Interaction of heparin and heparin fractions with human platelets

'ZIRONBOSiSRESEARCH 28; 275-280, 1982 0049-38&8//82/20027j-06$03.00/OPrinted in the L'SA. Copyright (c) 1982 Pergamon Press Ltd. All rights reserved.

BRIEF

INTERACTION

OF

R.Mastacchi,

Alfa

COMMUNICATION

HEPARIN

AND

L.Stanzani,

Farmaceutici

“Opocrin

HEPARIN

M.Barbanti

Research

Research

FRACTIONS

WITH

,L.Montecchi

Laboratories,

Laboratories,

HUMAN

and

Bologna,

Corlo,

Modena,

P

ltal

PLATELETS

Bianchini”

Y

Italy

(Received 13.4.1982; in revised form 24.7.1982. Accepted by Editor G. de Gaetanoj

INTRODUCTION

It

has

cement

recently

of

in

ction

(1,2,3),

(4,s).

The

been

vitro but

et

these

effects

conflicting

of

heparin

mixture

of

polysaccharide

Our

purpose

was

were

heparin and

remain could

a be

that

preparations,

induces of

the

subject

of

explained

are

formed

an

enhan

release

by by

rea

controversy the a

hetero

complex

chains. to

subfractions

fractions

that

aggregation

re sults

geneity

heparin

reported

plate1

found

investigate

on

in

vitro

to

interact

the

activity

of

human

platelet

aggregation.

with

platelets

to

different The

different

degrees.

MATERIALS

Blood ceiving

was

drawn

medication

from of

venipuncture.

Intestinal

were

from

obtained

anticoagulant ble

1.

solution cals

Co.

The

adult

any

various 8).

ADP

and

dissolved

in

least

whose

were

and

distilled

was

obtained

water.

275

days

denied

heparin are in

t-e

preceding

characteristics

dissolved

salt

who 10

weight

molecular

disodium

volunteers at

heparin

Labs

fractions

(pH

for

mucosal

and

METHODS

human

type

Opocrin

activity

AND

fractions such

reported phosphate from

as in

Ta

buffer Sigma

Chemi -

ii”c?ABIN

276

PRP

was

7.3% at

obtained

sodium 230

g

(PPP)

by

was

10

min

1500

g

for

100

.OOO

+

10.0003’

siliconised

with

PRP

for

mixture At

the

ml

was

aggregation

was

capped

ipuncture.

Oual

Channel

42

temperature

addition

of

4PP.

The

,~l

heparin

were

for

4

reaction

solution

or

transferred

submaximal

recorded

to in

room

the

of

stored

ven

ELVI

adjusted

at

samples

addition

after an

erythocytes

was was

blood)

plasma

incubated

IO-10

PRP,

incubation

after

hours

whole

poor

PKP

PRP

using

before

0.3.5 of

2

with

parts

remaining

in

PPP.

within

vehicle

the

x0.2

dose

to of

minutes

ADP

after

vehi_

the

Results

obtained

ments

of for

were

exposed

aggregation

each

substance).

significance

by

as

percent

obtained

in

Student’s

t

Characteristics

of

heparin

increase

the

Differences

of

esperiments between

addition

of

means

the (

maximal 3

were

experi

assessed

test.

TAGLE

1

and

heparin

anticoagulant

fractions

activity

(llJ/mg

1

S.D.)

mol.

Weight

(daltons +

Unfractioned mucosa

N.259

Heparan

Slow batch Fast batch

3.5 2 0.2.

N.HTS/dJj

Dermatan

Fast

157.5 2 4

13.958

+

894

19.608

+

890

?:.5oj

+ 850

AK

sulphate

batch

batch

j

S.D.

intestinal

heparin

batch

batch

aa

(0.4-0.9

stimulus.

amplitude for

or

minutes,

end and

f.c.)

the

JO

of count

performed

9

Platelet

autologous

used was

contained

gregometer #i

and

anticoagulated to

temperature. platelet

with

Hcparin

blood

anticoagulant

room The

,ul

oF

part

centrifugation

min.

aggregation

gregometer.

cle.

by

15 tubes

Platelet

at

prepared

at

1

(

Col.28.

PLA-CELETS

centrifugation

citrate for

XXI

sulphate N.

DES

moving N.

Fm

moving N.

Sm

moving N.

Fm

7-8/HF

6

._ o 2

0.3

heparin 1117

90.2

-+ 3

5.423

+ 96

heparin 1026 LMW

727

161.0

+

6.4

36.933

-+ 1160

heparin

57.9

2 0.3

5.740 + 40

I

259

Snl

AK

1026

50 40

HTS

30

30

20

20

10

10

50

50

Fm 111 7

435 w

40

= w

40

30

“0

30

20

z a9

20

*

10

10

.50

50.

I

A

I

DES

?-8/HF

Fm

FIG. Percent

increase

heparin

and heparin

of

PRP).

ments.

of

The results *

p<

human platelet fractions

at

+ S.D.

are

0.05

727

1 aggregation different the

by

unfractionated

concentrations

mean of 3 different ** p < 0.01

( pg/ml experi

BEPARIX MD

278

Anticoagulant to

the

with

3rd

the

performed in

international

using the

standard

institute

Heparin al

was

described

National

don). et

assay

procedure

for

sheep

United

plasma

States

was

according

Pharmacopoeia,

(established

Biological

fractionation

Vo1.28, No.2

PLATELETS

1973)

Standards

performed

furnished

by

Control

(Len

and

as

described

by

Nader-

(6). RESULTS

When were

to

gregation ce

of

was heparan

human

enhanced

at

shown

differences

in

Similar

at

results

tion

to

These

were

Ill/ml

of

findings

rimental

1.

Slow

moving

to

fast

is

significantly when

a2

the

without

presen

effect

sulphate AK)

less

reactive

heparin

shows

heparin

or

sub

with no

The

plate

significant

activity

than

converted

no

Maximal

and

heparins.

on

has

concentrations.

lower we

in

(259

moving

platelet

except

lower

heparin

fractions

min,

Dermatan

vivo

total

obtained

30

Sm

of

1026.

heparin

concentra

-

PRP. indicate

pro-aggregant

1).

significantly

iLg/ml,

heparin

for

surprisingly

be

respect 2

was in

with to

figure

with only

the

and

degrees,

(Figure

at

found

heparin incubated

different

which

obtained

were

727,

of and

aggregation

was

fractions

pug/ml PRP

activity

activity as

80

sulphate,

slight

lets

to

titrated

platelet

only

Fm

2

from

added

that

activity

of

molecular

heparin

weight

does

fractions,

at

not

parallel

least

in

these

expe-

conditions.

DISCUSSION

The

results

heparin

and

gregation

obtained

heparin induced

ted

in

the

te,

at

therapeutical

show

that

fractions by

ADP,

literature.

unfractioned

potentiate although

Indeed

in

at

different

heparan

concentrations,

intestinal vitro

platelet

degrees,

sulphate have

mucosal

human and

as

dermatan

no

effect

on

et

al

could

a2 indica

sulphd

platelet

as

gregation. The

different

buted

to

ctioned after

results the

heparin, the

longer

Although less

with

only

basis

obtained

different incubated time

of

aggregation lower upon

Salzman

for

conditions, 2

minutes,

incubation

weight we

can

select

is

heparins heparin

(7)

although gave

(data

enhancement

molecular which

by

experimental

not

the

be our

same

attri unfra

results

as

shown).

reported (8,9,10) fractions

to this

be

generally is

without

not

the pla_

Vo1.28,

HEPARIN

so.2

telet

reactivity,

can

also

let

aggregation

in

of

paran

sulphate

other

this

conclusion

basis

iar

because

influence

the

molecular

study

wheight,

do

not

or

have

AT

suggests

could

dermatan

like

III

presence

(Iii.

present

wheight and

factors,

activity

enhancement

279

AU.) PLAXTEZLE-X

not

be

that

anticoagulant

sulphate,

spite human

ia

Zucchini

on

and

of

with

plate

only

activity in

interaction

heparin

explained

the

that

elevated

he

molecu

platelets.

ACKNOWLEDGEMENTS

OF

The

Authors

the

manuscript.

wish

to

thank

Giul

Mrs.

for

preparation

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