Stabilization of the plasmin digestion products of fibrinogen and fibrin by calcium ions

STABILIZATION OF THE PLASMIN FIBRINOGEN AND FIBRIN

DIGESTION PRODUCTS BY CXLCIU>f IONS

OF

G.G. LINDSEY, G. BROWN and 3. FRANKS* L.R. PURVES, University of Department-of Chemical Pathology, 7925, Observatory, Cape Town Medical School, Republic of South Africa Cape Town, and "Veterans Administration Hospital and Clinical University of Colorado, Research Centre, United States of America. Denver, (Received

11.12.1977.

Xccepted

by

Editor

R.F.

Doolittle)

ABSTRACT The role of calcium in the stabilization of fibrinoD-monogen and fibrin plasmin digestion products, The largest D mer and D-dimer has been studied. are both stable to further fragment, Di", and D-dimer plasmin degradation in the presence of calcium ions. Chelation of calcium leads to further plasmin digestion due to sequential cleavage of peptides from the This stabilization C-terminal end of the y-chain. is due to calcium ions and not to occupation of the All the digestion products of Dcross-link site. dimer were also found to be stabilized by the readdition of calcium ions. INTRODUCTION

The ducts been

digestion

- fragments recognised

described linked geneous scent link

of

D and

lysine site

The

i.e.

fragment

(I).

It was

by

plasmin

be

yields

Heterogeneity

with

many

corresponding

D-dimer,

D can

analogue,

(2).

E

as a problem

(Z-5).

fibrin,

fibrinogen

subspecies

digestion

is always

prepared

by

having

product

homogeneous

proposed

into that

main

fragment

incorporation

dansyl-cadaverine therefore

of

two

the the

D has been

from

and of

pro-

cross-

a homothe

vacant

fluorecross-

*Dl refers to the product of plasmin digestion of fibrinogen the presence of Ca2+. Smaller D fragments are numbered in order of decreasing molecular weight, D2 to D5. 477

to

resistance in

475

CALCIW

further

plasmin

cross-link

D

(6,7)

had

showed

homogeneous in

the

this

fragment could

also

be

further

plasmin

we

the

showed

linking

and

formed

by

from

calcium in

the

mixture:

40 Ploug

units

This

digestion

of

that

a

fibrinogen

ions

with

D formed

in

to a terminal

smaller

fibrinogen

absen-

confirmed

fragment

93,000)

by

of EGTA.

fragment

digestion could

D and

effects

with

also

In

this

be report

D-dimer

due

D

in the

to cross-

ions. AND

from

METHODS fresh

of

5 mg

fibrinogen

human

(Leo)

1 minute

After

plasma

5 mM

by

Aliquots

were

reaction

stopped

an equal

volume

Trasylol

(Bayer),

100 mM

EGTA

Titration Radiometer

and

of

was

plasmin

pH

released

digestion 200

volume

mg

acids

ammonium

(Behring), 20 ml.

2X

the

the

was

of

CaC12,

clot,

7.4,

the

digestion

calcium

out

et

al

in a (IO).

fibrinogen 5 mM

prepara-

calcium.

bovine

0.15M formed

ions

5 mM.

carried

50 units

5 mM

and

Further of

pH

to avoid

concentration

presence

The

(Sigma), Tris,

containing

SDS,

a human

fibrinogen,

at 37'C.

intervals

by Mihalyi

clotting

in

added

at

chelation

- final

amino

by

O.IM

units/ml.

by

as described

prepared

concentrate 7.4,

out

(neutralised)

mixture:

F13

Kallikrein

carried

Autotitrator

D-dimer

Reaction

500

Dl was

was

taken of

out

plasminogen

NaCl,

CaC12

13 activity.

fragment

carried

2 mg

in 0.15M

2 ml.

with

was

fibrinogen,

urokinase

Factor

4 mg

the

between

calcium

the

of (9)

calcium

presence

volume

Tris,

the

D-dimer

to calcium

prepared

Timan

ions.

plasmin

of in

digestion

weight

directly

distinguish

digestion

Reaction

tion

of

fragment

to demonstrate

of

digestion

presence

D formed

of

fractionation.

Plasmin

with

of the

activity

and

chelation

80,000.

of

due

was

(8)

weight

timed

and

those

Fibrinogen

of

occupation

the

al

(molecular

digested the

in

Haverkate

MATERIALS

sulphate

by

fragment

et

be

plasmin

calcium

of EGTA

formed to

that

absence

investigate

presence

D

D could

prepared

in

x-01. 12.X0.3

anticlotting

Lugovskoi

D of molecular

plasmin

the

calcium.

to further of

on

relative

led

of

and

presence

is conferred

fragment

fragment

presence

led

the

that

and

finding

ECTA

out

activity

calcium

ST.~BILIZATION

(2).

carried

enhanced

ce of

degradation

site

Studies

ION

at

thrombin,

NaCl,

0.1M

37'C

after

18

'hours,

plasmin

was

for

at

39 plasminogen

(Sigma),

CaC12

NaCl,

in 0.153

digested

mixture

cellulose IO nY

phosphate

lar-sieved

on any

ted

from

at pH

volume

60

and

to

retitration

to pH

presence

column The

remo

NaCl,

0.

was

were 2%

of

same

separate

buffer, ?;0 by

adding

rapid

the

reaction

fibrinogen

subsequent the

Z-

Trasylol.

clotting

for

nolecu-

added.

and

a

equilibra-

5 n>! with

and as

to was

containing by

in

then

started

removed

SDS

prepared

the

was was

dansyl-cadaverine were

e E and

:f Tris

plasnin

The

on a DE 52

gradient

15 mg*

concentration

of

”

5 n?!

20 ml.

x 2 5 cm)

reaction

Aliquots

mM

dialysis,

(100

The

volume

Conditions

volume

to

preactivated

7.4.

7.4,

D-diner;

0.153

a final

2.45

(II)

with

1.1

(Leo),

after

NaCl

digested mixture:

uroXinase

a 0 - 0.5X

8.6

4B

D-dimer

of

digestion.

pH

pg

an equal

Fluorescent

units pH

using

occurred,

EGTA

the

colunn

in 5 millCaC12,

neutralised

with

Ploug

and

reaction

Digestion

Tris,

D-monomer.

7.4

extensively

chromatographed

buffer,

digestion

stopped

500

a Sepharose

1.5 ml

further

37'C.

0.1X

was

(Khatman)

diner

washed

homogenised,

18 hours

in

plasmin

non-fluorescent

D-dimer. Polyacrylamide SDS

(SDS-PAGE)

(gradient Blue

5-20%)

(Sigma).

state

with

gel

was

(12) The

and

of

optical

density.

fibrinogen

the method

out

on

the

presence

stained

bands

were

quantitated

flat-bed

was

Two-dimensional (13)

with

with

The

scanner.

regarded

SDS-PAGE

of 0.1%

slab

were

or D-dimer

of O'Farrel

in

density-gradient

thegals

protein

a Vitratron

tration

electrophoresis

carried

Coomassie in

the

initial as

was

wet

concen-

100%

of

carried

f3-mercaptoethanol

gels

in

total out

the

by

second

dimension. Analytical model were pH

ultracentrifugation

E ultracentrifuge centrifuged

7.4,

5 m>l calcium

homogeneous standards

D-dimer of

equipped

in buffers and and

was with

containing 50

U/ml

Dl-monomer

carried

UV

out

The

optics.

0.153

on

a Beckman samples

O.Ol?l Tris,

NaC1,

Electrophoretically

Trasylol. preparations

were

used

as

comparison.

RESULTS In the gen

yielded

presence

of 5 m?l Ca

a homogeneous

2+

fragment

plasnin Dl

even

digestion after

of

fibrino-

prolonged

perio-

ds of were

formed We

after

showed

min-mediated occurred moles et

by

uptake

of NaOH

fibrinogen fragment

consumed/mole

that

no

D-subspecies

that

Dl

no

the

further

plas-

digestion

E was

complete

in

agreement

with

with

excess

fibrinogen

and

to monitor

at 40 Mihalyi

(10). of

degradation to D4,

plasmin

action

calcium

occur lysis

chelators

result

of

those

in turn

in molecular

the

cleavage

due

from

were

(Fig. thus

2)

from

weight

of

of peptides

D dimer

that

DI.

these D3 We

fragments,

of

with and

other

BioRad

cleavages

arose

from

confirmed Dl

in

digestion.

obtained

fragments from

resulted

modification

phosphate

fragment

arose

EGTA

of smaller

to EGTA

citrate,

seen

order,

derived

results

EDTA,

be

to a series

not

similar

viz.

of D2 which

ions Dl

Dl was

It can

in a specific

decrease

to

of

since

100.

calcium

fragment

identical

digestion

Chelex

the

of

Further

the

of of

1) showed

digestion.

measuring

release

Chelation

02

(Fig.

48-hours

digestion

once

NaOH

al

the

SDS-PAGE

digestion.

hydrothat

to D4 was

the y-chain

FIG.

(see

a

later).

1

Fibrinogen SDS-PAGE of plasmin digestion of fibrinogen the presence of 5mN calcium ions

X Y

DI

Plasmin

E

Peptides

0

15

30

MINUTES

I

2

4 HOURS

48

in

X diner

result

similar further

SDS-PAGE smaller

after

(Fig.

4)

fragments,

(Fig.

3)

calcium: showed 01

was

ions that

to

obtained

had

by

.?igesting

been

chelated

5::

excess

xas

digested

to

a

D-dimer

HOURS

MINUTES

6.

mc

UM

EC

C.

I I

\

MINUTES

series

EGT.I. of

5.

FIG.

CAL

D-

Total D/ _/’ /.?

2

The effect of calciun on the digestion of 5 UM fibrinogen. Calcium was added one minute after plasmin in (_A) and (B) and 5 mhl EGTA in (C). The effect of calcium ion renoval on a comoleted digest is shown by EGTh addition after 60 minutes in Legend : (A). - fibrinogen, Fibg 1 ,2,3,k - various D Y and E specjes, digestion products.

vo1.12,50.3

EC

A

B.

C

FIG.

\

l\

Digestion of D-dimer in the presence of 5 mM EGTA (A) and its inhibition by the readdition of calcium ions (B). Note that the fragments D2 and D3 are also stabilized after calcium addition as well as the remaining D-dimer.

Total

‘Ddimer

1,

i

\ \

i

/l+-MINUTES

D-dimer

3

!alJRs

-

FIG.

Dl - 5

4

SDS-PAGE of plasmin digestion of D-dimer in the presence of EGTA.

0

5

IO 20

MINUTES

40

I,53

5

HOURS

7

one

or both

fijrin aui

conoaers

zation link

that by

in vbic‘n both t'ne stability

calcium

sites,

Labelled

we

substituted

on

digrstee?

3

(Fig.

ions

The

the

siniiar

the

of

s?are

our

that

than

was

carried

-32 r 2 cc:c?ierl

dans?l-zadav2rir.e 13.

-1 ‘l?-0. ROP.-i_L___2ZSC-

Yhis +

is

material r~~erlal

5j.

dimer

-

f-D' D2-5

XL-peptide

._.: . 0

2

_... ...’

510204090

0 FIG.

2

and

of 'both 5rojs-

usin_; fluor2scent---

Factor

tne

**‘e:e

of

qi r_de 2 3 d t>2 -2 stab iii-

to occa>ati~n

anaIogu2

to

stt;
sites

experiments

&In-14 by

cross-linking

our

D-dimer

lysin2

manner

natural

cross-link

rath2r

repeated

D dimer.

in

in

Ts ensure

(iLj.

on D-diner

to show

f-D

'be in-solved

may

5

10

20 40

WYINUTES

5

SDS-PAGE of plasmin digestion of fluorescent D-dimer. The stained gel is shs;_n in
dS

E

Dl-5

FIG.

D-dimer

-76ooo -42ooo -35oc0 -26500 -23ooo

12000

6

Two-dimensional SDS-PAGE of a partial D-dimer digest. The first dimension contained SDS, the second had B-mercaptoethanol added as well. a, B and y refer to the a, 6 and y chains. y-y and yl tu 5 refer to the y chain of Ddimer and Dl - 5 respectively. Arrows show fluorescent spots. A diagram of the first dimension gel (c.f. 20 minute digest in Fig. 4) is shown above the gel. The geminal appearance of some of the spots is an artefact. A schematic diagram is also shown (left>.

Two-dimensional and

the

various

molecular from

weight.

slightly

slightly dimer

The

larger

larger

(y-y)

cleavage, As

the

the

the

y-chain

were

fluorescent.

case

of Dl

confirmed

the

presence

(Fig.

by

the

cium

to a free

only

was

tion

products

3).

calcium

further

concentration

digestion

were

Analytical

also

equal

criterion

of

the

cleavage

of D-dimer

in size to

The

Y-chain of

occurred

role

5 mM

same

product

of

in

calcium

of D-dimer

readdition of

in

of excess

(Fig.

inhibited

cal-

Not

3B).

but

the

diges-

stabilized.

ultracentrifugation

containing

initial

digestion

by

the

daltons)

daltons).

the

plasmin

D-diner

of

decreased

protective

inhibited

that

3 chains

(43,000

from

The

that

was

showed

(12,000

digestion,

fact

of EGTA

S-chain

(~1)

Dl,

manner

6), c( and

progressively

a-chain

in the

was

(by the

(Fig. all had

y-chain

than

than

and

an ordered

sample

SDS-PAGE

D fragments

amounts

SDS-PAGE

revealed

that

the material

lization

with

EGTA

of

and

digested

Dl

10 minute

still

in

a partly

dimer

c.f.

was

resulted

of

and

D2 monomer

digest

in Fig.

Further

dimeric.

the material

D-dimer

becoming

38)

destabi-

completely

monomeric. DISCUSSION Our

results

stabilization recent

observation

3 high that

affinity

this

to Dl in

and

the

cium

and by

binding

(2-5)

concentrations

stability direct

13 activity ly due calcium mation

of

must the

cross-link

for

fibrinogen

suggests

of be

by

due

the

which

reduction

therefore

hold

C-terminal

end

are

to variable

of

of

y-chain

Dl

and

the

insusceptible

weight

(Fig.

D-diner

y-chain, to plasmin

The

(2) is the

required

in molecular

the

cal-

as plasmin

calcium.

Dl

also

binding

D reported

as well

absence

has

therefore

calcium

fragment

preparation

of

is

that

about

that The

(15)

fluorescently-labelled ions

(6)

ions.

calcium

in

Timan

calcium

therefore

its

digestion

site,

al

and

to

be brought

stored

and

The

(16).

ions that

in

calcium

to plasmin

et

could

used

of D-dimer

is due

heterogeneity

of samples

result

of Haverkate

sites may

The

D-dimer.

literature

those D-dimer

Marguerie

stabilization

contamination

the

confirm

of Dl

for

is entireThe

5). in

such

which

Factor

a confor

contains

attack.

Dl

ai-*

i I:s t p a r a 1;1 e 5 *y SDS-PA.GX

*qirtgslly

c u 1 a r ::e i p h t for The

fluorescent

ly larger

than

conpared 36,000

with

peptide

lently been

action

y chain

Eroo? the XOlE?

Dl-like

sita

for

lG,OOO

dsltons,

not

the

products

xeizht

cular

Dl nonomer,

from

3 Kinimum

around

together licked

cieaved

5t

be

y-chain 5 chain,

cross-link

cross-link (17)

il_,o le-

ii3,OOi) (Fig, molecular

the

i .i slight-

released which

6)

weight

cr;iss-lic:i.

agrees

cith

17t h.e r

(17).

The D-dioer

the

uould

criteria

the

Thus

(2).

th.us 5 " g g e 3 f I n .g a :2w

;!.e 2 r 3 :s.s - 7. j L n k peptice,

be

is present

still

necessary

in another

only

interaction

ultracentrifugation

demonstrated

peptide

is not is

since

dimer as

may

part

at a stage by

binds that of

shows when

tightiy

to

the

the non-covalent the

molecule.

that

the

Since

SDS-PAGE.

holding

;ile

a non-co.la-

cross-iir?:i :?s the

parent D-dimer

clea::ad molacuie inrer-

CALCTLTI

13.

O'FARREL, P.H.: electrophoresis 1975.

TO?; STXBTLIZATICT

Vo1.12,Xo.-i

High resolution of two-dimensional .I. Biol. Chem. 250, of proteins.

14.

DOOLITTLE, R.F.: fibrin conversion.

Structural aspects of Adv. in Prot. Chem.

15.

MARGUERIE, G., CHAGNIEL, G. and of calcium to bovine fibrinogen, 490, 94, 1977.

16.

TAKAGI, Purification T. and KONISHI, K.: properties of fibrin stabilizing factor. Acta 271, 363, 1972. Biophys.

17.

LINDSEY, G.G., BROWN, the fibrin cross-link Thrombosis Research.

the 27,

4007,

fibrinogen I, 1973.

to

The binding SUSCILLON, M.: Biochim. Biophys. Acta

G. and PURVES, L.R.: peptide. Manuscript

and some Biochim.

Isolation submitted

of to