THROMBOSIS Pergamon
RESEARCH 15; 199-207 Press Ltd.1979. Printed
in Great
Britain
BLEEDING TIME IN LABORATORY ANIMALS. III - DO TAIL BLEEDING TIMES IN RATS ONLY MEASURE A PLATELET DEFECT? (THE ASPIRIN PUZZLE)
Elisabetta DEJANA', Antonio QUINTANA'* , Antonella CALLIONIO and Giovanni de GAETANO Laboratory of Autonomic Nervous System and Laboratory of Cardiovascular Clinical Pharmacology Istituto di Ricerche Farmacologiche "Mario Negri" Via Eritrea, 62 - 20157 MILAN, Italy
(Received
19.2.1979.
Accepted
by
Editor
H.
Stormorken)
ABSTRACT The template bleeding time tested in the present study was not prolonged in rats given aspirin (single doses ranging from 1 to 200 mg/kg b.w.). In contrast, bleeding after the transection of tails immersed in saline at 37°C was significantly longer in aspirin treated than in control rats. This prolongation was maximal 1 hour after aspirin (200 mg/kg) and was back to normal within 24 hours, when platelet malondialdehyde formation was still completely inhibited. Both template and transection bleeding times were significantly prolonged in thrombocytopenic and thrombocytopathic rats and in normal rats given a pyrimido-pyrimidine compound (SH-869). These data suggest that platelets are important in the haemostatic process of the rat, but that the platelet defect induced by aspirin is not detectable by the bleeding time measurement techniques used. These may be sensitive not only to platelet dysfunction but also to modifications of vascular tone and blood flow and to blood coagulation and fibrinolysis systems. The complex and contrasting effects of aspirin on haemostatic factors other than platelets (for instance on vascular prostacyclin and fibrinolysis) may influence bleeding times measured by different techniques and contribute to the puzzling results obtained so far with this drug.
'At present at the Department of Pathology, McMaster University, Hamilton, Ontario, Canada. **Visiting Scientist from the Department of Pharmacology, University of Bilbao, Spain. o Reprint requests. 199
BLEEDING
200
TIME IN RATS AND ASA
Vo1.15,No.l/2
INTRODUCTION Contrastating results are reported
in the literature on the effect of
aspirin on bleeding time in different animal models.
Our group presented
evidence that in rats aspirin at doses between 25 and 200 mg/kg b.w.,orally did not prolong bleeding time, measured at room temperature by two techniques (1,2).
Minsker and Kling (3), using different experimental conditions but
also in rats, found a significant, dose-related prolongation of bleeding time after aspirin.
They recommended this technique for screening platelet active
drugs in rats. The purpose of the present study was to investigate whether methodological differences assessed in the preceding paper (4) might account for the different results obtained by various investigators, and whether bleeding time measurement techniques in rats are specifically sensitive to aspirin or to other anti-platelet-function drugs. MATERIALS
AND
METHODS
The techniques and experimental conditions used to measure tail bleeding time of unanaesthetized male rats (CD, Charles River, Italy, 250-300 g body weight) are described in the preceding paper (4).
For clarity's sake, they
are summarized in Table I. Platelet malondialdehyde generation on stimulation with thrombin was assessed as described (5). Student's t-test was used for statistical analysis. The following drugs were used : aspirin(Bayer, Milan, Italy) was given orally suspended in 0.5% carboxymethyl-cellulose, or as its soluble lysine salt (Flectadol, Maggioni, Milan, Italy) reconstituted with sterile redistilled water and injected intraperitoneally at different intervals before bleeding time was measured ;
SH 869 (2 piperazinyl-4-thiomorpholino-pyrido
(3-2-a)pyrimidine sulphate trihydrate from Karl Thomae, Biberach a/Riss,West Germany) was dissolved in sterile redistilled water just before the experiment and injected i.p. at the dose of 25 mg/kg b-w. one hour before bleeding time was measured ;
heparin
(Novo Industry A/S, Copenhagen, Denmark) was injected
i.v. at the dose of 100 units/kg b.w. 10 min before bleeding time was measured; stanozolol(Winstro1 , 17-Jl-hydroxy-17d-methyl-androstan/3,3-C/pyrazole from Zambon, BressoI Italy): rats were fed a diet containing 50 mg/kg of stanozolol for 16 weeks (6).
BLEEDING
Vo1.15,No.1/2
TABLE Effects of Aspirin (200
201
TIME IN RATS AND ASA
I
mg/kg,i.p.) Administered 1 hour Before Measurement of
Bleeding Times in Different Experimental Conditions. Each Value Represents Mean + S.E.M. of 10 Rats. Bleeding
Times
(sec.)
I
Group
I
Tail
A-+
Milieu
"C
TRANSECTION
TEMPLATE Control
Aspirin
Control
Aspirin
Air
23
133 + 8
129 2 3
390 t 27
417 t 47
Air
23
197 +17
144 +16+
424 5 58
450 5 68
B
4
C
.,& Saline
23
293 1t17
265 _t22
518 _ + 39
502 1t 97
0
G
37
113 -t12
1082
200 -t 28
507 -t 47
Saline
5
Position of the tail during measurements : -(horizontal) + PcO.01
;
(vertical).
vs control values.
The effect of these drugs on template bleeding time was determined on the same animals since previous experiments had shown no significant difference between two consecutive measurements made on the tail of the same control rats. As this was not the case for the transection bleeding times, different groups of control and treated animals were used (each animal being transected only once) (1). Fawn-Hooded rats with 'storage pool' disease (7) were obtained from Hoffman-La Roche, Basle, Switzerland, through the courtesy of Dr. T.B.Tschopp. Severe thrombocytopenia (less than 10,000 plateletslpl) was induced by injection of a specific rabbit antiplatelet serum (8). RESULTS 1. Effects of Aspirin on Bleeding Times in Different Experimental Conditions in Normals Rats. A. Effect of a Single High Dose of Aspirin
BLEEDING
202
TIME IN RATS AND ASA
Vo1.15,No.l/2
a) Template Technique Aspirin, administered at a relatively large dose (200 mg/kg b.w. i.p.) one hour before testing, did not modify template bleeding times in groups A, C and Cl (Table I).
It significantly shortened the bleeding times of rats from
group B (tail vertical in room air at 23Y). b) Transection Technique Aspirin treatment did not modify transection bleeding times in groups A, B and C but it significantly prolonged it in group D(tai1 vertical in isotonic saline at 37°C) (Table II). The duration of the effect of aspirin in the latter experimental condition was investigated in groups of rats at different intervals after drug administration.
Table II shows that bleeding time was maximally prolonged at one
hour, becoming progressively shorter thereafter until it had returned to nornel by 24 hours.
Platelet malondialdehyde production appeared to be completely
inhibited during the first 24 hours after aspirin treatment and returned to normal only after 96-120 hours.
The inhibition of platelet function by
aspirin was therefore clearly dissociated from its prolongation of bleeding time.
Table II also shows that template
bleeding times measured between 1
and 48 hours after aspirin administration remained constantly within the normal range. B.
Effect of Single Lower Doses of Aspirin Aspirin, administered at single (oral or intraperitoneal) doses between
1 and 100 mg/kg did not significantly modify bleeding times measured one hour after dosing.
A slight not significantlenghthening was observed in animals
from group D given 5.0 mg/kg, i.p. (template) or 100 mg/kg, i.p. (transection) (141 + 27 sec. and 302 + 45 set, respectively). 2.
Bleeding Times in Rats with Experimental Thrombocytopenia or Congenital 'Storage Pool' Defect Bleeding times measured by either the template or transection techniques
(tail vertical in saline at 37"C, group 0 of Table I) were extremely prolonged (> 600 seconds) in rats made thrombocytopenic and in rats with congenital 'storage pool' disease. 3.
Effects of Other Drugs Active on the Haemostatic System The effect on bleeding time of some drugs active on haemostasis was meas-
ured according to scheme D of Table I.
Vo1.15,No.1/2
BLEEDING
TIME
TABLE
203
IN RATS AND ASA
II
Time Course of the Effects of Aspirin (200 mg/kg b.w., i.p.) on Template and Transection Bleeding Times (Group D, Table I) and Platelet Malondialdehyde Generation. Mean + S.E.M. of Groups of 6-10 Rats. Malondiaidehyde
Bleeding Time (set)
Hours after administration
TEMPLATE
(nmol/l.4xlO
0
103 + 8
216 + 70
1
114 +lO
570 f 30
6
112 5 9 -
516 + 49
n.d.
12
380 + 54
n.d.
24
99 + 9
252 + 45
n.d.
48
platelets)
TRANSECTION
1.60 f 0.20 n.d.
72
104T8 -
147 T 21
0.53 + 0.06
96
-
1.28 5 0.18
120
-
1.62 ; 0.25
0.72 + 0.04
n.d. = not detectable ____________________~~~~~~~~~~ Administration of a single dose of SH 869 -
a pyrimido-pyrimidine deriva-
tive which inhibits platelet aggregation in rats (8,9) -
resulted in signifi-
cant prolongation (over 500 set) of bleeding times measured by both techniques. Heparin, the classical anticoagulant drug, slightly prolonged template bleeding time but invariably made transection bleeding time longer than 600 seconds. Transection bleeding time was significantly longer (488 t 31 set) in rats under chronic treatment with stanozolol, an anabolizing steroid with marked fibrinolytic activity (6). 4.
Effect of Aspirin on Tail Blood Flow in Normal Rats One hour after aspirin (200 mg/kg i.p.), blood flow in the tails of rats
from groups B and D was significantly reduced (Table
III).
The absolute
blood
flow values were significantly higher at 37'C than at 23'C, both before and after aspirin treatment.
204
BLEEDING
TIME IN RATS AND ASA
T A B L E
Vo1.15,No.1/2
III
Tail Blood Flow (ml/min) of Normal Rats Before and 1 hour After Aspirin (200 mg/kg b.w., i.p.). Each Value Represents Mean 2 S.E.M. of 8 Animals.
Before ASA
Temperature ("C)
After ASA
P
23
0.036 t 0.004
0.017 t 0.002
RESEARCH 15; 199-207 Press Ltd.1979. Printed
in Great
Britain
BLEEDING TIME IN LABORATORY ANIMALS. III - DO TAIL BLEEDING TIMES IN RATS ONLY MEASURE A PLATELET DEFECT? (THE ASPIRIN PUZZLE)
Elisabetta DEJANA', Antonio QUINTANA'* , Antonella CALLIONIO and Giovanni de GAETANO Laboratory of Autonomic Nervous System and Laboratory of Cardiovascular Clinical Pharmacology Istituto di Ricerche Farmacologiche "Mario Negri" Via Eritrea, 62 - 20157 MILAN, Italy
(Received
19.2.1979.
Accepted
by
Editor
H.
Stormorken)
ABSTRACT The template bleeding time tested in the present study was not prolonged in rats given aspirin (single doses ranging from 1 to 200 mg/kg b.w.). In contrast, bleeding after the transection of tails immersed in saline at 37°C was significantly longer in aspirin treated than in control rats. This prolongation was maximal 1 hour after aspirin (200 mg/kg) and was back to normal within 24 hours, when platelet malondialdehyde formation was still completely inhibited. Both template and transection bleeding times were significantly prolonged in thrombocytopenic and thrombocytopathic rats and in normal rats given a pyrimido-pyrimidine compound (SH-869). These data suggest that platelets are important in the haemostatic process of the rat, but that the platelet defect induced by aspirin is not detectable by the bleeding time measurement techniques used. These may be sensitive not only to platelet dysfunction but also to modifications of vascular tone and blood flow and to blood coagulation and fibrinolysis systems. The complex and contrasting effects of aspirin on haemostatic factors other than platelets (for instance on vascular prostacyclin and fibrinolysis) may influence bleeding times measured by different techniques and contribute to the puzzling results obtained so far with this drug.
'At present at the Department of Pathology, McMaster University, Hamilton, Ontario, Canada. **Visiting Scientist from the Department of Pharmacology, University of Bilbao, Spain. o Reprint requests. 199
BLEEDING
200
TIME IN RATS AND ASA
Vo1.15,No.l/2
INTRODUCTION Contrastating results are reported
in the literature on the effect of
aspirin on bleeding time in different animal models.
Our group presented
evidence that in rats aspirin at doses between 25 and 200 mg/kg b.w.,orally did not prolong bleeding time, measured at room temperature by two techniques (1,2).
Minsker and Kling (3), using different experimental conditions but
also in rats, found a significant, dose-related prolongation of bleeding time after aspirin.
They recommended this technique for screening platelet active
drugs in rats. The purpose of the present study was to investigate whether methodological differences assessed in the preceding paper (4) might account for the different results obtained by various investigators, and whether bleeding time measurement techniques in rats are specifically sensitive to aspirin or to other anti-platelet-function drugs. MATERIALS
AND
METHODS
The techniques and experimental conditions used to measure tail bleeding time of unanaesthetized male rats (CD, Charles River, Italy, 250-300 g body weight) are described in the preceding paper (4).
For clarity's sake, they
are summarized in Table I. Platelet malondialdehyde generation on stimulation with thrombin was assessed as described (5). Student's t-test was used for statistical analysis. The following drugs were used : aspirin(Bayer, Milan, Italy) was given orally suspended in 0.5% carboxymethyl-cellulose, or as its soluble lysine salt (Flectadol, Maggioni, Milan, Italy) reconstituted with sterile redistilled water and injected intraperitoneally at different intervals before bleeding time was measured ;
SH 869 (2 piperazinyl-4-thiomorpholino-pyrido
(3-2-a)pyrimidine sulphate trihydrate from Karl Thomae, Biberach a/Riss,West Germany) was dissolved in sterile redistilled water just before the experiment and injected i.p. at the dose of 25 mg/kg b-w. one hour before bleeding time was measured ;
heparin
(Novo Industry A/S, Copenhagen, Denmark) was injected
i.v. at the dose of 100 units/kg b.w. 10 min before bleeding time was measured; stanozolol(Winstro1 , 17-Jl-hydroxy-17d-methyl-androstan/3,3-C/pyrazole from Zambon, BressoI Italy): rats were fed a diet containing 50 mg/kg of stanozolol for 16 weeks (6).
BLEEDING
Vo1.15,No.1/2
TABLE Effects of Aspirin (200
201
TIME IN RATS AND ASA
I
mg/kg,i.p.) Administered 1 hour Before Measurement of
Bleeding Times in Different Experimental Conditions. Each Value Represents Mean + S.E.M. of 10 Rats. Bleeding
Times
(sec.)
I
Group
I
Tail
A-+
Milieu
"C
TRANSECTION
TEMPLATE Control
Aspirin
Control
Aspirin
Air
23
133 + 8
129 2 3
390 t 27
417 t 47
Air
23
197 +17
144 +16+
424 5 58
450 5 68
B
4
C
.,& Saline
23
293 1t17
265 _t22
518 _ + 39
502 1t 97
0
G
37
113 -t12
1082
200 -t 28
507 -t 47
Saline
5
Position of the tail during measurements : -(horizontal) + PcO.01
;
(vertical).
vs control values.
The effect of these drugs on template bleeding time was determined on the same animals since previous experiments had shown no significant difference between two consecutive measurements made on the tail of the same control rats. As this was not the case for the transection bleeding times, different groups of control and treated animals were used (each animal being transected only once) (1). Fawn-Hooded rats with 'storage pool' disease (7) were obtained from Hoffman-La Roche, Basle, Switzerland, through the courtesy of Dr. T.B.Tschopp. Severe thrombocytopenia (less than 10,000 plateletslpl) was induced by injection of a specific rabbit antiplatelet serum (8). RESULTS 1. Effects of Aspirin on Bleeding Times in Different Experimental Conditions in Normals Rats. A. Effect of a Single High Dose of Aspirin
BLEEDING
202
TIME IN RATS AND ASA
Vo1.15,No.l/2
a) Template Technique Aspirin, administered at a relatively large dose (200 mg/kg b.w. i.p.) one hour before testing, did not modify template bleeding times in groups A, C and Cl (Table I).
It significantly shortened the bleeding times of rats from
group B (tail vertical in room air at 23Y). b) Transection Technique Aspirin treatment did not modify transection bleeding times in groups A, B and C but it significantly prolonged it in group D(tai1 vertical in isotonic saline at 37°C) (Table II). The duration of the effect of aspirin in the latter experimental condition was investigated in groups of rats at different intervals after drug administration.
Table II shows that bleeding time was maximally prolonged at one
hour, becoming progressively shorter thereafter until it had returned to nornel by 24 hours.
Platelet malondialdehyde production appeared to be completely
inhibited during the first 24 hours after aspirin treatment and returned to normal only after 96-120 hours.
The inhibition of platelet function by
aspirin was therefore clearly dissociated from its prolongation of bleeding time.
Table II also shows that template
bleeding times measured between 1
and 48 hours after aspirin administration remained constantly within the normal range. B.
Effect of Single Lower Doses of Aspirin Aspirin, administered at single (oral or intraperitoneal) doses between
1 and 100 mg/kg did not significantly modify bleeding times measured one hour after dosing.
A slight not significantlenghthening was observed in animals
from group D given 5.0 mg/kg, i.p. (template) or 100 mg/kg, i.p. (transection) (141 + 27 sec. and 302 + 45 set, respectively). 2.
Bleeding Times in Rats with Experimental Thrombocytopenia or Congenital 'Storage Pool' Defect Bleeding times measured by either the template or transection techniques
(tail vertical in saline at 37"C, group 0 of Table I) were extremely prolonged (> 600 seconds) in rats made thrombocytopenic and in rats with congenital 'storage pool' disease. 3.
Effects of Other Drugs Active on the Haemostatic System The effect on bleeding time of some drugs active on haemostasis was meas-
ured according to scheme D of Table I.
Vo1.15,No.1/2
BLEEDING
TIME
TABLE
203
IN RATS AND ASA
II
Time Course of the Effects of Aspirin (200 mg/kg b.w., i.p.) on Template and Transection Bleeding Times (Group D, Table I) and Platelet Malondialdehyde Generation. Mean + S.E.M. of Groups of 6-10 Rats. Malondiaidehyde
Bleeding Time (set)
Hours after administration
TEMPLATE
(nmol/l.4xlO
0
103 + 8
216 + 70
1
114 +lO
570 f 30
6
112 5 9 -
516 + 49
n.d.
12
380 + 54
n.d.
24
99 + 9
252 + 45
n.d.
48
platelets)
TRANSECTION
1.60 f 0.20 n.d.
72
104T8 -
147 T 21
0.53 + 0.06
96
-
1.28 5 0.18
120
-
1.62 ; 0.25
0.72 + 0.04
n.d. = not detectable ____________________~~~~~~~~~~ Administration of a single dose of SH 869 -
a pyrimido-pyrimidine deriva-
tive which inhibits platelet aggregation in rats (8,9) -
resulted in signifi-
cant prolongation (over 500 set) of bleeding times measured by both techniques. Heparin, the classical anticoagulant drug, slightly prolonged template bleeding time but invariably made transection bleeding time longer than 600 seconds. Transection bleeding time was significantly longer (488 t 31 set) in rats under chronic treatment with stanozolol, an anabolizing steroid with marked fibrinolytic activity (6). 4.
Effect of Aspirin on Tail Blood Flow in Normal Rats One hour after aspirin (200 mg/kg i.p.), blood flow in the tails of rats
from groups B and D was significantly reduced (Table
III).
The absolute
blood
flow values were significantly higher at 37'C than at 23'C, both before and after aspirin treatment.
204
BLEEDING
TIME IN RATS AND ASA
T A B L E
Vo1.15,No.1/2
III
Tail Blood Flow (ml/min) of Normal Rats Before and 1 hour After Aspirin (200 mg/kg b.w., i.p.). Each Value Represents Mean 2 S.E.M. of 8 Animals.
Before ASA
Temperature ("C)
After ASA
P
23
0.036 t 0.004
0.017 t 0.002
