'WROMBOSIS Printed

RESEARCH in the

United

THE GENERATION

Vol. 6, Pergamon

States

OF C-TACTIC

LEUKOCYTES

ACTIVITY

BY THE ACTION

PP.

l-8,

Press

1975 ,

Inc.

FOR HUMAN

OF PLASMIN

ON

HUMAN FIBRINOGEN

Robin McKenzie, University Department and the South-East

(Received

ABSTRACT

and A-B. Kay

of Respiratory Diseases, City Hospital, Edinburgh Scotland Regional Blood Transfusion Service, Royal Infirmary, Edinburgh

l.lO.lC,,74.

Chemotactic

D.S. Pepper

activity

Accepted

by

Editor

for human peripheral

P.J.

Gaffney)

blood leukocytes

was

generated by the action of plasmin on human fibrinogen. When plasmin digestion was stopped at time intervals up to 24 hours, a small amount of activity was p resent at 15 and 30 min. corresponding to the transient appearance of fragment Y. The activity contained in the 24-hour digest k'as considerably higher and eluted from Sephadex G-75 with molecules of approximately 30,000 daltons. When purified X, Y, D and E were assayed individually for chemotaxis only fragment Y was active but in relatively high concentrations. Thus the chemo-tactic activity generated by the action of plasmin on fibrinogen was mainly associated with one or more lower molecular weight polypeptides and to a lesser extent with the Y fragment.

INTRODUCTION A number fibrinogen

of studies

yields

smaller peptide appear

four major

material

in body fluids

have a number

have shown that plasmic fragments

(l,2,3).

with various

activities activity

on human fibrinogen

This was subsequently

fibrinopeptide plasmin

(12).

B (13).

digestion

clinical

states

identified

to attract

to (FDP)

(4,5,6,7)

and

report we

by the action of thrombin as a property

study we have examined

fibrinogen

products

In a previous

can be generated

In the present

products,of

degradation

(8,9,10,11).

have shown that chemotactic

of human

X, Y, D and E in addition

Fibrin/fibrinogen

in association

of biological

designated

degradation

of

the capacity

human peripheral

blood

leukocytes. MATERIALS plasmin

Digestion

of Human Fibrinogen.

AND METHODS One gm of human

fibrinogen

(KABI,

of

CHEMOTAXIS

2

Grade L) was dissolved against phosphate

buffered

saline

to a concentration

10,000 units/ml)

reaction

was stopped

to 40 volumes

for chemotaxis

immediately

alone,

of the reaction

mixture.

prepared

in the same way with the addition

The digests were analysed

ether, concentrated

After

described

G-25 column

to a column

of Sephadex

and penicillin.

molecular

weight were also filtered

on the column

(Whatman)

(3).

UM-10 membrane described

vitamin

by Sephadex

Measurement Millipore 8.0 p

technique

gave single

weights

(13).

(100 x 2.5 cm) was

200,000;

58.

Fragments

X, Y, D

chromatography

by ultrafiltration

PBS with added antibiotics

on CM-52 on a as

bands on disc gel electrophoresis to their previously

(3). was assayed

The number

and the results

by a modification

of cells migrating

expressed

(x 40) fields or as the percentage control

plasmin

with the following

Products.

also corresponded

Chemotaxis

of Boyden

filter were counted

against

24-hour

G-75

chloride,

were concentrated

markers

molecular

used as the chemotactic

with

in 0.05 M pyridine

- Blue Dextran,

G-200 and ion exchange

The fragments

of Chemotaxis.

five high power

Digestion

(Amicon) and dialysed

monomeric

of 30% TCA was

The separation

Markers

1,357, and sodium

Fibrinogen

which using the appropriate described

B12,

The preparations

above.

For

(14).

was extracted

of a 10 times concentrated

collected.

and E were prepared

alone were

(13).

was applied

of Purified

Control

at zero time and 24 hours.

the supernatant

at 4OC and 5 ml fractions

Preparation

at -80Oc.

one fifth of the volume

on a Sephadex

in PBS with streptomycin

45,000;

The preparation

and streptokinase

performed

ovalbumin,

of Trasylol

disc gel electrophoresis

centrifugation

One millilitre

digest of fibrinogen

storage

of Trasylol

by polyacryamide

and desalted

Gel Filtration.

equilibrated

and plasminogen

(TCA) precipitation

added to the digest.

The

of one volume

of fibrinogen

acid

(Earl, 5 W/ml).

or following

diluted

(Streptase,

by the addition

mixtures

as previously

To 100 ml of fibrinogen

and 2 ml of plasminogen

at time intervals

(Bayer, 10,000 units/ml)

trichloracetic

50 units of

of 5 mg/ml was added 0.25 ml of streptokinase

Behring,

was assayed

for 18 hours at 4'C

(PBS), pH 7.2, containing per ml.

1

Vol.6,No.

FDP'S

in 100 ml of water and dialysed

and 5 pg of streptomycin

penicillin

AND

of the

through

as the mean cell count of

of a normal

human serum pool

(15).

RESULTS A 24-hour for peripheral

plasmin

digest of human

blood leukocytes

fibrinogen

in a dose-dependent

an

was found to be chemotactic fashion

(Fig. 1).

i’ol.5,?;0.1

CHEWOTAXIS

MD

FDP'S

Chemotarls (Mean cell count)

Volume

of

FIG.

fibrinogen-plasmin

was detected

the same conditions.

not inhibit or potentiate positive plasmin

chemotactic

In

Fig. 2 three

with plasmin patterns

experiments

appearance

was in the 24-hour

When a preparation of Sephadex

G-75,

of fragment

demonstrable

and did

serum used as the of a 24-hour

fibrinogen/

supernatant,

fibrinogen

and the chemotactic at intervals.

was digested activity

and

In two out of

was seen at 15 and 30 min. corresponding The major

Y.

chemotactic

activity,

digest.

of a 24-hour

size of approximately

Significant

under

with small peptides. in which

compared

fibrinogen

the major peak of activity

The chemotactic Fig. 4.

depicted

a small peak of activity

human

activity

was found in the desalted

up to 24 hours

on disc gel electrophoresis

to the transient

molecular

are

alone incubated

of chemotactic

precipitation

was not associated

for time intervals

three studies

however,

Following

The volume of

or plasmin

of normal

little activity

that the activity

digest.

was also devoid

the activity

control.

digest with TCA,

suggesting

with fibrinogen

Trasylol

(ml)

1

Chemotaxis of a 24-hour plasmin/fibrinogen digest was made up to 0.8 ml with PBS.

No activity

digest

activity activity

at concentrations

eluted

30,000 daltons

of purified

digest was passed over a column of an apparent

(Fig. 3)

fragments

was detected

with material

X, Y, D and E are shown in

only with fragment

of 0.5 mg and 1.0 mg per ml.

Y and was

CHEW:_ITAXI3

.L\SD FDP' s

FIG. 2 Disc gel electrophoresis and chemotaxis of plasmin/fibrinogen digests at The chemotactic results of three experiments various time intervals. are depicted along with the positions of fibrinogen and fragments X, Y, D and E on the disc gels. Chemotoxis (Meon

cell count)

Blue 7

Dex.

V&.8,,

OA

7

1

NoCi v

Ohanm

-2

FIG. 3 Sephadex G-75 chromatography of a 24-hour plasmin/fibrinogen digest. The optical density (O--0)of fractions was measured at 2.80 nm. Chemotaxis is indicated by vertical bars and the elution of molecular weight markers is indicated by arrows. Molecular markers are Blue Dextran (Blue Dex.), ovalbumin (OA), vitamin B 12 (Vit. Blo) and sodium chloride (NaCl).

AND

CHEcfOTAXIS

Vo1.6,No.l

FDP'S

80

60 Chemotoris c%or control) 40

of FDP (mg/+l

Conccntrotion

FIG. 4 Chemotaxis

of purified

fragments

X, Y, D and E.

DISCUSSION Chemotactic digestion

of fibrinogen

30 minutes, stimulus

activity

for human

(Fig. 1).

some activity

this fragment

at the concentration At 24 hours, chemotactic

The nature

having

of plasmin

band(s) which appeared bands were observed

digestion.

on fibrinogen

during

digests

by the larger Furlan

weight

prolonged

mobilities

digests

activity

but the activity whereas

neither

The failure

or possibly

corresponding

with

30,000

daltons.

generated

of these agents

alone

rests in unidentified

(Fig.

E (ca. 50,000 molecular

2)

and may have contained

to demonstrate

greater

of inhibition

a requirement

three polypeptide

plasmin

to a

eluted

was clearly

than fragment

at 30 min.

more

Thus on disc gel several

digest.

further

(Fig. 4).

digest was applied

of approximately

may have been a result fragments

A chemotactic

there was considerably

of the 24-hour

and Beck described

to D and E which survive electrophoretic

detectable,

the plasmin

material.

15 or

Y since in separate

that the activity

These bands first appeared

with the earlier

fragment

after

in a pure form and shown to be active

is unknown

which migrated

the later chemotactic

migration

molecular

It is possible

were chemotactic.

(Fig. 2).

G-75 the major peak of chemotactic

an apparent

by plasmin

was stopped

for the 15 or 30 minute

When a sample

of this material

by the action

weight).

was isolated

estimated

activity.

the reaction

was probably

when Y was no longer

column of Sephadex molecules

When

was generated

was found in these digests

in these preparations

experiments

leukocytes

digestion

to molecular

(3).

activity

of leukocyte

for further

fragments

in addition

These have disc gel

sizes of 25,000,

14,000

CHENOTAXIS

6

Whether

and 11,000.

these contain

AND

Vol.6,No.l

FDP'S

the chemotactic

described

property

herein

is unknown. Though

fibrinopeptide

latter stages of plasmin

B is cleaved digestion

Sephadex

G-75 column

fractions

peptide

(ca. 1,500).

However

in the fibrinogen

digest

100% of this peptide fraction

was cleaved

leukocyte

Barnhart

migration

reported

by plasmin.

fragment D was not chemotactic

attributing

chemotactic

contact with abraded and subsequent

properties

suggest

rabbit

leukocytes,

24-hour

Stecher

thus explain

to agents

which

were used as target

this apparent

(17).

cells;

include anticoagulant

activity

(8,9) and ability

products

of smooth muscle

to attract

pathological pulmonary

conditions

embolism

contraction

activities

human

factor

factors

effect of plasmin

(13,151.

digestion Using

activity

in a

investigation system may

discrepancy. associated

These biological

chemotactic

in

following

of Hageman

the use of a homologous

activity

(11).

cause cell migration

In the present

Other biological

potentiation

in

There are difficulties

found only weak chemotactic

digest

described

after 24 hours digestion.

produced

and Sorkin

to

granulocytes

the conditions

(Fig. 4).

that the main chemotactic

human plasmin/fibrinogen

human leukocytes

Under

of a number of recognised

lies in one or more small peptides

reported

of canine

skin as this may lead to the activation

generation

Our results

previously

in the

(13).

(18).

this report

0.3 )Imol/ml if

Thus the concentrations

D evoked migration

technique

of this

that the amount of B peptide

concentration

(0.1 pal/ml)

that fragment

in vivo using a skin window

weight

to the column was approximately

would be less than the minimum

stimulate

to the molecular

it was estimated

in the

was found in the

(171, little activity

corresponding

applied

of fibrinogen

from the p-chain

leukocytes

with fibrin/fibrinogen

to alter platelet

(10) and increased

together

(5), transplantation

intravascular

rejection

in which high levels of FDP's have been detected

products

function

capillary

with the capacity

may be of significance

such as disseminated

digestion

permeability

of plasmin

in a variety coagulation

(91,

digestion of

(41,

(6) and glomerulonephritis

(7)

in the blood and urine.

ACKNOWLEDGEMENTS This work was supported anonymous

by a grant from the British

gift to the Department

Mr. Edwin Lithgow discussions.

for technical

of Respiratory assistance

Diseases.

Heart Foundation

and an

We are grateful

and to Dr. John Cash for helpful

to

v01.t;,x0.1

CHEYOTAXIS

AND

FDP'S

REFERENCES 1. NUSSENZWEIG, V. and SELIGMANN, M. Analyse par des methodes immunochimiques, de la dggradation par la plasmine du fibrinogzne humaine et de la fibrine, a diff