Delayed fibrin elimination from the lungs of burned rats with endogenous inhibition of the fibrinolytic system

THROMBOSIS RESEARCH Printed in the United

DELAYED RATS

FIBRIN

WITH

vo1.2, pp. Pergamon

States

ELIMINATION

ENDOGENOUS

FROM

INHIBITION

THE OF

LUNGS

THE

365-376,

Press,

OF

1973

Inc.

BURNED

FIBRINOLYTIC

SYSTEM

L. Bagge, Department

C.

Busch,

of Forensic

(Received

L.

Rammer

Medicine,

8.2.1973; Accepted by

and T.

University

in revised Editor P.

Saldeen

of Uppsala,

Sweden

form 28.3.1973. Olsson)

ABSTRACT The elimination of fibrin from the lungs of burned rats with endogenous inhibition of the fibrinolytic system was studied after intravenous injection of thrombin. The fibrin content using labelled fibrinogen and alof the lungs was quantified, bumin. It was found that in burned rats the elimination of fibrin from the lungs was delayed. The lungs of these rats showed micro-atelectases which were more numerous than in rats without fibrinolysis inhibition.

INTRODUCTION Micro-emboli vessels

of patients

(1, 2, 3). damage

containing

fibrin

dying from

The assumption were closely

was supported

A retarding elimination

post-traumatic

that the occurrence

correlated

by the frequent

the blood of these

are frequently

patients

in the pulmonary

pulmonary of fibrin

insufficiency

and the pulmonary

to the state of the fibrinolytic finding of marked

fibrinolysis

system

(l),

inhibition

in

( 3, 4).

effect of an exogenous of intravascular

the development

observed

of pulmonary

inhibition

fibrin from damage

365

of fibrinolysis

upon the rate of

the lungs - and consequently

- has been demonstrated

upon

in animal

366

FIBRIN

experiments lysis

has,

(5,

6, 7).

however,

disappearance fibrinolysis

The

inhibition

ned by a quantitative The study also fibrinogen

effect

from

(48 hours method,

includes

using labelled

Stockholm)

animals. weighing

access

Under

of fibrino-

study the rate of

during the phase of

injury)

fibrinogen

( 8 ) was

ad

of the validities

albumin

exami-

(9).

of human and rat

for this method.

Experimental

Thermal

inhibition

rats

after the thermal

a comparison

Vo1.2,~0.4

In the present

the lungs of burned

MATERIAL

free

of the endogenous

not been investigated.

of fibrin

FROM LUNGS

ELIMINATION

AND

50 female

METHODS

Sprague-Dawley

200 + 5 g were used.

to tap water and food (Ewos

injury.

The method

ether anaesthesia produced

has

been

which

surface

but did not lead to death.

animals

allowed

in detail previously

immersed

a third-degree

were

Farm,

rat pellets). described

the rats were

seconds

The

rats (Anticimex

scald

Control

in water at,90°C

covering

rats were

(8).

for

20

14 % of the body

clipped

and immersed

in water at 38OC. Labelled

fibrinogen.

Blomback 0.

Tangen,

(11)s

Human

and Blomback

(10 ),

AB Pharmacia),

were used.

fibrinogen

and rat fibrinogen prepared

The fibrinogen

using an electrolytic

iodination

until use,

after

labelling,

bility

of 87 %.

(Dowex 1 - X8, iodide.

while

method

The

fibrinogen

50-100

Twenty-four

mesh)

ml and 5 pCi/ml

stable.

The

(about 9 pCi/ml

a tail vein under ether anaesthesia.

for

to

by dr.

human fibrinogen

Both preparations

before

was calculawas

was used immediately had a coagula-

an ion-exchange

injection,

the thrombin

solution,

(kindly supplied

The iodination

was run through

hours before

body weight of the fibrinogen

(12).

fibrinogen.

shortly

according

to Straugn och Wagner 125 I, in our laboratory with

the rat fibrinogen

since it was less

prepared

according

was labelled

ted to be about 0. 5 atoms/molecule freeze-dried

(AB Kabi),

containing

to eliminate

infusion,

0.25

about 0.8

the rat fibrinogen)

column

mg

free

ml/l00

g

protein/

was injected

into

FIBRIN

vo1.2,~0.4

Thrombin

injection.

ved in saline weight tail

vein

siliconized

albumin. serum

Sweden). to injection.

0.5

was

tubes

for

and the lungs cleaned

Radioactivity

spectrometer channels

obtained

counted

exactly

paper

and all the

ether

collected

(about (13 ).

1 ml),

The

free

column

mg

prior

protein/ml

anaesthesia

an 18-

at the bifurcation

in plastic triple

thoracic

dissected.

with

sacrifice.

into the aorta

was

removed,

Ellermann

microhaemato-

cavity from

was

opened

connective

tissue,

and weighed.

I and

Weighed

samples

in the plastic Autowell

131

for

samples

This

II).

contained

blood tubes

was

between

and dissol-

in a gamma-

equipped

and with digital

crossover

radionuclide.

of lungs,

Ellermann

I respectively

corrected

to the lower

with pre-

Studsvik,

0.2

before

Under

Blood

Nuclear,

were

g body

determined

an ion-exchange

5 minutes

introduced

measured

125

dissol-

into a

pump

was

containing

samples.

was

rapidly

(Picker

was

anaesthesia

I (AB Atomenergi,

solution

measurement

were

set for

the higher

saline

measurements.

ved fibrinogen

values

of a

determinations

with filter

ether

II B infusion

run through

abdomen.

were

Roche)

NIH units/100

of the lungs

131

with

was

needle

and fibrinogen

Unita

volume

labelled

radioactivity

under

a Braun

and organ

the opened

Twenty-five

injected

plasma

injected

of blood

siliconized

through

crit

ml

8 ,

367

syringes.

preparation

and 13 pCi/ml

gauge

The

albumin

The

Collection

using

FROM LUNGS

(Topostasin

injection. were

in 5 minutes,

Labelled

thrombin

before

of saline

cision-ground

human

Bovine

shortly

in 1 ml

ELIMINATION

with two

recording.

The

theho

channels

counts

were

More

than 3,000

more

than 10 times

from

always

the background

radioactivity. Calculations. been

described

groups,

The method

of calculating

in a previous

the amount

of fibrin

F=

paper

(9 ).

in the lungs

-

1

Q

(T -

the fibrin For

each

(F,

mg/g)

P -

EVc),

content

in the lungs

has

rat in the experimental was

calculated

as:

FIBRIN

368

ELIMINATION

vo1.2,~0,4

FROM LUNGS

where

Q

is the factor

sisting

of the mean

@asma

fibrinogen

is the total

P

is the plasma

relative

specific

in control

125

T

125

125

for converting

I in the plasma

into mg of fibrin,

radioactivity

(cpm x 103/mg)

conof

rats,

I radioactivity/g

125

I radioactivity

tissue,

I radioactivity/g

tissue,

and the organ plasma

calculated

volume

as the product

of

131 I ratio),

(=organ/plasma

and 125

is the extravascular

EVC

the induction

of intravascular

as the mean difference

I radioactivity/g coagulation

between

tissue,

(saline

calculated

infused

before

into control rats) 125 I radioactivity

the total and the plasma

of the organ.

The

1251 and

13’I radioactivities

in plasma

activity

were calculated 125 I radioactivity

in blood/(l-haematocrit), and the 125 I in plasma and fibrinogen. the difference between the concentrations citrate

were corrected

in serum

as

The fibrinogen

of haematocrit

upon the

dilution of plasma.

For the statistical

evaluation

Morphological

examination.

fixed material

were

PTAH

for

method

Experimental

the thrombin min 5 minutes

Labelled

respectively instead

before

paraffin

fibrinogen

of thrombin.

sections of formalinand by Mallory’s

and thrombin injury.

Groups

5 and 35 minutes

All the animals death.

was used,

of fibrin.

after the thermal

measurements

injection.

p. thick

Student’s t-test

with haematoxylin-eosin

the demonstration

with saline

for radioactivity

of the results, Five

stained

procedure.

24 and 48 hours infused

for the influence

as the radio-

were

were

Control

of rats were

injected

rats

were

sacrificed

after the termination

injected

with labelled

of

albu-

FIBRIN

ELIMINATION

369

FROM LUNGS

RESULTS The

results

are

the termination found the

shown

of the thrombin

in the lungs same

in Tables

I and

II and Fig.

injection,

of normal

and burned

human

rat fibrinogen

whether

or

equal rats

Findings

in the Groups

Five

amounts

a

Fibrinogen,

E p” I

Cpm

5 a

Lung,

cpm

Lung,

mg

of fibrin

(p > 0.1).

The

injected

with labelled

0’

5’

n

3

5

x 103/mg

plasma

fibrinogen

x 103/g

3. 51 t 14.7

0.24

r 3.7

12. 8 2

fibrin/g

were

results

were

Rat Fibrinogen,

Thrombin

T

mg/ml

after

I

Controls

a

minutes

was used.

TABLE Experirtnental Mean - S. D.

1.

1.7

0

1.74

i

35’ 6 0.50

1.50

1.9

15. 6 :

105. 6 :

20.2

24.1

4. 8 ?

1. 0

0.70:

16.4 :

+ 0. 55 5. 8

-t 14, 2 0.68

_-_---_--_-_-_---~-~_~~~_~~_~~__~--~-~~~-~_~~_~~_-~__-_-----------_-_-_____--____--~-~~~-~_~~_~~__~--~--~~-~~~~_~~_-~~_-_---------_-n a Q) c ;

Fibrinogen, Cpm

3

mg/ml

x 103/mg

plasma

fibrinogen

10. 61 :

4 1. 69

5.8

+ 0. 8

9.8

t

7.91

4

+ 0.13

6. 53 + 0. 83

5. 5 t 0. 6

5.7

f 0. 5

43. 8 2 23.8

44.9:

13.0

a Lung,

cpm

Lung,

mg

x 103/g fibrin/g

0

2.2

5.5

:

3.3

6.2t

2.9

FIBRIN

370

ELIMINATION

TABLE Experimental nogen. Mean

indings F - S. D.

in the Groups

vo1.2,~0.4

FROM LUNGS

II

injected

with labelled

Controls

a al

Fibrinogen,

0’

5’

n

3

4

plasma

4.03

: 0.37

Fibri-

Thrombin

T

mg/ml

human

35’ 4

1.66:

0.44

7.4:

1.2

7.3:

2.3

49. 5 + 3.4

7.8

t 4.3

0.64:

0. 50

E :

Cpm x 103/mg

fibrinogen

$ z

Lung,

cpm

Lung,

mg fibrin/g

x 103/g

9. 6 2 0.7 5.3

+ 0. 3 0

5. 5 t

1. 2

0.53

+ 0.59

~-~-~___-_-_-_--~-----~-~_-_------------_--___-___-_-_______-_____ -_---_____-_-_----~~~-~-~_-_--~------_--~_-_______-_-_-_-----_____ n a

Fibrinogen,

2 2 p9

Cpm

3

mg/ml

x 103/mg

plasma

fibrinogen

Lung,

cpm

x 103/g

Lung,

mg fibrin/g

-

9.81

2 0.74

2.8

+ 0.6

3. 6 2 0.7 0

5 7.80: 2.2

6 1.40

+ 0.45

2.8

+ 0.2

22.3

2 9.4

7.3

: 3.9

+O.l

19.8 2 8.3 6.6:

6.67

3.3

Non-burned rats ‘- -

Burned rats

8-

F 3

6-

FIG. /

1

The amount of fibrin in the lung after thrombin infusion in rats injected with labelled rat fibrinogen. Mean

+ L S. D.

FIBRIN

vo1.2,~0.4

In the non-burned considerably burned

reduced

rats

(p -

0. 001).

nogen

was

PTAH

killed

of fibrin

had a significantly

higher

Similar

results

sections

injection.

at the 35-minute the lungs numerous

were

Lung

after

the thrombin

was bund. fibrin

obtained

showed

from

vessels

It was, interval. small

in the burned

both normal

At this

content

injection,

time

a

point

the

than the normal

whether

human

and burned

rats

of burned staining.

rat,

2) at the

(Fig.

infrequent

however,

In the rats killed atelectases rats

in both

(Fig.

or

rats

rat fibri-

showing

fibrin

showed

two intervals

groups,

after

in the non-burned

at the 350minute but these

fibrin the rats

interval, were

more

3).

FIG. PTAH

371

FROM LUNGS

used.

in the pulmonary

thrombin

35 minutes

amount

-stained

located

rats

ELIMINATION

2

deposits

in medium-sized

arteriole.

lI'lBl
372

FIG. Lung of burned staining.

Tables

rat,

showing

I and II also

the plasma

in the various

fibrinogen.

The

to a higher

concentration

the thrombin the human

spread

show the relative

fibrinogen

burned rats

but non-significant

wide

micro-atelectasis.

specific groups

radioactivity

specific

fibrinogen.

in the relative

PTAH

(cpm/mg)

of

of rats given human or rat

showed a lower

of nonalabelled

decrease

infusion

3

specific

radioactivity, There

was a slight

radioactivity

both for the rat and - a little more

due

after

pronounced-

for

fibrinogen.

DISCUSSION Fibrin (5).

is eliminated In the present

surprisingly investigation

rapidly

from

a considerably

tion rate in the lungs was found after thermal

the lungs of normal reduced injury.

fibrin

rats

elimina-

FIBRIN

vo1.2,~0.4

The

burned

rats

inhibition

after

ministration

trauma

from

reduction

nolysis The

rat

in the lung

it has

( 5,

LUNGS

previously

been

6 ), it seems

reasonable

rate

in the burned

fibrinolysis

shown

markedly

elimination

373

of endogenous

inhibitors

examic

injection

acid),

that

delays

the ad-

the fibrin

to assume

rats

seems

the lungs

(14).

was

that

due to fibri-

to be mainly

burned

plasminogen

study

we

rise

in plasma

to a similar

results

have

found

responsible

for

the

increase

(tran-

in fibrin

re-

in the

inhibitors

fibrin

the

in plasmino-

to that

activation

impaired

that

inhibitor

equivalent

in plasminogen

(uroki-

activators

in a retardation

at least

of

bur-

thus

elimination

in the

rats.

conceivable

contributory

burned

rats

ments,

however, was

upon

RES

prolonged

coagulation

cause

of the

burned

and

non-burned the fact

5 to 35 minutes against

this

A third

possible

fibrin

rats

that

was

in the

experi-

administered

from

plays

elimination

In previous

of intravenously

probably

the

lung

a negligible

role

trypan

in the in this

rat

( 6)

res-

model. burned

in the

between

rats

fibrin

at the later

no difference

seen

fibrin

(15 ).

elimination

in the

difference

activity

effect

experimental

butory

lungs

the

inhibition

present

of the delayed

of the RES

no significant

observed

in the

However,

cause

is a reduction

and therefore

More

the

consists

activation

plasminogen-activation

giving

increase

post-burn

of plasminogen

In a recent

of a magnitude

The

2 days

whereas

of a synthetic

inhibition

rats

inhibitors

(8).

in an amount

from rats

in the

not changed

gen-activation moval

in the burned

and in antiplasmin,

are

intravenous

the

lung

increases

inhibitors)

pect

As

in the phase

fibrinolysis

inhibition

twofold

blue

( 8).

in fibrin

fibrinolysis

nase

One

the

were

FROM

inhibition.

about

ned

study

of synthetic

elimination the

in this

ELIMINATION

content

interval

in the the

is another of the

after

consumption

burned

possible lungs

contribetween

the thrombininfusion. of fibrinogen

and non-burned

rats

from argues

possibility.

in the

contributory burned

rats.

cause No

may

reduction

be a reduced in blood

flow

blood

flow

through

through the

liver

FIBRIN

374

in mice

(15)

or through

period,

but pulmonary

the kidneys blood

The increase

in the number

ned rats may

be explained

vascular

fibrin

deposits

administration

duced

Recently

impaired

to occur

in rats

inhibition for

In this

study the

quantification

slight

2 days

ted for

effect

of the lungs

to the intra-

been shown that the

in rats

changes

(17)

and dogs

in the lungs

after

may

be a factor

deposition

in organs

in-

that the radioactivity

radioactivity

from

(cpm/mg)

decrease

of fibrin

point of view,

human fibrinogen

may

also

It

quite

similarly.

showed

that both fibrinogens

tolerates

The

concentrations

by the method

both

after are

used.

is preferable,

a

sui-

From

a

but,

freeze-drying,

as

is not

as was shown in the present

be used,

the

of the two fibrinogens

of rat fibrinogen which

for

was compared.

of the two fibrinogens

in organs

rat fibrinogen,

of importance

method

the lungs

appears

the use

that the

and rat fibrinogen

at low fibrinogen

It therefore

been shown

in the kidneys.

by the present

eliminated

has also

and it is thus possible

in the use of human

I and II)

infusion.

in the kidneys

system

reliability

as a satisfactory

available,

of the bur-

inhibitors

a burn (18),

of fibrin

the quantification

theoretical long

after

but non-significant

the thrombin

as far as we know.

in the lungs

It has earlier

fibrinolysis

elimination

in and were

specific

studied

exposure

the morphological

fibrin

was found (Tables

relative

(16) is seen in this post-burn

not been

rats.

vo1.2,No.k

LUNGS

coagulation.

of fibrin

accumulated

has

in these

of the fibrinolytic

the injurious

in rats

by the longer

increases

intravascular

flow

FROM

of micro-atelectases

of synthetic

(7 ) markedly

ELIMINATION

in-

vestigation.

Aknowledgements Supported

by grants

40X-2035-08B), macia.

from

the Swedish

the Torsten

Medical

and Ragnar

Research

Sbderberg

Council

foundation

(No B73-

and AB

Phar-

FIBRIN

ELIMINATION

375

FROM LUNGS

References

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Saldeen, sinsk 217,

2.

Om

T.

forenings

Saldeen,

Virchows.

Lindquist,

O.,

Rammer,

L.

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and

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20-25,

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1967.

T.

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inhibition

Inhibition

insuffi-

in a post-trau-

138,

stand.

Diathes.

Experimental

T.

Saldeen,

T.

545-549,

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) 22,

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360-371,

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24,

Busch,

C., L.

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J.

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).

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376

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Vo1.2,~0.4

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16.

Renal Rammer, L. effect of intravascular press.

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Impaired fibrin Rammer, L. and Busch, C. kidneys of burned rats. To be published.

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