THROMBOSIS

vol.

RESEARCH

lo,

pp. 159-162, 1977

Pergamon

COMMUNICATION

BRIEF

ANTITHROMBIN Ian

David

Coagulation Watford * Present

Sas peaks

et

of

III

Mackie+,

(Received

al

(1)

that

highly

complexes towards the

in

found

the

binding

occupied

by

reduced

and

reduced

in

The

sites

on

containing to

The plasma Serum

and

was

crossed

disclosed produced

ml

for

Sas

III

20 mm

antiserum the

of

second

two

peaks

agarose.

wholly

or

lf

partly

heparin

to allow 108

sample

binding

is

III

the

was

& of

native

159

x 54 mm

aPplied

8 ml

anode.

(Nyegaard

labelled

greater

Co.,

of

AT

the

blue of

Oslo)

gel.

marker

1%

agarose,

was

added

electrophoresis. normal

III1

blood

glass

to

a bromophenol

dimension

of

distance

of antithrombin

peaks.

immunoelectrophoresis

.by clotting

(2)

heparin

plain

the

modified

until

from

to the

gel.

was

20 pl

are

factors,

crossed

a greater

in

mobility

continued

III

migrate III

the

added

suggested

antithrombin

agarose by

in was

They

they

precipitation

a point

plate

that

precipitation

obtained

if heparin

clotting

used the

0.2

the

Milica

separate

antithrombin

heparin

used,

reached

be

electrophoretic

Electrophoresis had

could

antithrombin

activated

were

three

charged

than

of

and

dimension.

and

anode

the

BrozoviE*

serum,

first

formed

method

separation plates

of

the

the

Howarth,

that

III

negatively are

PATTERN

27.10.1976; in revised form 19.11.1976. Accepted by Editor B. Blombgck)

antithrombin

gel

IMMUNOPRECIPITATION

Laboratory, Northwick Park Hospital, Rd, Harrow, Middlesex, England. address Central Middlesex Hospital, Acton Lane, London N.W.10

immunoelectrophoresis agarose

Press

and in

a

titrated

AT

1112.

glass

tube

160

ANTITHROMBIN III

Vol.10,No.l

AT-ill'AT-III

la

Al-Ill4

AT-Ill3

lb

Antithrombin III plasma; b) serum tube for 1 hour.

FIG 1 immunoprecipitation produced by clotting

peaks of: a) titrated native blood in a glass

2ta

AT Ill3

FIG

2

Antithrombin III immunoprecipitation peaks of: a) plasma, b) serum, produced from blood collected into EACA/citrate.

and

Vol.10,No.l

ANTITHROMBIN III

harvested

after

and

4 , as well

AT

(see

III

The

addition

or

change

the

factor

two

a small

over

factor

Xa

pattern

50 NIH

were

was

addition We

of Xa

exogenous (Diagen)

slowly

amount

moving

of AT

also

peaks

1111

AT

and

AT

of

observed

in

25mbl CaC12, to

III3 III*

shown

the

tion

was

the

ml

III,

of undiluted

plasma.

A plasma-like

obtained

CaC12

prothrombin

pattern

of

antithrombin

or

50mW and

in Table

1,

typical

after

the

the was

CaC12

the

obtained

with

consumption

collected

serum

pattern if

IJI.

thromboplastin,

serum

only

on

after of AT

prothrombin

were 1 hour.

III consump-

or more. TABLE

Prothrombin

titrated clotted

0.1

of

plasma

902

serum

to

antithrombin

effect

titrated

immunoprecipitation

or

titrated

or

failed

Reptilase.

studied

50mM

of

plasma of

thrombin,

ml.

or

(Parke-Davies,

titrated pattern

per

of ARVIN next

units

added

immunoprecipitation

added

thrombin to

immunoprecipitation

unless

As

showed

as

Fig.1).

Ortho)

the

1 hour,

161

Consumption

plasma with

and

AT

1 III

prothrombin consumption

Immunoprecipitation

AT III immunoprecipitation pattern

25mM

CaC12

60%

AT

III1

AT

III*

50mM

CaC12

90s

AT

1113

AT

III4

50mM CaC12 + thromboplastin

99%

AT

1113

AT

III4

1 It of

plasma

appears

from

serine

experiments achieved

proteases,

clotting

(as measured

addition

of

excess

these

by

prothrombin

proteolytic

enzymes

that either

thorough by

consumption) (thrombin

activation

efficient or by or

the

factor

Xa),

162

ANTITHROMBIN III

is

required

of

antithrombin

different

in

blood)

show

the as

proteases

from

absence

complexing not

inhibited

by

agreement

with

without

that

These

change results

interpretation

of

antithrombin

III,

deficiency,

clotting, gulants

involved

recent the

used

the

they

clotting,

of

to our

for

sera

in should

be

prothrombin

may

affect

the

are is (4)

in who

increased AT

ITI3)

peak.

consistent

families

due

of

Peljper thrombin

that

9Y';b,

interpreted

This

purified

moving

ml

is normally

which

and

lO':n,

Sera of

a variety

immunoprecipitation

by

T .lJ. per view.

(corresponding

particularly

(3,5),

(500

peak

and

Sas

into

this

or Trasylol. of

slowest

indicate serum

as measured are

report

peak

in

in

EACA

addition

collected

We have

moving III

of

consumption

(Fig.2).

antithrombin

of

blood

confirm

slowest

pattern

binding

ITT.

prothrombin

peak

the

antithrombin

any

with

addition

the

the

or Trasylol

Citrate,

TT14

directly the

demonstrated second

AT that

between

with

Citrate

blood,

of

immunoprecipitation

indicating

experiments

such

indicating

the

to antithrombin

3.13y~ Tri-Sodium

proteases

III

in

presumably

3.139, Tri-Sodium and

this

the

change

preliminary

obtained

to

the III,

serine

Our EACA

for

Vol.10,No.l

and

reliable

patterns with

obtained

antithrombin after

consumption. patterns

of

efficient

If

anticoa-

observed.

REFERENCES

1.

D.S., CASH, J.D. SAS, G., PEPPER, antithrombin III: differentiation Thrombosis Research, trophoresis.

Plasma and serum by crossed immunoelec6, 87, 1975a.

2.

SAS, G., PEPPER, D.S., CASH, J.D. thrombin III in normal plasma and of Haematology, 30, 265, 1975.

Investigations British serum.

3.

SAS, G., PEPPER, D.S., CASH, J.D. Further investigation on antithrombin III in the plasmas of patients with the Thrombosis, III Budapest'. abnormality of 'antithrombin Diathesis Haemorrhagica, (Stuttg.), 2, 564, 1975.

4.

D.S. SAS, G., PEPPER, III complex by crossed Research, 2, 95, 1976.

5.

GOMPERTS, E.D., FEESEY, M., VAN DER WALT, J.D. Twodimentional immunoelectrophoretic studies in antithrombin Thrombosis Research, 8, 713, 1976. III deficiency.

on antiJournal

Detection of thrombin-antithrombin Thrombosis immunoelectrophoresis.