The fibrinogenolytic pathway of fibrinogen catabolism: A reply

THRO>IBOSIS Printed

RESEARCH in the

LETTER

THE

States

TO

FIBRINOGENOLYTIC

THE

COLLEN,

N.

Laboratory

of

University

In it

has

logi

a

letter

been

co I

over”

and

for

this

pathway This

20

similar (3)

%

of those

; b) the 25

rivatives the

high

In

fibrinogen

which

may

lonamino

fied

et

caproic

Blomback material

and

is

balance

Murano

al acid

was

(EACA)

reduced

25

185,

under

1973) physio-

pathway” %

suggests

of

no

of

the less

not

the while

ethanol

turna

of in

such

role

to

and

may

not

when the

yield that

in

of of 491

intact

for

made

: a)

dethat

digestion b)

operates

that at

the

coa-

similar

valid.

glycine

degraded

occur

fibrinogen

and

rabbits

be

fibrinogen

to

proteolytic

precipitation

humans

fibrinogen

seems

were

partial

Ao(chains,

solubility

solubility

assumptions

ethanol

in

low

human

posesses

degraded

rabbits

high

: a)

precipitation,

digestion

due

that,

observations

containing

two

in

found

Blomback,

that

for

(2),

of

assumptions

(5)

2,

catabolic

following

fibrinogen

during

these

maior

humans

by

plasmic

reasoning,

occurred

However,

the

plasma

formation

heterogeneity have

Research

accounts for

23.1.1974.

evidence

a

rabbits

on

labeled

the

this

is

material by

of

gulation-fibrinolysis rates.

fresh

solubility

through

(4).

based

from

turnover %

(Thromb.

“conclusive

in

form Copley)

(1).

was

obtained

VERSTRAETE

Coagulation,

in revised Editor A.L.

information

man”

p re p ared

to

about

in

conclusion

fibrinogen, over

“available

M.

Belgium.

is

pathway

:

Leuven,

fibrinogenolysis

“this

that

there

CATABOLlsM

Department,

editors-in-chief

that

circumstances, that

of

L.euven,

the

claimed

fibrinogen,

Blood

10.12.1973; Accepted by

to

FIBRINOGEN

and

Research

3000

(Received

OF

REPLY.

SEMERARO

Medical

1974 Inc.

EDITORS-IN-CHIEF

PATHWAY A

D.

4, pp. 491-496, Pergamon Press,

vol. Vnir;ed

was

replaced

purification Aa(chains A
by method

in was

the

epsiof puri-

increased.

492

ON THE FIBRINOGENOLYTIC

Since

the

degradation

assumed trace

that

fibrinogen

quantities

completely

of

90,000

during

capable

to

convert

lysine of

is

low

much

p lasminogen particularly ring

more

susceptible

(10).

It

The of

data

But

derivatives. nificant The

finding

(11)

or

slower

gulation

as

a

studies

fibrinopeptide

A

the

fibrinogen

lease. than olysis

If in

cal nogen

pathway during

indicate

et that

in

could

also

be

before of the

is

possible much

purification

plasmino-

are

does

occur

du-

with

possibility

normal

of in

of

ar-

in

man

likely

higher

this

factor to

on

at that

a-

solubility

species

a

infusion

in

man

deficiencies

exclude in

in

(12)

vivo

man.

coa-

Further-

radioimmunoassays only

2-3

of

percent

fibrinopeptide a

much in

sig-

coagulation.

heparin

through

operates

significant

intravascular

conditions

mediated

a

fibrinogen

based

even

the

A

slower

vivo

of re-

rate

fibrinogen-

slower.

accepting

fibrinogen

of

be

and

in

coprecipitates

that

tends

(13)

are

plasminogen

rabbits

coagulation

al

been

native

the

during

pathway

coagulation it

by

conditions,

Nossel

of

the

which

in

revealed

turnover

metabolic of

vivo

rabbits

Thus,

normal

in

III

NH2-termi-

than

formation

consumed

congenital

turnover

in

may

in

maior

the

more,

with

than

is

fibrinogen

patients

that

through also

to

of

obvious.

indicate

study

approxi-

enhanced

plasmin

procedure,

catabolized

of

plasmin

lysine

plasminogen

becomes

case

have

of

acid

degradation, some

(4)

amounts

bonds

the

weight

must

markedly

to

not

fraction

molecular

NH2-terminal

some

fractionation

fibrinogen

the

is

in

Cohn

which

Trace

activation that

7).

a

to

may

weight

glutamic

and

proteolytic

same

of

that

in

conversion

EACA,

Sherman

the

amount

this

alone

occur

(6,

with

exposed

fibrinogen.

molecular acid

8).

-terminal

to

to

is

a

to

lysine,

NH2

degradation

fibrinogen

has

(6,

appears

the

of

seems

authors

be

of

plasminogen

Because

fibrinogen

not

thus

throughout

tefactual

mount

of

fractionation.

fibrinogen

is

amounts

constantly

degradation

NH2-terminal

(9),

abolished,the

EACA

glutamic

of

purified

susceptible

Cohn

-terminal

Vo1.4,No.3

However,

protection

fractionation

plasminogen

presence

fibrinolytic

and

the

may

enzymes.

amounts

82,000

generated

completely

plasminogen

NH2

significant

not

circulation

incomplete

and

approximately

the

vitro

Circulating

contains

gen

in

such

plasminogen.

nal

in

was

proteolytic

prevent

Actually

mately

process

PATHWAY

that

fibrinogenolysis

catabolism procedure

in

man,

is

a

proteolysis

employed

by

maior of Mosesson

physiologihuman et

fibrial.

03 THE FIBRISOGEXOLYTIC

vo1.4,?io.3

should and

be low

excluded

and/or

molecular

the

precursor-product

weight

fibrinogen

we

some

433

PATHWAY

relationship

should

also

be

between

demonstrated

in

high vivo

in

of

hu-

man. TO

this

man

fibrinogen

(14).

Human

end

isolated plasma

brinogen-substituted degradation X,

Y

fered

fibrinogen lulose

eluted was

protein

derivatives ded

Ati

the

second

is

(16)

of chains (BB

the

urea-O.025

alkylated (fig

&

in BB

chain)

and

Ao( third

and

thrombin-modified

a

MW

were the

M

acetate

its

chains

and

of

fiplasmic

100,000

washed

fibrinogen buffer

out

with

was

virtually

pH

separated

(fragments

5.0. by

buf-

The

CM-cel-

I).

three peaks, en A-LK chains

(designated

with

and

chromatography

fibrinogen

proteins EACA

composition

affinity

of

retain D

chain

by

column

columns

chromatography

eluted

a

fragment

8 M

and

chromatography

through

and

the

plasma

Not-adsorbed

with

reduced

on

frozen

Such

Trasylol

: CM-cellulose

Figure The

(15).

containing

quantitatively

passed

containing

D”)

experiments

fresh

agarose.

“big

saline

from was

products

and

did

+ (16)

of reduced corresponding of )

(Aachain)

are

and alkylated fibrinogen. to the carboxymethylated

fibrinogen, eluted peaks.

as in

the

indicated. valley

Degrabetween

ON THE FIBRINOGENOLlTIC

The 6

chromatography cm

column

0.025 tic

M

of

The

linear

gradient

buffer

and

The

M

75

%#

8

the

%

gradient

changed

at

changed

at

M

rate

of

as

the

ml

pH

20

the

%

per

24

8

M

hr as

hr

of

due

ace-

with

a

The

gra-

analysis

time. of

the

eluate

principle,

to

urea-

starting

the

(in

x

with

transmission

constant

of

cm2

4.3

4.3

transmission

kept

1

buffer.

using

when

rate

8

a

to

limiting

device,

was

reduced

lowered

5.2

When

to with

was

acetate

linearly

nm.

a

flow

pH

composition

linearly

a

app lied

equilibrated

pH

ultrograd

280

samples

urea-O.025

LKB

at

the

acetate

was

75

mg

CM-52)

which

M

M

with

the

of

of

urea-O.025

above

50

developed

composition

was

actually

was

obtained

on

(Whatman

buffer

composed

gradient

below

CM-cellulose

column

8

was

eluate

performed

sodiumacetate

acid.

dient

was

PATH-b-_&y

was but

the

electric

BB

and

circuiting). This chains, re

procedure

and

in

degraded

than

90

and

Ao(

between

a

very

minor

fraction

Thus

our

significant

should

without

(less

of

from

fresh

processed

gen

degradation

at

Further

as

a

major

experiments

brinogen, as

summary

well

as

of

sustain

(17)

third

the

half

a

that

the

fibrinogen

corresponded

in

has dozen

for

conditions A<

samples

was

by

We

presently

investigating

reproduaffinity

healthy

conditions

chains.

are

prepared from

protective

the

found

only

degraded

obtained

more

to

were

normal

plasma, are

better

different

accounted

that

fibrinogen on

whe-

peak.

view

far

the

Ao(

region

was

Ao(chains

they

Ao(

the

of

peaks

but

the

in

recovery

degraded

thus

frozen

material

protein

chains

the

blood

do-

whether

shows

any

fibrino-

all. our

contention

catabolic

along

metabolic

and

circulating

under

on

possibly

%)

precautions.

plasma,

genolysis

the

stressed

fresh

In

not

of

electrophoresis

Ao(

5

of

The

Some

and

than

do

be

special

amount

second

Bt3

separation

(16).

first,

obtained

chromatography nors

small

respectively.

results

chromatograms

clear

gel

the

amount

It

a

a

present

the

chains

region

cible.

only

are

that

the

The

in

SDS-polyacrylamide

in

a

of

chains

revealed Bi3

)51

elution

Ao(

%.

fractions

resulted

the

is

that

the

pathway

in

man

molecular

the

studies

lines

hypothesis still

composition described

similar

to

in

those

of

remains

in to

of ------_-circulating present

letter

performed

by

vivo be

fibrinoproven.

_ human are

Sherman

fi-

required in

rab-

bits. It degradation

was

proposed

products

of

to the

the

Subcommittee

International

on

Committee

fibrinogen of

and

Thrombosis

fibrir+gen) and

Hae-

ON THE

Vo1.4,No.3

mostasis forms

with

represent

degraded

(low

to

Ad

circulating

solubility such

1973)

(Vienna,

include

chains,

would

PATHWAY

the

definition

the

assumption

protein.

weight) be

in

under

coagulable

molecular

a decision

FIBRINOGENOLYTIC

Since

fibrinogens

it

may

495

of

fibrinogen

that

these

is possible be

also

the

would

that

preparation

also

these

high

artefacts

premature.

ACKNOWLEDGEMENTS _-~--That by

a better

CM-cellulose

could The been

of

of

chromatography

be obtained details

separation

the

was

with

mentioned

procedure,

S-Carboxymethylated the

to one

as worked

use of of out

the

us (D.C by

fibrinogen

Dr.

LKB .)

chains

ultrograd

by

Dr.

Murano

device

G. have

Murano. not

yet

published.

REFERENCES

1.

MOSESSON, M.W. The fibrinogenolytic holism. Thromb. Res., 2, 185, 1973.

2.

MOSESSON, M.W. of human fibrinogen T966.

3.

MOSESSO N, M.W., FINLAYSO N, J.S., UMFLEET, R.A. and GALANAKIS, D. Human fibrinogen heterogeneities. I. Structural and related studies of plasma fibrinogens which are high solubility catabolic intermediates. J. Biol. Chem. 247, 5210, 1972.

4.

SHERMAN, L.A. ways of catabolism.

5.

and SHERRY, of relatively

Fibrinogen J. Lab.

MURANO, G., WIMAN, : some characteristics Thromb. Res. 1, 161,1972.

9en

S. high

pathway

of

fibrinogen

The preparation and solubility. Biochem.

turnover : demonstration Clin. Med. 79, 710,

B. and BLOMBACK, of its S-carboxymethyl

cata-

properties 5, 2829,

of multiple 1972.

B. Human derivative

path

fibrinochains.

6.

WALLEN, P. and WIMAN, 8. Characterization of human plasminoI. On the relationship between different molecular forms of gen. plasminogen demonstrated in plasma and found in purified preparations. Biochim. Biophys. Acta 221, 20, 1970.

7.

COLLEN, D., ONG, E.B. and JOHNSON, plasminogen. Fed. Proc. 31, 229, 1972.

8.

WALLEN, P. and WIMAN, B. gen. II. Separation and partial lar forms of human plasminbgen. 1972.

A. J.

Native

Characterization of human characterization of different Biochim. Biophys. Acta,

human

plasminomolecu257, 122,

496

ON THE FIBRINOGENOLYTIC

H., MOLLA, glutamic acid Thromb. Res.

Vo1.4,No.3

PATHWAY

9.

CLAEYS, terminal plasmin.

A. and VERSTRAETE, to NH2 -terminal iysine (in press).

TO.

CLAEYS, properties Submitted

IT.

COLLEN, tabolism siological

12.

TYTGAT, G., COLLEN, D. and VERMYLEN, J. Metabolism and distribution of fibrinogen. II. Fibrinogen turnover studies in polycythae mia, congenital afibrinogenaemia and thrombocytosis, haemophi lia A, during streptokinase therapy. Brit. J. Haemat. 22, 701, 1972.

13.

NOSSEL, H.C., CANFIELD, R.E. and BUTLER, Jr., V.P. fibrinopeptide A concentration as on index of intravascular tion. IV Cong. Int. Sot. Thrombosis Haemostasis, Vienna, stract volume, p .237.

T4.

HEENE, tives on 2, 137,

15.

REUTERBY, J. and BLOMBACK, B. Adsorption of KYDRYK, D., plasmic fragment D to thrombin-modified fibrinogen-Sepharose. Thromb. Res. 2, 297, 1973.

16.

MURANO, G., WIMAN, B., BLOMBACK, M. B. Preparation and isolation of the S-carboxymethyl of human fibrinogen. FEBS Letters 14, 37, 1971.

17.

McDONAGH, J., MESSEL, and BLOMBACK, NO, G. gen and fibrin chains by an trophoresis method. Biochim.

H. and VERMYLEN, of human plasminogen. for publication. D., TMGAT, and distribution conditions in

and D.L. insulubilized 1973.

J.

Physicochemical and Effect of epsilonamino

G., CLAEYS, of fibrinogen. humans. Brit.

MATTHIAS, fibrinogen

M. Conversion of NH2human plasminogen by

F.R. and

proenzyme caproic acid.

H. and PIESSENS, R. MeI. Fibrinogen turnover in phyJ. Haemat. 22, 681, 1972.

Adsorption of fibrinogen fibrin monomer. Thromb.

and

Plasma coagula1973, Ab-

derivaRes.

BLOMBACK, derivative chains

H., McDONAGH, R.P., Jr., MURAB. Molecular weight analysis of fibrinoimproved sodium dodecyl sulfate gel elecBiophys. Acta, 257, 135, 1972.