Vol.

177.

No.

June

28,

1991

BIOCHEMICAL

3, 1991

AND

BIOPHYSKAL

RESEARCH

COMMUNICATIONS

Pages

1198-1204

THE REACTION OF THROMBIN WITH PLATELET-DERIVED NEXIN REQUIRES A SECONDARY RECOGNITION SITE IN ADDITION TO THE CATALYTIC SITE Andrew

State

Received

Ching

Chang+

and Thomas

C. Detwiler"

Department of Biochemistry of New York Health Science BrookJyn, N.Y. 11203

University

Center

at Brooklyn

May 7, 1991

SOMARY: A protease nexin released by activated platelets forms stable complexes with o-thrombin. Active-site-blocked thrombin does not form the stable complex, but it inhibits formation of the stable complex by active a-thrombin. 7Thrombin, which has a damaged substrate recognition site (the anion-binding exosite), did not form the complex and did not inhibit formation of the stable Complex formation was inhibited by the C-terminal complex by a-thrombin. dodecapeptide of hirudin, which has been shown to bind to the anion-binding A monoclonal antibody that blocks reactions of thrombin that involve exosite. the anion-binding exosite also inhibited formation of a stable complex of LTthrombin and the platelet-derived protease nexin. It is concluded that the anion-binding exosite of thrombin, a site that confers a high degree of specificity for substrates with a complementary site, binds to the platelet nexin 0 1991 Academic mess, Inc. prior to reaction of the catalytic site with the serpin.

Thrombin, actions

(2).

proteolytic cells

It

(6),

protease

exhibits

activity,

both

and it

as a mitogen

monocytes thrombin

the central

(8).

acts

for

Some of

of hemostasis

(1)

as an agonist

fibroblasts

these

(l),

procoagulant for

(7)

actions

biological

anticoagulant

platelets

and

involve

has multiple and

(5)

(3,4)

and endothelial

as a chemotactic

agent

a non-proteolytic

of

(9-12). When platelets

a serpin forms

(13),

are

referred

an SDS-stable

members +Current

of

the

address:

activated to

NIH,

by thrombin

as platelet-derived

complex

serpin

++To whom correspondence

with

family

of

NIAID,

Bldg

should

thrombin

(15)

proteins

(16).

4, Room 413,

or other

agonists,

protease

nexin

with

they (PN,)

characteristics PN, is

Bethesda,

considered

release

(14). of to

MD 20892.

be addressed.

Abbreviations: PN,, platelet-derivedprotease nexin; APMSFand APMS-thrombin, p-amidinophenyl-methyl-sulfonyl fluoride and itsthrombin derivative; Hepes, 4-(2-hydroxyethyl)-l-peperazineethanesulfonicacid;PAGE, polyacrylamidegel electrophoresis; SDS, sodium dodecyl sulfate. 0006-291x/91 Copyright All rights

for

domain

$1.50

0 1991 by Academic Press. Inc. of reproduction in any form reserved.

1198

PN, other be a

Vol.

No.

177,

secreted

3,1991

platelet

demonstrated

thrombin

that

of serpins

As with

all

the

that

reaction

the

is

its

(17),

RESEARCH

but

physiological

released

protease-serpin

catalytic

interaction,

site

of

it

has

target.

COMMUNICATIONS

not,

in

fact,

The function

is such a tiny

exosite"

inhibition

exosite,

of

a site

that

with

thrombin

evidence

a high

a complementary

that

fraction

from

degree

reaction

been

of PN, is

of the amount

paper

in addition, second

site

previously

II

cofactor

the catalytic

of thrombin

In this

the

a site

by heparin

R (23)

the

(14,18).

PN, requires,

(19,20,21),

18-20

assures

reactions, of thrombin

thrombin

and we present

"anion-binding

site

thrombin

BIOPHYSICAL

in plasma.

involves

contain

AND

inhibitor

because the amount

perplexing,

the

BIOCHEMICAL

site,

of specificity

shown

(22). is

with

a second

site

of

on thrombin

is

the

be involved

in

to The

anion-binding

a substrate

recognition

of thrombin

PN,

we demonstrate

for

substrates

that

site.

MTERIALS AND METHODS Highly purified human a-thrombin and y-thrombin were generously supplied by Dr. John W. Fenton II, Division of Laboratories and Research, New York State Department of Health, Albany, NY. Thrombin was labeled with 1251 or inhibited Monoclonal anti-a-thrombin antibody EST-6 was with APMSF as described (13). obtained from American Oiagnostica Inc., Greenwich, CT. Monoclonal antibody MT6 was the generous gift of Dr. Koji Suzuki, Institute for Enzyme Research, University of Tokushima, Tokushima, Japan. A tyrosine-sulfated dodecapeptide corresponding to the C-terminus of hirudin, Hir(54-65)SO,, was purchased from Bachem Feinchemikallen AG, Bubendorf, Switzerland. Human AT-III was obtained from Sigma Chemical Co., St. Louis, MO. The procedures outlined below have been described (13). Human platelets were obtained from fresh blood and washed in Hepes-buffered saline containing 1 mM EDTA. They were activated with the ionophore A23187 (Sigma) and separated from the supernatant solution, which contained secreted proteins. Thrombin-PN, complexes were formed by additon of labeled thrombin to the supernatant solution or to PN, that had been partially purified by heparin affinity chromatography. Formation of complexes was measured by SDS-PAGE chromatography by a modification of the method of Laemmli (24). Labeled complexes were identified by autoradiograpy. Bands were cut from the gel and counted to quantify the complex.

RESULTSANDDISCUSSION Covalently stable

complex

thrombin

with

inhibited with

a-thrombin

PN, (14,18),

PN, (Fig

(e.g. but

1; see also

it

ref

APMS-thrombin)

does not

inhibited the reaction We observed that 18).

form an SDS-

of active r-thrombin

awas

unable to form the SDS-stable complex (data not shown), but, in contrast to APMSthrombin, it was unable to inhibit formation of the stable complex of PN, and aWe hypothesized that i) APMS-thrombin (Fig 1). for a dissociable and essential interaction with

thrombin thrombin lacks

the

site

r-Thrombin, esterolytic

for

this

dissociable

a product

and amidolytic

of activity

competed

with

PN,, and ii)

active

T-thrombin

binding. limited

tryptic

(toward 1199

small

hydrolysis substrates)

of

a-thrombin,

comparable

has to that

BIOCHEMICALAND

Vol. 177, No. 3, 1991

52

BIOPHYSICALRESEARCH

COMMUNICATIONS

1.0

.AQ : .1./’ (, i’ 5 , ,-

.!5 z a

0.8

E s a if

0.6

0.4

I ..$

E

0.2

e E

0.0

01

15

10

Time (min)

Fig The effects of APMS-thrombin and 7-thrombin on the formation of an SDSstable complex of '251-labeled a-thrombin with PN,. The supernatant solution of ionophore A23187-activated platelets was incubated at 37°C with 5 nM labeled athrombin with either buffer (o), 50 nM APMS-thrombin (0) or 50 nM T-thrombin (A) mixed just prior to addition (all final concentrations). At the indicated times, aliquots were added to SDS sample buffer to stop the reactions. Samples were analyzed by SDS-PAGE/autoradiography. The radioactivity corresponding to a thrombin-nexin complex was measured in bands cut from the gels. u The effect of the C-terminal dodecapeptide of hirudin in formation of 500 PM Hir(54-65)SO, was incubated with 100 nM "'Ia thrombin-PN, complex. labeled a-thrombin for 10 min at 22%. An aliquot of this mixture was transferred to the supernatant solution of A23187-activated platelets to give final concentrations of 5 nM thrombin and 25 PM Hir(54-65)SO,. After the indicated times at 37“C, the amount of complex was measured as for Fig 1. The experiment was with thrombin alone (0) or thrombin with peptide (a).

of a-thrombin,

but it

susbstrates,

such

has been

exhibits

essentially

as fibrinogen

attributed

to destruction

anion-binding

exosite

cluster

of

cationic that

binding binding

amino

y-thrombin

exosite

(21).

exosite

could

Additional obtained

by demonstration

thrombin

with

Hir(54-65)SO, and X-ray

site it

from

because

derivatized only

inhibit

EST-6

with

site

T-thrombin

site

the

the

anion-

exosite

SDS-stable

of

of

hirudin,

inhibitor

of hirudin

was

complex

dodecapeptide thrombin

C-terminus

is

anion-

PN,.

anion-binding

of

a

It

(27). that

to

site,

of a modified

suggests for

affinity the

referred

(31-33), binds

to the

(34). also

EST-6

and with

1 thus of the

of T-thrombin site,

catalytic

C-terminal

a high that

amidolytic

thrombin weakly

Fig

some protein

recognition

because

formation

by the is

i)

the

binding

2) that

revealed antibody

reacts

of

of a-thrombin

of thrombin,

activity

fibrinogen

from

involvement

Hirudin

crystallography

does not

the

the

a-thrombin

secondary

inhibited

The monoclonal exosite but it

distant

(Fig

PN, was

exosite

acids

for

(28-30).

anion-binding

or

differs be the

for

of a substrate-recognition (19-21)

The experiment

support

activity

The modified

(25,26).

as the concluded

no catalytic

appears

inhibits

to react thrombin's

(35),

native

thrombin

with

and iii)

we found

1200

the anion-binding

action

activity (36)

ii)

with

EST-6 the

reacts

on fibrinogen, with

same affinity, that

EST-6

activebut reacted

Vol.

177,

No.

3, 1991

BIOCHEMICAL

0

AND

20

BIOPHYSICAL

40

60

RESEARCH

60

EST-6

COMMUNICATIONS

I 120

100

(nM)

Fig The effect of MoAb EST-6 on the formation of SDS-stable complexes of athrombin with PN, and AT-III. 100 nM lz51-labeled a-thrombin was incubated with EST-6 at twenty times the indicated concentrations for one hour at 4'C in 10 mM Hepes, 500 mM NaCl, 2 mM CaCl,, pH 7.4. An aliquot, sufficient to give 5nM thrombin and the indicated concentrations of EST-6, was incubated for 15 min at 37°C with AT-III (0) or with partially purified PN, (0) in 10 mM Hepes, 700 mM NaCl, 2 mM CaCl,, pH 7.4, containing 1 unit of heparin. Samples were analyzed

by SDS-PAGE/autoradiography. solution. in an enzyme-linked Arg77A

(the

inserted

immunosorptive

residues

are

residues

chain

(data

binding

not

shown), of

complex

a sequence

with

antithrombin

is

not

to be involved

formation (MT-6) the

complex

at still

another

thrombin-PN,

complex

(17).

The specificity residues there

is evidence

for

example,

of active in

proteases

which

reactive center of

the

reactive

center

loop

loop

a short

plasminogen other

plasma

(42).

(36)

site

also

inactive

with

serpins center

Similarly,

sites proteases

deletion

on the

activator

to

serpins

without

from

lip

of

be inhibited affecting

active

1201

reaction

of

exosite shown) that antibody closer

than

which

binds

on formation of the

strained

was

(16,40), has been

of complexes

in mutant

serpins

substituted follow abolished

activator activity

for

the

the reactive

of plasminogen lip

amino acid It

formation point,

of the

loop

be involved.

sequence

catalytic

a

a region

to the

site

of

Heparin,

center

serpin

anion-

(39),

a function

by plasminogen its

the

B-

27).

did not entirely

from the the

of the

by a monoclonal

may also

one

specificity

thrombin

anion-binding (data not

had no effect

More

with

formation

affect

can inhibit

(18,41). loop

in another,

the

not

(cf.

in a reactive

additional

that

segment

site

has been considered

a cleavage that

did

the

part

blocked

of thrombin

catalytic

site

of serpins

at and near

shown,

site

to the

to thrombin

EST-6

of

a large

EST-6

was not inhibited

to the thrombomodulin-binding exosite

represents

to Arg67-

chymotrypsinogen

a reaction in which the As a control, we found

(38).

of the thrombin-PN,

anion-binding

but

that

In contrast, III,

corresponding

with

A, B, and so forth)

(27,37).

3).

with the supernatant

a peptide

by equivalence

by letter

(Fig

were obtained

assay with

a-thrombin

thrombin known

results

numbered

indicated

exosite

thrombin-PN,

Similar

activator ability

inhibitor (43),

suggesting

of and

Vol.

177,

No.

3, 1991

involvment To our

of

other

knowledge

substrate

than the

recognition

binding

exosite

there

may possibly

be

site

This

raises

the possibility

wound

healing.

reactions into

would,

(44-47). into the

site

less

will

that

the function Either

a larger

may be

heparin

the

complex role

of actions

matrix,

with

(22).

anion-binding

serpin

for

PN, than

other

thrombin.

serpins,

proteases.

The

the

or a peptide

rather released

fibrinolysis

or

of protease-serpin

thrombin-PN,

platelet

by thiol-disulfide

complex.

of the complex

itself

and

secondary

of athrombin-PN,

of the products

secreted

the anion-

by the

in hemostasis,

that

a specific

II

cofactor

of that

other

of

involves

recognized

formation

significant

complex

extracellular

also

is production

an important

are examples

involvement

is

than assure

interaction.

the

be much less

thrombin

COMMUNICATIONS

reaction with

that

RESEARCH

protease-serpin

of specificity

nevertheless,

It

the of

reaction

degree

may have

There

in

report

its

of thrombin. formation

incorporated thus

in

there

recognition

its

previous

BIOPHYSICAL

in a protease-serpin

of a serpin

many circumstances

inhibition

site

active

is a high

Under

than

the

of thrombin

of thrombin

during

AND

only site

The significance exosite

BIOCHEMICAL

complex

thrombospondin, exchange

is and

(13,18).

ACKNOWLEDGMENTS We are grateful to Dr. John Fenton II for the gift of purified human aand r-thrombins and for helpful discussions and to Dr. Koji Suzuki for his cooperation in supplying his antibody. This research was supported by the United States Public Health Service, National Institutes of Health grant #HL-37250. REFERENCES Jackson, C.M., and Nemerson, Y. (1980) Annu. Rev. Biochem. 49, 765-811. Fenton, J.W. II (1981) Ann. N. Y. Acad. Sci. 370, 468-495. and Owen, W.G. (1981) hoc. Nat]. Acad. Sci. USA 78,2249-2252. Esmon, C.T., Marlar, R.A., Kleiss, A.J., and Griffen, J.H. (1982) Blood 59, 1067-1072. Davey, M.G., and Luscher, E.F. (1967) Nature (London) 216, 857-858. Weksler, B.B., Ley, C.W., and Jaffe, E.A. (1978) J. Clin. Invest. 62, 923-

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