Arachidonic acid was used after solubilization equimoles of sodium carbonate and dilution with saline. PAF, ristocetin and epinephrine did not cause aggregation of PRP. The dependence of ADP-, collagen- and arachidonic acid-induced aggregation on their concentrations was examined.
Effects of anticoagulants on platelet aggregation of normal rats (experiment 2). The different effects of citrate, oxalate and heparin on aggregation were studied, and that of calcium chloride on platelet aggregation was also examined.
Effects of BHT or BHT quinone methide on platelet aggregation in vitro (experiment 3). The effects of BHT on citrated or heparinized PRP were studied in vitro. For citrated PRP, methanol and dimethyl-
sulphoxide (DMSO), at final concentrations of 5%, inhibited ADP-induced aggregation partially, and acetone, Tween 20 and linoleic acid (5%) did so completely. Collagen-induced aggregation was completely inhibited by 5% methanol and DMSO. To determine the effects of BHT or BHT quinone methide on ADP-induced platelet aggregation in vitro, BHT or its metabolite dissolved in methanol or DMSO was used. To determine the effects of BHT or BHT quinone methide on collagen-induced aggregation, a portion of the methanol solution of these compounds was transferred to a reaction tube; after evaporation of its solvent, PRP was poured into a tube containing dried BHT, and platelet aggregation was measured. For arachidonic acid-induced aggregation, the effects of BHT in methanol were measured.
Effects of BHT feeding on platelet aggregation (experiment 4). Rats were fed a diet containing 1.2% BHT for either 4 or 7 days. After PRP was prepared from heparinized blood, ADP-, collagen- and arachidonic acid-induced aggregations were measured.
Effects of feeding warfarin or aspirin on ADP- and collagen-induced platelet aggregation (experiment 5). After rats were fed diets containing 1.2% aspirin or 0.001% warfarin for 7 or 4 days, respectively (Takahashi and Hiraga, 1979b and 1981), ADPand collagen-induced platelet aggregations were measured using heparinized PRP. Statistics. Data were routinely expressed as mean_+ SEM and were analysed statistically by Student's t-test. Significance was assumed at P < 0.05 (Gad and Weil, 1982). RESULTS
Effects of aggregating agents on the aggregation of platelets from normal rats (experiment 1) Maximum platelet aggregations (%) of both titrated and heparinized PRPs taken from normal rats caused by ADP, collagen, ristocetin, epinephrine, arichidonic acid and PAF are shown in Table 1. ADP and collagen obviously induced aggregation in both PRPs (Figs 1 and 2). ADP at a concentration of 9.5/~M provoked 62-66% of maximum aggregation after about 2 min. However, calf-tendon collagen at about 10 #g/ml could not always induce aggregation in citrated PRP. Curves obtained from platelets capable of aggregating by chance are shown in Fig. 2. A large amount of calf-skin collagen also induced aggregation with a different pattern in citrated PRP (Fig. 2C). Collagen at lower levels caused more platelet aggregation in heparinized PRP than in citrated PRP (Fig. 2D). Arachidonic acid induced aggregation only in heparinized PRP (Fig. 3). Ristocetin, epinephrine and PAF did not cause notable aggregation.
Effects of certain anticoagulants on platelet aggregation of normal rats (experiment 2) Platelet aggregation by ADP and collagen was compared using citrated, oxalated and heparinized PRP. In both ADP- and collagen-induced aggregation, heparinized PRP produced much greater aggregation in response to the lower concentrations of the aggregants than did citrate and oxalate (Table 2).
BHT and platelet aggregation Table
1. Activity of certain aggregating alatelets in PRP from normal
Maximum platelet aggregation (“/)
Aggregating agent and concn Citrated
agents on rat rats
PRP
ADP (9.52 PM) ADP (31.7 PM) ADP (95.2 HIM)
62 59 50
ADP ADP ADP ADP
14 31 65 66
(0.952 PM) (4.76 PM) (9.52 PM) (9.52 PM)
Collagen* Collagen*
(4.8 Kg/ml) (20.0 pg/ml)
9 63
Collagen* Collagen’ Collagen* Collagen*
(9.0 pg/ml) (13.0 fig/ml) (16.7yg/ml) (20.0 fig/ml)
9 21 29 33
Collagen* Collagen* Collagen*
(13.0 pg/ml) (16.7 pg/ml) (20.0 rg/ml)
48 41 61
Collagen’
(9.1 pg/ml)
69
Collagen? Collagen? CoIlagent Collagen?
(95.2 fig/ml) (I 8 1.8 fig/ml) (260.8 pg/ml) (333.3 fig/ml)
0 0 89 95
CoIlagent Collagen?
(190.5 pg/ml) (400.0 pg/ml)
0 74
Ristocetin
(7.1 mg/ml)
Epinepbrine
0 0
(47.6 PM)
0
AA (2.0 mhi) Heparinized
PRP
ADP (4.88 FM) ADP (9.52 PM)
63 65
ADP (0.0952 PM) ADP (0.952 PM) ADP (9.52 /LM)
19 66 72
Collagen* Collagen*
(4.8 pg/ml) (9.1 /~g/ml)
10 76
Collagen* Collagen* Collagen*
(1.2 pg/ml) (2.5 pg/ml) (4.8 pg/ml)
18 41 58
Ristocetin Ristocetin
(7.14 mg/ml)
(I 3.6 mg/ml)
5 IO
Ristocetin
(30.0 mg/ml)
0
Epinepbrine Epinephrine
(4.76 pM) (16.7 phi)
4 24
Epinephrine Epinephrine Epinepbrine
(9.09 PM) (20.0 PM) (90.9 PM)
0 0 0
AA (0.5 rnMj AA i2.0m~j AA (3.9 mM)
0 78 21
AA (3.9 mhf)
59
AA (3.9 mM)
48
AA AA AA AA
(0.2 (0.5 (2.0 (3.9
mM) mM) mM) mhf)
62 68 69 65
AA AA AA AA
(0.2 (0.5 (2.0 (3.9
mM) mM) mM) mM)
0 69 68 66
PAF (8.67 /‘M) PAF (86.7 /LM)
0 0
*Calf-tendon collagen obtained from Niko Bioscience Inc. TCalf-skin collagen obtained from Sigma Chemical Co. or Sigma Diagnostics. Data in each column were obtained from the same PRP of a single rat.
175
ADP-induced aggregation of titrated PRP was increased by the addition of calcium chloride (Fig. 4) while the low reactivity of titrated PRP to collagen could not be raised by the addition of Ca*+. Arachidonic acid caused aggregation in heparinized but not in titrated PRP. Ca*+ addition restored the low reactivity of titrated PRP to arachidonic acid to a very limited extent (Fig. 3). Eflects of BHT or BHT quinone methide on platelet aggregation in vitro (experiment 3) Effects on ADP- and collagen-induced rat platelet aggregations of normal PRP by BHT or BHT quinone methide (Table 3 and Fig. 5) show that BHT inhibited both types of aggregations at concentrations of more than about 10m3M (Fig. 5). Inhibition was observed in both titrated and heparinized PRP. BHT quinone methide also inhibited these aggregations to a lesser extent. BHT at the lo-‘M level did not inhibit PRP aggregation induced by arachidonic acid (Table 3). BHT over 0.455 x 10e3 M also inhibited the disaggregation at 5-10min after ADP-induced aggregation (Fig. 6 ana Table 4) while somewhat activating platelet aggregation with the maximum at c. 1.0 min over 4.55 x 10m3M (Fig. 6 and Table 5). Effects of BHTfeeding on platelet aggregation (experiment 4) Body-weight gains, food intakes and BHT intakes of rats given 1.20% BHT for 4 or 7 days are shown in Table 6. Haemorrhages in epididymal adipose tissues were observed in all animals maintained on the BHT diet for 7 days. Bleeding in the peritoneal cavity and testes was found in one rat of this group and haemorrhagic anaemia in another. A conspicuous decrease in haematocrit, probably due to bleeding, was found in two out of seven rats given BHT for 7days. Maximum platelet aggregations of heparinized PRP induced by ADP and collagen are shown in Table 7. The mean maximum aggregation induced by 0.95 PM-ADP was increased significantly in animals given BHT for 4 days, while no significant differences were observed in other values. Rats fed 1.20% BHT for 7 days to measure the arachidonic acidinduced aggregation developed haemorrhages in the epididymis and some other organs. There was no significant difference in aggregation between control and BHT-administered rats at the 2.0-mM concentration of arachidonic acid but BHT produced a significant increase in aggregation at the 0.5-mM concentration of arachidonate and a significant decrease in aggregation at the 3.9-mM concentration of arachidonate. Effects of feeding warfarin or aspirin on platelet aggregation (experiment 5) Body-weight gains, food intakes and drug intakes of rats given warfarin for 4 days or aspirin for 7 days are shown in Table 8. In the warfarin group, five out of 12 animals died of bleeding, and all seven survivors had haemorrhages in the peritoneal cavity, epididymis, testis, muscle and around the inferior vena cava. Haemorrhagic anaemia and bleeding in the stomach or caecum was found in five rats administered aspirin for 7 days. Feeding warfarin or aspirin
100 -
(A)
0= g
50
g
<{
0
~
=;
b
I
~ I
0
I
5
10
100
(B)
50 g
0 I
o
100
I
5
I
10
15
(c)
_8 50 g
<
0
w I
0
I
I
5
10
t
15
Tim(= (min) Fig. 1. Platelet aggregation in citrated rat PRP (A) and heparinized rat PRP (B and C), induced by ADP. Concentrations of ADP (/~u) in (A), 0.952 (a), 4.76 (b), 9.52 (c) and 31.7 (d); in (B), 4.88 (a) and 9.52 (b); in (C), 0.0952 (a), 0.952 (b) and 9.52 (c). 176
BHT and platelet aggregation
177
100
"100I
(A)
50
50
0 i
1o 1oo
(D)
(8)
g
50
50
g
C
0 0 I
I
I
I
I
0
5
10
0
5
Time
(min)
Time
lOI (rain)
Fig. 2. Platelet aggregation in citrated rat PRP (A, B and C) and heparinized rat PRP (D), induced by collagen. Calf-tendon collagen (Niko Bioscience) was used for (A), (B) and (D), and calf-skin collagen (Sigma Diagnostics) was used for (C). Concentrations of collagen (~g/ml): in (A), 4.8 (a) and 20.0 (b); in (B), 9.0 (a), 13.0 (b), 16.7 (c) and 20.0 (d); in (C), 181.8 (a), 260.8 (b) and 333.3 (c); in (D) 1.16 (a), 2.34 (b) and 4.80 (c). did not significantly affect ADP- and collageninduced platelet aggregation of heparinized PRP from rats (Table 9). DISCUSSION
Platelets are believed to carry out their role in human haemostasis is by adhesion to injured vascular sites, by stacking on adhered platelets (aggregation), by accelerating the aggregation by releasing ADP from the platelet-dense body, and by platelet aggregation and vascular contraction, induced by thromboxane A2 made from the arachidonic acid of platelet membranes. It is well known, and was reconfirmed in the present experiments, that the aggregation properties of rat platelets are very different from those of human platelets. Rat platelets did not react to ristocetin, suggesting that they might lack the receptor to yon FCT 2 9 / ~
Willebrandt factor and that the mechanism of platelet adhesion to vascular tissue might not be as developed as in humans. Rat platelets also lack receptors to epinephrine and serotonin. The secondary aggregation is generally considered as the aggregation by substances released from platelet organella following the primary aggregation. The ADP-induced aggregation of rat platelets did not have this secondary step. A large dose of calf-skin collagen induced irreversible aggregation but calf-tendon collagen caused reversible aggregation with a lag time of about 15 sec (Fig. 2). Irreversible natural aggregation was observed 24 hr after guinea-pig PRP had stood at room or refrigerator temperature. Such aggregation was not found in rat PRP (data not shown). These latter observations have also been described by other authors (Dodds, 1978). Rat platelets are also less sensitive than human platelets, or than guinea-pig platelets, to several aggregation-inducing stimuli.
O. T A K A H A S m
178 100
g
lOO
(A)
(B)
50
50
O
b
I
I
t
I
I
!
0
5
'10
0
5
10
Time
(mln)
Time
(mln)
Fig. 3. A r a c h i d o n a t e - i n d u c e d platelet a g g r e g a t i o n o f h e p a r i n i z e d P R P (A) a n d the differences in a g g r e g a t i o n b e t w e e n c i t r a t e d a n d h e p a r i n i z e d P R P (B). In A , the c o n c e n t r a t i o n s o f a r a c h i d o n i c a c i d (raM) a r e 0.2 (a), 0.5 (b), 2.0 (c) a n d 3.9 (d). In (B) h e p a r i n i z e d P R P (a); c i t r a t e d P R P (b); a m i x t u r e o f c i t r a t e d P R P , 10 U h e p a r i n a n d 0.9 mM C a 2÷ (c) w e r e a d d e d w i t h 2.0 rnM a r a c h i d o n i c acid.
Table 2. Effects of certain anticoagulants and calcium on rat-platelet aggregation induced by aggregating agents Ca 2+ (0.9 mM)
Anticoagulant
Maximum platelet aggregation (%)
ADP (0.952 #M) A D P (0.952 pM) ADP (0.952 #M)
----
Heparin Citrate Oxalate
70 27 16
ADP (0.952/~M) ADP (0.952 gM) ADP (0.952 gM)
----
Heparin Citrate Oxalate
66 25 47
ADP -ADP ADP ADP ADP
-+ + + +
Citrate Citrate Citrate Citrate Citrate Citrate
63 0 84 59 66 63
-+ + ---
Oxalate Oxalate Oxalate Oxalate Citrate Citrate
47 0 30 69 25 68
-
Heparin Citrate Oxalate Oxalate Oxalate
70 4 8 0 0
Collagen (4.8 #g/ml) Collagen (4.8 #g/ml) Collagen (4.8 pg/ml)
-
Heparin Citrate Oxalate
73 4 11
Collagen (4.8 #g/ml) -Collagen (4.8/~g/ml)
+ +
Citrate Citrate Citrate
0 0 0
Collagen (9.1 gg/ml) -Collagen (9.1/~ g/ml)
+ +
Citrate Citrate Citrate
0 0 0
Arachidonic acid (2.0 mM) Arachidonic acid (2.0 mM) Arachidonic acid (2.0 mM)
--+
Heparin Citrate Citrate
78 0 14
Aggregating agent and concn
(9.52/~M) (9.52 #M) (4.88 #M) (4.88 #M) (2.47 # i )
ADP (0.952 pM) -
-
ADP ADP ADP ADP
(0.952 gM) (9.52 # i ) (0.952 gM) (9.52 #M)
Collagen Collagen Collagen Collagen Collagen
(4.8 #g/ml) (4.8 #g/ml) (4.8 #g/ml) (9.1 # g/ml) (16.8 gg/ml)
Data in each column were obtained from the same PRP of a single rat.
BHT and platelet aggregation 100
179 (B)
(A)
'°°I _g g
d 50
50
g
f
9
•
0
o Time
I
I
I
I
5
to
o
5
(mln)
Time
I
10 ¸ (mJn)
Fig. 4. Enhancement of ADP-induced platelet aggregation of citrated PRP by calcium chloride (A and B). (a) 0.9/~M ADP; (b) 10 U heparin+0.9mM Ca2+; (c) 10 U heparin+0.9pM ADP; (d) 10U heparin+0.9mM Ca 2+ + 0 . 9 # i ADP; (e) 10 U h e p a r i n + 0 . 5 # i ADP; (f) 10 U heparin+0.9mM Ca 2+ + 0.5/~i ADP; (g) l0 U heparin + 0.9 mM Ca 2+ + 0.2 pM ADP.
These results cast doubt on the critical role of platelets in rat haemostasis. Rat platelets may have high Ca-dependency when aggregation occurs. Heparinized PRP is more sensitive to ADP and particularly to collagen on aggregation than is citrated PRP. It has also been recognized that arachidonic acid cannot induce aggregation of citrated rat platelets, but can induce this in heparinized PRP as well as in citrated human platelets (Hwang, 1980). ADP-induced aggregation of citrated PRP can be increased by Ca 2÷, to the level of heparinized PRP (Table 2). Collagen- and arachidonic acid-induced aggregations of citrated PRP, however, are not restored to the level of heparinized PRP by Ca 2+ addition. Presumably an irreversible change in the surface structures of the platelet mem-
brane occurs when Ca 2+ is chelated by citrate; in any event, beparinized PRP is preferable for aggregation measurement when rat platelets are used; its preparation, however, is very difficult. Although after their separation from erythrocytes and leucocytes, platelets are stable, when they are together with those blood cells they are extremely sensitive, reacting with small bubbles in test tubes and probably with other blood components; the blood-collecting technique used is thus important in the collection of undamaged platelets from heparinized blood. BHT and BHT quinone methide inhibited ADPand collagen-induced platelet aggregation in vitro. Administration of BHT caused bleeding in rats because of inadequate blood coagulation. When bleeding had already occurred, platelet aggregation
Table 3. Inhibition of aggregation of platelets, in heparinized PRP from normal rats, by BHT or BHT quinone methide in vitro Treatment and concn (mM)
Maximum platelet aggregation (%)
Inhibition of platelet aggregation (%)
Aggregation induced by ADP (9.52/~M) Vehicle control BHT (0.303) BHT (3.03) BHT quinone methide (0.524) BHT quinone methide (5.24)
71 22 12 42 58
0 69 83 41 18
Aggregation induced by collagen (4.8/Jg/ml) Untreated control BHT (0.318) BHT (3.18) BHT quinone methide (0.549) BHT quinone methide (5.49)
71 74 36 59 52
0 -4 49 17 27
Aggregation induced by arachidonie acid (2.0 raM) Vehicle controlt BHT (0.560) Vehicle control* BHT (1.06) *Methanol (4.8%). tMethanol (2.4%).
64 66 38 59
0 - 3 0 - 55
180
O. TAKAHASHI Table 6. Body-weight gain, daily BHT intake and number of rats with haemorrhage after administration of 1.2% BHT in the diet to groups of six rats for 4 or 7 days
loo!
o=
Group
BHT intake (mg/kg/day)
Control BHT
-648
~" 50
I/"L -
i-,_
0
/l
¢ x
. . . . .
Concentration
of
BHT
--~
[raM)
Table 4. Inhibition of disaggregation of ADP-indueed aggregated platelets in PRP from normal rats by BHT in vitro
Untreated control Vehicle control* 0.455 4.55
Disaggregation after I0 min (%)
Maximum platelet aggregation (%)
98 103 56 55
50 39 39 39
*Methanol (4.55%). Data are the means of two experiments.
capacity was normal. This was similar to the finding in rats administered warfarin or aspirin. Although massive haemorrhages were observed, platelet aggregation capacity was normal. Aggregation by 3.9 m i arachidonic acid was greatly inhibited in the group given BHT. These results suggest differences in the properties of platelets and/or plasma between control and BHT-administered rats. Platelet aggregation induced by the optimal level of arachidonate (2 mM) however, was quite normal. Platelet properties are known to vary greatly between rats and man, and the roles of rat platelets in haemostasis have not always been clearly described. Dodds (1978) reported that heparin caused aggregation of human platelets but not of rat platelets. There is considerable evidence that warfarin and aspirin affect platelet aggregation and capillary function in humans and other animals (Balazs et al., 1986; Table 5. Activation of ADP-induced platelet aggregation in PRP from normal rats, by BHT in vitro Concn of BHT (mM)
Activation after 0-5 rain (%)
Maximum platelet aggregation by ADP (%)
Vehicle control* 0.455 4.55 45.5
O? 07 14~ 21~
39? 517 34~ 12~
*Methanol (4.55%).
No. of rats with haemorrhage
Four-days feeding 11.1 -+ 0.3 - 6 . 6 -+ 0.7***
0 0
Seven-days feeding (ADP-/collagen-induced aggregation) Control -24.5 -+ 0.8 0 BHT 738 -0.1 -+ 1.6"** 6?
-
Fig. 5. Inhibition of ADP- and collagen-induced platelet aggregation of rat citrated PRP by butylated hydroxytoluene (BHT) in vitro. BHT was dissolved in methanol (a) or dimethylsulphoxide (b) (final concentration of solvents was 4.55% by vol). Maximal aggregation of PRP with added solvents only was taken as 100%. In (c), an aliquot of methanol solution of BHT was dried in a test tube, PRP was added and stirred, and then aggregation by collagen was measured. In curve (a) the numbers of samples were 6, 10, 4, 8, 4, and 6 for each consecutive point, respectively; in curves (b) and (c) each point represents a single sample. Concentrations of ADP and collagen (Sigma) were 0.95 #M and 333.3 #g/ml, respectively.
Concn of BHT (raM)
Body-weight gain (%)
Seven-days feeding (araehidonie acid-induced aggregation) Control -21.6 _+ 1.1 0 BHT 668 - 2 . 0 -+ 1.1"** 6:~ ?Sites of haemorrhage: epididymal adipose tissue 6/6; abdominal cavity and testis 1/6; anaemia 1/6. :~Sites of haemorrhage: epididymal adipose tissue 6/6; abdominal cavity and stomach 1/6. Values (for body-weight gain) are means + SEM; those marked with asterisks differ significantly (Student's t-test) from the corresponding control value (***P < 0.001).
Levine, 1975; Maekewa, 1975; Smith, 1986). Dejana et al. (1982) however, state that in rats bleeding time
cannot be prolonged by aspirin administration. The present results corroborate the view that warfarin and aspirin do not necessarily inhibit platelet aggregation activity, and that bleeding caused by these drugs may not be related to changes in platelet aggregation capacity (Takahashi and Hiraga, 1985). This may also be so in the case of bleeding caused by BHT. The lack of formation of platelet thrombi is noted not only with natural bleeding but also with bleeding caused mechanical cutting in rats (Thilo and Bohm, 1973). Thilo and Bohm also found an accumulation of red blood cells in the bleeding area. This, together with our observation that rat platelets may be extremely active before PRP is separated, requires that the roles of whole-blood-cell components and coagulation in rat haemostasis be reconsidered. We previously reported the effects of BHT on certain properties of platelets (Takahashi, 1985 and 1986; Takahashi and Hiraga, 1981 and 1984). Those results suggested that platelets did not play a direct role in BHT-induced haemorrhage although the drug did change some platelet properties. The present Table 7. Platelet aggregation of heparinized PRP from groups of six rats fed 1.2% BHT for 4 or 7 days Maximum platelet aggregation (%) Aggregating agent and concn
Control
BHT
Four-days feeding ADP (0.0952 pM) 16.2 + 10.6 ADP (0.952 #M) 60.2 + 1.30 ADP (9.52#M) 68.5--+ 1.61 Collagen (2.4 #g/ml) 56.7 -+ 1.69 Collagen (4.8 #g/ml) 65.7 + 2.42
2.4 + 0.24 65.3 +_ 1.84" 73.0_+ 2.35 60.2 + 2.29 68.7 _+ 2.76
Seven-days feeding ADP (0.952 #M) 62.5 -+ 2.99 ADP (9.52/~M) 70.2-+3.82 Collagen (2.4 lzg/ml) 58.7 -+ 3.85 Collagen (4.8 #g/ml) 65.7 _ 3.26 Arachidonic acid (0.5 mM) 69.8 -+ 1.70 Arachidonic acid (2.0 raM) 69.5-+ 1.12 Arachidonic acid (3.9 raM) 63.2 -+ 1.25
64.4 + 2.52 75.3-+1.15 65.9 _+0.99 71.7 :i: 1.30 76.2 _+ 1.25" 74.0 -+ 1.84 13.7 -+ 2.56***
Values are means + SEM; those marked with asterisks differ significantly (Student's t-test) from the corresponding control value (*P < 0.05; ***P < 0.001).
BHT and platelet aggregation
181
_ o
~
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O. TAm~xsrn Table 8. Body-weight gain, drug intake and numbers of deaths and survivors with haemorrhage among rats given 1.20% aspirin or 0.001% warfarin for 7 or 4 days, respectively No. of rats Drug No. with haemorrhage Treatment intake Body-weight of group (mg/kg/day) gain (%) rats Deaths Survivors Control -34.4 _+ 1.0 6 0 0 Aspirin 500 -20.6 -+ 1.1"** 6 0 5t Control -17.5 -+ 1.4 6 0 0 Warfarin 1.05 -0.7 -+2.2*** 12 5:~ 7§ tSites of haemorrhage: stomach and caecum 6/6; anaemia 5/6. ~Sites of haemorrhage: epididymal adipose tissue 5/5; abdominal cavity 5/5. §Sites of haemorrhage: epididymal adipose tissue 7/7; abdominal cavity 7/7; muscle 3/7; testis 1/7. Values (for body-weight gain) are means -+SEM; those marked with asterisks differ significantly(Student's t-test) from the corresponding control value (***P < 0.001). Table 9. Platelet aggregation of heparinized PRP from rats fed 1.20% aspirin or 0.001% warfarin for 7 or 4 days, respectively Maximum platelet aggregation (%) Aggregating agent Control Aspirin Control Warfarin ADP (0.95/aM) 67.0 __.2.2 (6) 70.3 _ 2.4 (6) 69.6 + 8.0 (5) 73.4 + 3.0 (7) ADP (9.5/aM) 72.2--+2.0(6) 78.0+3.6(6) 72.4-+3.3(5) 73.6_+5.1(7) Collagen (2.4/ag/ml) 64.0 + 1.3 (6) 55.8 -+ 5.8 (6) --Conagen (4.8/ag/ml) 71.2-+1.2(6) 65.2-+3.1(6) 71.2-+4.9(5) 77.9_+1.9(7) Numbers of rats are shown in parentheses.
p a p e r describes the re-examination of the effect o f BHT-feeding o n aggregation, which is one o f the m o s t i m p o r t a n t functions o f platelets. The results showed n o inhibitory effect of B H T or o f warfarin a n d aspirin, o n platelet aggregation, while B H T a n d B H T q u i n o n e methide were s h o w n to inhibit A D P - a n d collagen-induced aggregations in vitro. T h e r e are reports describing the in vitro inhibitory effect o f B H T o n A D P - , arachidonic acid-, t h r o m b i n a n d calcium i o n o p h o r e A:31s7-induced aggregation o f platelets f r o m h u m a n s ( M u r a n o v et al., 1986; P a n g a n a m a l a et al., 1977), b u t it was concluded t h a t these effects were non-specific a n d were due to platelet m e m b r a n e s stabilization. This speculation is consistent with the present results of the effect o f B H T or B H T q u i n o n e methide o n aggregation o f rat platelets in vitro. The differences between the effects o f B H T in vivo a n d in vitro m a y be due to the low c o n c e n t r a t i o n s o f B H T or B H T q u i n o n e methide t h a t develop in the p l a s m a in vivo (less t h a n 0.04mM q u i n o n e methide; B H T c a n n o t be detected). In conclusion, n o inhibition o f platelet aggregation was observed using the standardized m e t h o d when rats were fed BHT, a n d the effects o f B H T o n platelet aggregation capacity in vitro m a y not be i m p o r t a n t in the bleeding that occurs in rats fed BHT.
REFERENCES
Balazs T., Hanig J. P. and Herman E. H. (1986) Toxic responses of the cardiovascular system. In Casarett and Doull's Toxicology. Edited by C. D. Klaassen, M. O. Amdur and J. Doull. pp. 387-411. Macmillan, New York. Born G. V. R. (1962) Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature, London 194, 927-929.
Dejana E., Villa S. and de Gaetano G. (1982) Bleeding time in rats: a comparison of different experimental conditions. Thrombosis and Haemostasis 48, 108-111. Dodds W. J. (1978) Platelet function in animals: species specificities. In Platelets: A Multidisciplinary Approach. Edited by G. de Gaetano and S. Garattini. pp. 45-59. Raven Press, New York. Gad S. C. and Weil C. S. (1982) Statistics for toxicologists. In Principles and Methods of Toxicology, Edited by A. W. Hayes, pp. 237-320. Raven Press, New York. Hwang D. H. (1980) Aggregation and inhibition of rat platelets, and the formation of endoperoxide metabolism. Prostaglandins Medicine 5, 163-173. Levine W. G. (1975) Anticoagulant, antithrombotic and thrombolytic drugs. In The Pharmacological Basis of Therapeutics. 3rd Ed. Edited by L. S. Goodman and A. Gilman. pp. 1350-1368. Macmillan, New York. MacMillan D. C. and Sim A. K. (1970) A comparative study of platelet aggregation in man and laboratory animals. Thrombosis et Diathesis Haemorrhegica 24, 385-394. Maekawa T. (1975) Pharmacology of anticoagulants. In
An Outline of Clinical Pharmacology (Rinsho Yakurigaku Taikei). Vol. 1 I(A). Editorially supervised by H. Kumagai. pp. 224-261. Nakayama Shoten, Tokyo (in Japanese). Muranov K. O., Gashev S. B., Smirnov L. O., Shvedova A. A., Ritov, V. B. and Kagan V. E. (1986) Inhibition of platelet aggregation with antioxidants. Biulleten Eksperimentalnoi Biologhii I Meditsiny (Moskva) 101, 337-339 (in Russian). Panganamala R. V., Miller J. S., Gweba E. T., Sharma H. M. and Cornwell D. G. (1977) Differential inhibitory effects of vitamin E and other antioxidants on prostaglandin synthetase, platelet aggregation and lipoxidase. Prostaglandins 14, 261-271. Smith R. P. (1986) Toxic responses of blood. In Casarett and Doull's Toxicology. Edited by C. D. Klaassen, M. O. Amdur and J. Doull. pp. 223-244. Macmillan, New York. Takahashi O. (1985) Reduced aggregation capacity of washed platelets and dysfibrinogenaemia in rats given butylated hydroxytoluene. Food and Chemical Toxicology 23, 937-939. Takahashi O. (1986) Feeding of butylated hydroxytoluene to rats caused a rapid decrease in blood coagulation
BHT and platelet aggregation factors II (prothrombin), VII, IX and X. Archives of Toxicology ~8, 177-181. Takahashi O. (1987) Decrease in blood coagulation factors II (prothrombin), VII, IX and X in the rat after a single oral dose of butylated hydroxytoluene. Food and Chemical Toxicology 25, 219-224. Takahashi O. (1988a) 2,6-Di-tert-butyl-4-methylene-2,5cyclohexadienone (BHT quinone methide): an active metabolite of BHT causing haemorrhages in rats. Archives of Toxicology 62, 325-327. Takahashi O. (1988b) Inhibition of phylloquinone epoxide reduetase by BHT quinone methide, salicyclic acid and a-tocopherolquinone. Biochemical Pharmacology 37, 2857-2859. Takahashi O., Hayashida S. and Hiraga K. (1980) Species differences in the haemorrhagic response to butylated hydroxytoluene. Food and Cosmetics Toxicology 18, 229-235. Takahashi O. and Hiraga K. (1978) Dose-response study of hemorrhagic death by dietary butylated hydroxytoluene (BHT) in male rats. Toxicology and Applied Pharmacology 43, 399--406. Takahashi O. and Hiraga K. (1979a) 2,6-Di-tert-butyl-4methylene-2,5-cyclohexadienone: a hepatic metabolite of
183
butylated hydroxytoluene in rats. Food and Cosmetics Toxicology 17, 451--454. Takahashi O. and Hiraga K. (1979b) Preventive effects of phylloquinone on hemorrhagic death induced by butylated hydroxytoluene in male rats. Journal of Nutrition 109, 453-457. Takahashi O. and Hiraga K. (1981) Haemorrhagic toxicosis in rats given butylated hydroxytoluene. Acta Pharmacologica et Toxicologica 49, 14-20. Takahashi O. and Hiraga K. (1984) Effects of dietary butylated hydroxytoluene on functional and biochemical properties of platelets and plasma preceding the occurrence of haemorrhage in rats. Food and Chemical Toxicology 22, 97-103. Takahashi O. and Hiraga K. (1985) Differences in the haemorrhagic toxicity of aspirin between rats and mice. Acta Pharmacologica et Toxicologica 56, 6-13. Tanimoto Y. (1982) Hematology, Intersection of Man and Experimental Animals. p. 689. Seishi Shoin Ltd, Tokyo (in Japanese). Thilo D. and B6hm E. (1973) A 3.5-second phenomenon in haemostasis. A scanning electron microscopic study. Thrombosis et Diathesis Haemorrhagica 30, 363-370.
0278-6915/91 $3.00+ 0.00 Copyright© 1991 PergamonPress plc
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SOME PROPERTIES OF RAT PLATELET AGGREGATION A N D EFFECTS OF BUTYLATED HYDROXYTOLUENE, W A R F A R I N A N D ASPIRIN O. TAKAHASHI Department of Toxicology, Tokyo Metropolitan Research Laboratory of Public Health, 24-1 Hyakunincho 3-chome, Shinjuku-ku, Tokyo 169, Japan (Received 20 July 1990; revisions received 29 November 1990)
Abstract--The platelet aggregation characteristics of male Sprague-Dawley (JcI:SD) rats were investigated. Epinephrine, ristocetin, serotonin and platelet-activating factor were ineffective in rat platelets. Heparinized platelet-rich plasma (PRP) was more sensitive than citrated PRP to three aggregating agents, ADP, collagen and arachidonic acid. Butylated hydroxytoluene (BHT) and BHT quinone methide (2,6-di.tert-butyl-4-methylene-2,5-cyclohexadienone) inhibited ADP- and collagen-induced aggregation at concentrations over 10-3M in vitro. The ADP-, collagen- and arachidonic acid (0.5-2.0mM)-induced aggregations of PRP obtained from rats given 1.20% BHT in the diet for 7 days were normal, while arachidonic acid (3.9 mM)-induced aggregation of PRP from BHT-fed rats was significantly lower than control. PRP from rats given aspirin and warfarin also aggregated normally with ADP or collagen addition. These results suggest that heparinized PRP may be preferable in platelet aggregation analyses in rats and reaffirmed that effects on platelet aggregation may not play a key role in BHT-induced bleeding. Platelet aggregation capacity also does not necessarily reduce in haemorrhages induced by aspirin or warfarin. INTRODUCTION It is well known that the food antioxidant butylated hydroxytoluene (BHT) can cause haemorrhagic death in rats (Takahashi and Hiraga, 1978). This effect is both strain- and sex-dependent. Compared with other strains, Jcl:SD male rats had the highest death rate when exposed to BHT, and females were less sensitive to BHT than males (Takahashi et al., 1980). We have reported that a central event in BHT-induced bleeding may be defective blood coagulation due to a deficiency in coagulation factors II, VII, IX and X following the inhibition of the biosynthesis of those four vitamin-K-dependent factors by 2,6-di-tertbutyl-4-methylene-2,5-cyclohexadienone (BHT quinone methide), an active metabolite of BHT, in the liver (Takahashi, 1986, 1987 and 1988a,b). We have also examined the effects of BHT on some properties and functions of platelets: particle distribution, fatty-acid composition, factor-3 availability and aggregation activity, and have concluded that those factors probably do not play such a critical role as inadequate coagulation in the early stages of this bleeding (Takahashi, 1985 and 1986; Takahashi and Hiraga, 1981 and 1984). However, the method used to assay platelet aggregation capacity described in our previous reports was not necessarily a standardized one because we had no platelet aggregation analyser. Since we have now obtained this instrument, we now report our re-examination of the effects of BHT on platelet aggregation. It has been established that the aggregation properties of rat platelets differ greatly from those of man Abbreviations: BHT = butylated hydroxytoluene; DMSO =
dimethylsulpboxide; PAF = platelet activating factor; PPP = platelet-poorplasma; PRP = platelet-rich plasma.
and other species; rat platelets do not show the secondary aggregation that human platelets do (MacMillan and Sim, 1970). The doses of A D P and collagen required to induce aggregation in rat platelets are much higher than in human platelets (MacMillan and Sim, 1970). The relationship between the aggregation of rat platelets and drugs is yet more complicated, the reactions have different features from those of human platelets (Tanimoto, 1982). Furthermore, although it is unequivocal that platelets are a prerequisite for human haemostasis and a haemostatic model made from the study of human platelets is tentatively used for an interpretation of the rat haemostatic mechanism, the role of rat platelets in stopping bleeding is extremely obscure. It is questionable whether the human model is entirely applicable to rats. It is believed that platelet thrombi play a key role in stopping bleeding. The formation of platelet thrombi can actually be observed at an injured site of a vessel in a human model. Thilo and Bfhm (1973), however, did not find this formation in a rat model. There is, in fact, one report of the possible formation of white thrombi at injured tissues, which may be a crucial stage in human haemostasis (Thilo and Bohm, 1973). To shed further light on the behaviour of rat platelets, we have also examined the effects of warfarin and aspirin on the aggregation of rat platelets, since these drugs are known oral anticoagulants and antiaggregants for human platelets. MATERIALS AND METHODS
Chemicals. Platelet aggregation reagents, adenosine diphosphate (ADP) and calf-tendon collagen were obtained from Niko Bioscience Inc. (Tokyo, Japan), and arachidonic acid from porcine liver
173
174
O. TAKAHASHI
(purity c. 90%), fl-acetyl-7-O-(octadec-9-cis-enyl)L-~t-phosphatidylcholine (platelet activating factor: PAF; purity c. 99%), ristocetin, epinephrine and calf-skin collagen were from Sigma Chemical Co. or Sigma Diagnostics (St Louis, MO, USA). Sodium salts of citric acid, oxalic acid and heparin were purchased from Wako Pure Chemical Industries (Osaka, Japan), and 0.02 M-calcium chloride solution was from International Reagent Corp. (Kobe, Japan). BHT, 3-(~t-acetonylbenzyl)-4-hydroxycoumarin (warfarin) and acetylsalicylic acid (aspirin; Japan Pharmacopoeia) were from Tokyo Kasei Kogyo Co. (Tokyo, Japan), Sigma Chemical Co. (St Louis, MO, USA) and Tsukishima Yakuhin K. K. (Tokyo, Japan), respectively. BHT quinone methide was synthesized as described previously (Takahashi and Hiraga, 1979a). Animals and treatments. Male Sprague-Dawley (Jcl:SD) rats, purchased when 4 wk old, were used for all experiments. For experiments 1, 2 and 3, rats (10-20wk old) raised on a laboratory ration were killed to collect platelets after starvation for 18 hr. In experiments 4 and 5, rats (5-6 wk old) were given BHT, warfarin or aspirin in a purified diet for 4-7 days. The composition of the diet was as described previously (Takahashi and Hiraga, 1978 and 1979b). Preparation ofplatelets. Whole blood was collected (under ether anaesthesia) from the inferior vena cava into plastic syringes containing 0.124M-sodium citrate solution, 0.124M-sodium oxalate solution or 1000 units/ml sodium heparin (in saline) solution (anticoagulant solution-blood, 1:9, v/v), carefully avoiding formation of a bubble. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared by centrifugation of blood at 60-80g for 10 min and 1550 g for 25 min, respectively, at room temperature. Platelet aggregation analysis. Platelet aggregation of PRP was determined by the optical method described originally by Born (1962) using an NBS eight-channel platelet aggregometer (NBS Hema Tracer, Niko Bioscience Inc., Tokyo, Japan). PRP (200/zl) was placed in a siliconized cuvette, containing a siliconized stirring bar, placed in an aggregometer at 37°C and stirred at 1000 rpm. After the light-transmittance of PRP was adjusted at 0% against PPP of 100%, aggregation was observed as the change in turbidity.
Effects of aggregating agents on the aggregation of platelets from normal rats (experiment I). Following the addition of ADP, collagen, arachidonic acid, PAF, ristocetin or epinephrine to PRP, platelet aggregation was recorded. Arachidonic acid was used after solubilization equimoles of sodium carbonate and dilution with saline. PAF, ristocetin and epinephrine did not cause aggregation of PRP. The dependence of ADP-, collagen- and arachidonic acid-induced aggregation on their concentrations was examined.
Effects of anticoagulants on platelet aggregation of normal rats (experiment 2). The different effects of citrate, oxalate and heparin on aggregation were studied, and that of calcium chloride on platelet aggregation was also examined.
Effects of BHT or BHT quinone methide on platelet aggregation in vitro (experiment 3). The effects of BHT on citrated or heparinized PRP were studied in vitro. For citrated PRP, methanol and dimethyl-
sulphoxide (DMSO), at final concentrations of 5%, inhibited ADP-induced aggregation partially, and acetone, Tween 20 and linoleic acid (5%) did so completely. Collagen-induced aggregation was completely inhibited by 5% methanol and DMSO. To determine the effects of BHT or BHT quinone methide on ADP-induced platelet aggregation in vitro, BHT or its metabolite dissolved in methanol or DMSO was used. To determine the effects of BHT or BHT quinone methide on collagen-induced aggregation, a portion of the methanol solution of these compounds was transferred to a reaction tube; after evaporation of its solvent, PRP was poured into a tube containing dried BHT, and platelet aggregation was measured. For arachidonic acid-induced aggregation, the effects of BHT in methanol were measured.
Effects of BHT feeding on platelet aggregation (experiment 4). Rats were fed a diet containing 1.2% BHT for either 4 or 7 days. After PRP was prepared from heparinized blood, ADP-, collagen- and arachidonic acid-induced aggregations were measured.
Effects of feeding warfarin or aspirin on ADP- and collagen-induced platelet aggregation (experiment 5). After rats were fed diets containing 1.2% aspirin or 0.001% warfarin for 7 or 4 days, respectively (Takahashi and Hiraga, 1979b and 1981), ADPand collagen-induced platelet aggregations were measured using heparinized PRP. Statistics. Data were routinely expressed as mean_+ SEM and were analysed statistically by Student's t-test. Significance was assumed at P < 0.05 (Gad and Weil, 1982). RESULTS
Effects of aggregating agents on the aggregation of platelets from normal rats (experiment 1) Maximum platelet aggregations (%) of both titrated and heparinized PRPs taken from normal rats caused by ADP, collagen, ristocetin, epinephrine, arichidonic acid and PAF are shown in Table 1. ADP and collagen obviously induced aggregation in both PRPs (Figs 1 and 2). ADP at a concentration of 9.5/~M provoked 62-66% of maximum aggregation after about 2 min. However, calf-tendon collagen at about 10 #g/ml could not always induce aggregation in citrated PRP. Curves obtained from platelets capable of aggregating by chance are shown in Fig. 2. A large amount of calf-skin collagen also induced aggregation with a different pattern in citrated PRP (Fig. 2C). Collagen at lower levels caused more platelet aggregation in heparinized PRP than in citrated PRP (Fig. 2D). Arachidonic acid induced aggregation only in heparinized PRP (Fig. 3). Ristocetin, epinephrine and PAF did not cause notable aggregation.
Effects of certain anticoagulants on platelet aggregation of normal rats (experiment 2) Platelet aggregation by ADP and collagen was compared using citrated, oxalated and heparinized PRP. In both ADP- and collagen-induced aggregation, heparinized PRP produced much greater aggregation in response to the lower concentrations of the aggregants than did citrate and oxalate (Table 2).
BHT and platelet aggregation Table
1. Activity of certain aggregating alatelets in PRP from normal
Maximum platelet aggregation (“/)
Aggregating agent and concn Citrated
agents on rat rats
PRP
ADP (9.52 PM) ADP (31.7 PM) ADP (95.2 HIM)
62 59 50
ADP ADP ADP ADP
14 31 65 66
(0.952 PM) (4.76 PM) (9.52 PM) (9.52 PM)
Collagen* Collagen*
(4.8 Kg/ml) (20.0 pg/ml)
9 63
Collagen* Collagen’ Collagen* Collagen*
(9.0 pg/ml) (13.0 fig/ml) (16.7yg/ml) (20.0 fig/ml)
9 21 29 33
Collagen* Collagen* Collagen*
(13.0 pg/ml) (16.7 pg/ml) (20.0 rg/ml)
48 41 61
Collagen’
(9.1 pg/ml)
69
Collagen? Collagen? CoIlagent Collagen?
(95.2 fig/ml) (I 8 1.8 fig/ml) (260.8 pg/ml) (333.3 fig/ml)
0 0 89 95
CoIlagent Collagen?
(190.5 pg/ml) (400.0 pg/ml)
0 74
Ristocetin
(7.1 mg/ml)
Epinepbrine
0 0
(47.6 PM)
0
AA (2.0 mhi) Heparinized
PRP
ADP (4.88 FM) ADP (9.52 PM)
63 65
ADP (0.0952 PM) ADP (0.952 PM) ADP (9.52 /LM)
19 66 72
Collagen* Collagen*
(4.8 pg/ml) (9.1 /~g/ml)
10 76
Collagen* Collagen* Collagen*
(1.2 pg/ml) (2.5 pg/ml) (4.8 pg/ml)
18 41 58
Ristocetin Ristocetin
(7.14 mg/ml)
(I 3.6 mg/ml)
5 IO
Ristocetin
(30.0 mg/ml)
0
Epinepbrine Epinephrine
(4.76 pM) (16.7 phi)
4 24
Epinephrine Epinephrine Epinepbrine
(9.09 PM) (20.0 PM) (90.9 PM)
0 0 0
AA (0.5 rnMj AA i2.0m~j AA (3.9 mM)
0 78 21
AA (3.9 mhf)
59
AA (3.9 mM)
48
AA AA AA AA
(0.2 (0.5 (2.0 (3.9
mM) mM) mM) mhf)
62 68 69 65
AA AA AA AA
(0.2 (0.5 (2.0 (3.9
mM) mM) mM) mM)
0 69 68 66
PAF (8.67 /‘M) PAF (86.7 /LM)
0 0
*Calf-tendon collagen obtained from Niko Bioscience Inc. TCalf-skin collagen obtained from Sigma Chemical Co. or Sigma Diagnostics. Data in each column were obtained from the same PRP of a single rat.
175
ADP-induced aggregation of titrated PRP was increased by the addition of calcium chloride (Fig. 4) while the low reactivity of titrated PRP to collagen could not be raised by the addition of Ca*+. Arachidonic acid caused aggregation in heparinized but not in titrated PRP. Ca*+ addition restored the low reactivity of titrated PRP to arachidonic acid to a very limited extent (Fig. 3). Eflects of BHT or BHT quinone methide on platelet aggregation in vitro (experiment 3) Effects on ADP- and collagen-induced rat platelet aggregations of normal PRP by BHT or BHT quinone methide (Table 3 and Fig. 5) show that BHT inhibited both types of aggregations at concentrations of more than about 10m3M (Fig. 5). Inhibition was observed in both titrated and heparinized PRP. BHT quinone methide also inhibited these aggregations to a lesser extent. BHT at the lo-‘M level did not inhibit PRP aggregation induced by arachidonic acid (Table 3). BHT over 0.455 x 10e3 M also inhibited the disaggregation at 5-10min after ADP-induced aggregation (Fig. 6 ana Table 4) while somewhat activating platelet aggregation with the maximum at c. 1.0 min over 4.55 x 10m3M (Fig. 6 and Table 5). Effects of BHTfeeding on platelet aggregation (experiment 4) Body-weight gains, food intakes and BHT intakes of rats given 1.20% BHT for 4 or 7 days are shown in Table 6. Haemorrhages in epididymal adipose tissues were observed in all animals maintained on the BHT diet for 7 days. Bleeding in the peritoneal cavity and testes was found in one rat of this group and haemorrhagic anaemia in another. A conspicuous decrease in haematocrit, probably due to bleeding, was found in two out of seven rats given BHT for 7days. Maximum platelet aggregations of heparinized PRP induced by ADP and collagen are shown in Table 7. The mean maximum aggregation induced by 0.95 PM-ADP was increased significantly in animals given BHT for 4 days, while no significant differences were observed in other values. Rats fed 1.20% BHT for 7 days to measure the arachidonic acidinduced aggregation developed haemorrhages in the epididymis and some other organs. There was no significant difference in aggregation between control and BHT-administered rats at the 2.0-mM concentration of arachidonic acid but BHT produced a significant increase in aggregation at the 0.5-mM concentration of arachidonate and a significant decrease in aggregation at the 3.9-mM concentration of arachidonate. Effects of feeding warfarin or aspirin on platelet aggregation (experiment 5) Body-weight gains, food intakes and drug intakes of rats given warfarin for 4 days or aspirin for 7 days are shown in Table 8. In the warfarin group, five out of 12 animals died of bleeding, and all seven survivors had haemorrhages in the peritoneal cavity, epididymis, testis, muscle and around the inferior vena cava. Haemorrhagic anaemia and bleeding in the stomach or caecum was found in five rats administered aspirin for 7 days. Feeding warfarin or aspirin
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Tim(= (min) Fig. 1. Platelet aggregation in citrated rat PRP (A) and heparinized rat PRP (B and C), induced by ADP. Concentrations of ADP (/~u) in (A), 0.952 (a), 4.76 (b), 9.52 (c) and 31.7 (d); in (B), 4.88 (a) and 9.52 (b); in (C), 0.0952 (a), 0.952 (b) and 9.52 (c). 176
BHT and platelet aggregation
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Time
lOI (rain)
Fig. 2. Platelet aggregation in citrated rat PRP (A, B and C) and heparinized rat PRP (D), induced by collagen. Calf-tendon collagen (Niko Bioscience) was used for (A), (B) and (D), and calf-skin collagen (Sigma Diagnostics) was used for (C). Concentrations of collagen (~g/ml): in (A), 4.8 (a) and 20.0 (b); in (B), 9.0 (a), 13.0 (b), 16.7 (c) and 20.0 (d); in (C), 181.8 (a), 260.8 (b) and 333.3 (c); in (D) 1.16 (a), 2.34 (b) and 4.80 (c). did not significantly affect ADP- and collageninduced platelet aggregation of heparinized PRP from rats (Table 9). DISCUSSION
Platelets are believed to carry out their role in human haemostasis is by adhesion to injured vascular sites, by stacking on adhered platelets (aggregation), by accelerating the aggregation by releasing ADP from the platelet-dense body, and by platelet aggregation and vascular contraction, induced by thromboxane A2 made from the arachidonic acid of platelet membranes. It is well known, and was reconfirmed in the present experiments, that the aggregation properties of rat platelets are very different from those of human platelets. Rat platelets did not react to ristocetin, suggesting that they might lack the receptor to yon FCT 2 9 / ~
Willebrandt factor and that the mechanism of platelet adhesion to vascular tissue might not be as developed as in humans. Rat platelets also lack receptors to epinephrine and serotonin. The secondary aggregation is generally considered as the aggregation by substances released from platelet organella following the primary aggregation. The ADP-induced aggregation of rat platelets did not have this secondary step. A large dose of calf-skin collagen induced irreversible aggregation but calf-tendon collagen caused reversible aggregation with a lag time of about 15 sec (Fig. 2). Irreversible natural aggregation was observed 24 hr after guinea-pig PRP had stood at room or refrigerator temperature. Such aggregation was not found in rat PRP (data not shown). These latter observations have also been described by other authors (Dodds, 1978). Rat platelets are also less sensitive than human platelets, or than guinea-pig platelets, to several aggregation-inducing stimuli.
O. T A K A H A S m
178 100
g
lOO
(A)
(B)
50
50
O
b
I
I
t
I
I
!
0
5
'10
0
5
10
Time
(mln)
Time
(mln)
Fig. 3. A r a c h i d o n a t e - i n d u c e d platelet a g g r e g a t i o n o f h e p a r i n i z e d P R P (A) a n d the differences in a g g r e g a t i o n b e t w e e n c i t r a t e d a n d h e p a r i n i z e d P R P (B). In A , the c o n c e n t r a t i o n s o f a r a c h i d o n i c a c i d (raM) a r e 0.2 (a), 0.5 (b), 2.0 (c) a n d 3.9 (d). In (B) h e p a r i n i z e d P R P (a); c i t r a t e d P R P (b); a m i x t u r e o f c i t r a t e d P R P , 10 U h e p a r i n a n d 0.9 mM C a 2÷ (c) w e r e a d d e d w i t h 2.0 rnM a r a c h i d o n i c acid.
Table 2. Effects of certain anticoagulants and calcium on rat-platelet aggregation induced by aggregating agents Ca 2+ (0.9 mM)
Anticoagulant
Maximum platelet aggregation (%)
ADP (0.952 #M) A D P (0.952 pM) ADP (0.952 #M)
----
Heparin Citrate Oxalate
70 27 16
ADP (0.952/~M) ADP (0.952 gM) ADP (0.952 gM)
----
Heparin Citrate Oxalate
66 25 47
ADP -ADP ADP ADP ADP
-+ + + +
Citrate Citrate Citrate Citrate Citrate Citrate
63 0 84 59 66 63
-+ + ---
Oxalate Oxalate Oxalate Oxalate Citrate Citrate
47 0 30 69 25 68
-
Heparin Citrate Oxalate Oxalate Oxalate
70 4 8 0 0
Collagen (4.8 #g/ml) Collagen (4.8 #g/ml) Collagen (4.8 pg/ml)
-
Heparin Citrate Oxalate
73 4 11
Collagen (4.8 #g/ml) -Collagen (4.8/~g/ml)
+ +
Citrate Citrate Citrate
0 0 0
Collagen (9.1 gg/ml) -Collagen (9.1/~ g/ml)
+ +
Citrate Citrate Citrate
0 0 0
Arachidonic acid (2.0 mM) Arachidonic acid (2.0 mM) Arachidonic acid (2.0 mM)
--+
Heparin Citrate Citrate
78 0 14
Aggregating agent and concn
(9.52/~M) (9.52 #M) (4.88 #M) (4.88 #M) (2.47 # i )
ADP (0.952 pM) -
-
ADP ADP ADP ADP
(0.952 gM) (9.52 # i ) (0.952 gM) (9.52 #M)
Collagen Collagen Collagen Collagen Collagen
(4.8 #g/ml) (4.8 #g/ml) (4.8 #g/ml) (9.1 # g/ml) (16.8 gg/ml)
Data in each column were obtained from the same PRP of a single rat.
BHT and platelet aggregation 100
179 (B)
(A)
'°°I _g g
d 50
50
g
f
9
•
0
o Time
I
I
I
I
5
to
o
5
(mln)
Time
I
10 ¸ (mJn)
Fig. 4. Enhancement of ADP-induced platelet aggregation of citrated PRP by calcium chloride (A and B). (a) 0.9/~M ADP; (b) 10 U heparin+0.9mM Ca2+; (c) 10 U heparin+0.9pM ADP; (d) 10U heparin+0.9mM Ca 2+ + 0 . 9 # i ADP; (e) 10 U h e p a r i n + 0 . 5 # i ADP; (f) 10 U heparin+0.9mM Ca 2+ + 0.5/~i ADP; (g) l0 U heparin + 0.9 mM Ca 2+ + 0.2 pM ADP.
These results cast doubt on the critical role of platelets in rat haemostasis. Rat platelets may have high Ca-dependency when aggregation occurs. Heparinized PRP is more sensitive to ADP and particularly to collagen on aggregation than is citrated PRP. It has also been recognized that arachidonic acid cannot induce aggregation of citrated rat platelets, but can induce this in heparinized PRP as well as in citrated human platelets (Hwang, 1980). ADP-induced aggregation of citrated PRP can be increased by Ca 2÷, to the level of heparinized PRP (Table 2). Collagen- and arachidonic acid-induced aggregations of citrated PRP, however, are not restored to the level of heparinized PRP by Ca 2+ addition. Presumably an irreversible change in the surface structures of the platelet mem-
brane occurs when Ca 2+ is chelated by citrate; in any event, beparinized PRP is preferable for aggregation measurement when rat platelets are used; its preparation, however, is very difficult. Although after their separation from erythrocytes and leucocytes, platelets are stable, when they are together with those blood cells they are extremely sensitive, reacting with small bubbles in test tubes and probably with other blood components; the blood-collecting technique used is thus important in the collection of undamaged platelets from heparinized blood. BHT and BHT quinone methide inhibited ADPand collagen-induced platelet aggregation in vitro. Administration of BHT caused bleeding in rats because of inadequate blood coagulation. When bleeding had already occurred, platelet aggregation
Table 3. Inhibition of aggregation of platelets, in heparinized PRP from normal rats, by BHT or BHT quinone methide in vitro Treatment and concn (mM)
Maximum platelet aggregation (%)
Inhibition of platelet aggregation (%)
Aggregation induced by ADP (9.52/~M) Vehicle control BHT (0.303) BHT (3.03) BHT quinone methide (0.524) BHT quinone methide (5.24)
71 22 12 42 58
0 69 83 41 18
Aggregation induced by collagen (4.8/Jg/ml) Untreated control BHT (0.318) BHT (3.18) BHT quinone methide (0.549) BHT quinone methide (5.49)
71 74 36 59 52
0 -4 49 17 27
Aggregation induced by arachidonie acid (2.0 raM) Vehicle controlt BHT (0.560) Vehicle control* BHT (1.06) *Methanol (4.8%). tMethanol (2.4%).
64 66 38 59
0 - 3 0 - 55
180
O. TAKAHASHI Table 6. Body-weight gain, daily BHT intake and number of rats with haemorrhage after administration of 1.2% BHT in the diet to groups of six rats for 4 or 7 days
loo!
o=
Group
BHT intake (mg/kg/day)
Control BHT
-648
~" 50
I/"L -
i-,_
0
/l
¢ x
. . . . .
Concentration
of
BHT
--~
[raM)
Table 4. Inhibition of disaggregation of ADP-indueed aggregated platelets in PRP from normal rats by BHT in vitro
Untreated control Vehicle control* 0.455 4.55
Disaggregation after I0 min (%)
Maximum platelet aggregation (%)
98 103 56 55
50 39 39 39
*Methanol (4.55%). Data are the means of two experiments.
capacity was normal. This was similar to the finding in rats administered warfarin or aspirin. Although massive haemorrhages were observed, platelet aggregation capacity was normal. Aggregation by 3.9 m i arachidonic acid was greatly inhibited in the group given BHT. These results suggest differences in the properties of platelets and/or plasma between control and BHT-administered rats. Platelet aggregation induced by the optimal level of arachidonate (2 mM) however, was quite normal. Platelet properties are known to vary greatly between rats and man, and the roles of rat platelets in haemostasis have not always been clearly described. Dodds (1978) reported that heparin caused aggregation of human platelets but not of rat platelets. There is considerable evidence that warfarin and aspirin affect platelet aggregation and capillary function in humans and other animals (Balazs et al., 1986; Table 5. Activation of ADP-induced platelet aggregation in PRP from normal rats, by BHT in vitro Concn of BHT (mM)
Activation after 0-5 rain (%)
Maximum platelet aggregation by ADP (%)
Vehicle control* 0.455 4.55 45.5
O? 07 14~ 21~
39? 517 34~ 12~
*Methanol (4.55%).
No. of rats with haemorrhage
Four-days feeding 11.1 -+ 0.3 - 6 . 6 -+ 0.7***
0 0
Seven-days feeding (ADP-/collagen-induced aggregation) Control -24.5 -+ 0.8 0 BHT 738 -0.1 -+ 1.6"** 6?
-
Fig. 5. Inhibition of ADP- and collagen-induced platelet aggregation of rat citrated PRP by butylated hydroxytoluene (BHT) in vitro. BHT was dissolved in methanol (a) or dimethylsulphoxide (b) (final concentration of solvents was 4.55% by vol). Maximal aggregation of PRP with added solvents only was taken as 100%. In (c), an aliquot of methanol solution of BHT was dried in a test tube, PRP was added and stirred, and then aggregation by collagen was measured. In curve (a) the numbers of samples were 6, 10, 4, 8, 4, and 6 for each consecutive point, respectively; in curves (b) and (c) each point represents a single sample. Concentrations of ADP and collagen (Sigma) were 0.95 #M and 333.3 #g/ml, respectively.
Concn of BHT (raM)
Body-weight gain (%)
Seven-days feeding (araehidonie acid-induced aggregation) Control -21.6 _+ 1.1 0 BHT 668 - 2 . 0 -+ 1.1"** 6:~ ?Sites of haemorrhage: epididymal adipose tissue 6/6; abdominal cavity and testis 1/6; anaemia 1/6. :~Sites of haemorrhage: epididymal adipose tissue 6/6; abdominal cavity and stomach 1/6. Values (for body-weight gain) are means + SEM; those marked with asterisks differ significantly (Student's t-test) from the corresponding control value (***P < 0.001).
Levine, 1975; Maekewa, 1975; Smith, 1986). Dejana et al. (1982) however, state that in rats bleeding time
cannot be prolonged by aspirin administration. The present results corroborate the view that warfarin and aspirin do not necessarily inhibit platelet aggregation activity, and that bleeding caused by these drugs may not be related to changes in platelet aggregation capacity (Takahashi and Hiraga, 1985). This may also be so in the case of bleeding caused by BHT. The lack of formation of platelet thrombi is noted not only with natural bleeding but also with bleeding caused mechanical cutting in rats (Thilo and Bohm, 1973). Thilo and Bohm also found an accumulation of red blood cells in the bleeding area. This, together with our observation that rat platelets may be extremely active before PRP is separated, requires that the roles of whole-blood-cell components and coagulation in rat haemostasis be reconsidered. We previously reported the effects of BHT on certain properties of platelets (Takahashi, 1985 and 1986; Takahashi and Hiraga, 1981 and 1984). Those results suggested that platelets did not play a direct role in BHT-induced haemorrhage although the drug did change some platelet properties. The present Table 7. Platelet aggregation of heparinized PRP from groups of six rats fed 1.2% BHT for 4 or 7 days Maximum platelet aggregation (%) Aggregating agent and concn
Control
BHT
Four-days feeding ADP (0.0952 pM) 16.2 + 10.6 ADP (0.952 #M) 60.2 + 1.30 ADP (9.52#M) 68.5--+ 1.61 Collagen (2.4 #g/ml) 56.7 -+ 1.69 Collagen (4.8 #g/ml) 65.7 + 2.42
2.4 + 0.24 65.3 +_ 1.84" 73.0_+ 2.35 60.2 + 2.29 68.7 _+ 2.76
Seven-days feeding ADP (0.952 #M) 62.5 -+ 2.99 ADP (9.52/~M) 70.2-+3.82 Collagen (2.4 lzg/ml) 58.7 -+ 3.85 Collagen (4.8 #g/ml) 65.7 _ 3.26 Arachidonic acid (0.5 mM) 69.8 -+ 1.70 Arachidonic acid (2.0 raM) 69.5-+ 1.12 Arachidonic acid (3.9 raM) 63.2 -+ 1.25
64.4 + 2.52 75.3-+1.15 65.9 _+0.99 71.7 :i: 1.30 76.2 _+ 1.25" 74.0 -+ 1.84 13.7 -+ 2.56***
Values are means + SEM; those marked with asterisks differ significantly (Student's t-test) from the corresponding control value (*P < 0.05; ***P < 0.001).
BHT and platelet aggregation
181
_ o
~
I,--
o
0
~,~
l 0
O,
~
~,~
~...
00
B
I/ ~° I
~
'° ~ ~
~.= ~..~
0
=~'~
~-
v
~)
~ .2
. ~ ~'~ ~-~.~
~'~
•
Ov-~
~
~ ".2 (%)
UOl~O~e~B~V
(%1
UOl~Ol~eJ I ~ V
~J=
182
O. TAm~xsrn Table 8. Body-weight gain, drug intake and numbers of deaths and survivors with haemorrhage among rats given 1.20% aspirin or 0.001% warfarin for 7 or 4 days, respectively No. of rats Drug No. with haemorrhage Treatment intake Body-weight of group (mg/kg/day) gain (%) rats Deaths Survivors Control -34.4 _+ 1.0 6 0 0 Aspirin 500 -20.6 -+ 1.1"** 6 0 5t Control -17.5 -+ 1.4 6 0 0 Warfarin 1.05 -0.7 -+2.2*** 12 5:~ 7§ tSites of haemorrhage: stomach and caecum 6/6; anaemia 5/6. ~Sites of haemorrhage: epididymal adipose tissue 5/5; abdominal cavity 5/5. §Sites of haemorrhage: epididymal adipose tissue 7/7; abdominal cavity 7/7; muscle 3/7; testis 1/7. Values (for body-weight gain) are means -+SEM; those marked with asterisks differ significantly(Student's t-test) from the corresponding control value (***P < 0.001). Table 9. Platelet aggregation of heparinized PRP from rats fed 1.20% aspirin or 0.001% warfarin for 7 or 4 days, respectively Maximum platelet aggregation (%) Aggregating agent Control Aspirin Control Warfarin ADP (0.95/aM) 67.0 __.2.2 (6) 70.3 _ 2.4 (6) 69.6 + 8.0 (5) 73.4 + 3.0 (7) ADP (9.5/aM) 72.2--+2.0(6) 78.0+3.6(6) 72.4-+3.3(5) 73.6_+5.1(7) Collagen (2.4/ag/ml) 64.0 + 1.3 (6) 55.8 -+ 5.8 (6) --Conagen (4.8/ag/ml) 71.2-+1.2(6) 65.2-+3.1(6) 71.2-+4.9(5) 77.9_+1.9(7) Numbers of rats are shown in parentheses.
p a p e r describes the re-examination of the effect o f BHT-feeding o n aggregation, which is one o f the m o s t i m p o r t a n t functions o f platelets. The results showed n o inhibitory effect of B H T or o f warfarin a n d aspirin, o n platelet aggregation, while B H T a n d B H T q u i n o n e methide were s h o w n to inhibit A D P - a n d collagen-induced aggregations in vitro. T h e r e are reports describing the in vitro inhibitory effect o f B H T o n A D P - , arachidonic acid-, t h r o m b i n a n d calcium i o n o p h o r e A:31s7-induced aggregation o f platelets f r o m h u m a n s ( M u r a n o v et al., 1986; P a n g a n a m a l a et al., 1977), b u t it was concluded t h a t these effects were non-specific a n d were due to platelet m e m b r a n e s stabilization. This speculation is consistent with the present results of the effect o f B H T or B H T q u i n o n e methide o n aggregation o f rat platelets in vitro. The differences between the effects o f B H T in vivo a n d in vitro m a y be due to the low c o n c e n t r a t i o n s o f B H T or B H T q u i n o n e methide t h a t develop in the p l a s m a in vivo (less t h a n 0.04mM q u i n o n e methide; B H T c a n n o t be detected). In conclusion, n o inhibition o f platelet aggregation was observed using the standardized m e t h o d when rats were fed BHT, a n d the effects o f B H T o n platelet aggregation capacity in vitro m a y not be i m p o r t a n t in the bleeding that occurs in rats fed BHT.
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183
butylated hydroxytoluene in rats. Food and Cosmetics Toxicology 17, 451--454. Takahashi O. and Hiraga K. (1979b) Preventive effects of phylloquinone on hemorrhagic death induced by butylated hydroxytoluene in male rats. Journal of Nutrition 109, 453-457. Takahashi O. and Hiraga K. (1981) Haemorrhagic toxicosis in rats given butylated hydroxytoluene. Acta Pharmacologica et Toxicologica 49, 14-20. Takahashi O. and Hiraga K. (1984) Effects of dietary butylated hydroxytoluene on functional and biochemical properties of platelets and plasma preceding the occurrence of haemorrhage in rats. Food and Chemical Toxicology 22, 97-103. Takahashi O. and Hiraga K. (1985) Differences in the haemorrhagic toxicity of aspirin between rats and mice. Acta Pharmacologica et Toxicologica 56, 6-13. Tanimoto Y. (1982) Hematology, Intersection of Man and Experimental Animals. p. 689. Seishi Shoin Ltd, Tokyo (in Japanese). Thilo D. and B6hm E. (1973) A 3.5-second phenomenon in haemostasis. A scanning electron microscopic study. Thrombosis et Diathesis Haemorrhagica 30, 363-370.