Collagen-Induced Pulmonary Thromboembolism in Mice

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THROMBOSIS RESEARCH

Printed in the United States

Vol. 1, pp. 233-242, 1972

Pergamon Press, Inc.

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 1Jpjohn ompany

Kalamazoo, Michigan 49001

(Received 25.5.1972. Accepted by Editor A.L. Copley)

ABSTRACT:

Injection of an aqueous collagen suspension into the caudal-

caval vein of normal rats or mice resulted in about 75% mor-

tality due to the formation of platelet aggregates which sub-

sequently lodged in the pulmonary circulation.

Animals pre-

treated with inhibitors of platelet aggregation [aspirin (ASA),

phenylbutazone] or animals made thrombocytopenic were protected

from death. Based on these observations a 2-stage,group sequen-

tial 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 non-

traumatized, 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 re-

_-

latively 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 adeno-

sine 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

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THROMBOEMBOLISM IN MICE

Vol.l,No.3

ficiency.

It seemed reasonable, therefore,

to investigate this system as a pos-

sible model for studying platelet thromboemboli formation in rats or mice.

Intravenous injection of ADP into the tail vein of either rats or mice re-

sults in platelet aggregates which subsequently can become trapped in the

pulmonary circulation.

However, we found ADP injections to be ineffective

in causing death. This lack of effect may be due to vasodilatation induced

by ADP (5) as well as to the reversibility of ADP-induced platelet aggrega-

tion 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 throm-

bus formation.

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

thrombotic agents based

on the above observations and the results of its ap-

plication 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@, B rroughs Wellcome and Co., Tuckahoe, N.Y.) (8).

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

Engineering Corp., Cambridge, Mass.).

After nine days the platelet count

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.

Care was taken not to overheat the

contents of the blendor.

The homogenate was either centrifuged at 3,000

RPM (International Model PR.3) r 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.


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Platelet Counts. Platelet counts on whole blood samples were deter-

mined microscopically.

The Coulter Counter, Model B

(Coulter Electronics,

23 5

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. For screening, the

dose was arbitrarily set at 100 mg/kg. 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 solu-

tion (without Ca*) to g ve 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

compound or with vehicle alone. After two hours,

the mice were given col-

lagen intravenously.

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

lows:

Accumulated Deaths No.

Stage

Accept Reject Mice

1 ND

4

6

2 6

7 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


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Vol.l No.3

or more of the animals die.

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.

Thus,

the expected false negative rate for such a drug is 0.02.

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 clas-

sification while those with activity equivalent to

quire an average of 12 animals.

To monitor the performance of the test system,

eludes a group of animals treated with vehicle and

that of aspirin will re-

each screening run in-

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 plate-

let 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.


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THROMSOEMSOLISM IN MICE

Vol.l,No.3

Microscopic examination of the lung sections from normal animals in-

jected with collagen showed many aggregates of platelets which had oc-

cluded 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 col

lagen. 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).


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THROME OEME@LISM

IN MICE

239

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)

Tyrode's

Collagen

No.

10

10

Platelet

Count

x 103/mm3

1,674$59

157f14

The maximal effect of the drug obviously depends on the rate of absorp-

tion 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 admin-

istration,

we decided to inject collagen at this time.

TABLE V

Effect of Phenylbutazone and Aspirin on Thromboembolism in Mice

Phenylbutazone (300 mg/kg)

Aspirin (300 mg/kg)

Time No.

Survival (X)

No.

Survival (W)

Control 30 10 3 (30) 10 3 (30)

Treated 30 -- 10 5 (50)

60 10 10 (100) 10 5 (50)

120 10 7 (70) 10 6 (60)

\ 180 9 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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241

This report describes a screen for anti-thrombotic agents which over-

come the objections listed above.

Furthermore,

the model uses non-anesthetized

and non-traumatized animals.

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 plate-

let 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 Forma-

tion 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 Platelet-

Aggregating 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 Thrombo-

cytopenia 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.

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Collagen-Induced Pulmonary Thromboembolism in Mice