THROMBOSIS RESEARCH Printed in the United

Vol. States

6, PP- 195-200, Pergamon Press,

15375 Inc.

COMMUNICATION

BRIEF

THE EFFECT OF OEFISRASE ON ARTERIAL THROMSUSFORMATION* R. H. SOURGAIN and F. SIX= Laboratoriumvoor Fysiologieen Fyslopathologle Eenheid voor Hart- en Bloedvatenonderzoek Vrije UniversiteitBrussel, Belgium

(Received 2.8.1974; in revised form 4.10.1974. Accepted by Editor M. Verstraete. Received by Executive Editorial Office 25.12.1974)

INTRODUCTION In a previous publicationa method for inducing a white platelet thrombus in a mesenteric artery of the rat by electrical stimulationfollowed by ADP perfusionwas described [II. By microprojectionof the arterial segment on a series of 30 LOR's [Light DependingResistances)it thus became possible to register the thrombus formation continuously. In fig. 1 the set of 30 numbered LDR's with the projected arterial segment and thrombus IS representedschematically. The variations in light intensityappearingwhen the thrombus develops are registeredon magnetic tape as analog signals. By electronicaveraging It is possible to obtain a "Total Thrombus Value" as formerly indicated. In this paper we examined the effect of intravenouslyadministereddefibrase on arterial thrombus formation in the rat. The no

longer

electmnlcally

analog

signals

are

averaged but directly analysed by computer, METHODS AND MATERIALS

Preparationof the animal, dissectionof the artery and the microprojec-

L

This investigationwas supportedby F.G.W.O. Contract No. 20459. With the technical assistanceof R. ANORIES and F. VEREECKE. 195

DEFIBRASE

196

tion

techniques

Defibrese

have already

was isolated

it behaves

off

any changes analog

is

rapidly

in heart

punch reader

a thmmbln-like

after

digitised

pressure.

effect

in

The registered

and the results

appropriate is

as

It did not cause

with disc-drive,

values

(21:

molecule.

of the rat.

or blood

(equipped

facilities)

of these

vein

by A-D conversion

16 K computer

storage

for

Basle

ultracentrifuge

A from the fibrinogen

respiration

rate,

and scope

venom by Pentapharm,

responsible

In the tail

are digitlsed

a Hewlett-Packard

atrox

and in the analytical

the fibrlnopeptide

signals

Further

from Bothrops

Deflbrase

One ml was injected

v01.6,~0.2

FORMATION

been published.

electmphoretically

a pure substance. splitting

AND THROMBUS

teletype,

Fortran

performed

analysed

by

paper

IV progruwning.

on digital

magnetic

tape. The computing

techniques

enable

11 the total

thrombus value

21 the total

surface

us to develop

four

independent

parameters

:

of the thrombus

31 the duration

of the thrombus

41 the mobility

of the maximum intenSity

Vi?dUe.

REGISTRATIONPROCEDURE After

electrical

occasionally Wheatstone

stimulation occurring

bridge

at the site

connected

der to establish tely

and disappearance of

application

to each LDR is

of the electrode,

brought

These values

the base-line.

of the small mural thrombus

into

each

equilibrium

are registered

for

in orapproxima-

10 seconds.

After

of the artery

perfusion

projection

with ADP a thrombus then develops

on the LDR’s modifies

A momentary value A-D converter

for

their

each channel

and computer

gives

and its

resistance.

[LDRI is

a value

thus registered

KtIl

proportional

which via

the

to the increase

in light. In order

to avoid

1000 and ordered lest All

negative according

values

all

to their

30 K(I) highest

values

values,

are incremented starting

with

with the smal-

ones. the ordered

[from

potentials

with a value

number 1 to p Inclusive1

Potential

Is just

smaller

p being

than 900.

smaller

than 900 are then discarded

the number of the LDR where the

vo1.6,No.2

DEFIBRASE

Computerized

this

In

then

flow

blood

reason

smeller voltages

100 mV ere

of ell

voltage

eventually

(IBASE]

is

the

voltage

The IBASE Is then given

of

has to be calculated.

calculated

from

p*l

account

ordered

the

p*l

by the formula

to

by taking

to

channel

Klfl variations.

Important

too smell

consldered

LDR channels

equals

induce

base-line

a computerized

The base-line

tion.

197

FORMATION

Base-Line

Differences For

AND THROMBUS

for

the

Deviations thrombus

sum [Cl

channel

incremented

with

fonsa-

of

the

number n whose

100 mV.

: -fl KU+

t j-P+1

IBASE -

. n-p

The Thmmbus

Value

Each LDR covered

by the

thrombue

TRV The sum [Xl second1

of

the

TRV’s

over

=

STRV values then

The surface all

of

LDR’s

sumnated

where

a value

tb

taken

the the

is

then

TRV defined

thrdmbus

by the

by the

are sumnatsd all

the

total

ISUR

-

represents

duration

?

the

LDR’s

thrombus of

positive es

or zero.

STRV [every

TRV.

second

on the

TRV is

identified

“TOTAL THROMBUS VALUE” (TTV)

covered

over

every

\ )

=

if

- IBASE

LDR’s

so that

fomation,

of

KtIl

the

STRV the

gives

:

equation

If

projection

(ISURI

at

the thrombus

obtained.

corresponds

projection the

is

through

each

to

second

the

sum [Ll

end then

thrombus.

30-n

beginning

and t,

the

end of

the

thrombus

respec-

tively. Changes

in

second]

are

location calculated

of

the

LDR containing

according

to

the

the formula

maximum intensity

:

(each

DEFIBRASE

198

where ht is

the total

where htk is

AND THEOMBUS

duration

the number of

of the presence

seconds

that

v01.6,~o.z

FORMATION

of the induced

the maximum intensity

thrombus and is on the

kth LOR. I and j are mean values

in the set

excluding

and the one below

the one above

These 28 tthrombus where 1 varies

exploring]

Fifteen four

the maximal possible by M a value

white

Wistar

above given

administration

of 30).

by the couple

1.j.

by

and

ikbt

On dividing

[total

LDR’s

from 1 to 14 and j from 1 to 2.

Atk

for

the set

registering

LDR’s are designated

The means i and 3 are then given

M stands

of 20 effectively

value

between

rats

respectively.

jkz

of

. At

0 and 1 is

obtained.

have been used as experimental

parameters

explored

of defibrase.

before

Each animal

animals

and the

and 15 minutes

after

the

thus serving

as its

own

is

control.

RESULTS The intravenous

platelet

The fibrinogen within

of

defibrase

to

content

on the

As can be seen

decreased

to less

does

than 5 % of

from

four

parameters

these

are systematically

are

observations, smaller

after

given

its

ficant. The "S" values examined to be significant

by the

in table

the TTV. total

not

influence

original

the

value

form. duration

the administration

A "sign test" on these results was performed proved

rats

6 hours of the administration.

The results

surface

administration

count.

and found

of the drug.

to be highly signi-

Wilcoxon test for paired

at the 0.02 level.

and total

results

DEFIBRASE

vo1.6,~0.2

AND THROMBUS

199

FORMATION

TABLE TN

(mV x eecl, Duration [set), before (8) and after

Total Surface (LDR x sec1 and “S” Values [Al Administration of Oefibrase Duration

TTV

Rat

Bj

A

I

27,013

1

2

22,573

3

12,933

4

51,005

5

149,665

6

60,045

7

20,523

6

27,614

9

31,630

10

22,915

11

67,527

12

26,015

13

51,563

14

36,039

15

43.540

B:

: I ; I , 1 I I : I,

Total

A

B

I I 371 ; 266

11.460

Surface ;

i 1,181 I

“S-

A

B

998

0.57

I

353 I I 341 I I 361 i 421 ; I 391 : I 211 i I 351 i

6,309 6,166 37,669 51,240

23,695 I, 14,257 ,I 12,652 I 23,571 I I 9,713 I ; 60,544 I ! 16,616 I I 25,924 I ; 12,766 I 6,743 I I

211

1,739

i

517

0.24

211

1,197

0.30

351

2,430

: 622 I ; 2,391

301

4,474

I 3,054

0.60

231

4,307

,: 1,794

0.37

211

1,613

0.35

221

2,120

211 : 231 I 261 : 121

2,309

;

A

) 0.54 1 : 0.40 I I 0.80 I

0.55

I 0.44 I 0.56 I ; 0.89

0.62

i 0.70 I, 0.76

0.79

: 0.76

0.54 0.30

; 0.62 I ; 0.68

~ 361 i 351 I 261 i 231

3,662

,I 1,315 I 1.324 I I 2,491 t : 724 I : 3,716

1,616

,I 1,243

0.37

i 0.57

406 It 294 I 487 ; 272 I I 391 161

3.410

: 1,567

0.40

2,152

: 1,131 I : 808 I

0.80

I 0.40 I I 0.80

0.90

I 0.90

1,654

4,206

I

I

DISCUSSIONAND CONCLUSIONS In our Investigation zyme defibrase

the intravenous

in a series

of 15 rats

thrombus duration

and the total

was significantly

increased.

zyme when injected lasts

for

Not only

the volume

also

structure

its

deed we can assume within its

the thrombus

decreased

thrombus surface1

furthermore

These experimental exhibits

the “S” value

data Indicate

that effect

the enwhich

one hour.

is markedly itself

en-

the TTV, the

an antlthrombosing

of the thrombus and Its that

of a thrombin-like

significantly

intravenously

approximately

administration

greater

duration

changed as judged ‘S” values

and reflect

by the value

correspond

a greater

are thus decreased of

“S”.

to a greater

inherent

but

instability

In-

mobility of

architecture.

The mechanisms underlying

the antithmmbotic

are not yet

One can imagine

clear

to us.

effect that

induced

pmteolysis

by defibrase of the fibrlno-

200

DEFIBRASE

gen mOlecUleS of then 18SS. sible

understood In for

fibrinogen field

is

our

the

Platelet

why this opinion

membrane

activity

split

being

products continued

in

not

order

to

only

plasma in

found

FORMATION

responsiblei

for

of

activity were

is

lasts

products

an anti-aggregating split

AND THROMBUS

to

however,

one hour

proteins

thrombus

this

it

is

not

and sometimes

are

mainly

formation.

be active.

8lUCidat8

V01.6,No.2

Research particular

respon-

HOWeVer

in

this

aspect.

REFERENCES 1.

EOURGAIN, R.H. mental arterial

2.

Reptilase as a deflbrinogenating STOCKER, K. and EGBERG, N. Thromb. Oiath. haemorrh. (Stuttg.1 : Suppl. 54, 361, 1973.

and SIX, F. thrombosis

A continuous in the rat.

registration method in experiThromb. Research : 4, 599, 1974. agent.