The effects of cell-released protease nexin on the measurement of thrombin binding to mouse cells

Vol. 105, No. 2, 1982 March 30. 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 667-673

THE EFFECTS OF CELL-RELEASED PROTEASE NEXIN ON THE MEASUREMENT OF THROMBIN BINDING TO MOUSE CEILS David Department

Received

A. Low and Dennis

of Microbiology,

February

15,

0. Cunningham

College of Medicine, University Irvine, California 92717

of California,

1982

We previously showed that fibroblast-like cells release protease nexin into their growth medium. Protease nexin links to thrombin and mediates the cellular binding of thrombin via the protease nexin part of the complex to a site different from that for unlinked thrombin (1,Z). To determine the effect that cell-released protease nexin had on the measurement of total cell-bound thrombin, we separately measured the cellular binding of both 1251-thrombin and 1251-thrombin-protease nexin complexes. Scatchard analy i of our binding data indicates that the cellular binding affinit of linked ?*$I-thrombin is about 19-fold higher than that of unlinked l**I-thrombin. We show that protease nexin acts to increase the apparent affinity of 1251-thrombin for cellular binding sites. Introduction Thrombin

is

mouse embryo on their tease

mitogen

for

(3,41.

These

cells

cells

cell nexin

surface (2).

ages with

thrombin

complexes,

formed

the

a potent

complex

thrombin-protease the apparent

Protease

nexin

is

serine

in the medium,

bind

different

measure nexin

affinity

and also

and other

to a site

Here we separately

(5,6,71

a variety

the

of cells

in culture

have high

affinity

thrombin

have cell

surface

released

by cells

proteases

(2,8).

to cells

from that cellular

complexes

via

which binding

and show that

of 1251-thrombin

for

cellular

binding

including binding sites

and forms

for

covalent

the protease unlinked unlinked

protease

nexin

binding

nexin

nexin

part

thrombin

of both

thrombin acts

prolink-

Thrombin-protease

binds

sites

of

(2). and

to increase

sites.

Materials and Methods Highly purified human thrombin (about 3100 National Institutes of Health units/mgl was generously supplied by Dr. John W. Fenton II (9). Ovalbumin (B grade) was from Calbiochem, PhAsO was from Aldrich, and lactoperoxidase (320 un/mg) was from Worthington. We purchased DV medium from Flow Laboratories; antibiotics were from Gibco and calf serum was from Irvine Scientific (Santa Ana, California). Na I251 was obtained from Amersham and Iodogen was from Pierce.

Vol. 105, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Secondary mouse embryo fibroblast-like cells were cultured as described Binding studies were performed as described (2,7) except where noted. (41. Thrombin was iodinated using a scaled-down lactoperoxidase procedure (9) and retained full activity in a fibrinogen clotting assay at less than 0.4 mole I/mole thrombin (7 x 105 - 1 x 106 cpm/pmol) (7). Hirudin was iodinated using Iodogen and the hirudin assay for measuring cell-bound thrombin was performed as described (71. Levels of Iz51-thrombin and 1251-thrombin-protease nexin in the medium were determined using Weber-Osborne sodium dodecyl sulfate-polyacrylamide gels Samples were analyzed using 10% polyacrylamide as described except that (111. th y were not heated prior to electrophoresis. Cell-bound I*51-thrombin and 12E I _ thrombin-protease nexin were measured using a bis-tris sample buffer (2) and mono-tris/bis-tris sodium dodecyl sulfate polyacrylamide slab gels as described (12). I251-thrombin-protease nexin, formed in the medium or bound to cells, was stable under the conditions of analysis described above. After electrophoresis, quantitation of radioactivity in gel slices (2 mm each) was Lines were drawn through data points in Figures carried out as described (131. 1 and 2 using least squares analysis. Results To measure amounts

the cellular

of 1251-thrombin

thrombin-protease dodecyl that

complexes

for than

nexin

1 shows that

its

binding

that

of total

thrombin

binding

medium

shows that

cellular

the apparent lower

affinity

was taken

cells

via

ligand

for

its

(linked

binding plus

sites.

unlinked

of 1251-thrombin-protease

binding

of 1251-thrombin

amount

for

cells

nexin

19-fold

higher

analysis

using

cells

nexin

by about

of

ConcentrationI.

Importantly, thrombin)

of

used in the

to be the

medium was about

indicated

binding

of I251-thrombin-protease

in fresh

sodium

1.5-fold

in fresh

increased (Fig.

1,

panel). A potential

thrombin-protease this

of free

and 1251-

our results

was formed

the amount

affinity

increasing

using

in the medium at each 1251-thrombin

using

of 1251-thrombin

Since

nexin

of complexes

the apparent

sites

and quantitated

electrophoresis.

(21,

binding

1251-thrombin-protease Figure

gel

thrombin,

and 1251-thrombin

separated

1251-thrombin-protease

of cellular

and unlinked

to mouse cells

were

in the medium to cells

determination

of linked

added

polyacrylamide

most cell-bound

complexes

were

nexin

sulfate

binding

step

is

rate nexin

dependent

limiting complexes on levels

step

in the

is the

formation

formation

of protease

nexin

of cell-bound of complexes in the medium.

1251in the medium; Since

re-

1251-thrombin-protease nexin lWe have found that the amount of cell-bound nexin but not to levels pggportional to levels of I251-thrombin-protease Also, our results indicate that blockage I thrombin in the medium. of formation of 1251-thrombin-protease nexin complexes in the medium almost completely inhibits the formation of cell-bound complexes (not shown). ;;

668

Vol.

105,

No.

2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

Bound

~molecules/cell

RESEARCH COMMUNICATIONS

x lO-31

Scatchard analysis of the binding of I*51-thrombin and 1251nexin to mouse cells. Mouse cells (9.0 x lo5 cells/35 mm dish) were washed and incubated in serum-free Dulbecco-Vogt modified Eagle's After five days medium from some medium containing ovalbumin (50 pg/mll. of the dishes was removed and replaced with fresh medium. Increasing amounts of 1251-thrombin were then added to cultures, with and without the of unlabeled thrombin (280 nM1. After 1 hr at 37"C, the amount of fjfi!:F:ombin and I*51-thrombin-protease nexin both in the medium and cellbound, was determined as described in Materials and Methods. The amount of radidactivity in each sample was normalized based on the fraction: (total radioactivity recovered on gel/total radioactivity in sample) which was about 0.8. The amount of nonspecific binding (binding in the present subtracted to yield the amount of specifically-bound thr m in-protease nexin at each 1*51-thrombin concentration. The amount of ?!L* I thrombin-protease nexin in the medium was used as the "free" concentratransformation (14) of binding data shown in the upper

lease

of

tion

in

nexin as

protease conditioned

(2). many

fresh tioned measured

nexin

At

is

gradual

medium, maximal

which

medium

(not the

increased

total by

As number

about

shown of

12-fold.

we

(14

Figure

there

formed

in

1 (upoer

669

incubaprotease

ahout

conditioned panel), nexin

the

of

were

1251-thrombin-orotease However,

binding

amounts

nM)

complexes in

performed

substantial

levels nexin

shown).

shown),

contains

Iz51-thrombin

1251-thrombin-protease medium

(not

apparent

usinq binding cellular

8-fold versus condisites affin-

Vol. 105, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Figure 2. Disso iation of 1251-thrombin and thronbin from mouse cells. 11 6.5 x 10 s cells/35 mm dish) received thrombin (2.8 nM) or w I thrombin ce s ( (2.8 nM1. with and without the addition of thrombin (280 nM). in 2 ml of serum-free Dulbecco-Vogt modified Eagle's medium containing Hepes (10 mM) and ovalbumin (0.1%). After a 30 min incubation at 37'C, cells were rinsed twice using the serum-free medium described above and 2 ml of this medium was added to each dish. At increasing time intervals after this second incubation, cells were cooled to 4Y and were rinsed 3 times in ice-cold Dulbecco's phosphate buffered saline (D-PBS) and once in D-PBS containing phenylarsine oxide (0.1 mM). After a final rinse in D-PBS the amount of specifically bound I251-thrombin (linked plus unlinked1 remaining on one set of cell cultures was determined using sodium hydroxide solubilization as described (1,5). Also, the fraction of specifically-bound 1251-thrombin that was linked to protease nexin was determined immediately following the first 30 min incubation as described in Figure 1 and was equal to 0.20. The amount of unlabeled thrombin remaining on dishes was determined using the hirudin assay (-1, total cell-bound 1251-thrombin (I251-thrombin as described (5). plus I251-thrombin-protease nexin); (-1, cell-bound 1251-thrombin (after subtraction of the amount of cell-bound 1251-thrombin-protease nexin); (-1, cell-bound thrombin.

ity to

of

complexes

in

incubations

number

conditioned

in

of

fresh

cellular

thrombin-protease

medium These

medium.

hinding

sites

were

nexin

was

and

results the

by about

reduced

showed

apparent

dependent

on

a method

called

the

that

both

cellular conditions

70-fold

compared the

apparent

affinity used

of

for

1251-

binding

incubations. We previously picogram

amounts

ciation

rate

was about used

in

this

of twice

developed of

cell-hound

thromhin that

experiment

from for

thrombin the

cell

20%

(71. surface However,

1251-thromhin. ahout

the

of

specifically

hirudin

Figure

assay 2 shows

(measured at

for that

with the

cell-hound

the

thromhin thromhin

measuring the

disso-

hirudin

assay)

concentration was

BIOCHEMICAL

Vol. 105, No. 2, 1982 linked

to protease

that

only

about

nexin

16% of total

cell-dissociated tracted

after

the amount

point

in the

after

correction

tion

rate

for

thrombin

(hinding

data

used for

an incubation

at 37'C

dissociation

We showed

for

3 h (7).

curve.

I251-thrombin

was

Therefore, nexin

we sub-

from

each

As shown in Figure

(.066/min)

previously

nexin

I251-thrombin-protease

undissociable

unlinked

1).

I251-thrombin-protease

of cell-bound

for

Fig.

RESEARCH COMMUNICATIONS

1251-thrombin-protease

cell-hound

1251-thrombin

for

AND BIOPHYSICAL

nexin,

was about

2, the

dissocia-

the same as that

(.069/mini.

Discussion We recently preparations

showed

contained

diiodotyrosine site

for

taininq

that

diodotyrosine

was impaired

thrombin.

the hirudin

was measured.

fraction

Laemmli

gels

used to analyze

appears

to measure

ability

affinity

to be about

twice

affinity

appears

not linked

to be the

thrombin

l251to protease

Tris-containing

(7),

the hirudin

assay

much of the apparent

containing

of the different

for

linked

Because

of Iz51-thrombin

result

concentrations

of thrombin

(1).

the binding

sites

on the alkaline,

measured 1251-thrombin

mediated

of binding

formation

con-

cell-bound

nexin

amount

hindina

thrombin,

at low 1251-thrombin

the

affinities

for

total

11, protease

surface

of lP51-thrombin that

experiment

unstable

but

containing

to the cell

the binding

complex

in 1251-thrombin

Iz51-thrombin

to bind

apparent

were

unlinked

in the binding

of thrombin

(71.

we underestimated

the complexes

and thrombin

residues

(Figure

the

because

difference

of the molecules

of 1251-thromhin

increased

Previously

fraction

In the latter

As we show here

and significantly

nexin

its

appeared

assay.

of a significant

thrombin.

in

Additionally,

monoiodotyrosine

using

a large

methods

monoiodotyrosine of measurement

hinding.

If the

above hypothesis

thrombin

and thromhin

tracting

the undissociable

the

dissociation

the

cellular

by the hirudin

rates

should

then

be similar. fraction

rate (71,

of 1251-thromhin and thrombin

of unlinked

measurement

the dissociation

In Fiqure

of l251-thrombin

dissociation assay

is correct,

but not

of thrombin

accurate. 671

rate

2 we show that linked

dissociation

after

to protease

are almost linked

of I25I-

equal.

thrombin

subnexin, Because

is measured

from cells

is

Vol. 105, No. 2, 1982

BIOCHEMICAL

The reduced

binding

in conditioned binding

versus

sites

nexin

are

binding

for

formed

site

protease

apparent

in fresh

the apparent

via

competitive

binding.

medium

versus

that

presence nexin

since

he due to the presence

Since

lower

medium than

This

be linked

phenomenon

would

the

time

the high

affinity

much of the

to cell-released

occur

nexin

proteases

during

of 2 or more

proteases,

of 1251-thrombin-protease

of protease

to cell-released

medium,

Alternatively,

affinity

nexin

of 1251-thrombin-protease

in conditioned

medium might cellular

of Iz51-thrombin

levels

he measured.

the fraction

linked

that

to a lesser

linked

would

nexin extent

in

to Iz51-thrombin

he increased

cellular

release

due to the of protease

occurred. In summary,

affinity that

medium could

in conditioned

lowering

fresh

of Iz51-thrombin-protease

preferentially

thus

RESEARCH COMMUNICATIONS

affinity

complexes.

would

nexin

fresh

AND BIOPHYSICAL

results

presented

of lz51-thrombin of thromhin.

thrombin

containing

Our results

must be discriminated

of thrombin

with

cell

here

surface

indicate

that

monoiodotyrosine

show that in order binding

cellular

the cellular (71 is

binding

to accurately

binding

similar

of linked

determine

the

to and unlinked interaction

sites.

Acknowledgments This

work was supported

Institutes

of Health.

GM-07134.

We gratefully

technical

D.A.L. thank

by Research

Grant

was supported J.

W. Fenton

CA-12306

from the National

by predoctoral II

for

thrombin

training

grant

and G. Ryan for

assistance.

References D. 0. (1980) . Baker, J. B., Low, D. A., Simmer, R. L., and Cunningham, Cell 21, 37-45. D. D. (19811 2. Low, D. A., Baker, J. B., Koonce, W. C., and Cunningham, Proc. Nat. Acad. Sci. USA 78, 2340-2344. 3. Chen, L. B., and Buchanan, J. M. (19751 Proc. Nat. Acad. Sci. USA 72, 131-135. 4. Carney, D. H., Glenn, K. C., and Cunningham, D. D. (1978) J. Cell Physiol. 95, 13-22. 5. Carney, D. H., and Cunningham, D. 0. (1978) Cell 15, 1341-1349. 6. Glenn, K. C., Carney, D. H., Fenton, J. W. II, and Cunningham, D. D. (1980) J. Biol. Chem. 255, 6609-6616. D. D. (19821 J. Biol. Chem., in press. 7. Low, D. A., and Cunningham,

Vol. 105, No. 2, 1982 8.

9. 10. 11. 12. 13. 14.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Low, D. A., Cunningham, D. D., Scott, R. W., and Baker, J. B. (1981) in Receptor-Mediated Binding and Internalization of Toxins and Hormones, eds. Middlebrook, J. L., and Kohn, L. D., pp. 259-270, Academic Press, New York. Fenton, J. W. II, Fasco, M. J., Stackrow, A. B., Aronson, D. L., Young, A. M., and Finlayson, J. S. (1977) J. Rio]. Chem. 252, 3587-3598. Martin, B. M., Wasiewski, W. W., Fenton, J. W. II, and Detwiler, T. C. (1976) Biochemistry 15, 4886-4893. Weber, K., and Osborne, M. (1969) J. Biol. Chem. 244, 4406-4412. Knauer, D. J., and Cunningham, 0. D. (1982) Proc. Nat. Acad. Sci. USA, in press. Baker, J. B., Simmer, R. L., Glenn, K. C., and Cunningham, D. D. (1979) Nature 278. 743-745. Scatchard,-G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672.

613