Effect of dextran on fibrinolysis inhibition activity in serum

THROMBOSISRESEARCH Vol.12.~p.1165-1175. % Pergamon Press Ltd.1978. Printed inGreSBrirain.

EFFECT OF DEXTRAN ON FIBRINOLYSIS I?ZIBITIOIJACTIVITY IN SEWf4 Gunnar Carlin

and Tom Saldeen

Institute of Forensic >Iedicine, University of Uppsala and Department of EXperimental Eledicine, Pharmacia AB, Uppsala, Sweden

form (RCCeivaCI 10.7. 1977: in revised Accepted hy Editor 6. Ikkala. Received hv FxPcutivP Editorial Office

5.9.1977. 17.4.1978)

AESTXCT Clots derived from blood taken from rabbits infused with dextran were lysed much faster than normal clots. The fibrinolysis inhibition activity of serum was decreased and the clot lysis effect of dextran seemed to be due to a serum factor rather than to an altered fibrin network. The effect of dextran on the fibrinolytic activity of serum was present both after in vivo infusion to rabbits and after in vitro addition of dextran to human and rabbit sera.

INTRODUCTION Delayed fibrin elimination from the lungs is an important mechanism in the pathogenesis of the delayed microembo1is.m syndrome. Increased fibrinolysis inhibition activity ( FIA ) in the blood plays a major role in this delay of fibrin elimination ( 1 ). Substances which alter this activity and thus the lysability of fibrin would seem to be of interest in studies of the pathogenesis of the delayed microembolism syndrome. In the present investigation we have studied the effect of dextran on the lysis of blood clots and especially on FIA in the blood.

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XATELIAL Experimental animals: Babbits weighing 3-4 kg were used. The animals had ---------free access to food and tap water. Dextran: Dextran with mean molecular weights of 20 000, 40 000, 70 000 and ---_ 150 000 dissolved in saline to a 6 % concentration ( Pharmacia AB, Uppsala ) was infused into a marginal ear vein. In one ir.vitiro experiment 20 % dextran 70 000 was used. Chemicals: Human fibrinogen grade L ( AB Kabi, Sweden ) ----Urokinase ( LBwens, Denmark ) Thrombin, Topostasin@ ( Roche, Switzerland ) 4-amino-methylcyclohexanoic acid ( AXCA >, ( AB Kabi, Sweden ) Human plasminogen ( AB Kabi, Sweden > S-2251 Buffer: ---

( kindly supplied by Kabi Diagnostica. Sweden )

Verona1 buffer

pH 7.35 ( 2 ).

METHODS Sampling of blood: Blood was collected from an ear artery in rabbits or from ----_---_ a cubital vein in humans. Preparation of serum: The blood was coagulated at room temperature. After 2 ---------h it was centrifuged for 20 min at 3600 g. The serum was immediately frozen ( - 20 'C > and stored until use within one month. The freezing and storage of serum induced a slight decrease in FIA. There was no difference in this decrease between normal serum and serum containing dextran. Preparing -----------and lysis of blood clots: One ml of blood was drawn into a plas---tic tube and 0.1 ml urokinase ( 500 Plough units ) was added. The blood was allowed to coagulate at 37 'C in a water bath. The time between addition of urokinase and complete lysis of the clot was recorded. Lysis of washed clots: One ml of blood was collected in a glass tube and 0.1 ----------ml of lz5I-fibrinogen and 0.05 ml saline or 20 % dextran 70 000 were added. The blood was allowed to coagulate at 37 OC. The clot was then washed twice in saline for a total of 10 min and incubated with 2 CU plasminogen in 1 ml Verona1 buffer at 37 'C. One hundred units of urokinase were added and the

FIBRINOLYSIS

DESTRAX:

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I?ZlIBITIOX

1167

amount of radioactivity in the lysate was measured 1, 4 and 24 h after addition of urokinase. 125 I-fibrinoge2: Human fibrinogen was labelled with ----_loride ( 3 ).

125 I by iodine monoc;l-

Fibrinolytic inhibition activity ( FIA ): A clot lysis method slightly modi-------_----_---__ fied ( 4 ) from a method described by Paraskevas et al. ( 5 ) was used for determination of FIA. FIA is expressed in the rabbit experiments as the concentration ( in % ) of AKA

giving an equal inhibition effect. In the expe-

riments with human serum FIA is expressed as % of the inhibition effect of normal pooled human serum. Addition of dextran alone without serum had no effect in this clot lysis system. Plasminogen and antiElasmin were determined by means of an amidolytic assay -------_-_ _-( 6 ); a chromogenic substrate for plasmin ( S-2251 ) was used. The release of the coloured cleavage product from this substrate is proportional to the enzymatic activity. Plasminogen activators: The fibrinolytic activity of resuspended euglobulin --------precipitate ( plasminogen activators ) was measured on unheated bovine fibrin plates as described by Nilsson and Olow ( 7 ).

RESULTS

In vitro lysis of blood clots derived from rabbits intravenously infused with dextran Nine rabbits were intravenously infused with 1.2 g dextran 70 OOO/kg b.w. for 1 h.

Blood was taken for clot lysis determination before the dextran

infusion, immediately after, 1 and 24 h after the end of the infusion. The results are given in Fig. 1.

As can be seen, only 3 of 9 clots formed from

blood taken before the dextran infusion were completely lysed, whereas 6 of 9 clots formed from blood taken immediately after the dextran infusion were lysed. Dextran still had an effect on the clot lysis in blood taken 1 h after the end of the dextran infusion, but not in blood taken 24 h after the infusion. This experiment thus showed that infusion of de::tran increased the lysability 02 blood clots.

1168

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The next experiment was performed to investigate whether the increased lysability was an effect of an altered clot structure due to the dextran infusion.

50

. .. .. . .. . ,_..... t

.. . .. . . . ,...... .’ _.,....

. . . . . . . . .

.

0~ 0

1

*

lyris lime fkrts)

FIG. Increased lysability of whole-blood clots after infusion of dextran in nine rabbits. The figure shows the number of completely lysed clots as a function of the lysis time. Note the increased lysis immediately after the end of the infusion.

4

.’

.

. .

. .

rJh

l--TF--2.

Lysis of washed blood clots. Filled symbols represent lysis after addition of 2 CU plasminogen and 100 Plough units of urokinase. Open symbols represent lysis after addition of urokinase only. Mean of four clots.

Effect of dextran on the lysability of washed blood clots Two rabbits were infused with 1.2 g dextran 70 OOO/kg b.w. in 40 min. Fivenl of blood were taken before and after the dextran infusion for determination of lysis of washed clots by the radioactivity method. The results are given in Fig. 2. It is seen that about 24 % of the clot was lysed after 1 h. There was no difference between clots from blood taken before and after the dextran infusion. After 4 h the clots to which urokinase had been added lysed to about 40-50 % and slightly more after 24 h. In clots to which only urokinase and no plasminogen had been added there was only minor lysis. No significant difference was found between clots formed from dextran infused rabbits and clots from normal blood at any determination point. This result indicated that the dextran effect was not due to an altered clot. The next experiment was undertaken to find out if the increased lysis activity of dextran was an effect of a changed serum factor.

I-01.12,so.h

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FTBRISOLYSIS

Effect of destran infusion o;,FIA in

I\-HTBITIOS

L169

serun

Effect of various amounts of intravenously infused dextran 70 000 ----------__--------------------Six rabbits were infused with 0.5 g, 9 rabbits with 1.2 g and 3 rabbits with 2.0 g dextran 70 OOO/kg b.w. in 30 min. Blood was taken for measurements of FIA in serum before the dextran infusion and 1, 4 and 24 h after its start. The rabbits infused with 0.5 g dextran/kg b.w. showed no significant change of serum FIA. The rabbits of the other two groups, however, showed a marked decrease of FIA 1 and 4 h after the infusion. Twenty-four hours after the dextran infusion the decrease was less obvious. There was no significant difference between the effects of 1.2 and 2.0 g dextran/kg b-w. ( Fig. 3 ). Effect of dextran infusion rate ---------------Twenty-eight rabbits were infused with 1.2 g dextran 70 OOOfkg b.w. at different infusion rates. Ten of the rabbits received the dextran in 15 min, 9 rabbits in 60 min and 9 in 180 min. The serum FIA was measured 30 min and 1, 2, 4 and 24 h after the start of the dextran infusion. The results are shown in Fig. 4. It can be seen that 1 h after the start of the infusion the serum FIA had decreased markedly. Thirty minutes.after the start of the infusion the FIA in the group given dextran at the highest infusion rate was already at the minimal level. This level had not yet been reached in the other two groups; the rabbits given dextran at the slowest infusion rate showing the smallest decrease of FIA in serum. Effect of various molecular weights of dextran ----------------------Twenty-six rabbits were infused with 1.2 g dextran of various molecular weights per kg b.w. in 30 min. Five rabbits received dextran 20 000, 6 rabbits dextran 40 000, 9 rabbits dextran 70 000 and 6 rabbits dextran 150 000. The serum FIA was measured before the dextran infusion and 1, 4 and 24 h after its start. A slight, but significant decrease of FIA in serum was noted in rabbits given dextran 20 000. In the other three groups there was a large decrease of FIA; this being most pronounced following infusion of dextran 70 000 ( Fig. 5 ). These experiments thus showed that infusion of dextran markedly reduced the serum FIA level as measured by the clot lysis method. The next experiment was performed to determine which fibrinolytic parameters changed after infusion of dextran.

DEYTR;\S:

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FIBRINOLYSIS

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eT

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I

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s

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FIG.

Vol.l2,No.6

I?v%IBITIOS

4

Ibwrl

FIG.

3.

4.

Decrease in FIA in serum as measured by a clot lysis method after infusion, at different rates, of 1.2 g dextran 70 OOO/kg b.v. in rabbits.

Decrease in FIA in serum as measured by a clot lysis method after infusion of various amounts of dextran 70 000 in rabbits. FIA in Figs. 3-5 is expressed as described in "Methods".

,_,..__... -4 ,..’

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,_..” .= ,_.’ . ..’

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FIG.

5.

Decrease in FIA in serum as measured by a clot lysis method after infusion of dextran of various molecular weights in rabbits.

FIBRINOLYSIS

DEXTRAS:

v01.12.50.6

1.171

I?;HTBTTT!??;

Effect of dextran infusion on FIA, plasminogen, plasnicogen activators and antiplasmin in blood Four rabbits were given 1.2 g dextran 70 OOOfkg b.w. in 30 min. Blood was taken before the dextran infusion and 60 ruin after its start and was exanined for FIA, plasminogen, plasminogen activators and antiplasmin. The results are given in Table I. FIB and plasminogen activators in the blood showed a significant decrease after the dextran infusion but plasminogen and antiplasmin did not alter significantly.

TABLE

I

Effect of dextran infusion to rabbits on fibrinolysis inhibition activity ( FIA ), plasminogen ( PLC ), plasminogen activators ( PA f and antiplasmin ( AP ). N = 4.

FIA

PLG

PA

AP

(% AMCA)

(CU)

(mm2)

(Cu)

Before dextran

X

0.098

3.23

96

1.53

Sd

0.050

1.29

16

0.06

After dextran

x

O.O32xxx

2.20

60x

1.42

Sd

0.003

0.93

21

0.06

Effect of dextran in vitro

on FIA in serum from rabbits and man

Five rabbits were examined for FIA by the clot lysis method before and after the addition of 0.05 ml 20 % dextran 70 000 to 1 ml of blood and 1 h after the start of a 30-min intravenous infusion of 1.2 g dextran 70 OOO/kg b.w. The results are given in Table II. It can be seen that addition of dextran to the blood in vitro

resulted in a decrease of FIA down to the

sarle level as was seen in the rabbits given dextran in viuo. the same amount of dextran to human blood in vitro ked decrease of FIA (Table III).

Addition of

also resulted in a mar-

DEXTRAS:

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FIBRISOLYSIS

TABLE

Vo1.12,No.6

I~;HIBITTO?i

II

FIh of rabbit serum before and after ir.vitro and
After dextran Before dextran

NO.

I‘r, vitro

-Tr. v?z,Ja 0.06

0.06

0.15

0.01(

0.06

0.17

0.04

0.12

0.06

0.06

5

0.12

0.03

0.04

Mean

0.13

o.o4xx

O.O6xxx

SD

0.03

1

0.12

2 3 4

TABLE

0.013

0.006

III

FIA of human serum before and after addition of dextran ;n vitro mal)

( % or nor-

Before dextran

After dextran

1

230

137

2

100

5

3

190

45

4

324

12

Patient NO.

DISCUSSION The present investigation shows that clots derived from blood taken from rabbits infused with dextran are lysed much faster than clots formed from normal rabbit blood. One possibility is that the increased lysability is due to an altered fibrin network. It has been shown that dextran makes the fibrin network coarser ( 812 ), this effect

being unrelated to the molecular weight of the dextran

( 8 ). Tangen et al. ( 12 ) found that the altered fibrin was more suscepti-

Vo1.12,No.4

ble

to

this

DEITRAS:

plasmin

finding

dextran

digestion, and

were

while

showed

under

FIBRINOLYSIS

that

certain

KopeE

fibrin

et

1173

I?XKBTTIOlr;

al.

clots

( 13 ) were

prepared

circumstances,

more

from

unable

to

confirm

fibrinogen

resistant

to

and

plasmin

diges-

tion. In

our

experiments

normal

washed

with

dextran,

role

in

These

clots

the

ran,

due

clot

lysis

by

plasminogen Dextran

in

the

effect

the

amount to

creased

of

to

the

on

of

that

the

factor;

digestion

rabbits

altered

of

infused

fibrin

seems

to

no effect

on

the

infusion.

be

influenced

plays

not

This

a

is

not

( 4 ).

The

biological

This

primarily

activity

of

Further

dextran

effect

of

weight

dextof

dext-

but

it

fib-

is

also

concentration

of

significantly. might

due

inhibitors

plasminogen.

clots

by a special -

on antiplasmin.

on FIA

the

given

molecular

mostly

change

of

rabbits

inhibitor

present

plasminogen-plasmin

of

from

of

did

lysability

activity. and

rate

however,

have

a decreased

serum

inhibition

the

dextran

activation

plasmin

from

that

increased

thus

plasminogen

blood,

of

the

blood

a plasminogen-plasmin

amount the

from

possibility

concentration

used -

appeared

that

due

not

method

the

formed the

fibrinolysis on the

inhibitor

affected

be

a serum

apparently

rinolysis

clots

between

lysability.

to

dependent

but

of

demonstrated

a decreased

was

no difference

against

increased

experiments

showed

The

and

arguing

was probably

ran

we found

in these

to

the

indicate

a decrease

blood,

but

inhibitors

studies

on

this

in

or

might

to

problem

an inare

in

progress. It

was

as

that

interesting infused

dextran showed

are

in

)

the

from

blood

taken

maximum effect dextran

in

concentrations

vivo.

adhesiveness,

to

They

factor,

as with

has

been

blood

of from

assumed infusion

in

to

shown

artificial

patients about

in

vitro

earlier of

had

that

dextran. thrombi

infused 4 h after

that

to

the

effect

dextran

maximum 4 h after

the

is

same

al.

of

and the

effect

on

concentration

of

dextran

could

known

to

( 15,

be

decrease 16 ).

found

the

highest

also

expe-

infusion.

the

infusion

of

( 14 )

loop

dextran, end

effect

effects

et

( Chandler

with the

the

certain .&berg

vitro had no increasing

corresponding

of

serum

infusion

occurred

of

in

added

v*ivo

lysability

sability tained

It

on ir.

Addition

platelet

dextran

uivo.

dependent

an increased

riments that

that

lyob-

due

to

platelet

a

11-r,

DESTRAIS:

FIBRISOLYSIS

IXMIEITIOS

vo1.12,so.6

Our result does not seem to be due to a platelet factor, since serum contnins very few platelets. It could be argued that FIA found in serum might be released from platelets. However, we found the same amount of FIA

in

normal and thrombocytopenic dogs after infusion of norepinephrine ( 17 ), which increased the serum FIA, speaking against this possibility. Few investigations on the effect of dextran on fibrinolysis have been published so far. Nilsson and Eiken ( 18 ) found that dextran had no influence on the euglobulin clot lysis time and the fibrinolytic activity in the euglobulin precipitate

whereas Deutsch ( 19 ) and Fischer and Wimmer

( 20 ) reported a decreased euglobulin clot lysis time in patients infused with dextran. Cronberg et al. ( 16 ) found a slight decrease though not significant, in urokinase inhibitors after administration of dextran to human volunteers. The reason for the discrepancy between their results and ours is not known. It should not be due to species differences since we found a decrease in FIA not only in rabbit but also in human serum after addition of dextran to blood in vitro, and in preliminary experiments also after infusion of dextran to patients ( 21 ).

REFERENCES The microembolism syndrome. Microvasc. Res. 11: 227,

1.

SALDEEN, T.: 1976.

2.

HJORT, P.G., RAPAPORT, S.I. and OWREN, P.A.: A simple, specific onestage prothrombin assay using Russels viper venom in cephalin suspensions. J. Lab. Clin. Med. 46: 89, 1955.

3.

131 I-labelling of HELMKAMP, R.W., CONTRERAS, M.A. and BALE, W.F.: proteins by the iodine monochloride method. Int. J. AppZ. Radiat. Isot. 18: 737, 1967.

4.

WALLIN, R., BAGGE, L. and SALDEEN, T.: Viewpoints on the determination of fibrinolysis inhibition in blood. Forens. Sci. 10: 154, 1977.

5.

PARASKEVAS, M., NILSSON, I.M. and MARTINSSON, G.: A method for determining serum inhibitors of plasminogen activation. SC&. J. Ctin. Lab. Invest. 24: 138, 1962.

6.

BAGGE, L., BJijRK, I., SALDEEN, T. and WALLIN, R.: Purification of a plasminogen activation inhibitor in serum from posttraumatic patients. Thrombosisand Haemostasis39: 97, 1978.

7.

NILSSON, I.M. and OLOW, B.: Fibrinolysis induced by streptokinase in man. Acta Chir. Scand. i23: 247, 1962.

~01.1.2.No.6

DESTRr\?i: FTBRINOLYSIS

I\-MIRITIQN

1175

3.

CARLIS, G., WIK, K.O., ARFORS, K.-E., SALDEE?;, T. and TANGEN, 0.: Influences on the formation and structure of fibrin. Thrombosis &s. 3: 623, 1976.

9.

GOLLUB, S. and SCHAEFER, C.: Structural alterations in canine fibrin produced by colloid plasma expanders. %rcj. SynecoZ. Obstet. 127: 783, 1968.

10.

MUZAFFAR, T.B., YOUNGSON, G.G., BRYCE, W.A.J. and DHALL, D.P.: Modifications of fibrin structure with dextran. An ultrastructural study. Micro~asc. Res. 4: 466, 1972.

11.

TANGEN, 0. and WIK, K.O.: Influence of dextrans on the formation and structure of fibrin as studied by thrombin time measurements and scanning electron microscopy. :.l~crovasc.Res. 4: 466, 1972.

12.

TANGEN, O., WIK, K.O., ALMQUIST, I.A.M., ARFORS, K.-E. and HINT, H.C.: Effects of dextran on the structure and plasmin induced lysis of human fibrin. ThrombosisRes. 1: 487, 1972.

13.

KOPEe, M., WEGRZYNOWICZ, Z., ZAJDEL, M., SAWECKA, J. and SZUMIEL, I.: Effects of histones and dextran on some properties of fibrin, particularly on its susceptibility to plasmin. Thrombosis Res. 5: 359, 1974.

14.

ABERG, M., BERGENTZ, S.E. and HEDNER, U.: The effect of dextran on the lysability of ez viva thrombi. Am. Surg. 181: 342, 1975.

15.

BYGDEMAN, S., ELIASSON, R. and GULLBRING, B.: Effect of dextran infusion on the adenosine diphosphate induced adhesiveness and the spreading capacity of human blood platelets. Thromb. Diath. Haemorrh. 15: 451, 1966.

16 :

CRONBERG, S., ROBERTSON, B., NILSSON, I.M. and NILCHN, J.E.: SuPPressive effect of dextran on platelet adhesiveness. Thmmb. Diath. Haemorrh. 16: 384, 1966.

17.

LINDQUIST, O., BAGGE, L. and SALDEEN, T.: Induction of endogenous fibrinolysis inhibition in the dog. Acta Chir. Stand. 242: 20, 1976.

18.

NILSSON, I.M. and EIKEN, 0.: Further studies on the effect of dextran of various molecular weight on the coagulation mechanism. Thromb.

Diath. Hawnorrh. 11: 38, 1964. 19.

DEUTSCH, E.: Die Antikoagulatien in der Therapie der peripheren arteriellen Durchblutungssttirungen. Wien ktin. Wschr. 76: 151, 1964.

20.

FISCHER, M. and WIMMER, H.: Aktivierung des fibrinolytischen Systems durch nieder-molekulares Dextran. Bibl. Haerzat.23: 1278, 1965.

21.

CAP.LIN,G., :IODIG, J. and SALDEEN, T.:

To be published.