THROMBOSIS

Printed

RESEARCH
in the United

Vol. 1, pp. 233-242,

1972
Pergamon
Press, Inc.

States

COLLAGEN-INDUCED PULMONARY THROMBOEMBOLISM IN MICE

E. E. Nishizawa, D. J. Wynalda, D. E. Suydam, T. R. Sawa and J. R. Schultz

Research Laboratories, The 1JpjohnCompany
Kalamazoo, Michigan 49001

(Received

ABSTRACT:

25.5.1972.

Accepted

by

Editor

A.L.

Copley)

Injection of an aqueous collagen suspension into the caudalcaval vein of normal rats or mice resulted in about 75% mortality due to the formation of platelet aggregates which subsequently lodged in the pulmonary circulation. Animals pretreated with inhibitors of platelet aggregation [aspirin (ASA),
phenylbutazone] or animals made thrombocytopenic were protected
from death. Based on these observations a 2-stage,group sequential screen for antithrombotic activity was developed in which
6 mice were tested in each stage. Compounds possessing little
or no activity could be eliminated after testing in the first
stage. Aspirin was used as a positive standard to monitor the
test system. A secondary test for platelet aggregation in rat
platelet-rich plasma was used to confirm activity.

Introduction
Ideally, a screen which is capable of detecting anti-thrombotic agents
would be one in which platelet thrombi formation could be evaluated in nontraumatized, unanesthetized animals.

It would also be advantageous to test

in small animals, such as mice, since quite often only small amounts of test
compounds are available.

Furthermore, the screen should be rapid and inex-

pensive to operate.
Previous thrombus models _in vivo (1,2) were expensive and required a relatively large amount of compound, since rabbits or pigs were the animals of
choice.

Moreover, these systems involved the use of anesthetized animals sub-

jected to surgical trauma.

In 1968 Nathaniel and Chandler (3) reported that rats infused with adenosine diphosphate (ADP) died as a result of massive pulmonary congestion caused
by platelet aggregates.

It

was also reported that ADP injections into the car

onary arteries of pigs (4) caused death in these animals due to coronary insuf
233

THROMBOEMBOLISM

234

IN MICE

Vol.l,No.3

ficiency.
It seemed reasonable, therefore, to investigate this system as a possible model for studying platelet thromboemboli formation in rats or mice.
Intravenous injection of ADP into the tail vein of either rats or mice results in platelet aggregates which subsequently can become trapped in the
pulmonary circulation.
in causing death.

However, we found ADP injections to be ineffective

This lack of effect may be due to vasodilatation induced

by ADP (5) as well as to the reversibility of ADP-induced platelet aggregation and rapid degradation of ADP.
Collagen infusion appeared to be more meaningful, since collagen-induced
platelet aggregation more closely resembles the physiological situation (6)
where exposure of subendothelial tissue may serve as focus to initiate thrombus formation.

This report deals with the development of a screen for anti-

thrombotic agents based on the above observations and the results of its application in one series of 120 compounds.
Methods
Male Upj:TUC(SD)spf rats weighing around 250 g and male Upj:TUC(ICR)spf
mice weighing around 20 g were used in these studies.

Thrombocytopenia was

produced by whole body X-irradiation (7) or by treatment with busulfan

(Mylera@,

Burroughs Wellcome and Co., Tuckahoe, N.Y.) (8).

Rats were exposed to 520 R from a VandeGraaf Generator (High Voltage

After nine days the platelet count

Engineering Corp., Cambridge, Mass.).

had decreased to l/10 normal.

Mice were given 50 mg busulfan/kg in a single oral dose.

These animals

were used lo-12 days after dosing when the platelet count was l/10 normal.
Collagen.

Collagen suspension was prepared by homogenizing 2 g tendon

collagen (Sigma, St. Louis, MO.) in 120 ml of modified Tyrode's solution

(without Ca*)

in a Waring blendor.

contents of the blendor.

Care was taken not to overheat the

The homogenate was either centrifuged at 3,000

RPM (International Model PR.3) or filtered through several layers of paper

tissue.

Enough suspension of collagen (usually 0.2-0.3 ml) was administered

(i.v.) to give a survival rate of about 20% in normal fasted mice.

For rats,

the dose was about ten times as great.

Histology.

Lung tissues were fixed in buffered 10% formalin, sections

were cut at 6p, and stained with hematoxylin and eosin or vanGiesen's stain.

THROMBOEMBOLISM

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Platelet Counts.

23 5

IN MICE

Platelet counts on whole blood samples were deter-

mined microscopically.

The Coulter Counter, Model B (Coulter Electronics,

Hialeah, Fla.) was used for counts of platelet-rich plasmas.

Drugs.

All compounds to be tested were dissolved or suspensed in a

0.25% aqueous methyl cellulose vehicle or in water.

dose was arbitrarily set at 100 mg/kg.

For screening, the

Drugs were given orally to fasted

animals 2 hours prior to the injection of collagen.

Control animals were

given water or methyl cellulose vehicle.

Platelet Aggregation.

Platelet aggregation in platelet-rich plasma

(PRP) was measured in blood samples obtained from the aorta of rats or
mice.

Citrated blood (1 part of 2.2% sodium citrate to 9 parts blood) was

centrifuged at 1500 RPM (International Centrifuge, Size 2).

The PRP was

separated and the platelet count was determined on the Coulter Counter
Model B.

The remainder of the red cells was centrifuged at 3,000 RPM for

platelet poor plasma (PPP).

Platelet counts were adjusted to 1 X 106/mm3

with the corresponding PPP and further diluted with modified Tyrode's solution (without Ca*)

to give a final count of 500,000/mm3.

Platelet aggregation was measured using either the Payton Aggregation

Module (Payton Assoc., Buffalo, N.Y.) or the Chronolog instrument (Broomall,
Pa.).

To a 0.95 ml sample of PRP warmed for 3 minutes at 37OC, 0.05 ml of

collagen suspension was added and the platelets allowed to aggregate.

The

concentration of collagen used was adjusted to give less than maximum extent
of aggregation.
Screen.

Mice that had been f&ted

overnight were orally dosed with a

After two hours, the mice were given col-

compound or with vehicle alone.

lagen intravenously.

The testing procedure in a two-stage sequential screen (9) was as follows:

Stage

Accumulated Deaths
Accept
Reject

No.
Mice

ND

12

A test compound is declared inactive in the first stage if 4 or more of the

6 animals tested die.

If less than 4 animals die, no decision (ND) is made

and the compound is then tested in the second stage with another 6 animals.
At this stage the compound is declared active if 6 or less of the 12 animals
(total of first and second stages) tested die or it is declared inactive if 7

THROMBOEMBOLISM

236

or more of the animals die.

Vol.l,No.3

IN MICE

With this procedure a compound may be de-

clared inactive at either stage, but it must he tested in both stages to

be classified as active.

This concentrates testing on the more active a-

gents.
In a series of studies with this animal model it was found that the
survival rates for animals treated with vehicle and 300 mg/kg aspirin were
16.3% (371227) and 80.6% (87/108), respectively.

This information provides

a basis for judging the expected performance of the screen.

It gives

the probability of declaring a compound active as a function of the true

(but unknown) probability (p) of animals surviving in this test system.
This curve shows that a compound with activity equivalent to that observed
with aspirin (p = 0.80) will be declared active in 98 of 100 tests.
the expected false negative rate for such a drug is 0.02.

Thus,

Compounds with

activity similar to that found with vehicle treatment (p = 0.16) would be

declared active in only 4 of 100 tests , giving a false positive rate of 0.04.
It was found the expected number of animals required to classify a drug
as a function of the true probability of reducing mortality.

Compounds with

activity similar to vehicle will require an average of 6.3 animals for classification while those with activity equivalent to that of aspirin will require an average of 12 animals.
To monitor the performance of the test system, each screening run ineludes a group of animals treated with vehicle and a group treated with a
positive standard, aspirin.

This test system is considered out of control

if 4 or more of the 6 animals die.

Active drugs are further tested in rats.

The animals were orally dosed

in groups of 6 and after 1 hour, aortic blood samples were removed and platelet aggregation studies on PRP were done as described above.
Results
In our initial study using rats, it was shown that collagen injection had
no effect on thrombocytopenic rats (Table I).

All normal rats collapsed and

a majority of them died but the thrombocytopenic animals were unaffected.

Since one of the animals that collapsed in the thromhocytopenic group had a
platelet count of 730,F00/mm3, it was not included in the average for the
platelet count.

THROMBOEMBOLISM

Vol.l,No.3

IN MICE

237

TABLE I
The Effect of Irradiation (500 R) on Thromboembolism in Rats
Platelet
count
x 103/mm3

No.
Control

6
,

Treated

Collapse

1,495+260

1052 24*

Survival (X)

1 (17)

6 (100)

*Average of 5: the animal that collapsed had platelet

count of 730,300/mm3.
Oral administration of aspirin (ASA) inhibited collagen-induced platelet aggregation in PRP prepared from these animals (Table II).

There was

no change in platelet count and following collagen infusion a significant

number of the animals (90%) pre-dosed with ASA survived whereas in the control the survival rate was 30%.

TABLE II
Effect of ASA (400 mg/kg) on Platelet Aggregation
and Thromboembolism in Rats

No.

Platelet
Count
x 103/mm3

Collagen
Aggregation

No.

Collapse

Survival
(X)

Control

1,793+77

51.520.9

10

10

3 (30)

Treated

1,732+68

-0.5f0.4

10

10

9 (90)

p<O.OOl

A similar pattern of survival of thrombocytopenic animals was seen when

mice were used.

In these animals it was more convenient to produce thrombo-

cytopenia with busulfan.

After about 10 days, the platelet count in these

animals was reduced to l/10 normal.

Collagen injection into these animals

were without effect while in the control animals, only 33% survived.
TABLE III
Effect of Busulfan (50 mg/kg) on Thromboembolism in Mice

No.

Platelet Count
x 103/mm3

Control

12

2,542 2 180

Treated

12

260 3- 35

Survival
(X)
4 (33)
12 (100)

THROMSOEMSOLISM

238

Vol.l,No.3

IN MICE

Microscopic examination of the lung sections from normal animals injected with collagen showed many aggregates of platelets which had occluded and distended the capillaries (Fig. la).

Most of the aggregates

were located in capillaries at the periphery of the lung.

Lung sections

from thrombocytopenic animals given collagen showed normal histology (Fig.

lb).

Fig. la

Fig. lb

Lung sections from mice 3 minutes after intravenous administration of collagen. In normal mice the capillaries are occluded with platelet aggregates
and distended (Fig. la), but when the animals are thrombocytopenic the vessels
are not occluded (Fig. lb), vanGiesen's (X 1500).

THROME!OEME@LISM

Vol.l,No.3

239

IN MICE

When collagen was injected into normal animals, the platelet count in
circulation after 3 minutes was decreased to about l/10 that seen following
infusion of Tyrode's solution (Table IV).
TABLE IV
Platelet Count in Mice 3 Minutes After 'Infusion
at Tyrode's Solution or Collagen
Substance
Infused
(0.3 ml)

No.

Platelet
Count
x 103/mm3

Tyrode's

10

1,674$59

Collagen

10

157f14

The maximal effect of the drug obviously depends on the rate of absorption and the rate of inactivation.

The effect of phenylbutazone and aspirin

at various times following oral administration is shown in Table V.

The max-

imal effect for phenylbutazone appears to take place sooner than for aspirin.
Since both these compounds were active in the mouse at 2 hr after oral administration, we decided to inject collagen at this time.
TABLE V
Effect of Phenylbutazone and Aspirin on Thromboembolism in Mice

Time

Phenylbutazone (300 mg/kg)

Survival (X)
No.

Control

30

10

Treated

30

--

60

10

120
180

3 (30)

Aspirin (300 mg/kg)

No. Survival (W)
10

3 (30)

10

5 (50)

10 (100)

10

5 (50)

10

7 (70)

10

6 (60)

6 (67)

10

3 (30)

In order to determine the dose required for our positive standard, the
effect of phenylbutazone and aspirin were studied at various dosages.
Table VI shows that 300 mg/kg gave adequate nrotection.

Aspirin was selec-

ted as our standard on the basis of its reproducibility in the test system
and its ready availability.

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Vol.l,No.3

THROMBOEMBOLISM

IN MICE

241

This report describes a screen for anti-thrombotic agents which overcome the objections listed above.
and non-traumatized animals.

Furthermore, the model uses non-anesthetized

Thus it is an improvement over existing testing

methods.
This screen has been used successfully to find potential anti-thrombotic
compounds.

However, since the screen may also respond to compounds which

have vasodilatory effects, a secondary test, involving measurement of platelet aggregation in rat PRP following oral administration of drug is used to
confirm platelet involvement.

References
1.

J. F. Mustard, H. C. Rowsell, H. A. Smythe, A. Senyi and E. A. Murphy.

"The Effect of Sulphinpyrazone on Platelet Economy and Thrombus Formation in Rabbits." Blood -29:859 (1967).

2.

E. E. Nishizawa, T. Hovig, F. Lotz, H. C. Rowsell and J. F. Mustard.

"Effect of Natural Phosphatidyl SerineFraction on Blood Coagulation,
Platelet Aggregation and Hemostasis." Br. J. Haematol. -16:487-499
(1969).

3.

E. J. Natheniel and A. B. Chandler. "Electron-Microscopic Study of

Adenosine Diphosphate-Induced Platelet Thrombi in the Rat." J.
Ultrastruct. Res. -22:348-359 (1968).

4.

L. Jorgensen, H. C. Rowsell, T. Hovig, M. G. Glynn and J. F. Mustard.

"Adenosine Diphosphate-Induced Platelet Aggregation and Myocardial
Infarction in Swine." Lab. Invest. 17:616-644 (1967).

5.

J. Swedenborg, G. Taylor and P. Olsson. "Hemodynamic Changes of

Adenosine Diphosphate and Thrombin in Relation to Their PlateletAggregating Activity." Stand. J. Lab. Invest. -27:213-219 (1971).

6.

J. F. Mustard. "Platelet Aggregation in Thromboembolic Disease."

Adv. Cardiol. 4:131-142 (1970).

7.

S. Mitra. "The Effects of Irradiation on the Megakaryocytes of Bone

Marrow and Relationship with the Platelet Values in Peripheral Blood
and Post-Irradiation Anemia." Indian J. Med. Res. -48:710-713 (1960).

8.

S. A. Evensen, M. Jeremic and P. F. Hjort. "Experimental Thrombocytopenia Induced by Busulphan (Myleran) in Rabbits." Thromb. Diab.
Hemorrh. X1X:570-577 (1968).

9.

J. R. Schultz, F. R. Nichol, G. L. Elfring and S. D. Weed. "Multiple

Stage Procedures for Drug Screening." Biometrics -27~772 (1972),
Abstract.