molecular weight kininogen (Fitzgerald factor) by ...

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1983 62: 457-463

Characterization of the procoagulant chain derived from human high molecular weight kininogen (Fitzgerald factor) by human tissue kallikrein M Maier, KF Austen and J Spragg

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Characterization High Molecular

of the Weight

By Human

high

chain urinary

mol

mol

loop

as

whereas

the

present

together

as

64,000.

The

64.000

chain had

size.

amino

acid

procoagulant cleavage antiserum

K

HMWK that

was

that

retains

all

that

is

equal

in

molecule.

Cleavage

a reduction

in size of

the

mol

was

nonfunctional

procoagulant to

that by

kallikrein by

upon

elicited

Ouchterlony

an

analysis

as kallikreins two distinct

limited

(EC 3.4.21.8). forms of kininogen,

may and

be complexed

is thought

with

to be the

Human desig-

(HMWK) (LMWK).

prekallikrein preferred

and The in plas-

substrate

for

plasma kallikrein.23 Human HMWK has been shown not only to be a source of kinin but also to serve, together with plasma prekallikrein or with factor XI,4 as an important component in the initial step of contact activation of the Hageman-factor-dependent pathways.59 Plasmas deficient in HMWK have a prolonged in vitro partial thromboplastin diminished kaolin-activatable kinin fibninolysis.

Purified

all the abnormalities single polypeptide HMWK is cleaved sites in the interior moiety and yield free

The

protein.

chain,” shares with LMWK, “light tively

chain,” retains

HMWK,

time,

as well

generation

which

is able

to correct

in such plasmas, consists of a chain with mol wt l20,000.’#{176}’ by purified plasma kalliknein at two of the molecule to liberate the kinin a two-chain disulfide-linked kininamino-terminal

extensive while the

chain,

immunologic carboxy-terminal

is antigenically the procoagulant

unique activity

on

“heavy

determinants chain,

Blood,

Vol. 62.

the

size

No. 2 (August).

by

approximately

1983:

pp. 457-463

l0,000,’’

the site,

that the

representing vasoactive and

the

the

of

initial

from of

and

the

chain, the

the

bradykinin

procoagulant would

chain. contain,

in

C regions.

reduction

of

procoagulant

which glands

functionally, kallikrein,

are and

glandular

kalliknein,

by a procedure

has also been rat2223 plasma

extracted

from

included

that

found in in their

and antigenibut are related

other.’62#{176} Glandular kallikrein antigenically in human2’ and

the

initial

products

B-fragment.

containing

secretions, are structurally, cally different from plasma

plasma

the

released

cleavage

the

Glandular (tissue) kallikneins, kidney, pancreas, and salivary

and

is

HMWK

accompanying

to each identified

procoagulant for

kinin

for

kallikrein

amino-terminal

intact

A, B. and

the

The

chain

plasma

carboxy-terminal

Thus,

chain.

appropriate

A-chain,

for chain

“light”

with

peptide

the

respectively.

and

when

Thus,

permitted,

nonfunctional

is not

formed

released

human

immunoaffinity

chromatography, cleaved both synthetic substrates and HMWK.24 A single study of the interaction of a glandular kallikrein, namely, human salivary kallikrein, with human plasma HMWK substrate25 described limited time-dependent proteolysis but did not assess residual procoagulant activity. Highly punfled HMWK has been employed in the present study as a substrate for highly purified human urinary kallikrein to examine functionally and structurally the kinin-free kininogen formed by this tissue kallikrein. From

the

Department

the Department of Women s Hospital, Supported MM.

or

and

HMWK.

has

procoagulant

degradation

chain

C-chain, sequence.

the “heavy”

obtained

be designated

and

was

ofthe

Submitted

Address Building. ©1983

Harvard

by

and

Grants

with

the

University January

reprint

the Fellow

ofthe

Max

of

Medical

Vienna.

1 983;

requests

Vienna,

accepted

to Dr. Jocelyn

Room 625. 250 Longwood by Grune & Stratton. Inc.

Avenue,

and

Brigham

AI-07722.

National

Institute of

21,

MedicalSchoo/,

Immunology.

HL-22939,

RR-05669from

a Postdoctoral

he is presently Faculty

ofMedicine. Rheumatology Boston. MA.

in part

AM-05577.

and quantitaof the intact

HMWK.’#{176}’5 Plasma kalliknein appears to cleave carboxy-terminal chain of HMWK at a second diminishing

as and

to

continued

of

by HUK

of a stable

of

It is proposed

sequence.

by

proteolysis

high molecular weight kininogen molecular weight kininogen

former

that

size

product

HMWK.

activity.

by

proteins

two-chain

without

known contains

PLASMA

smaller

others and

the

HMWK

chain of

isolation

kininogen

carboxyterminal wt

activity

kinin-free

function

of HMWK

terminology

reflects

protein

with

the

to

unreduced

reduced

band

common

tam the amino acid sequence of the vasoactive polypeptides. Bradykinin or lysyl-bradykinin are

enzymes plasma

ma’

protein

isolated

kininogens

procoagulant

the

con-

from

the proteolysis

time,

similar

monospecific

inhibited first

from

plasma

limited

the

procoagulant

chains

the

Spragg

a

homogeneous

ARE

released

nated low

The

with

ININOGENS

kinin

with

and

human

Jocelyn

within

broad

composition

(“light”) of

single

and

from

of

separated

of similar an

a

Austen,

a singleby

SDS-PAGE

chains

chain

chromatographically

by

two

Frank

Derived From Human Factor) by Human

kinin

with

assessed

protein,

K.

(HMWK).

native

is associated

1 1 5.000.

wt

release

of the

Maier,

is cleaved

a two-chain

activity

by HUK

120.000. to

and form

Manfred

kininogen

wt

(HUK)

procoagulant

of HMWK

weight

with

kallikrein

disulfide the

molecular

protein

Procoagulant Chain Kininogen (Fitzgerald Tissue Kallikrein

AI-10356,

Institutes Kade

and

of Health.

Foundation.

Physiology,

Inc.; Medical

Austria. March

17.

Spragg. Boston.

/983. Seeley MA

G. Mudd 021/5.

0006-4971/83/6202---0035$OI.OO/O

457


MAIER.

458

MATERIALS

AND

METHODS

prior

Reagents and

Sephadex

Piscataway, dium

QAE G-100

NJ;

dodecyl

CA.

Kunitz

was

purchased

Indianapolis,

fluorophosphate

(DFP), from

brene

WI; MA.

anti-inter-a-trypsin

(plasma

from

Seratec,

prekallikrein)

deficient

Overland

and

Park,

plasmas

KS;

and

New

from

George

antiserum

component ofcomplement Elkhart, IN. lodoacetamide Rochester, NY.

and

against

(CIINH)

acetic

anti-a,

the

inhibitor

was purchased

MuCo.,

Eastman

acid

Na2EDTA, at room same

first

One

Inc.,

4#{176}Cagainst

buffer

screened

Organic,

During

kininogen

fractions

were

kallikrein on

purification,

incubated

for 5 mm guinea

the

bradykinin.

terminal

In studies

kallikrein,

the

otherwise

and

stated,

time

M CaCI2,

serial

dilutions

undiluted ty.

(Fort

Lee,

protein

NJ).

were

without

For

made

0.1

incubation

M

0.2

electrophoresed For

lant chain and

acid

was

analysis

urinary

by affinity

gel filtration single

HCI in

containing

18-32

1% in SDS, 20

D500

mm

at

0.5

buffer,

by then

of HMWK prior 24

pH

hr. 2.2,

The

sample subjected

band

analyzer.

kallikrein

(HUK)

was

purified

from

pooled

fresh

on aprotinin-CH-Sepharose

G-l00.#{176}The

wt of48,000

final

on SDS-PAGE

product with

revealed without

4.9-mI

was

dialyzed

with

eluted

and

0.5-mI

assayed pg/mI,

HMWK

dialyzed

NaCI,

M

concentrated

buffer.38 dialyzed

and

0.01

M Tris-HCI,

the

QAE

first

Sephadex

the

washing, 0.0

M to 0.4 were

M

dialyzed

at a conductivity

pooled,

concentrated

phosphate and

bags

One

containing

fractions

aliquoted

in dialysis

to

a linear

same

from

M sodium

0.003

applied

with

extensive

were

mM

dialysis

were

appeared

fractions

against

0.15

were

functions

These

in 0.05

ofQAE

gradient

and

0.5

was

column

column

mm

After

for

After

salt

of

addition

dissolved

against

buffer.

funcpooled

Fractions

indicated

of 2.8-mI

20 mS.

containing

same

clotting

90

eluted

in the

to a 10-mI

a linear

samples

as above. I 5 and

Samples

the

with

and

extensively

and applied

the were

DFP.

column.

as

IgG

M NaCl,

fractions

QAE

rabbit

both

for

0.075

M NaCI

inhibitors

column, was

the

and

purified

by the

precipitate

column

for

pooled,

equilibrated

stored

(mol

to

buffer, at

pH

-70#{176}C.

wt cutoff

3,500)

covered with sucrose. The overall

(Table

I ).

recovery The

U/mg Km

with for

purified

HUK

on

rate

constant migrated

apparent

mol

minor seen

wt

contained

HUK.

At

HMWK

5.2

pg

37#{176}C and

was

5.0

x

k,,

-

(k.,,/km)

was

8.6

x l0

is considered

with present (Fig.

the

or without in the

appropriate

a,-antitrypsin,

activity

the

sec’, M’

sec’.

deficient antithrombin

The

purified

band

with

Ill,

1).

was

no

urinary

of HMWK.

plasmas;

of

(Fig.

samples

XI was

x

5.2

second-

protein

incubation

aggregates

of as

(8.8),

=

the

reduction

unreduced

or factor

pH V,,,

and

major

33.9%

HMWK

optimum

prior

1 ) or after factor,

was

M,

as a single

to represent

Hageman

the

0.43

activity clotting kinin/mg

l06

the

band

with

a specific

enzyme,

reduction

prekallikrein,

a2-macroglobulin,

and

120,000,

HMW

assay

had

on SDS-PAGE

after and

on kinin-generating

HMWK

of protein

HMWK

clotting

based

purified

mole/mm/mg

order

plasma a

of

containing

Sephadex

kallikrein

and and

were

8.0,

longer

chromatography a mol

HMWK pH

The

on Sephadex with

as described

l0

to

0. 1 5 M to 0.5

analyzed

the was

from portions

assessed

procoagu-

and

gradient

18.1

to lyophilization

precipitate

5.3, containing

of SP-Sephadex

milliliter

7.4,

M urea.

water

I 10#{176}C for

at 60#{176}C,and

g; the

pH

at

NaCI for

and

stirred

C50

740

or

buffer,

x 25 cm column

2.5

between

pg of with

60#{176}C,followed

I 5 mm

sample

distilled

incubated

I hr at 2,000

was

the

dialyzed

M with

sulfate saturation

A50

M NaCl.3

were 0.1

buffer

a

and

was per-

samples

for

citrate

(PAGE)

M NaCI

pool

0.35

exhibiting

1 .0 mM

NaCI,

factor-

Instruments

containing

at

that activi-

the 0.35

4#{176}C.The

QAE

to correct

Na2EDTA, 0.5 mM benzamidine, and against the same buffer, the resolubilized

column

Purification

Human urine

assuming procoagulant

acetate

A50

with

plasmas.

a 680-pg

against

in 6 M

on a Durrum

Protein

gels

temperature

developed

by Buchler

for

analysis,

redissolved

was

(I:lS-l:lO0),

and

of

of 25

supplied

M iodoacetamide

dialyzed

hydrolysis

lyophilized,

M in urea

in 7.5%

amino

at room

gel electrophoresis

dithiothreitol

with

addition

Hageman-factor-deficient,

SDS-PAGE,29 6.6

p1)

25

for

M sodium

salt

kininpartial

a kaolin-phospho-

prekallikrein-deficient

to the directions

according

at

immediately.

After

of HMWK

included

plasma

polyacrylamide

Alkaline formed

controls and

and

curve

plasma

5 mm

37#{176}C,unless

(usually

continued

I U/mI

for at

centrifuged

ability

M

applied

employed

a substrate

Fractions

0.01

0.001 and

assay

as

in A total

against

M and

in a Lowry for

mg/liter

elution

with

plasma.

made

3 wk.

of Sephadex

8.0,

serve

was

50 than

fractions

pH

and

that

Polybrene,

Step-wise

to

with

at

released

benzamidine,

column

to 55% ammonium

(NH4)2SO4

as they

an excess of

temperature M

ml column

capacity

eluted

brought

enzyme from

and

50 mg/liter

Tris-HCI,

kallikrein,

DFP,

0.001

concentration for

5ec’

ethylenediaminetetra-

M, 0.1 2 M, 0.18

of 500 M

urinary

solid

urinary

and purified two-stage

volumes

A standard

contains

synthetic

was assayed

fractions,

of pooled normal

Negative

‘

kinin

was

measured

purified

mm

5

at 37#{176}C for 2 mm.

formed.

was with

incubated

another

column

incubation clot

plasma

XI-deficient,

and

separately

Equal

were incubated

an adherent

generated

0.075

samples

activities

and

urinary

in

to a 2-liter

0.05

M,

iO