Bleeding time in laboratory animals. III - Do tail bleeding times in rats only measure a platelet defect? (The aspirin puzzle)

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

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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

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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

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TIME IN RATS AND ASA

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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.

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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

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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


37

0.110 + 0.010

0.070 t 0.006

<0.0025

DISCUSSION This study confirms and extends previous observations (1,2) that aspirin does not

prolong template tail bleeding time in normal rats.

bleeding after transection

In contrast,

of a tail immersed in saline at 37°C lasted

significantly longer in rats given aspirin (1 hour before) than in control rats, a finding in agreement with that of Minsker and Kling (3).

However,

transection bleeding time was back to normal 24 hours after aspirin when platelet malondialdehyde formation was still completely inhibited (see also ref. 5).

These data indicate that the platelet defect induced by aspirin is

not detectable by either the template or the transection technique. Conversely, the template bleeding time used in the present study was sensitive to reduced platelet count and to the effect of a vasodilator and antiaggregating drug of the dipyridamole type (SH 869); in addition, it was abnormal in rats with 'storage pool' disease. by heparin.

It was only slightly affected

The transection bleeding time was significantly longer not only

in thrombocytopenic or thrombocytopathic rats, but also in rats anticoagulated by heparin or with fibrinolysis activated by chronic treatment with stanozolol. Aspirin has been shown to inhibit prostaglandin I2 (PG12) generation in the vessel wall of several animal species, including the rat (10,ll). is a potent

Since PG12

endogenous inhibitor of platelet aggregation (lO,ll), its in-

hibition by aspirin treatment could facilitate the haemostatic process, thus counteracting the aspirin-induced platelet function defect.

Vascular PG12

activity was normal in rats made thrombocytopenic by antiplatelet antiserum (12) or with 'storage pool' disease and was potentiated by the dipyridamole like compound SH 869

(unpublished observation).

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TIME IN RATS AND ASA

This observation could explain the marked effect on bleeding time of this drug which is also a potent vasodilator.

pG12 is another powerful vasodilator (11) and the reduction of blood flow observed in the tail of aspirin-treated rats could, at least in part, be due The effect of aspirin on bleeding time could there-

to its inhibition (13).

fore also reflect a modification of blood flow in the microcirculation (14,15). This could be of little importance in the larger vessels injured by the transection technique at 37Y

when reflex vasodilation is predominant.

Our finding that stanozolol, a drug active on the rat fibrinolytic system (6), prolonged transection bleeding time may be of interest in view of the observation that aspirin can activate fibrinolysis in whole human or rat blood (16,17). In conclusion, the template

bleeding time measured in the present study

(group D, Table I), was sensitive to defects of platelet number and of some platelet functions (such as those induced by 'storage pool' disease or dipyIn contrast, the test was unable to detect aspirin-

ridamole-like drugs).

induced platelet dysfunction, possibly because of the complex and contrasting effects this drug has on the haemostatic system of the rat.

It has been

suggested that platelets are more sensitive than vascular cells to in vitro inhibition by aspirin ( 18 ).

If this had been the case

in our in vivo

system, bleeding times should have been prolonged by low doses of aspirin. But this was not found, although template bleeding times were slightly longer after administration of 2.5-5 mg/kg aspirin than after 200 mg/kg.

In this

context is of interest to recall that Villa et al. (19) were unable in rats to find any dose of aspirin (between 1 and 200 mg/kg b.w., i.p.) which inhibited platelet malondialdehyde production without to some extent affecting the generation of vascular prostacyclin activity. The transection

technique was sensitive to high doses of aspirin in well

defined experimental conditions (group D, Table I), but did not necessarily reflect the defective platelet function induced by the drug.

This technique

cannot therefore be used to screen aspirin-like drugs, because it was also sensitive to drugs inhibiting blood coagulation or activating fibrinolysis. Addendum:

Data have recently been published (20) confirming -

anaesthetized rabbits -

in

suggestions made in previous reports (1,2,14,15) and

in the present study that modification by aspirin of factors other than platelets (vascular PG12?) may play a crucial role in formation of the experimental haemostatic plug.

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TIME

IN RATS AND ASA

Vol.lfi,No.l/2

REFERENCES

1.

STELLA, in time

2.

de

L.,

DONATI, M.B., animals. I.

laboratory in

rats.

Thromb.

GAETANO,

and

G.,

Res.

7,

CAVENAGHI,

platelets.

II.

aggregation

and de Aspirin

and

709,

1975.

A.E.,

Effect

of

bleeding

GAETANO, G. Bleeding time does not prolong bleeding

and

STELLA,

ditazole

time

in

on

rats.

L.

in

Ditezole

vivo

platelet

Haemostatis

6,

190,

1977. 3.

4.

MINSKER,

D.H.,

prolonged

by

DEJANA,

time

different

5.

1, 6.

hi.,

Bleeding Res.

QUINTANA, animals.

S.,

in

and

de

in

time

10,

A.,

conditions

A.,

rats

is

de

GAETANO,

A comparison

Thromb.

GAETANO,

rat:

and

II.

rats.

in

1977.

619,

G.

Res.

this

Aspirin,

A dilemma

G. of issue.

thrombo-

resolved?

Lancet

1978. EC,

M.,

KORNBLIHTT,L.,

fibrinolytic

with

,

prostacyclin

1307,

P.J.

Thromb.

laboratory

VILLA,

and

MYSLIWI of

in

assay

LIVIO, xane,

KLING,

CALLIONI

E.,

Bleeding

and aspirin,

an

anabolic

Chemical

and

potential

in

steroid,

Fibrinolysis

DONATI,

rats

by

stanozolol. and

M.B.

Enhancement

long-term In:

Thrombolysis,

treatment

Progress vol.

4,

in

1978,

in

press. 7.

TSCHOPP,

T.B.,

serotonin

in

and

WEISS,

young

storage-pool

H.J.

platelets

disease.

Decreased of

Thromb.

ATP,

fawn-hooded

Diath.

ADP

rats

Haemorrh.

and with

32,

670,

1974. 8.

DEJANA,

E.,

CASTELLI

Contribution ADP

9.

in

of

the

rat.

to

Thromb.

and

infusion

of

Effect Haematol.

VILLA, S., rat vascular

and

GAETANO,

treatment

with

14,

1977.

1117,

lysine

POGGI,

J.

Stand.

de

39,

ADP.

drugs.

Fast,

G.

G.,

and

BONACCORSI ,A.

cardiovascular 135, A.,

haemolysis

thrombotic

tissues.

GAETANO,

the

Haemost. I.,

aggregation

intravenous

de

M.G.,

REYERS-degli-INNOCENTI, Platelet

10.

,

platelets

and

induced of

effects

de in

GAETANO, rats

potentially 13,

331,

acetylsalicylate.

G.

by anti-

1974.

Prostacyclin-like

long-lasting

of

1978.

activity inhibition

Prostanlandins

by

in

BLEEDING

Vo1.15,No.1/2

11.

MONCADA,

and

S.,

aggregation

and

disciplinary

VANE,

New York:

VILLA,

S.,

14.

induces

D., of

Haemostasis

BLAJCHMAN,

M.A.,

The

of

L.A.

M.,

and

M.L.,

human

fibroblasts

Commun. 19.

VILLA,

S.,

effect

of

in

rats

1979, 20.

78,

AMEZCUA, metabolites of

the

294,

the

of in

micro-

some

haemostasis

and

MUSTARD,

the

suppl.

296,

Effect

1,

525, of

J.F.

bleeding

time

259,

activity

Med.

1977.

after 1977.

aspirin

on

the

Cultured

preparation.

M.J.,

MAJERUS,

P.W.

arterial

cells

produce

prostaglandin.

Biochem.

a

labile

Biophys.

Res.

1977.

LIVIO, aspirin

cannot in

J.

and

platelet-inhibitory

J.

50,

fibrinolytic

DILLENDER,

G.

the

1978. in

on

Engl.

In

in

1976.

HI RSH,

M.B.

GAETANO,

921,

interest 74,

Blood

rats.

de

out-put

Effects

blood

in

BAENZIGER,

and

inhibitors

DONATI,

system

skin

5,

A.F.,

N.

A.,

from 1977.

Haemostasis

current

rabbits.

Increased

fibrinolytic 18.

of

SENYI,

ingestion.

MYSLIWIEC,

K.E.

prostaglandin

thrombocytopenic

701,

mesentery.

agents

aspirin 17.

rabbit

Normal

11,

15,

and

MOROZ,

Res.

Prostaglandins

ARFORS,

thrombosis.

G. tissues

cardiac

and

Garattini

239.

of

the

S.

GAETANO,

CALLIONI,

pharmacological

in 16.

rat.

p.

vascular

redistribution

conscious

effect

de

in

A Multi-

and

1978,

and

A.,

platelet

Gaetano,

Thromb.

QUINTANA,

E.,

207

ASA

Platelets:

Press,

rats.

DEJANA,

vasculature

15.

de

activity

Aspirin

BERGQVIST,

In:

A.,

AND

Prostacyclin,

G.

CALLIONI,

thrombocytopenic

intact

J.R.

Raven

prostacyclin-like

13.

IN RAT6

thrombosis.

Approach,

(Eds.).

12.

TIME

be

M.,

and

on

platelet

de

completely

GAETANO, and

G.

The

vascular

dissociated.

inhibitory

prostaglandins Br.

J.

Haematol.

press. J.L.,

PARSONS, of

arachidonic

haemostatic

plug.

M.,

and acid,

Thromb.

MONCADA, aspirin Res.

S.

Unstable

and 13,

477,

the

formation

1978.