Collagen-Induced Pulmonary Thromboembolism in Mice
Transcript of 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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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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THROMBOEMBOLISM IN MICE
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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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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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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THROMBOEMBOLISM IN MICE
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).
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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
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(1969).
3.
E. J. Natheniel and A. B. Chandler.
"Electron-Microscopic Study of
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Ultrastruct. Res. 22:348-359 (1968).
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6. J. F. Mustard.
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Abstract.