Stimulation of human granulocyte elastase by platelet factor 4 and heparin

Vol. 85, No. 3, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December 14, 1978

Pages

STIMULATION

1113-1118

OF HUMAN GRANULOCYTE ELASTASE BY PLATELET FACTOR 4 AND HEPARIN

Stewart

A. Lanky*,

Department San Diego Hospital, Received

October

James Marsh,

and Herbert

Wohl

of Medicine, University of California Medical Center, an d the Veterans Administration La Jolla, California 92161

27,

1978 SUMMARY

Highly purified platelet factor 4 (PF4) was found to be a potent stimulator of human granulocyte elastase activity against native elastin aa solubilized a elastin. Heparin neutralized this stimulation of elastolysis by PF4, but independently stimulated granulocyte elastase activity. Chondroitin sulfate, a constituent of the PF4 carrier molecule, also stimulated granulocyte elastase activity. The stimulation of granulocyte elastase by PF4 occurs at known serum concentrations of PF4. INTRODUCTION Human granulocytes anionic

contain

glycoprotein

from various against

with

activity

(1).

This

tissues

elastic

tissue elastase

emphysema

The granulocyte

(4),

and by synthetic

compounds effects

(5).

granulocyte cationic and after anticoagulant

elastase

anionic

elastase. protein

effect

*To whom requests

is

or cationic factor

released

damage

(7).

of heparin reprints

available

capable

the enzyme

(2),

of human

weight

concerning

on the

activity

during

weight

clotting

of neutralizing been

the of

is a low molecular

(8) , and has recently should

with

low molecular

from platelets PF4 is

active

by serum antiproteases

directed

4 (PF4)

also

an

isolated

the pathogenesis

inhibited

molecules

is

elastin

is

treated in

currently

is

for

elastase

animals

active-site

Platelet

which

mechanical

granulocyte

enzyme is

which

pH against

has been implicated

No information

of small

(EC 3.4.21.11)

at neutral

in experimental

and granulocyte (3).

an elastase

(6) the

shown to

be sent. 0006-291X/78/0853-1113$01.00/0

1113

Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved

BIOCHEMICAL

Vol. 85, No. 3, 1978

inhibit

human skin

collagenase

purified potent

and granulocyte

and elastase

the pathogenesis

AND BIOPHYSICAL

are

stimulator

(8).

proteases

which

serine

of human disease,

PF4 on granulocyte

collagenase

we examined

elastase.

of granulocyte

We have

elastase

MATERIALS

RESEARCH COMMUNICATIONS

Since

both

may play

the effects found

a role

in

of highly

PF4 to be a

activity.

ANDMETHODS

Platelet factor 4 was isolated from human platelets by affinity chromatography as previously described (10). No phenylalanine or methionine was found by amino acid analysis, and the protein displayed a single band on soidum dodecyl sulfate polyacrylamide gel electrophoresis. PF4 was dialyzed against .OlM phosphate (pH 7.4) with 0.30 M NaCl prior to experiments with granulocyte elastase. Granulocytes were collected from freshly drawn human blood by dextran sedimentation of the white-cell rich fraction (11). Following grsnulocyte lysis, the lysosomal granules were collected by centrifugation. These granules were lysed by repeated freeze-thawing, and purified granulocyte elastase was isolated from the granular extract by affinity chromatography using an elastin-sepharose column (12). Esterase activity of the column effluent was measured using N-t-BOC-L-alanine-p-nitrophenyl ester (NBA1 as described by Visser and Blout (13). The peak containing the NBA esterase activity displayed a typical granulocyte elastase isoenzyme pattern on polyacrylamide gel electrophoresis, and a single band on sodium dodecyl sulfate polyacrylemide gel electrophoresis. Human u-l antitrypsin was prepared from freshly drawn blood as previously described (14). The purity of this preparation was confirmed by immunoelectrophoresis and by polyacrylomide gel electrophoresis. Elastolytic activity was measured using elastin-agar plates (15) containing 7.75 ug of bovine ligamentum nuchae elastin per mm2. Reactants were mixed in a test tube and allowed to incubate for 30 minutes before being added to the plate. Plates were incubated at 37Oc for 24 hours. The areas of clearing were read by 2 observers, using a comparator (Bausch and Lomb), and the results averaged. Elastolytic activity was also measured using oxalic acid treated bovine ligament elastin (16) as a substrate according to the method of Keller and Mandl (17). PESULTS AND DISCUSSION We found activity

platelet

(figure

1).

granulocyte

elastase

c elastin.

This

200 llg/ml.

Using

of as little in an increase

factor

4 to be a potent

Concentrations activity

stimulation solubilised

as 20 pg/ml of elastolysis

stimulator

of elastolytic

of PF4 as low as 10 ug/ml

against was linear a elastin

both

insoluble

and soluble

up to PF4 concentrations as the

substrate,

PF4 to 5 ug of granulocyte of 38%, an d the

1114

elastin

addition

the

elastase

stimulated

of addition resulted

of 100 us/ml

BIOCHEMICAL

Vol. 85, No. 3,1978

ol z”

25

I

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a

50

75

I

’

100

PF4ADOE0

125

I

150

I

175

’

200

(ug/mlI

Figure 1: ug of elastin solubilized by humangranulocyte elastase in 24 hours as PFa is added to reaction tube. Open circles represent data for 5 pg elakase plus PF4; closed circles represent data for 5 ug and PF4. Each point is the meank S.E. elastase, 6.6 pg a-l antitrypsin, of 3 separate determinations on an elastin-agar plate.

PF~ to 5 ug of granulocyte activity

elastase resulted in a doubling of elastase

(not shown). Figure 1 also demonstrates that this effect

of

PF4 on granulocyte elastase occurs in the presence of alpha-l-antitrypsin. The addition of 6.6 pg of a-l-antitrypsin lowered the elastolytic but the addition

activity

fashion (figure

1).

levels,

of the non-inhibited

Whenthe elastolytic

activity

elastase was completely abolished by a-1-antitrypsin,

of

no

was restored by the addition of up to 500 vg/ml PF4. The

reported concentration the reported effect levels,

elastase

of the enzyme to 35%of control

of PF4 resulted in the stimulation

enzyme in a linear granulocyte

activity

to 5 pg of granulocyte

of PF4 in humanserum is 8-15 us/ml (18).

Therefore,

of PF4 on granulocyte elastase occurs at serum

and higher.

In view of the known binding of PF4 to heparin neutralization

of heparin's anticoagulant

effect,

resulting

the effect

in a of heparin

on the granulocyte elastase - PF~ system was examined. We found that the addition

of

more

than 0.5 units of heparin alone to granulocyte

resulted in the stimulation open circles).

of enzyme activity

This stimulation

not been previously

against elastin

elastase (figure

2,

of granulocyte elastase by heparin has

reported , and the possible physiologic

significance

Vol. 85, No. 3, 1978

BIOCHEMICAL

I 0

2

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

4 6 UNITS OF HEPAAIN

2 10

8

Figure 2: Experiment showing effect of added heparin on 5 Ug of granulocyte elastase alone (0) or 5 pg of granulocyte elastase plus 20 ug/m.l PF4 (A) as measured by elastin plate assay. Each point is the average ug elastin solubilised per 24 hours of 2 separate determinations.

of this stimulation

is unknown. Furthermore, it

that the stepwise addition elastase and until

the

pF4

PF~

can be seen

figure

of heparin to a mixture of granulocyte

results in a stepwise neutralization

stimulation

only the heparin effect

from

of granulocyte remains (figure

shows no such neutralization

of the PF4 effect

elastase has been abolished and 2, open triangles).

of the inhibitory

effect

granulocyte

collagenase (8).

granulocyte

elastase and 1 unit of heparin , no further

elastolysis

was seen until

Heparin

of PF4 on

When PF4 was added to a mixture of

the PF4 concentration

stimulation

of

reached 100 ug/ml

(not shown). Since PF4 has been shown to exist in both a free and a bound form in humanserum, and since the carrier molecules of chondroitin chondroitin

sulfate

sulfate

protein

is complexed with 4

(19), we also studied the effect

of

on granulocyte elastase , and on the pF4 stimulation

1116

2

BIOCHEMICAL

Vol. 85, No. 3, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE1 STIMVLATION OF GRANULOCr E ELASTASE BY PF4: aFECTS CF CBONBXZTIB SULFATE

Reaction

Enzyme activity+

Mixtw%

Gtanulocyte

elastaee

5 )lg

110.4

GrMulocyta

elaetaee

5 pg + 30 ug/ml PF4

164.4

Granulocyte elaetase 5 ug + 30 vg/ml PF4 + 0.5 mq/ml chondroitin SO4

255.7

Granulocyte elastase chondroitin SO4

173.9

5 ufu + O.Smg/ml

% Stimulation

49% 132% 58%

t Enzyme activity expressed as ~9 elastin solubilized in 24 hours. Reaction mixtures of 0.1 ml were incubated 30 minutes at 22'c before being added to elastin-agar plates. Condroitin sulfate was made up in O.OlM phosphate (pli 7.4) with 0.30M NaCl.

of elastolysis. sulfate

As is

(0.5

mg/ml)

shown in table

to granulocyte

stimulation

of elastolysis

chondroitin

sulfate

stimulation

of elastolysis.

sulfate

effects Eagan,

elastase

sodium

dodecyl

pancreatic which

been enape. tissue

for

their

Because injury,

attraction interactions

or bile

the

acids

are

(20).

in a

of the

same amount

in

further

the PF4 and chondroitin

proteins,

effect

on either

and since

such injury

an important

it

activity

anionic

added

These

like

to reaction

anions

attach

of

to elastin,

of cationic

enzyme. previously

or granulocyte during

inflammation

clotting with

is possible

that

PF4-granulocyte

physiologic

role

in tissue

1117

such as mixtures

PF4, have not

activated induces

of

molecules

the pancreatic

are normally

of qranulocytes,

elastolytic

the attraction

cationic

platelets

play

that

when small

and increase

weight

tested

that

and elastin

amphiphilic,

and the addition

appears

increased

sulfate

Low molecular

It

chondroitin

results

of GE and PF4 results

reported

is

elastase

is

(58%),

alone

of

additive.

et al have

pancreatic

elastase

to a mixutre

are

1, the addition

injury.

and

the elastase

of

Vol. 85, No. 3, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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