xeul.

T'rl.RO?-tBOSI3RESEARCH Printed in the

THE

INHIBITION CLEMENT

!:nited

OF HUMAN

PLASMA

E. BURROWES,

Division

;, pp. IiS-133, Pergamon Press,

States

FlAVlO

of Experimental

KALLIKREIN

BY ANTITHROMBIN

M. HABAL**,

Pathology,

and Institute

HENRY

Department

1975

Inc.

III”

Z. MOVAT***

of Pathology,

of Immunology

University

of Toronto

Toronto

M5.S lA8

Canada (Received 1.4.1975; Accepted by

ABSTRACT

Prekallikrein,

prekallikrein

gen and antithrombin kal I ikrein

to kal I ikrein

had arginine

from kininogen. concentrations

kininoThe pre-

by preka I I ikrein activator. and genemted kinin by antithrombin

3 of that of plasma.

III

Physiological

of heparin were required for the mpid and complete In the absence of heparin only partial and

inhibition

activity

esterolytic

weight

from human plasma.

were inhibited

of approximately

of kallikrein.

cleaving

high molecular

estemse activity

Both activities

in concentrations

much slower

activator,

form 22.j.1975. Packham)

III were isolated

was converted

The kallikrein

inhibition

in revised Editor N.A,

was achieved.

of kallikrein

The proteolytic,

was more readily

i.e. kinin-

inhibited

than the

activity. INTRODUCTION

Human plasma kallikrein hibitors

is readily

of serine estemses (1,2).

tinguishing

between plasma, urinary

point of view the natural plasma kallikrein was reviewed kallikrein This

inhibited

by two of these inhibitors,

recently

communication

*Supported and the Medical **Recipient ***Holder

inhibitors

by Harpel

(5).

by the Atkinson Research Council of a Studentship

of an Associateship

kallikreins

recent review

in-

used in dis-

From an in vivo

namely a2-macroglobuIin

In another

Inhib_ition of and Cl-inactivator,

plasma prekallikrein

and

in man were discussed (6).

of antithrombin

Charitable

(3,4).

of plasma are important.

deficiency

deals with the effect

particularly

pattern was one criterion

and glandular

proteinase

and plasma prekallikrein

by a number of inhibitors,

The inhibition

Foundation,

IIIon human plasma kallikrein.

the Ontario

Heart Foundation

of Canada (MT-1251). from the Medical of the Medical

Research Council Research Council

of Canada.

of Canada.

MATERIALS Plasma Kallikrein

Partially

purified

tography and gel filtration, chromatograpkd

With

appeared

The plasma (containing against

exchange

chroma-

10 pg of soy bean tryp-

the equilibrating

in the effluent,

concentration

sections of a Phatmacia

less plasma smaller columns were used.

to the column (eluting

buffer,

again

low molecular

weight

0.1 M Tris-

Varying

aliquots

The prekallikrein

buffer.

prekallikrein

The rechromatographed

equilibrated 10 &ml

columns of Sephadex

with 0.1 M Tris-HCl,

SBTI.

eliminated

(MW

further eliminated

eluting

It was either

It was linked

column,

pH 8.0.

IgG

x

84 cm)

M EDTA and

in rabbits.

effluent.

eluted with

and gel filtration

recovered against

and factor IgG was

on Sephadex

through this immunoadsorbent With this procedure

When arginine

was attached

pH 7.4) the prekallikrein containing

converted

the kalli-

to Sepha-

became adsorbed

0.14 M NaCl

from the arginine-Sepharose

the residual zymogen

was dialysed

by

C-50

into a 1.5 x 5.0 cm column equilibrated

0.04 M Tris-HCI,

About 20% of the material

lOO,OOO),

This rabbit anti-human

which adsorbed to the column.

with 0.04 M Tris-HCI,

(MWu

was further purif’ed

G that was free of prekallikrein

By passing the prekall ikrein

rose 48 (equilibmted

On standing,

0.003

of the prekallikrein

chromatography

in the non-binding

or kallikrein

of 150 ml through

(Pharmacia i , which (8), or it was passed through one of two affinity

antibody

and was subsequently kallikrein.

0.5 M NaCI,

The prekallikrein

to Sepharose 48 and packed

it was freed of IgG,

krein was recovered

ahead

Immunoglobulin

from serum by QAE-Sephadex

G-200.

equilibmtea’

(10 x 80 cm and 10

passed through SP-Sephadex

most of the residual columns (7).

with 0.04 M Tris-HCI,

were re-

eluted with the equilibrating

superfine

(MWM 22,000).

XI (8) was used to produce anti-IgG isolated

(SBTI) remained adsorbed

and factor XII when the NaCl

was then passed in aliquots containing

30 cm) with IgG

step almost all of the IgG and all the factor XI was

160,000-170,000)

whereas the SBTI was retarded

chromatography

x

of crude prekallikrein

always

G-200

pH 8.0,

In this purification

one of three methods.

kininogen

(37

together

in the presence of SBTI, on smaller columns of QAE,

with 0.05 or 0.02 M Tris-HCl. two interconnected

KS/370

Prekal I ikrein,

whereas the soy bean trypsin inhibitor

with

was raised to 0.12 M).

chromatographed,

kallikrein

by anion

Up to four liters of plasma was

M ethylene

column consisted of two interconnected column.

EDTA.

(7).

diamine tetmacetate; EDTA), until the conWhen 4 liters of plasma were used the of the plasma was that of the buffer.

ductivity

0.003

was prepared

before

Sephadex A-50.

pH 8.0 (containing

METHODS

kallikrein

as described

per ml of plasma) was dialysed

sin inhibitor

HCI,

on QAE

AND

to the active

and 0.003

M

column was enzyme.

PBS (0.02 M phosphate buffered

The pre-

saline,

pH

7.4) before use. In most assays a prekallikrein mp moles of BAEe (benzoyl

prepamtion was used which upon activation hydrolysed 260 arginine ethyl ester) per minute per ml, by a spectrophoto-

metric method described before (2). The prekallikrein had been converted to kallikrein by incubation with PKA or prekallikrein activator, isolated as described elsewhere (9). One and incubated for 30 tenth ml PKA (6.0 pg) was added to 0.9 ml prekal likrein (30 &ml) minutes.

In addition

to hydrolysing

BAEe, kallikrein

also generated

kinin

(7). The kininogens were adjusted to approximately 100 pg protein/ml. parations the kallikrein released 1.0-1.2 pg of bmdykinin equivalents.

from kininogen From such pre-

Kininogen High molecular weight (HMW)-kininogen was isolated from human plasma, as described in detail before (7,lO). More recently some of the material eluting with QAESephadex was precipitated first with 4% (final cont.) polyethylene glycol (Union Carbide, Carbowax 4000, Toronto, Qnt.), the precipitate discarded and the supematant precipitated

III

by raising the concentmtion dissolved

in PBS (l/5

of polyethylene

of the starting

removed by dialysis oqinst Sephadex

PBS.

Antithrombin

III

antithrombin-heparin

eluates

on elution

with ammonium

adsorbs to aluminum

phosphate buffer,

pH 8.1,

(Pevicon)

plasma was freed of the prothrombin (50 mg/ml

Laboratories,

plasma) and after Penna.).

hydroxide

plasma with 0.15 M NaCl.

The crude antithrombin

After

by positive

centrifugation

III (a2-

and the to

To obtain

buffer (12) or step.

Thus, the

for 15 minutes with barium

(Amphogel,

One ml Amphogel

for 15 minutes.

8.1 (11).

preparative

by adsorption

stirred at room temperature

three washes (25% of the starting

in barbital

final

removal of the barium carbonate

with aluminum

Philadelphia,

i.e.

can be chromatographed

electrophoresis

complex

hydroxide

disc gel electrophoresis.

focusing (11) was found to be the essential

carbonate

glycol

before,

by Rosenberg and Dcrmus (I 1) antithrombin cofactor)

it is homogeneous by polyacrylamide

the plasma was incubated

was re-

(7,lO).

a high degree of purity preparative isoelectric

The precipitate

plasma volume) and the residual polyethylene

As shown recently

the point where

to &j?G.

The subsequent steps were those described

G -200 and CM-Sephadex

antithrombin,

glycol

177

by centrifugation,

unflavoured,

Wyeth

was added to IO ml plasma and

The AI

was washed III was eluted

free of entrapped

from the Al(OH)3

by

plasma volume) with 0.36 M ammonium phosphate,

at 27,000

pressure ultrafiltration

g to clear the washings,

(Amicon

Corp.,

Lexington,

pH

they were concentrated

Mass.) and passed sequen-

tially through QAE-Sephadex, Sephadex G-200 and CM-Sephadex (7). Further purification was achieved with Concanavalin A-Sepharose, to which the antithrombin III became adsorbed

(13).

After

an ampholine Scientific,

Toronto,

at pH 5.10-5.15. diffusion Assays

Inhibition capable incubated

of pH 4-6.

Ont.)

The instructions

were followed,

The concentmtion

(M-Partigen, Arginine

Behringwerke,

by kallikrein

Studies

As described

of hydroiysing for varying

(BAEe hydrolysis)

(2).

above the kallikrein

by mdial

Toronto,

of the kallikrein

was diluted

focusing

mixture was incubated

(PBS), pH 7.4.

For the arginine

Ont.).

and bioassay of the in detail

before

read in a spectrophotometer

For the kinin assay, 0.1 ml reaction ml HMW-kininogen,

adjusted

bath to inactivate

the kallikrein.

pH 8.0,

ml of kallikrein

to 1.0 ml with After

with 0.5 ml of 0.003

contain

was incubated

i

ng 0.1 M NaCl

intervals

M BAEe

and the ex-

for 12 minutes

(2).

at 37” for 5 minutes with 0.1

PBS and then boiled

dilution

was

or phos-

estemse assay the enzyme-inhibitor

at 253 mp at 3 minute mixture

(2).

to about 30.0 &ml,

One-tenth

at room tempemture

to 2.5 ml with 0.1 M Tris-HCI,

III focused immuno-

lengths of time with 0.1, 0.2 and 0.4 ml of the inhibitor

saline

in

(LKB, Fisher

Antithrombin

were done as described

260 mpmoles of BAEe per minute.

or enzyme-PBS tinction

before

was determined

Hoechst Pharmaceuticals,

phate buffered and adjusted

as described

from kininogen

to isoelectric

of the manufacturer

of the inhibitor

estemse activity

kinir;r-genemted

III was subjected

this step the antithrombin

gmdient

for 5 minutes in o water

with de Jalon solution,

the genemted

kinin was assayed on the estrous rot uterus (2,7). Kininogen, kallikrein or inhibitor incubated with PBS served as controls. The arginine esterase activity was expressed as mpmoles of &4Ee hydrolysis, arginine.

based on a calibmtion

The k inin activity

standardized

with synthetic

curve made with dilutions

was expressed as bradykinin

equivalents.

of benzoyl

The uterus was

bmdykinin. RESULTS

The antithrombin

III (125 &ml)

induced

part ia I inhibition

of both the esterolytic

and

OL

I

I

I

I

I

I

I

5

10

15

20

25

30

60

Incubation (minutes) FIG. One-tenth

1

(30&ml)

ml kallikrein

was incubated

with 0.1 ml anti-

thrombin III (125&ml) with or without 0.1 ml heparin (1.5 units/ The kollikrein and antithrombin III had been ml) for 2-60 minutes. dialysed kallikrein

against

ml of benzoyl HCI,

PBS before

was incubated

pH 8.0,

arginine containing

intervals

BAEe as the blank.

The O.D. per minute

known amounts of benzoyl

activity

In the control

Following

0.1 M NaCI,

of BAEe hydrolysed

concentmtions

PBS.

tubes 0.1 ml

incubation

with 0.5

ethyl ester (BAEe) and 2.2 ml of 0.1 M Tris-

read at 253 mp at 2 minute

proteolytic

incubation.

with

the increments

of O.D.

were

for 12 minutes with buffer and

readings were converted per ml, from a calibration

into mpmoles curve of

arginine.

This process was markedly enhanced by physiological of kallikrein. The concentmtion of heparin was 1.5 units per ml and 0.1 ml

of heparin.

Figure 1 shows the effect of 0.1 ml of antithrombin was used in the reaction mixtures. with and without heparin on the esterolytic capacity of kallikrein and Figure 2 the ability Figure 3 is a kymogmph tmcing illustmof the enzyme to cleave kinin from kininogen. ting the inhidition

of kallikrein

incubated

with

PBS or antithrombin

III and heparin for

15 minutes. DISCUSS ION The inhibition

of plasma kallikrein

by a2-macrogIobuIin

and CT-inactivator

is well

L-01

.7,SO.

KALLIKREIX

1

ASD tiTIT!riROYBIS III

179

70 m

Antithrombin+Heparin

60 -~&tithrombir+Eiuffer 50

40 30

20

10 !

I- 2(j

10

incubation (minutes)

FIG. Kallikrein

was incubated

(or PBS) as described cubation

2

with antithrombin

in Figure

(100 pg/ml).

Th e reaction

boiling

for 5 minutes.

water

hibitor

(5).

Inhibition

After

postulated

cooling

the reaction

Cl-inactivator

by in-

kininogen

with synthetic

kallikrein,

was inhibitory

dilution

were assayed on the bmdykinin.

pig (15) plasma kallikrein

nor a2-macroglobulin

pig plasma inhibited

that the same inhibitor

followed weight

and appropriate

mixtures

of -bovine (14) and of guinea

which was neither from guinea

(or PBS) and heparin

was stopped by immersing the tubes into

estrous rat uterus, standardized

inhibitor

III

minutes,

for 5 minutes with 0.1 ml high molecular

with de JaIon solution,

established

1 for 5-60

thrombin,

was reported.

by an The in-

trypsin and plasmin.

in both bovine and guinea

It was

pig plasma

(15). A plasma inhibitor thrombin activity

of both trypsin and thrombin

has been known for some time (12).

of human plasma was first reported

(17) first noted that heparin was effective

by Mamwitz

as an anticoagulant

(16).

Anti-

Brinkhous et al.

only in the presence of a

plasma factor. Subsequently, a close relationship was described between the antithrombin activity of plasma and heparin, since heparin was found to enhance markedly the thrombin neutmlizing inhibiting

preparations not

activity

activated

of antithrombin

factor X (20,21,22)

of antithrombin

had been available

been prepared until fairly recently

were reported

(18,19).

Antithrombin

and factor XI (23).

acts in a similar way in

Concentrated

for some time (24),

but impure

but pure inhibitor

has

(25) and in the past four years good recoveries

(11,12).

Last year evidence

was presented that human antithrombin

111 in the presence of hepclrin

i.

FIG. Kymogmph

tmcing

Schultz-Dale

reaction

mixture

added to the Schultz krein, antithrombin dilution was l/400

with de Jalon

of kininogen

antithrombin

inhibits

solution

the esterolytic

and proteolytic

dilution.

incubated

The data reported activities

mixture

of kininogen.

kalli-

in Fig. 2), and

At “C” the dilution

“E” represents the reaction

before applying

was

“B” re-

for 20 minutes,

(as described

III and heparin

plasmin (26) and preliminary (27).

inAt “A”

PBS and kininogen)

with de Jalon solution.

also kallikrein

in ns/ml.

to the bath of the reaction

minutes before the addition

readily

(kallikrein,

and at “D” l/300.

ture kallikrein,

contractions

bradykinin,

III and heparin

by addition to l/600

represent

Dale bath in a l/2400

presents the application following

of the estrous mt uterus in a

The numerals

duced by standards of synthetic the control

3

of contractions

bath.

i ,.._.:

incubated

mix-

for 30

It was diluted

l/20

to the Schultz-Dale

bath.

data have been presented that it inhibits

in this paper support this view. of kallikrein

are inhibited,

As shown, both

the latter more

efficiently. Yet another enzyme inhibited by antithrombin III is a neutml protease of human neutrophil leukocyte lysosomes. This enzyme too is more efficiently inhibited in the presence of heparin. During inhibition there seems to be partial proteolysis of the antithrombin

III

(28).

t-0 1 .;,s0.1

KALLI~I?;

AXD AXTITIiRO>IBIS III

181

ACKNOWLEDGEMENTS The authors wish to thank Dr. D.M.

Wrobel

Cross for generous supplies of plasma. of Mrs. Otti

Freitag

and Mrs. I. MacDonald

The skillful

and Miss Marica

Michael

chnical

of the Canadian

and secretarial

+ are gratefully

Red

assistance

acknowledged.

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