THROMBOSIS Printed

vol.

RESEARCH in the

United

BRIEF

States

6,

pp.

Pergamon

87-91,

1975

Press,

Inc.

COMMUNICATION

- III: PLASMA AND SERUM ANTITHROMBIN DIFFERENTIATION BY CROSSED IMMUNOELECTROPHORESIS

G&A

SAS,*

DUNCAN

S. PEPPER

and

JOHN D. CASH

S-E Scotland Regional Blood Transfusion Edinburgh. Royal Infirmary,

(Received

11.11.1974; Accepted by

in revised Editor H.C.

Centre,

25,11.1974.

form

Godal)

INTRODUCTION Antithrombin serum which factors,

exerts

its biological

such as thrombin

gel filtration

available

and activated

of inactive

are found in normal

serum

studies

During dimensional

This technical

AT-III,

and it was demonstrated

containing heparin

is added to the agarose

positions

Present

(6) we observed

in similar

states.

into the studies

on

is used for the first in the antibody However,

serum.

(for the first electrophoresis

87

or semi-

for those clinical

was incorporated

plasma and native

are

that improved

peaks developed

Postgraduate Medical School, First Department of Medicine, 1389 Budapest, Hungary.

techniques

factor VIII using the two

of the plasma and serum Peaks were significantly

address:

Although

of hypercoagulable

that when plain agarose

gel with both titrated

coagulation

this is achieved

can be detected

was continued

precipitation

and

and that these

time no simple

when heparin

innovation

then coincident

complexes,

on coagulation

technique

of peaks was achieved

gel.

electrophoresis

weight

with the diagnosis

the course of studies

agarose

activated

might be of some importance

concerned

in plasma

IX, X and XI (l,2,3).

of these complexes

immunoelectrophoresis

visualisation

present

that, at least for thrombin,

(4,5), at the present

Such a development

and experimental

2 -globulin

factors

high molecular

by which the presence

quantitated.

is an%

effect by neutralising

studies have shown

by the formation

*

III (AT-III)

when

run) the

different.

Pl,AS>lA &

88

SERUM ANTITHROHBIN-III

MATERIALS ANDMETHODS Blood

Citrated

sampling:

using plastic citrate

syringes

venous blood was obtained

and stainless

(1 ml anticoagulant

collected

taken place to different

Serum degrees

venous blood was distributed coagulant, 60 min,

incubated

samples

in which

were obtained

in 1.8 ml aliquots

at +37OC and at various

at 2,000 g for 15 minutes

subjects

of 3.8% sodium

citrate

the thrombin

was

generation

in glass tubes without

times after coagulation citrate

incubated

had

20 ml of fresh

as follows:

Each sample was then further

centrifuged

a solution

Blood without

120 min and 360 min) 0.2 ml of 3.8% sodium

to each aliquot. +37Oc,

steel needles,

to 9 ml blood).

in glass tubes.

from healthy

solution

anti-

(j min, was added

for 15 minutes

and the supernatant

at

studied

(vide infra). Human thrombin Elstree,

(kindly

U.K.), Heparin

immune serum

supplied

(Weddel Pharm.

by Dr. D. Ellis,

Ltd. London,

(Nyegaard and Co. A/S Oslo, Norway)

Lister

Institute,

U.K.) and Anti-AT-III

were applied

in the course of

the experiments. Two dimensional

crossed

of AT--III with various electrophoresis. for 2 hours, containing identical lactate

immunoelectrophoresis concentrations

of heparin

The first electrophoresis

the second electrophoresis

1% antiserum

for 4 hours.

to that described

was excluded.

in the agarose

was carried

for the first

out at 20 V/cm at +4OC

at 20 V/cm at +4'C in an agarose The buffer

by Laurel1

Coomassie

(6) was used for the investigation

used for this technique

(6) with the exception

Brillant

Blue staining

was

that the calcium

was used.

RESULTS When plain agarose crossed

immunoelectrophoresis

with both titrated coagulation).

plasma

and native

different

This phenomenon

precipitation

(incubated

with the agarose,

peaks developed

for 6 hours after

the plasma

and serum petis

(Fig. 1). with maximum

peaks being achieved

examination

of three distinct

IAT-III11

III31 in Fig.

in the course of the

separation

between

at and above a heparin

of 16.6 units per ml of agarose.

More detailed

IIIl;

serum

was dose dependent,

the plasma and serum AT-III

presence

then coincident

On mixing heparin

were significantly

concentration

was used for the first phase

lc.

peaks:

and two smaller

of the normal

plasma

a fast and large peak increasingly

run revealed

the

(immunoantithrombin-

slower peaks

(IAT-III2

and IAT-

PWS>f&

??3SERLX

a9

JL_YTITHRO?~BIX-III

Se-*

rgseosa

f-

i-

‘3

b

i

C d FIG.

1

(a) and (b) represent the crossed immunoelectrophoretic patterns observed in plasma and serum, respectively, in the absence of heparin in the agarose. (c) and (d) are identical to (a) and (b) but with the addition of heparin (16.6 units/ml) in the agarose for the first electrophoretic separation. In (c) the letters x, y and z represent IAT-1111, IAT-III' and IAT-III? respectively.

When incubated

venous blood was withdrawn,

at 37OC, aliquots

centrifuged

removed

and the supematant

merge

increase

in IAT-III'

(Fig. 2a, b and c).

thrombin

(kindly supplied

at various

to a glass tube,

time intervals

run in an agarose

units per ml) there was a gradual corresponding

transferred

which were

gel containing

heparin

(16.6

in size in IAT-III' and a 3 and IAT-III that appeared at 2 hours to

diminution

When titrated by Dr. D. Ellis,

plasma Lister

was clotted Institute,

with excess human London)

and the

PLASHA

90

clot liquor examined, slower components

b

SERUM

AXTITHRO?_IBIN-111

a single peak was obtained

(IAT-1112,

IAT-III31

which was similar

to the two

(Fig. 2d).

FIG. (a),(b),(c)

(d)

Immunoelectrophoretic antithrombin III patterns from sera obtained at different intervals after spontaneous clotting of native blood (crossed immunoelectrophoresis with heparin). The serum specimens were taken as follows: (a) after 5 min, in (b) after 60 rain and in (c) after 120 min of spontaneous clotting of native blood. Antithrombin III in liquor obtained by clotting normal plasma with human thrombin (100 u per ml). (Crossed immunoelectrophoresis with heparin).

DISCUSSION Although for this phenomenon unbound AT-III

at the present we believe

may be detected

Thus it seems reasonable

time we are unable

it provides

a simple method

and semiquantitated

to conclude

to find an explanation

that IAT-III'

by which bound and

in biological represents

fluids.

the normal

Vol.6,No.l

biological AT-III explore

PLASMA de SERUY ANTITHROMBIN-III

active AT-III

- activated

molecule,

coagulation

this possibility,

and management

whereas

factor(s)

and IAT-III3

complexes.

and to introduce

of hypercoagulable

IAT-III2

91

Studies

this new approach

states are currently

are the inert

designed

to

for the diagnosis

in hand.

ACKNOWLEDGEM&TS This work was funded by a Research

Grant

award by the Scottish

Home

and Health Department.

REFERENCES 1.

DOMBROSE, F.A., SEEGERS, W.H. and SEDENSKY, J.A.

2.

YIN, T.K. and WESSLER, S.

Antithrombin-inhibition of thrombin and autoprothrombin (F-Xa) as a mutual.depletion system. Thrombos. Diathes. haemorrh. 26, 103, 1971.

with antithrombin

Identity of plasma-activated Factor-*X inhibitor J. Biol. Chem. 246, 3712, III and heparin cofactor.

1971. 3. DAMUS, P.S., HICKS, M. and ROSENBERG, R.D. Anticoagulant Nature, 246, 355, 1973.

action

of heparin

4. ABILDGAARD,u.

Stand.

J. Clin.

Binding of thrombin 24, 23, 1969.

Lab. Invest.

5.

BINDER, B.

to antithrombin

On the complex formation of antithrombin

Gel filtration haemorrh. 30,

studies on human plasma 280, 1973,.

6. LAURELL, C.B. Antigen-antibody 2, 358, 1965.

Reprint

requests

crossed

and serum.

III.

III with thrombin. Thrombos. Diathes.

electrophoresis.

should be made to John D. Cash.

Anal.

Biochem.