Vol.

THE

74,

No.

1, 1977

INHIBITION

BIOCHEMICAL

OF

ACTIVATED

THE John

Y. C.

EFFECT

Chan2,

Received

FACTOR OF

OTHER

Clement

Division of Experimental University of Toronto,

October

AND

XII

RESEARCH

(HAGEMAN

PLASMA

E. Burrowes,

Pathology, Medical

BIOPHYSICAL

FACTOR)

PROTEINASE

Flavio

M.

COMMUNICATIONS

BY ANTITHROMBIN

III:

INHIBITORS’

Habal

and

Department of Pathology, Sciences Building, Toronto,

Henry

Z.

Movat3

and Institute of Immunology, Ontario, M5S lA8, Canada

11,1976 Summarv

Human factor XII was activated by adsorption onto kaolin in the presence of high molecular weight kininogen. The washed kaolin-containing precipitates activate prekallikrein to kall ikrein. When antithrombin III was added to the reaction mixture, the conversion of prekallikrein to kall ikrein was inhibited, the degree of inhibition depending on the concentration of antithrombin and the time of incubation. Heparin had a slight enhancing effect with low concentrations of antithrombin and short incubation times. However, the inhibition of the generated kall iAntithrombin III inhibited also the krein by antithrombin III was markedly enhanced by heparin. effect of activated factor XII on the partial thromboplastin time, using factor XII-deficient plasma Of other plasma proteinase inhibitors used (al-antitrypsin, a2-macroglobulin, CT-inactivator) only CT-inactivator inhibited activated factor XII. Introduction The lytic

contact

systems.

There

pathological the

kinin

states system,

is inhibited

kallikrein.

prekall

ikrein.

converted

Canada

Copyright All rights

of blood

arising

from

formation

vasoactive

intermediate

Th is enzyme

(2) and

to its active

by

form

the

Student Associate

0 1977 by Academic of reproduction in any

by activated

Ontario supported of the

Heart

in the

inhibited

by III

factor

the (3).

XII,

Foundation

150

bradykinin

cascade

and

the

and

inhibition

is the is cleaved

is the plasma The

the

with

Research Council

proteinase

being

Medical

and

last

fibrino-

of these In the

from

kininogen

inhibitors

of

peptide by

of kallikrein

from a2-macro-

prekallikrein

in turn

activated

by

Research

Council

Council of Canada. of Canada.

systems,

cascade This

step.

generation

zymogen

latter

the

kinin-

inhibition.

Bradykinin

by the Medical Medical Research

Press, Inc. form reserved.

activation

peptide

antithrombin

linked

or inadequate

N (1). step

is readily

between

activation

by carboxypeptidase The

is intimately

balance

excess

of the

CT-inactivator

‘Supported (MT-1251). 2Graduate 3Research

coagulation

is a homeostatic

primarily

plasma

globulin,

phase

negatively

of

is

Vol.

charged

74, No.

surfaces

cular

weight

vity

of factor

activator

isolation before

and

XIIa

paper of factor

and

requiring

kininogen

(PKA) This

BIOCHEMICAL

1, 1977

that

for

catalytic its full

is inhibited

is also

and

rapid

the

inhibition

activation

inhibitor

is described Materials

high

RESEARCH

and

(4, 5,6,

small

COMMUNICATIONS

quantities The

7, 8).

Fragmented

(9).

factor

XII

of high

mole-

clot-promoting

acti-

or prekallikrein

(10).

of contact and

BIOPHYSICAL

of kallikrein

by CT-inactivator

XII (1 l), kallikrein of the

amounts

by CT-inactivator

inhibited

describes

AND

activated

molecular

in the and

factor weight

accompanying

XII kininogen publication

by antithrombin (12)

was

The

III. described

(13).

Methods

Activated factor XII was assayed by its capacity to activate prekallikrein to kall ikrein and by the partial thromboplastin time (PTT), which measures the ability to correct the prolonged Th e activation clotting time of factor XII-deficient plasma (8, 11). of prekall ikrein to kall ikrein was determined indirectly. Activated factor XII converts the zymogen prekallikrein to its active form kall ikrein and the latter, being an arginine esterase hydrolyses benzoyl arginine ethyl ester Briefly, 0.1 ml factor XII was incubated with 0.1 ml kaolin (10 mg/ml) for 2 minutes. (BAEe). To this mixture was added 0.1 ml of high molecular weight (HMW)-kininogen and incubated for 10 minutes. The reaction mixture was cooled to 4”, centrifuged at that temperature for 10 minutes at 30509, then resuspended in cold 0.1 M Tris-HCl buffer (pH 8.0, containing 0.1 M NaCI) and recentrifuged. The kaolin-containing precipitates to which the activated factor XII was adsorbed (8) was added to 0.1-0.2 ml prekallikrein, incubated for 10 minutes, the precipitates removed by centrifugation and the supernatant (containing the kallikrein) added to 0.5 ml 3 x 10m3M BAEe, made up to 3 ml with Tris-buffer and the extinction read at 253 nm at 2-minute intervals for 10 minutes. A mixture of 0.5 ml BAEe and 2.5 ml buffer served as the blank and the increase in absorbance was converted to nmoles of BAEe hydrolysed per ml of sample per minute, using a standard curve of known amounts of BA. The kaolin The PTT assay was based on the procedure described by Ratnoff and Davie (14). precipitates described above, to which the activated factor XII was adsorbed, was resuspended in Th is was incubated 0.1 ml of Tris-buffer containing 0.1 M NaCl (pH 8.0). in duplicate with 0.1 ml crude soybean phosphol ipid (Centrolex P, Chemurgy Div., Central Soyal, Chicago, II I. ) for 2 To this reaction mixture 0.1 minutes at 37’ in polystyrene test tubes (8 mm internal diameter). ml factor XII-deficient plasma (15) was added, immediately recalcified with 0. 1 ml 0.02 M CaC12 and the time required for a solid clot-formation was ascertained. Inhibition of activated factor XII was carried out by incubating the kaolin precipitates which factor XII and HMW-kininogen were adsorbed with either increasing concentrations thrombin III (with or without heparin) for 30 minutes or with a constant amount of inhibitor varying lengths of time. Several preparations of antithrombin were assayed in this manner. experiment was carried out using al-antitrypsin, a2-macroglobulin and CT-inactivator.

to of antifor One

Having observed differences between the effect of heparin in earlier studies (3, 16) and those described below, we compared the effect of heparin in one experiment, in which the inh ibitor was aded to the kal I ikrein-contain ing supernatant. As described above the activated factor XII when added to prekall ikrein converts it to kallikrein.

151

Vol.

74,

No.

BIOCHEMICAL

1, 1977

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

0 8.5

17

34

Concentration

Figure

1.

68

176

of AntithrombinlII(ug)

One tenth ml (10 mg/ml) k ao I in was incubated with 0.1 ml factor XII (1.5 pg) 0.1 ml HMW-kininogen (0.45 pg; 4.8 ng bradykinin equivalents) and increasing In the amounts of antithrombin III (0.1 ml) with or without heparin (4 units). control the antithrombin was replaced with 0.1 M Tris-HCl (pH 8.0), containing 0.1 M NaCl. As described in the Materials and Methods the conversion of prekall ikrein to kall ikrein by the kaolin-containing precipitates was assayed as BAEe hydrolysis. In the control 218 nmoles per ml per minute of ester was hydrolysed. The incubation with antithrombin was for 30 minutes. The data are expressed as percent inhibition.

This procedure was modified by The isolation of factor XII was described before (11). using Sephadex G-200 instead of G-100 and having used a larger starting volume of plasma (8 liters), the partially purified factor XII obtained by CM-Sephadex was rechromatographed on the same cation exchanger. The concentration of factor XII and other proteins was determined HMW-kininogen (85by the method of Lowry et al. (17); that of factor XII being 15 &ml. were purified as reported earlier (12). The prekal I i132 &ml) and prekallikrein (85.5 pg/ml) krein was adjusted so that on conversion it hydrolysed 200-300 nmoles of BAEe per minute per ml. The antithrombin was prepared as described in detail in the accompanying paper (13), which To obtain a2-macroglobul in the procedure of also describes the isolation of al-antitrypsin. Harpel (18) was followed. Partially purified CT-inactivator was a by-product of the purification of HMW-kininogen (12) and a highly purified preparation was supplied by Dr. Jack Pensky The inhibitors were quantitated by radial immunodiffusion (Case-Western Reserve University). (M-Partigen, Behringwerke, Hoechst Pharmaceuticals, Montreal, Que. ) and by their ability to inhibit certain enzymes (thrombin, trypsin,kallikrein) (see Ref. 13). Results As indicated, vated

form

and

incubation addition

of kaolin

of increasing

with

factor

concentrations

152

XII slowly

converts

of HMW-kininogen

the

latter

markedly

into

its acti-

enhances

the

Vol.

74,

No.

BIOCHEMICAL

1, 1977

AND

BIOPHYSICAL

15

10

20

Incubation

2.

Figure

Conditions antithrombin

as in Figure concentration

RESEARCH

30

COMMUNICATIONS

45

(Mm)

1, except varying was 68 pg.

the

time

of incubation

as shown.

The

lncubatlon (Mm) Figure

rate

3.

of this

cubated activated lower

Inhibition of kall (see Fig. 1) was without inhibitor activated factor Concentration of

reaction

with

(8).

increasing

factor concentrations

XII,

ikrein by antithrombin III. The inhibitor with or without heparir The kall ikrein added to the kall ikrein-containing supernatant. Note that unlike with hydrolysed 218 nmoles BAEe/min/ml. XII heparin had a marked enhancing effect on the inhibition. antithrombin was 68 pg.

When

the

kaolin-factor

concentrations which

no longer

of antithrombin

XII-HMW-kininogen

of antithrombin can

III

convert heparin

III prekall

enhances 153

there ikrein the

reaction is gradual to kall inhibition.

ikrein

mixture

inhibition (Fig. With

fixed

is inof the

1).

With amounts

Vol.

74,

No.

1, 1977

BIOCHEMICAL

AND

TABLE Effect

of Antithrombin Partial

III

on Activated

Thromboplastin

RESEARCH

COMMUNICATIONS

1

Test

Factor

with

III (pg)

Antithrombin

BIOPHYSICAL

Factor

PTT (sec.

0

XII

as Measured

XII-Deficient

by the Plasma

% Inhibition

)

0

124 141 150 154 190

8.5 17.0

34.0 68.0

14 19 22 38

The PTT was carried out in duplicates as described in Materials and Methods. When the PTT was done by the conventional method (incubation of factor XII with kaolin for 2 minutes, followed by incubation with factor XII-deficient plasma for 8 minutes, followed by addition of CaC12) a solid clot formed in 91 seconds. The factor XII-deficient plasma without any additive clotted in 564 Time of incubation with antithrombin III was for 20 minutes. seconds.

TABLE Effect the

of Antithrombin Partial

Incubation Antithrombin

III on Activated

Thromboplastin

Test

When

(Fig. the

enhancing

were

XII

Here

too

antithrombin effect

XII-Deficient

by

Plasma

(min.

)

PTT (sec.

% Inhibition

)

identical

to those

0 18 24 35 41

in Table

1.

The

concentration

of anti-

III was 68 pg,

factor 2).

Factor

as Measured

124 150 161 186 208

Conditions thrombin

tion

with

XII

Factor

with

III

Control 5 10 20 30

of activated

2

on the

and with

antithrombin, shorter

increasing times

was added

to the

inhibition

(Fig.

the

of incubation kallikrein

that

3). 154

time

of incubation

heparin had

been

further generated,

enhances enhanced heparin

the the

inhibi-

inhibition. had

marked

Vol.

74,

No.

BIOCHEMICAL

1, 1977

AND

TABLE Effect

of Plasma

Measured

by

Proteinase

al -antitrypsin

(275

a2-macroglobul

in

Ci-inhibitor

(84

RESEARCH

on Activated

to Convert

Prekallikrein

BAEe-hydrolysis (nmoles/min/ml

)

pg)

COMMUNICATIONS

3

Inhibitors

its Ability

Inhibitors

BIOPHYSICAL

Factor

XII

as

to Kallikrein

% Inhibition

282

(532

pg)

285

0

285

0

pg)

Buffer

One tenth ml of inhibitor was added to the activated factor XII-containing kaolin precipitates, incubated for 30 minutes at 37” and the washed precipitates added to prekall ikrein as described in Materials and Methods and the BAEe-hydrolysing activity of the generated kol I ikrein assayed.

Using

the

modified

PTT,

as described

of antithrombin

(in the

absence

of heparin)

constant

of antithrombin,

amount

of ontithrombin

and

Factor globulin

XII

(Table

the

activated

was

factor

inhibited

also

in Materials

and

lengthened

PTT was XII

the

lengthened

(Table

Methods,

increasing

PTT as shown proportionally

concentrations

in Table to the

1. time

With

a

of incubation

2).

by CT-inactivator,

but

not

by al-antitrypsin

and

a2-macro-

3). Discussion

Antithrombin markedly (22,

enhanced

23).

Since

it has been clotting

shown

factors, There

seems

has been

shown

by heparin

(19,

antithrombin

III

to inhibit namely

also

activated

to be a definite

to be probably 20,

21),

has become

inhibitor

alth ough

a2-macroglobulin

availoble

in highly

plasmin

(16,

28),

factor

XI

(31),

difference

the principal

pl asma factor

in the

155

likewise purified

kallikrein IX (32,

co-factor

of thrombin,

33)

effect

form

(3,

16, 29,

and

factor

of heparin

a process

inhibits (24, 30)

25, and

thrombin 26,

27)

certain

X (33). in the

inhibition

Vol.

74, No.

of the

above

enzymes.

vated

clotting

factors

on the

inhibition

activated of the

enzymes

The

on the

antithrombin that

both

form

complexes

factor

as shown

reason

X, ond for

these

of which

activated of the

activated

to study

complex

III failed.

(34),

of thrombin, augmented

to antithrombin

nature

attempts

inhibition

IX is markedly

inhibition

inhibited

minary

AND BIOPHYSICAL

plasmin,

kallikrein

by heparin, in this

differences heparin

RESEARCH COMMUNICATIONS

are

paper,

this

co-factor

also

on the

presently

has an effect

and

of the has little

effect

inhibition

unknown,

but

(thrombin,

acti-

of

at least

plasmin,

some

kallikrein)

esters.

In addition

The

the

XI and

XII.

arginine

inactivator

Whereas

of activated

factor

hydrolyse

BIOCHEMICAL

1, 1977

There

intact

activated

with

antithrombin

III

other

proteinase

factor

XII,

factor

which XII

inhibitors

were

is in keeping

adsorbed

with

to kaolin

formation

between

factor

is however,

a recent

communication,

factor

XII

III,

and

fragmented

as shown

XII

gel

Of

earlier

remains

only

CT-

(9,

10).

observations

by ellagic reported

XII

these

to be ascertained.

activated

factor

by SDS-disc

tested.

Preli-

acid

in abstract

or prekallikrein

and form

activator

electrophoresis.

Acknowledgements The authors wish to thank Mrs. Otti Freitag and Ms. Marica Michael and secretarial assistance. The pl asma used in these studies were supplied Red Cross through the courtesy of Dr. D. M. Wrobel and Mrs. I. Macdonald.

for technical by the Canadian

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Erdlis, E.G., Nakajima, T., Oshima, and Biology of the Kallikrein-Kinin K. F. Austen, eds., Fogarty Internat. Office, Washington, D. C.

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Harpel, P.C. (1976) Chemistry and Disease, J. J. Pisano and No. 27, U.S. Gov. Printing

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Burrowes,

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Kaplan, A. P., Meier, Hemostasis, -3, 1.

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Meier, H.C., and Kaplan,

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