Degradation products and heterogeneity of bovine fibrinogen

vol. 4, pp. 89-101, 1974

RESEARCH

THROMBOSIS Printed

in the United

DEGRADATION

PRODUCTS OF

Paturel,

L. Laboratoire

G.

05

B.P.

AND

BOVINE

FIBRINOGEN

and

D.R.F.,

Centre

de

Press,

HETEROGENEITY

Hudry-Clergeon

d'Hematologie,

Grenoble,

Pergamon

States

M.

Suscillon

Centre d'Etudes Nucleaires 38041 Grenoble Cedex, France.

Tri,

de

(Received 6.6.1973; in revised form 3.9.1973. Accepted by Editor M.J; Larrieu. Receive'd Executive Editorial Office 19.11.1973) ABSTRACT Plasmin a

proteolysis

function

and

of

of

time

by

DEAE-cellulose

shows

two

found

to

be

DEAE-cellulose bovine

different

gave

ratios. to

The

chromatography

fibrinogen the

This y

studied

to

Dl

early

and

0

product

of

was obtained

gradient1 D2

would,

heterogeneity

as

electrophoresis

(Tris-CaC12

rise phenomenon

chain

was gel

The E product and 02. The three fractions

01

heterogeneous.

by

fibrinogen

chromatography.

sub-fractions

of

related

bovine

polyacrylamide

products

in

therefore,

be

fibrinogen.

INTRODUCTION The polypeptidic several been

chains disulfide

observed

plasmin by

WARDER

et in

in

have

phoresis

in

bridges.

(31

(61. (X,

been sodium The

Y1

of

composed

and

21.

and

sequence

al.

B@

(1.

bovine

is

Ao.

bridges

early

products

molecule called

degradation

sified

fide

fibrinogen

A

y Y

human

of

which

are

chain (4,

51

formed

fragments

and

late

(0.

studied

(8,

dodecyl

sulphate

results

are

91

in

by

El

linked

been

products

accordance

has

have [6.

cleavage with

The

determined

polyacrylamide after

of

by

fibrinogen.

has

main

pairs

heterogeneity

fibrinogen

The

three

been 7).

These

gel

electro-

of the

clas-

the schema

disul-

Inc.

PO

DEGRADATION

proposed

by

further

MARDER.

degradation

final

0

chain

portions

very

and

E

it

is

observed

ral

up

to

0

phy

ten

of

product

tions this

in

to

the

This

KAY

Plasmin 3

:

the

CU-Sgouris/mg

Iniprol

:

CHOAY

mg

buffer,

pH

8.5.

is

carried

volume various by

out

of

the

addition

from

is

10, of

six

11,

this

12, phe-

to

the

further

showed

organizes

specify

the

early

a seve-

aspects

of

degradation

DEAE-cellulose that

also

into

certain

studied

we

chromatogra-

each

of

these

Cl products;

frac-

the

origin

AN0

METHODS blood

protein KABI

protein

(1

in

according

is

98%

CU-Sgouris

Q

KECKWICK

and

clottable.

glycerol

50%

to

1

containing

about

CTA).

Laboratory

of

prepared

:

fibrinogen

at

2O'C

10 in

a

Iniprol.

g/l

in

sodium

dialysis

mentioned

digestion

by

dissolved

containing

of of

a

showed

calf

plasmin

above

times

and

discussed.

products

per

The

product

(8.

a

chains.

methods.

which

we

we

as

obtained

Fibrinogenolysis plasmin

0

cleaved,

three

origin

we

to

from

and

was

human

and

tends

issued

prepared

1141;

The

the

Y

in

authors

product

MATERIAL

Mac

several

be

the

chromatographic

E

work

differently

:

to

composed

Furthermore.

phenomenon

Fibrinogen

then

sub-fractions;

fibrinogen

behaves

chains,

heterogeneity

fractions

bovine

first

bridges.

and

heterogeneities. three

the

Vol.4,No.l

discussed.

heterogeneity

the

08

disulfide

according

sub-fractions.

these

by

are

respectively

electrophoretic

the

similar

chains the

are

linked

still

By

of

products

involves

nomenon

Aa

affects

heterogeneous;

131

of

The

OF BOVINE FIBRINOGEN

bag

buffer. and

proteolysis

adding a

0,015

Tris-HCl

The

immerged

is

in are

of

0,05

chloride.

Samples

CTA

a

M reaction

large

withdrawn

immediately

at halted

Vol.4,No.

DEGRADATION

1

Polyacrylamide system

gel

of

trating

:

Resolving pH

32,

7,2;

FIBRINOGEN

gel

:

by

a

discontinuous

3.75

to

9%.

pH

10 to

40

ug;

amido-Schwartz

:

sample

91

performed

:

electrophoresis

buffers.

gel

OF BOVINE

6.9;

concen-

staining. DEAE-cellulose dialysed cmL

1

chromatography

against

the

initial

by

following

a

The device

gradient

-

is

of

250

two are

ml

used

of

protein1

applied

on

equilibrated

performed

0.04M;

: Tris-HC1

by

a

parallel

according

a

with

cm

system The

bottles. the

30 the

gradient

to

are

two

sample

to

be

pH

7,5

---)

Tris-HCl

0,04M;

CaC12

7,5

fibrinogenolysis

Hcl

then

and

mg

:

fibrinogen pH

(80

previously

elution

types

chromatographied

0.06M;

buffer

column

buffer.

realized

-

initial

OEAE-cellulose

samples

:

0,lM;

products

CaC12

O.lM;

: Tris-HCl

0,04M;

PH

6,2

----)

Tris-

Pi-l 5.5.

RESULTS l-

Lysis

study

trophoresis

as

and

Electrophoresis We different allow

of

SMITH

(151;

further the

unseparated the

different

the were

permitted the

weights

of

the

lysis steps

the

X

and

gels and

0

of

to met

are are Y

of

the

lysis

elec-

in shown

described transient

HEORICK

product

Fig. in

Some 1

(9%

Fig.

2.

species.

7,5%

and

identifica-

litterature. in

of

molecular

to

the

gels

above

The

according

confirm

in

concentration

products.

estimated us

to

of

Y

gel

products steps

Only of

intermediary

appearance

polyacrylamide

lysis

this

steps

by

of

products

according

time.

chromatography.

observed

the

of

OEAE-cellulose

separation

weight

teristic

function

concentrations. the

tion

a

The

charac-

gels1 We X

and notice product

x

92

DEGRADATION

is

divided

into

dualized

in

a

6%

(Fig.

Y

products;

gel .'

a

9%

formed

tity.

The

A

weak

product.

0

the

product

appears

groups,

Dl

and

The

estimated

weights

of

these

100

groups

are

furthermore

number

and

the

lysis

stage;

ly

000

75

fragment

their

new

more

anodic

The

molecular

47

000

the

gives

an

0,

the of

of

degradation corresponds

is

which, as

the on

a

the

lysis

time.

well

is to

during

clotting

time

shown

the

as

in

greatest

Fig.

from The shown the of

38;

are

and

1

0

on

the

approximatethis

organizes

into The

lysis less 52

E

time,

anodic 000

ones.

to

densitometric in

Fig.

313,

plasma

T'he in

presence

maximum

concentration

of

inhibithe

Y

product. Electrophoresis The

of

the

chromatographic

products

after profile

chromatographic obtained

at

2

species,the

lysis.

the

bands.

later;

the of

loss

ratios

HEORICK

stronger. of

vary

gels,

to D

quanunder

two

is

then

are

species

thrombin

products

band,

the

in

of

slightly

E

fragment

the

formed

function by

progres-

depending weight

and

appro-

lysis

moving

which

four

polyacrylamide the

the

The

molecular

appearing of

of

faster

of

are

decrease

approximately. into

is

X

deriving

E

according

diffused

develops

depending

prolongation

tion

among

weights

idea

a

the

in

the

species

composed

fragments

product

HEDRICK.

Y

then

one

0

indivi-

between

beginning

each

product as

species

of

02,

final E

firstly

daltons

integration

of

The

sub-fractions, like

the

intensities

average

a

fully

the

products

transformed

daltons.

product,

represent

daltons

relative

appears

several

000

to

to

product,

at

different

identical,

0

is

observed

intermediate

two

quite

be

Vol.4,No.l

product

according

may

The

whereas

can

weight, it

Y

superimposes

band

daltons,

X

the

it

molecular

000

early

sively

whereas

11.

190

an

sub-fractions,

gel

its

ximately from

two

OF BOVINE FIBRINOGEN

separation an

early

time

DEGRADATION

Vol.4,No.l

OF BOVINE

FIBRINOGEN

93 -.,, (I

:

Oh

UKlh

13h

2h

0,5h

* FIG. Polyacrylamide the

gel

plasmin

allow

the the

gel,

Y

the

case, 40 6%

full E

of

separation

and

prod?lct 2,s

I.lg; pH 6.9; gel, 2h.

of

has

showing

bovine

products

0

1

electrophoresis

degradation

few

steps

fibrinogen.

all

are

products.

In

superimposed,

migrated

mA/gel;

out

migration

of

9%

the

right

[in the

gels

this

gel).

:

time

of

The

6% last

Sample

9% gel,

3

: h,

i

F

h

0

0,s

1

1.5

2

3

5

5

13

24

72

100.

t Iysit’hoUrS)

FIG. Diagram Fig.

representing 1

and

some

the

other

9%

," % . .z

2 polyacrylamide

intermediary

steps

of

gels

the

shown

lysis.

in

94

DEGRADATION

OF BOVINE

FIBRINOGEN

Vol.4,No.l

Integration of gels

. . 150

.

100

A

50

t\k____’ t

0 set 40

:,____

20 12

3

4

5

8

12

18

20

24 hours

FIG.3 A- The relative amounts of the products as a function of the lysis time: the ordinate values [arbitrary units1 are obtained by density scanning of the stained polyacrylamide gels represents a The so-called Y' product shown in Fig.-2. band of very weak intensity situated between the X and Y products. B- The anticlotting activity of products formed at various This activity is tested by measuring times of proteolysis. the thrombin clotting time of a plasma- degradation products mixture. of digestion different

(30 minl and the electrophoretic

fractions

eluted without

appearance

are shown in Fig. 4A and 48.

gradient

at the beginning

analysis

represent

of the lysis.

The first peaks

the small fragments

Further,

of the

released

we note the successive

of the 01, X, Y, O2 and E products.

Only the Ol and

Vol.4,No.l

DEGRADATION

OF BOVINE FIBRINOGEN

50

95

fracth.nb.

-..,?

.

t

10

20

30

FIG. A-

B-

C-

40

50

fraction

nb.

4

chromatography (Tris-CaCl gradient) of lysed for 30 min. The zrrow indicates the start of the gradient. Polyacrylamide gel electrophoresis (9% gels1 of the 30 min lysate (total) and of the chromatographic fractions Cl-51 pointed out in Fig. 4A. DEAE-cellulose chromatography of the lysates obtained for lysis periods of 1 h 30. 4 h and 72 h. DEAE-cellulose bovine fibrinogen

_. .

96

DEGRADATION

02

products

are

well

OF BOVINE FIBRINOGEN

the

located,

other

three

wide

ctiromatogram zone. The E product _. We notice the important charge difference . For increasing lysis the 02 products. matograms

evoluate

chromatograms gel

and

01 the

02 in

to

initial

the

more

and

Tris-CaC12

The of

three

pooling

the

fractions

results

obtained

with

lysis

rate slowing to

neutralize

inhibitor partly

as

follows:

into

the

mobilities

lysate

chro-

of

these

the

progressive

intensification

of

a decrease’of

of

this

and

polyacrylamide

evidence

and

Dl

analysis by

a

apparent.

the the

furthermore.

initial

which

process,

are the

the near

to

E product

heterogeneity

heterogeneity

chromatography the

the

great

three

the

in

previously for

a and

down

in

traces small made

lots,

Fig.

5A.

c

lot.

are

Fig.

,

was

This part,

amounts

of

plasmin.

polyacrylamide

a

bovine the

We by

58

doses

shows

from

used.

Indeed, there

phenomenon

is can

sufficiently

gels

the

[O,OlS

coming

identical,

this

the

of

obtained

gel

in

in

with

SA shows

plasmin

amount

b lots

the

c

right

this

Fig.

0 product.

importance.

used the

twice

the

in

b and

indicated

the

heterogeneity

decreasing a,

of

of

(performed

represented of

where of

the

on

slightly

periods,

chromatographic

except

experiment,

Observations are

the

as

fibrinogen]

considerable

to

the

reveals

of

present

anodic.

fractions

lysis

still

fractions

put

to

chromatogram

the

attributed

the

are

between

4C;

Y species

DEAE-cellulose

studied

the

shown1

We note,

and

gradient1

fibrinogen.

if

the

between

The

another

of

X and

more

fibrinogen

of

study

Comparatively

Correlation

CTA/mg

Fig.

products,

02.

existence

in

E products.

favour

bovine

shown

(not of

and

becomes 2-

the

electrophoresis

disappearance 9

as

is

Vol.4,No.l

a be

enough

(Fig.

58)

DEGRADATION

Vol.4,No.l

OF BOVINE

00280nm

FIBRINOGEN

97

.(I,

..

10 b

a

30

20 c

-

a

-

F=.-

40

‘-

b;

x G

-

a

c:

G

fraction

i

b’

nb.

c

-I--

“i-1P

Y

.

50

‘b-D1

W

J

Oh

lh FIG.

A-

B-

the

fibrinogen

-

the

X and

and

slightly

we can the b

5h

5

DEAE-cellulose chromatography of bovine fibrinogen gradient). CaCl Polyzcrylamide gel electrophoresis [9% gels) of ducts obtained during the lysis of the a, b and As the lysis rate of the indicated in Fig. SA. is inhibited (see text) the right gel was chosen ther experiment where a higher amount of plasmin (twice the amount generally used).

-

-

^ 5h

increases

Y transient

faster

notice

early gives

mobility

0

in

species

in

to

these

the

D2

compared

The

D1

and

the to

lJ2 groups

are

three

lots

Only D1 an

quite

from

a

identical

the proc lots c lot in anowasused

to

c.

in

a

and

b

c,

products.

rise

slightly

(Tris-

and

the D2

an D1

groups

unfractioned are

also

important

group

is

with

difference present

in

a, while

intensification

fibrinogen present

in

in

(see c,

the

D2

of Fig. group

11. being

98

DEGRADATION

OF BOVINE FIBRINOGEN

Vol.4,No.l

predominant, -

there the

-

are three

the-E to

no

as

lysis

time,

stated

(see

b and

fibrinogen

in

must

a lot

the

quite

seems

to

b,

be

c

respectively. duct

it

the

of

need

to

lots

seem

to

separation

9% gels.

product

We

and

E product lysis

of

by

into

one

already

lysis

of

the

01

manner,

to

the

b lot

trail. but

Ol.

only the

late

group

occur

the

the

obtained account

of

in

01

the

existence

Experiences

it Ol

the

the

into

Oi! proportions

same

the

in

during

Taking

and

lots,

taking

b peaks

discussed. 01

the in

amountis

by

the

to

AN0

seems

that

+ O2 and at

the

0 products

each

of

O2 products

early

derived

0 profrom

the

the put

CONCLUSION

electrophoresis

degradation

recognized

polyacrylamide

according

identical.

all

several

done

of

rise

gel

we moreover

be

purity

the

differences

Polyacrylamide

in

from

therefore,

O2 relative

a and

c

that

resides.

the

b and

gives

be

the

compared

In

DISCUSSION

good

issued

show,

to

the

and

be

that

attributed

considering

lots

Ol

The

purified

of

must

trail

seems

These

level

three

of

not

0 products

peaks

identical.

on

b and

a,

E products

identical

products

does

in

done

early

aspect

probable

are

be

the

21.

chromatography.

a trail

c

the

chromatographic

a very

the

in

evolution

1 and

c degradation

account

b lot

an

Observation

0 product.

the

as

characteristic

fibrinogen

from

well

Fig.

The

a,

differences

lots,

product

the

three

apparent

products well

into

known

evidence

sub-fractions. gel

electrophoresis

at

pH

of

bovine

8.9

gives

fibrinogen

heterogeneity the

of

organization

A kinetic and

study

a

the of

of

OEAE-cellulose

the

0 the

DEGRADATION

Vol.4,No.l

indicates

chromatography, under two

two

sub-groups

groups

are

nearly

different.

Further

fractions.

The

then amount

and

lysis

stage.

by

using

studied lysis to

appropriated

the

lysis

stages,

the

Dl,

gous.

Ill

analogous

+

D

the D

(4,

51

shown

[8)

that

time

into

side

of

ring

the

and

a

C-terminal

chains

are

fibrinogen

to

new

as

a

CaC121

b

and

products,

and

D2

early

c

faster

on

E

products

and In

seems

products are

the

DEAE-cellulose

obtained.

the

and the

on

system

fractions

sub-

band

depend

elution

fractions

are

position,

bands

E

these

charges

and

The

and

of

diffused

fibrinogen

02

D

have (3)

of

reported

we

the

to

early

give

being

rise

analo-

transformed

the

to

into

heterogeneity enough

to

of

of

explain and

of

the

y

the

Dl

chains.

and

the

then

existence

be Two

of 0

2

the

has

the

conside-

chromatographic attributed

a,

sub-groups

to

of b in

a

=

(AU),

[EBl

2

Ylyl

---j

E

+

fibrinogen

b

=

(Aa

[BBl

2

'fly2

---j

E

+

the

2Dl

Ol

+

y

and

products:

fibrinogen

been

lysis

and

types

the

in

C-terminal

results

here,

could

the

it to

on

these

observed

fibrinogen

heterogeneities

according cleavages

basis

heterogeneity bovine

chain

Otherwise.

evoluates owing

On

y

fibrinogen.

product

fractions

degradation

a.

species

D

heterogeneity

0

and

chain.

early

weights

electrical

rise

bovine

the

bovine the

y

the

three

authors

smaller the

their

firstly

products.

Several human

2

appears

molecular

appears

(Tris,

of

Dl

late

of

the

product

sub-fractions.

the

of

each

Then

first

intensity

an

but gives

several

fractioned

0

The

same

product

We

the

02.

the

into the

and

cleavage

E

organizes

that

Dl

99

OF BOVINE FIBRINOGEN

O2

c early

100

DEGRADATION OF BOVINE FIBRINOGEN

fibrinogen The

Dl

and

02

acid

lacks

carbohydrates).

two

the

bands

phenomenon

by

[a612

being

[the It

inside

each

be

of

variations

in

accessibility

early

of

A the

the to

also

and

final

0

electric

resides

of

Dl

the

the

0

existence

sub-groups

and

E

considered

sites

of

and

products;

superimposition

of

their

product

the D2

in

this

of

heterogeneity

order

be

then

202

their

explained

conformational

cleavage

could

be

+

by

portion

to

the

E

chains

chain

attributed

heterogeneities.

y

leaves

e

recognized

the

Y

heterogeneity could

~2~2

only

of

composition

persistant

chain

(Aal

heterogeneity

amino

of

=

products,

the

charges,

c

Vo1.4,No.l

another

induced

nearly

the

same

(161.

REFERENCES

1.

BLONB~CK, in

2.

B.

HENSCHEN.

and

Biophys. 4.

YOSHIKAWA,

134,

:

HENSCHEN,

A.

67.

and

boxymethylated

occurence

6.

1958.

299,

disulfide

: -22,

T.

and

MONTGOMERY,

of

the

y-chains.

y-chain

Large

P. of

human

variants.

M.W..

fibrinogen

heterogeneities.

WARDER, weight

7.

EDMAN,

MOSSESSON. variants.

6.

amino

: -12.

bonds

355,

acids

in

1964.

R.

Bovine

Arch.

Biochem.

scale

preparation

fibrin Biochim.

and

of

S-car-

fibrinogen and the Biophys. Acta: 263.

1972.

351,

5.

of

Kemi.

N-terminal

1969.

chains

of

the

Kemi.

reactivity Arkiv.

Heterogeneity

fibrinogen.

On

Arkiv.

and

fibrin.

C.M.,

L.

fibrin.

Number

A.

GERBECK,

YAMASHINA,

and

fibrinogen 3.

and

fibrinogen

J.

V.J.,

FINLAYSON,

Biol.

Chem.

SHULMAN,

derivatives

I.

Physicochemical

J.

Biol.

of

:

244,

: N.R.

human

and

Chem.

J.S.

2111.

V.,

Les produits plasmine. 1. Inst. Pasteur

de degradation Separation et

PIZZO, effect

SELIGMANN,

100,

S.V., SCHWARTZ, of plasmin on

and UMFLEET. Identification

,

and

377,

the

5223,

R.A. of

Human y-chain

1972.

CAROLL,

W.R.

fibrinogen

immunological

NIJSSENZWEIG,

:

III. 247

High

produced

molecular by

plasmin.

characterization.

1969. M.,

PELMONT,

J.

and

GRABAR.

du fibrinogene humain par proprietes physico-chlmiques.

P. la Ann.

1961.

M.L., HILL, R.L. subunit structure

and

MCKEE, of human

P.A. The fibrino-

Vo1.4,No.l

gen.

DEGRADATION

3.

MILLS,

9.

Biol.

Chem. A

D.A.

fibrinogen

by

:

OF BOVINE

FIBRINOGEN

247,

1972.

636,

molecular

model

plasmin.

Biochim.

for

the

101

proteolysis

Biophys.

of

Acta

: 263,

human 619,

1972. 10.

G.A.

JAMIESON, molecular

12

DUDEK,

.

G.A.,

Jr.. of

Biophys.

KLOCZEWIAK,

Acta

M..

plasmin.

Biochim. A., and

: _’ 30

Proc. GARDLUND, 8.

vivo

B.,

relation

KECKWICK, The

8..

products

"disulfide

15.

proteolysis 44.

HEDRICK,

J.L. and

disc

gel

155,

1968.

T.S.

The

fragment

GRDNOAHL, human

fragments

knots".

KAY.

of

protein

and

SMITH,

estimation

G.A.

products.

and

NANCE,

fractions 671,

Fed.

N.J.

and

BLOMBACK

E

and

D

I. and

Research:

Size

molecular Arch.

PEPPER, Thromb.

N.H.

and

from

human

1 _'

RECORD,

Iso-

their 371,

E3.R.

plasma

with

1955.

A.J. of

electrophoresis.

JAMIESON. 1970.

subunit D.

fibrinogen.

Thrombosis

M.E.,

: -' 60

J.

digestion 197,

by

1970.

fibrinogen

of

of

MC

Biochem.

ration

16.

R.A..

preparation

ether.

Z.S.

macromole-

1972. 14.

high human

LATALLO, of

fibrin

EDGINGTON, of

KOWALSKA-LDTH.

characterization to

and

of 1966.

A.Z.,

214,

:

the

1971.

degradation

and

J. behavior

96,

comparison and

Acta

STRATHERN,

339,

Plasmic

lation

Biophys.

in

154.

:

and

of

digests

BUOZINSKI,

Characterization M. products of fibrinogen

CATANZARO,

Nature

P.J.

fibrinolytic

and KOPEC, cular end

structure

13

GAFFNEY, fraction

Biochim.

fibrinogen. 11

and

weight

and charge weights of

Biochem.

D.S. Diath.

isomer proteins

Biophys.

Heterogeneity Haemorrh.

:

:

sepaby 126,

of fibrinogen Suppl. 39, -