Thrombin activity in pulmonary emboli obtained at autopsy

THROMBOSIS RESEARCH 59; 879-882,199O 00493848/90 $3.00 + .OO Printed in the USA. Copyright (c) 1990 Pergamon Press pk. All rights reserved.

COMMUNICATION

BRIEF

THROHBIN

ACTIVITY

D.W.T.

Farstad*,

I.N.

IN

PULMONARY

Nielsen

**

EMEOLI

, R.

OBTAINED ***

Ruyter

and

AT

AUTOPSY

H.C.

Department of Department of,$athology, Rikshospitalet*, and Haemato&@cal Research Laboratory, Aker Hospital Hospital , Oslo, Norway

(Received

23.3.1990;

accepted

in revised form 4.7.1990

Godal

***

Medicine, Ullevsl

by Editor U. Abildgaard)

INTRODUCTION Thrombin is continuously generated during thrombus formation The rapidly immobilised by the fibrin meshwork (1,2,3). part of fibrin-immobilised thrombin is gradually released essentially by inactivated by circulating antithrombins, and antithrombin III (4). However, a minor fraction of thrombin is retained by the thrombus and may induce further fibrin formation This minor fraction of enzymatically active thrombin (5,6). as fibrinolytic degradation seems to be tightly bound to fibrin, releases thrombin linked to fibrin fragments (5).

and major

Below, obtained A (FPA)

we at upon

have studied thrombin activity in autopsy through analysis of generated incubation with fibrinogen.

AND

MATERIALS

pulmonary emboli fibrinopeptide

METHODS

Reagents. Buffers: Diemal-Na (2)

(1) 0.028

Tris-HCl-buffer,

Buffered

saline

volume of one solution (0.15

Key

Modified mol/l,

words:

diemal NaCl

0.15

solution modified M).

Pulmonary

M, (pH diemal

emboli,

buffer 0.126 pH

(pH mol/l

7.35) consisted of and HCl 0.1 mol/l.

7.40. 7.35, buffer

thrombin

879

0.15 to

M) nine

activity.

obtained volumes

by of

adding saline

Vol. 59, No. 5

THROMBIN IN PULMONARY EMBOLI

Soybean

trypsin

inhibitor

Sigma (R) was dissolved solution and stored at

Aprotinin kusen,

(NaN3),

Hirudin

2000 to

Bentonite

(R))

Lyophilized

preparation

diemal

buffer

20.000

KIE/ml,

to

from

obtain

Bayer

a

1%

Lever-

Germany.

Na-azid

solution

modified

(1 rasylol

sulphate

West

(SBTI):

in 4'C.

Merck,

Darmstadt,

Sigma concentration

AT-U, a

50

mg

of

Germany.

dissolved 20 AT-U/ml.

Kebo-Grave,

sulphate,

Oslo, Norway; Tris-HCl-buffer.

(R), of

West

bentonite

in

physiological

Yellowstone sulphate

Western

dissolved

saline

Bentonite, in 0.5

ml

was prepared by precipitation of titrated plasma with beta-alanine as described by Jakobsen and Kierulf (7) followed extensive dialysis buffered solution, to against saline by remove contaminating FPA, and subsequently stored in aliquots of 5 ml at -7OOC.

Fibrinogen

Pulmonary

Experimental Pulmonary 12-36 hours All

the

emboli

from

four

patients

obtained

autopsy.

at

procedures. emboli were post mortem. patients

died

obtained from

from

embolization

four

patients of

at

pulmonary

None had received anticoagulant or fibrinolytic with a diameter of 0.5-1.0 cm were cut into slices washed in buffered saline solution and stored at experiments; frozen sections from such slices Five to ten sections of 25 micrometer thickness each test tube to make a volume of thrombus 0.1 ml. Samples were washed in saline solution 3000 rpm for 5 centrifugation at followed by

autopsy arteries.

therapy. Emboli 0.5 cm thick, -2O'C. Prior to were prepared. were added to material about for ten minutes minutes. This

procedure was repeated three times altogether. of test solution containing buffer, Appropriate portions washed was added to Na-azid aprotinin sulphate and SBTI, Controls to addition of fibrinogen. thrombus material prior Final volume of the preparations was 0.65 ml; contained hirudin. were 0.66 mg/ml of SBTI, 2666 KIE/ml of final concentrations 1.5 g/l (4.4 pmol/l) of fibrinogen, 1.33 ATaprotinin sulphate, U/ml

of hirudin and 0.02% NaN3. Test tubes were incubated at 37'C adding interrupted by Reactions were Generated FPA was sulphate preparation. Nossel et al. (8).

for 2, 4 1.5 ml estimated

and 24 hours. bentonite of according to

881

THROMBIN IN PULMONARY EMBOLI

Vol. 59, No. 5

RESULTS

AND

COMMENTS

TABLE FPA

2h

1

generation

(umol/L) 24h

4h

0.867

2.467

0.002

0.001

3.927 0.005

3.001 0.004

4.722 0.001

3

1.313 0.010

2.617 0.011

5.433 0.019

4

2.341 0.014

2.951 0.018

10.358 0.038

Patient Control

1

Patient Control

2

Patient Control Patient Control

0.413 0.004

inin FPA concentration upon Table 1 depicts the increase of 0.1 ml of washed thrombus material in 0.65 ml cubation solution (4.4 umol/L) for 2, 4 and 24 hours refibrinogen of FPA generated rose markedly during spectively. The amount In all experiments the incubation in 3 of the 4 experiments. rise in FPA was effectively prevented by hirudin. No rise in FPA concentration occurred during incubation of fibrinogen circumstances,

in

the it

absence was not

of thrombus possible

to

material. estimate

Due the

to practical concenFPA

trations at zero time. Since, however, only traces of FPA were observed in incubation mixtures containing hirudin, this implies the added fibrinogen was the only source of FPA generation. that Our findings are in accordance with those of Francis et al (5) studied thrombin thrombi who retained activity in arterial obtained during vascular Most likely, surgery. the effect of remaining thrombin activity in purified test systems is amplified, due to the absence of natural thrombin inhibitors in the incubation mixture. The question of whether thrombin exerts its enzymatic activity whilst bound to fibrin, or whether this requires slow liberation from its fibrin attachment cannot be fully answered at present. Liu et al (3) have described a spontaneous release of thrombin from fibrin clots until equilibrium, while the experiments of Francis et al (5) may suggest that a minor fraction of fibrin retained thrombin cannot be released from its fibrin attachment during physiological conditions, and that this thrombin fraction remains bound to fibrin residues after fibrinolytic digestion, without loss of activity. Moreover, the enzymatically active site of thrombin is apparently not engaged in its fibrin binding (31, suggesting a proteolytic potential whilst bound to fibrin. If this holds true, the present observation of hirudin completely preventing release of FPA during incubation of slices of thrombi for 24 hours might

THROMBIN IN PULMONARY EMBOLI

882

offer indirect evidence are not occupied during studies are required to

that its give

hirudin fibrin a final

Vol. 59, No. 5

binding adsorption. answer.

sites of However,

thrombin further

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