Mechanical recording of reptilase-clot retraction = effect of adenosine-5′-diphosphate and prostaglandin E1

THROMBOSIS RESEARCH Printed in the United

4, pp. 189-192, Pergamon press,

vol. States

BRIEF

1974 Inc.

COMMUNICATION

MECHANICALRECORDINGOF REPTILASE-CLOT RETRACTION = EFFECT OF ADENOSINE-5’-DIPHOSPHATE AND PROSTAGLANDINE 1 G.de

Gaetano,

R.Franco,

Istituto

di Via

recording is

of

that

bath

clot

bivalent

out

at

papaverine

and theophylline

the

of

known to

clot

the

or

ted

clots

ful

stimulator

not

surprising.

during

enhance were

the used of

coagulant

should

of

those

been

clot

advantages to

the

therefore on such

extracted

from

process.By relaxants

this

system,

such

as

inhibit

contraction

or

such failed

a system

are

not

study

obtained

venom of

compounds as to

is

mentioned

be used;this

reverse

accele-

thrombin-indu-

and thrombin

platelets

system

this

a number of

above

the

of

isolated-organ

shown to

experinents

in which

mechanical

retraction;as

retraction,the

observations enzyme

I.M. Nilsson. 20.11.1973)

or noradrenaline

A system

clotting

preliminary

clot

the

muscle

Negri”

of

contrast,

smooth

process in

the

muscle

have

rdenosine-5’-diphosphate(ADP) rate

of

retraction.In

stimulate

Editor

Office

can be added

and some smooth

“Mario

possibility of

any stage

and S.Garattini

MILANO,Italy

by

Editorial

shown the

chemicals

cations

course

Accepted

retraction.One

or

or washed

has

20157

62 -

Executive

(1,3)

drugs

Farmacologiche

Eritrea,

Received

work

Ricerche

10.9.1973.

(Received

Recent

M.B.Donati,A.Bonaccorsi

finding

is

activated reports with

Botrops

a power-

the

atrox

,

reptilase. Human platelet-rich reptilase,ADP conditions 3.0

plasma

and prostaglandin previously

described

Cylindrical-shaped clots ml PRP in a siliconized

(PRP),platelet-poor El

(PGE1)

were

plasma used

(PPP),

in the

(2).

were obtained by adding 0.3 ml reptilase to Plexiglas container (height 100 mm,internal

REPTILASE

190

diameter sealed

10 mm) formed of two halves

with paraffin

hooks were inserted

before

20 minutes

were separated

and the clot

bath containing

recorder

in the sample.Two metal

fish-

of the sample before

at room temperature,the transferred

20 ml of a calcium of the clot

two halves

to an isolated-organ

and magnesium free

was suspended

(Type DY l,Basile,Milano,Italy) (Basile,Milano,Italy),while

a hook at the bottom repeated

and

Tyrode

pH 7.4 at 37’C.

One extremity transducer

pouring

Vo1.4,No.l

which were put together

at the two extremities

clotting.After

solution

CLOT-RETRACTION

at least

fed the other

of the bath.

3 times)

to an isometric into

a pen

was connected

Most of the experiments

to

(each

are summarized in the figure.

gmns 10

0.8. 0.6.

O30

60

7 UOminut*

90

Figure 1 Recording of the strength produced during retraction of PRPreptilase clots.Effect of ADP and PGE1. Before the addition of by the arrows),the solution each compound to the bath (indicated surrounding the clot was carefully replaced several times. The following

results

have been obtained

:

1) PRP clotted by reptilase neither retracted nor developed any mechanical strength during at least three hours. 2) When ADP (4.10m6 to 4.10-5M , final concentration) was added to the bath,retraction started almost immediately;the strength produced by the clot increased progressively for about 40 minutes,reached a plateau and then slightly decreased.

REPTILASE

Vol.4,No.l

In the maximal 3)

only

ADP added

If

ADP was washed

to

the

out

the

upward

the

curve

pg/ml

course falling

of

from

again,although

bath

to

a level

more

occurred.

If

reduced

the

PPP clotted

added

the

plateau

strength

of

was

continued

in the

the

that

test

of

of not

system

retraction

with

in the

ADP has been

present

study

suggested

that

activates

the

platelets

do not

the

as careful

clot,

course

of

reptilase

clot

has

platelet

PGEl can

of

the

been

course

of

confirmed;

PGEl is clot

be counteracted

clot

any mechanical not

modify

our

These PRP-clots;

experiments of

the

clot

reptilaseit

clot

does

not

of

observed

by further

of

where

alter

in a test the at

relaxing

addition

,

the

PGEl on ADP-

reversing

the

it

ADP outside

in addition,

capable

is

activated of

effect

,

reptilase;

induce

presence

retraction;

observa-

reptilase

into

the

previous

system;once the

the still

formation;

retraction,previously

shown that

moment the

any more

after

addition

The inhibitory

also

started

reversal

shown to

diffuses

washing

clots

was reversed

previous

clot

contractile

need

retraction.

(Z),has

after

ADP rapidly

PGEl had

PPP-clots.

of

PRP before

when added

after

clot.

with

ADP was preincubated retraction

the

in our

,

ADP , PGEl strongly

and extend

used

clot,

increased

the

develop

obtained

ADP induces tube

before

the

baseline.

again

ADP; PGEl did

confirm

the

PGEl strongly

and slower

contraction

line

results

original

of

to

reversed

PGEl was added

bath did

of

new contraction

partial

straight

the

of

PGE1. If

addition

preliminary

near

bath,contraction

to

added

contraction

ADP was added

by reptilase

horizontal

of

concentration

was invariably

slowly;this

maximal

strength,after

(2)

tracing

addition

of

contracted,a

study

before

maximal

the

had fully

system

degree

the

concentration)was

of

the

by a new addition

clot

now,the

to

mechanical

final

effect.If

washed

tion

related

the of

ADP before

inhibitory

been

until

any modification.

Removal

5)

of

at any moment before

its

191

system.

development

When PGEl (2-4 bath

performed

was directly

of

without 4)

experiment

strength

reached,the

CLOT-RETRACTION

any

effect of

tube

present

ADP

REPTILASE CLOT-RETRACTION

192

.

indicating

that

in certain

esctively

stimulated

In conclusion,clots and placed

in an isolated

model for

activate

the platelet

can be

by addition

to PRP of reptilase

organ bath are an original

a dynamic evaluation contractile

and

of compounds able

system,thus

promoting

to

clot

ion.

At our best

knowledge,

system. In addition,substances studied;an

important

compared to clots

induced

by different

this

is the first

inhibiting advantage

the previously

induced

described

substances

of their

systems

(e.g.

specific

smooth muscle

retraction

of studying

using clot

on such a can also

be

here as

thrombinretraction

ADP, adrenaline,collagen)

inhibitors.Studies

under way to compare the behaviour isolated

clot

report

of the system presented

is the possibility

in the presence of other

platelets

by ADP more than once. obtained

suitable retract

circumstances

Vol.4,No.l

of reptilase-clots

are now with that

systems.

ACKNOWLEDGEMENTS - The experienced technical assistance of Miss Carla Roncaglioni was highly appreciated. Reptilase-reagent was a gift of Pentapharm,Basle,Switzerland.PGE was kindly provided Prof.D.A.Van Dorp,Unilever Research, Th& Netherlands. REFERENCES and clot ,D. and Fantin, G.P. : Platelets 1. Bottecchia retraction :effect of divalent cations and several drugs. Thrombos -Diathes -Maemorrh.,in the press. Bottecchia, D. and Vermylen, J. : Retraction 2. de Gaetano,G., of reptilase-clots in the presence of agents inducing or the platelet adhesion-aggregation reaction. inhibiting Thrombos.Res. 1973,2,71. 3. Majno,G., Bouvier, C.A., Gabbiani, G., Ryan, G.B. and Statkov, P. : Kymographic recording of clot retraction:effects of papaverine,theophylline and cytochalasin B . Thrombos-DiathesHaemorrh., 1972,%,49.