Calcium binding to human fibrinogen — Localization of two calcium specific sites

RESEARCH Vol. 13 Press Ltd. 1978.

THROMBOSIS @Pergamon

CALCIUM

BINDING

TO HUMAN

OF

G.G.

pp. 345-350 Printed in Great Britain 00~~-~8~8/~8/0~01-03~j

TWO

CALCIUM

LtNDSEY,

G.

Department University

of

L.R.

Medical Cape

South

Accepted

PURVES

Pathology,

Town 7925,

Republic

9.6.1978.

and

Chemical

Cape

Observatory

(Received

FIBRINOGEN - LOC,4LIZATlON SPECIFIC SITES

BROWN

of

of

$02.00/o

School,

Town,

Africa.

by

Editor

R.F.

Doolittle)

ABSTRACT In this

report

calcium Dd

we

specific

omains.

sites

were

9 x

10-6M.

have

sites

Scatchard equivalent

fragment

demonstrated

binding

analysis

and

No

high

that

on

had

The

role

molecule

of

binding

of

cross-link

thermal the

sites

fibrinogen

et that

binding

not

a-chain,

the

The

initial

a-chain

C-terminal are

stabilized

(4)

we

have

portion by

high

are

affinity

located

showed

constant

sites

in

that of

were

in

all

the

the

approximately

demonstrated

for

remnant (7). calcium

investigated

of

of

the

plasmin

on

in

fragments

Since

we

ions

against

whether

D1

the

product

Dl

The

has initial

reduced

concluded molecule

well

recently

occupation and

of

the

3 high

the

fibrin

that

the

involving

affinity

reaction

by addition

of

calcium the

velocity

of

calcium

ions

binding

C-terminal

part

of

(6).

Dl and D-dimer shown

further and

have

than

fibrinogen

slightly

have

been

fibrinogen

(4).

(5).

action

We

rather

that

calcium

Marguerie

the

denaturation.

digestion

has

protect

degradation

was

a zone

fibrinogen

to

ions

demonstrated

plasmin

site

(3)

of

shown

calcium

plasmin

(3).

located

acid

of

specific

with

been

fibrinogen

have

al.

by magnesium were

3

ION

polyrnerisation also

and

further

are

digestion

centres

(2) the

from

Mzrguerie

the has

presence

stabilizes

D-dimer

binding

in

Calcium

that site

product

but

calcium

against

the

a dissociation

affinity

the

E.

(1).

demonstrated

of

fibrinogen

INTRODUCT

documented

2

human

345

is devoid

both

proteolytic

D-dimer

sites.

that

have

of its

fragments digestion

specific

Dl

and

D-dirrer

with

plasmin

calcium

binding

3i6

MATERIALS Fibrinogen sulphate linked

fibrin

column

(4).

8.6

0.005

M Tris,

0.15

from

purification

from

Chelex

100

DE

(BioRad)

immediately glassware

the

prior that

7.4 with

samples

treatment

to the

had been washed

with

(Elgastat).water

(calcium

Chelex-treated

(5) before

Equilibrium Tris

HCI

buffer

Calcium to 4.7

dialysis

et al.

7.5

was added as CaCl2 Protein

x 10-4M.

constant

for

each

over

calcium

(0.1

diluted

ml)

were

10 minutes

removed,

on an LS

Further and for

233

aliquots

to confirm

the

out

by atomic

nitrous

oxide/acetylene

Radiochemical

Centre

calcium

taken

as described

for

lnstagel

protein

out

in

with

distilled

The

prepared

the

range 7-15

in 5 mM

Donnan

effects.

of 3.5

mg/ml

x 1(r6

but was

dialysis

buffer

24 hours

after

which

(Packard)

and counted

samples for

(Beckman). concentration

in the

determinations

presence

of SDS

Calcium

samples.

spectrometry

(Varian

45Ca

by

in duplicate

Chelex-treated

out

10 ml

flame.

out

prepared

10-7M).

to overcome

curve. carried

of the dialysed absorption

carried

rinsed

in the range

counter

for

added of Dl

the Sephadex

were

concentration

was

gel electrophoresis

integrity

carried

the

scintillation

were

polyacrylamide

was only

was carried

M NaCl

with

from

and thoroughly

0.5

was

was similarly of calcium

the same buffer.

solutions

out essentially

concentration Dialysis

Final

cm) in

use.

concentration

was used as a control.

(10) All

Dialysis

containing

elution

concentration

was carried

(5) but at 4OC. pH

calcium

EGTA

Dl

against

dialysis.

E.

x 2.5

52

buffer,

E was a by-product

After

dialysed

solutions

Marguerie

Trasylol.

Fragment

affinity

on a DE

to remove (100

in the presence

column.

and deionised were

units/ml

containing

to remove

of cross-

4B-lysine

10 mM phosphate

column

were

equilibrium

in

G200

had started.

52 cellulose

a Sepharose

gradient

plasmin

by ammonium digestion

chromatographed

2 Kallikrein

pH

digestion

by plasmin

dialysis,

M NaCl

digested

the protein

column,

after

on a Sephadex

M NaCi,

after

bank plasma

was passed through

(Bayer),

out

fibrinogen

minutes

aged blood

(9) and,

Trasylol

METHODS

was prepared

an 0 - 0.5

using

was carried

prepared a few

digest

plasmin

containing

purification

G200

The

colu*nn

from

D-dimer

(8).

to remove

cellulose pH

was prepared

fibrinogen

AND

CaCl2

(11)

(SDS-PAGE)

determinations

Techtron

AA5)

was obtained

were

using

from

a

the

(Amershom). RESULTS

Fragments criterion that

human,

These high

sites 3 sites

constant Marguerie

bovine

(5),

were

specific

for

were

was 8.7 et al.

Similar

and

E were

purified

fibrinogen calcium

of magnesium

to homogeneity

Calcium has 3 high

as calcium

ions

(Fig.

1).

by the

binding

studies

demonstrated

affinity

binding

sites

binding

was not affected

Scatchard

analysis

(Fig.

I).

by

showed

that

equiv lent, since a linear plot was achieved. The dissociation -8 x 10 M (Fig. 2) compared with 2 x 10-6M reported by at pH 7.5

studies

had one (fig.

binding

D-dimer

band on SDS-PAGE.

like

concentrations

these

Dl

Dl,

of a single

1).

was unaffected

showed These

(5). that high

D-dimer

had 2 highaffinity

affinity

by the presence

sites of

were

also

binding specific

10 mM magnesium

ions.

for

sites

and

calcium The

and

Scatchard

Vol.l3,No.3

plots for sites

cALCIb?l BISDIYG

(Fig. D-dimer were

2)

were

was

linear

9.5

demonstrated

x

and

1r6M on

parallel and

fragment

to

that E

for (Fig.

EC FIBRIXOGEN

The

fibrinogen. Dl

347

was

10.6

dissociation

x

ccnstant

1r6M.

No

binding

1).

FIG. Dependence

of

fibrinogen E on ions

calcium

(Fibg.),

the

present

in

the

weights and

Dl

180,000

and

90,000

Dialysis

was

were

(

and

calcium medium.

for

fibrinogen,

340,000, respectively.

0

10

to Dl

dialysing

carried

of of

of

used

D-dimer

absence

binding

D-dimer,

concentration

Molecular

presence

1

mM

out

in

the

) and

in

the

magnesium

ions

( 0 ).

FIG. Scatchord

analysis

dependence per free for

mole

of on

calcium fibrinogen

of

the ions.

Data

(upper), Dl

calcium

bound/mole ion

ions

bound

concentration

and

calcium

the

calcium

(middle) free

2

is

of shown

D-dimer

(lower).

r

protein concentration.

-

moles c

-

148

CALCIPI

BIh-DI?;CL FIBRIINOGEX

DISCUSSION The

high

exhibited

affinity

parallel

products

are derived

ccnformational obtained

6 seporate This

fibrinogen. of

; our work

constant

temperature

used by Marguer ie.

Dl

and O-dimer Our

(4).

to high

affinity

C terminus the hold

sites

of the

plasmin

fragment

on the

of the

The

abnormal

the

y chain

correction

products

concentration

may play

a more

prateolytic

thrombin time

important

degradation

role

since

activity

as inhibitors

biological

activity

correlates

After

digestion

of fragment

Readdition (IS). restoring its ability

of

Thus

in situ.

structure

in a strained

Plasmin D (7).

Of

molecule, the

C-terminal

wed

3 high

affinity

portion

found

3 binding

sites

on human

mojor fibrinogen

destroyed

by plasmin

solubility the

Marguerie

allows

binding

sites

Although

We

The

product have

rise

fragment

the

fragments the

to occur. of fragment

in the fibrinogen

we purified

isolated

D without

D may hold

do not

involve

fibrinogen

of the a-chain E,

(14).

attached

the digested reaction

little

monomer still

to 2 moles

remaining

from

Their

to fibrin

present

ionised

site

(13).

and therefore

fragment

partial

D have

is nevertheless

gelling

gives

fibrinogen.

treatment.

cross-link

digested

the

that

plosma

of the molecule

on fragment

in heterogeneity

degradotion

the

of fragment

though

(4). of

site

(16),

from we s_tilI

is not present

and may well

NDSK

fragment

on

be of

to determine whether the third calcium binding site involves the N of the 3 chains. This work has been unsuccessful to date due to problems with the isolated NDSK. If the remoining site does involve

C-terminal

towards

site

in the D domains

resulting

the other fibrinogen terminals

binding

that

calcium

to proceed

to note

to bind

even

of fibrinogen

located

the

of insteod

C terminus

D region

peptide

the partially

of the a-chains.

blood bank blood,

obility

monomer,

calcium

1 mole

2 of these are

in the

sites

the

that

conformation.

by increasing

products

their

stabilises

conformation

digestion the

the

insusceptible

protecting

cross-link

to fibrin

site

These

monomer polymerisation

with

the

calcium

to bind

are still

well

cross-link

It is interesting

merely

of fibrin

Dl

the (4).

of the

sites

involve

shown

has an elongation

the plasminolytic

biological

ions

by calcium of calcium

we have

y chain

binding than

sites

of the

time.

to room

is conferred

since

contain

can be achieved

The

(12).

as opposed

do not

digestion

I which

in the or the

These

in a plasmin

plasmin

Paris

has a prolonged

do not

difference

binding

by calcium

y chain

allows

fibrinogen

which

but differ

of temperature

digestion

products.

of

bovine

is due to direct

of cross-linking

stabilised

of the

calcium

plasmin

stability

degradation

themselves

of the thrombin

calcium

this

- the site

y chain

C terminus

Removal

that

as a result

one another

to a species

out at 4’C

Dl

constants

(5) for

and to the effect

to further

show

degradation

D have

the

stability

results

than

with

et al.

and

on the degradation

dissociation

in ogreement

was carried

sites

rather

The

can be attributed

D-dimer

the

molecule

by Marguerie

fibrinogen

dissociation

ions

are

reported

ions for

that

digestion.

experiments

difference

calcium

on fibrinogen,

suggests

the parent

plasmin

those

sites

This

from

during

of 4 from

binding

plots.

directly

change

from

by a factor affinity

calcium

Scatchard

portion

the same site.

of the a-chains,

This is not and Stuhrmann (17 ).

then

both a-chains

incompatible

The extended y chain digestion by plasmin extracellularly on account of the plasma calcium

with

the

must “bent

contribute banana”

is of course unable concentration.

model

to occur

of

Vol.13,Xo.3

CALCIUM

BIXDIKG

& FIBRINOGEX

349

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