Tryptase from rat mast cells converts bovine prothrombin to thrombin

Vol.

132,

No.

October

30,

2, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1985

Pages

613-619

TRYPTASE FROM RAT MAST CELLS CONVERTS BOVINE PROTHROMBIN TO THROMBIN Hiroshi

Kido*, Takashi

Naomi Morita**

Fukusen*, Nobuhiko Katunuma* and Sadaaki Iwanaga**

*Department of Enzyme Chemistry, Institute School of Medicine, The University Tokushima 770, Japan **Department Kyushu Received

September

9,

SUMMARY:The effect

for Enzyme Research, of Tokushima,

of Biology, Faculty of Science, University, Fukuoka 812, Japan

1985

of tryptase

purified

from rat peritoneal

mast cells on

bovine prothrombin was examined. Tryptase activated prothrombin, as evidenced by the increase in thrombin activity with a synthetic substrate, t-butyloxycarbonyl-Val-Pro-Arg-4-methylcoumaryl-7-amide. The apparent-p value toward bovine prothrombin and the kcat value were 2.3 uM and 46.3 s , respectively. Studies on the time course of prothrombin activation by tryptase and by activated factor X (Xa), and analysis of the activation products on sodium dodecyl sulfate gel electrophoresis showed that the process of activation of prothrombin by tryptase was similar to that by Xa except that an intermediate of 67,000 daltons was formed. 0 1985 Academic Press, Inc.

Mast cells

are widely

most vertebrates, and

protease

are postrated

mmatory

(tryptase)

to be important is

little

known

reaction.

in the

of

their

Recently,

and

chymostatin,

cause up to 100% inhibition

leupeptin

by anti-rat

IgE,

but

suggest

in the process

that

cause that of

in

F(ab')z

little

if

of

infla-

release

tryptase,

such as (6).

granules

degranulation,

of

chymase,

any inhibition

chymase in mast cell IgE-mediated

the

fragments

of histamine of

(2,5)

reaction.

inhibitor

inhibitors

of

(l-4)

granules

functions

that

tissue

(chymase)

cells

inflammatory

low-molecular-weight

and aprotinin,

These results involved

the

mast

concrete

we found

connective

protease in

anti-chymase

induced

in the

and chymotrypsin-like

trypsin-like

However,

distributed

is whereas

Abbreviations: IgE, immunoglobulin-E; Boc-, t-butyloxycarbonyl-; MCA, 4-methylcoumaryl-7-amide; SDS, sodium dodecyl sulfate. 0006-291X/85

613

$1.50

Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 132, No. 2, 1985

tryptase

BIOCHEMICAL

is not.

the biological histamine

Several function

cleavage

(9).

(9)

substrate

of activated

proteases protein

that

tested, of

inhibits

tryptase

its

and is

activity,

ties

with

with

a substrate

(2).

Therefore,

prothrombin

activator

activation,

prothrombin

a B-chain

of 259 residues

with

study

prothrombin trypstatin

with

inhibits

to

kininogen

is it

from

little

work we examined

rat

the

plasma

whereas

(lo),

ability

and

is

mast

of

pH

tryptase

The physiologfactor

Xa.

On

composed of

of 49 residues rat

activi-

at neutral

to a-thrombin,

from

mast cells,

tryptase

(EC 3.4.4.13).

in blood

tryptase

thrombin,

serine

an associated

Boc-Phe-Ser-Arg-MCA,

and A-chain

(7).

a specific

Boc-Val-Pro-Arg-MCA

is converted

showed that

or pre-

of trypsin-type

co-purified

of prothrombin

and

Boc-Ile-Glu-Gly-Arg-MCA

which

to thrombin

effects

(7,8)

(8)

hydrolyzes

substrates

it

with

to cause no apparent

trypstatin,

of trypsin,

along

on the

kininogen

of thrombin,

in this

studies

reported

X (Xa>,

whereas

a substrate

activate

verted

is

tryptase

other

and that

released

high-molecular-weight

factor

than

is

on plasma kininogen

Tryptase

we found

more rapidly

present

which

including

cells

and to destroy

Recently,

ical

tryptase, (6),

RESEARCH COMMUNICATIONS

have been made to determine

of human low-molecular-weight

kallikrein

to

of

from human mast

on prekallikrein

(lo),

attempts

by degranulation

of tryptase

AND BIOPHYSICAL

(11).

cells

the complex

also

of

The con-

tryptase

was ineffective.

MATERIALS AND METHODS The complex of tryptase with trypstatin was purified to homogeneity from rat peritoneal mast cells as described previously For separation of the complex into tryptase and trypstatin, (2). the complex eluted from arginine-Sepharose 4B was applied to a column of trypsinogen-CH-Sepharose 4B previously equilibrated with 50 mM Tris-HCl, pH 7.5. Tryptase free from trypstatin was recovered in the unabsorbed fraction and trypstatin was adsorbed to the column. Trypsinogen-CH-Sepharose 4B was prepared by coupling activated CH-Sepharose (Pharmacia.Fine Chemicals) with trypsinogen (Sigma Chemical Co.)(12). The preparations of a-thrombin (3,500 NIH units/mg, 93% titratable with p-nitrophenyl pl-guanidinobenzoate), prothrombin and factor X from bovine plasma were as 614

Vol. 132, No. 2, 1985

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

described previously (13). Bovine factor Xa was isolated by gel filtration on Sephadex G-100 from the reaction mixture for activation of factor X with factor X-activating coagulant protein, as described previously (14). Boc-Val-Pro-Arg-MCA was purchased from the Protein Research Foundation, Osaka, Japan. All other chemicals were the highest quality available. Prothrombin was converted to cc-thrombin at 37'C in 50 mM TrisHCl buffer, pH 8.0, containing 100 mM NaCl and 10 mM CaC12 in a total volume of 2.0 ml. Final concentration of 0.6 mM Boc-Val-ProArg-MCA and 2-50 uM prothrombin were added to the reaction mixture. The reaction was started by addition of tryptase and the fluorescence of 7-amino-4-methyl-coumarin liberated from the substrate was monitored in a Hitachi fluorescence spectrophotometer, model 650-lOS, equipped with a recorder. Emission at 460 nm was measured with excitation at 380 nm. SDS-gel electrophoresis of reaction products of prothrombin with tryptase or factor Xa was performed by the method of Laemmli (15), as modified by Ames (16). Protein concentrations were determined by the method of Lowry et al. (17). RESULTS AND DISCUSSION The kinetics complex thrombin thrombin,

of

tryptase

of prothrombin

activation

with

trypstatin

had no amidolytic

activity

Boc-Val-Pro-Arg-MCA,

by tryptase

are shown in Fig. on the

synthetic

and no autoactivation

Incubation

Time

and by a 1.

Pro-

substrate of pro-

(min)

Fig. 1: Kinetics of prothrombin activation by tryptase and the were started by complex of tryptase with trypstatin . Reactions addition of 1.8 rig/ml of tryptase () or of 1.8 ngfml of the complex of tryptase with trypstatin (----) in 2 ml of Tris-HCl containing 100 mM NaCl, 10 mM CaClz and 0.6 mM buffer, pH 7.5, Boc-Val-Pro-Arg-MCA. After incubation for 45 s, indicated by the "arrow", 1.6 uglml of prothrombin was added. Activity of thrombin was measured as described under Materials and Methods. 615

of

Vol.

132,

No.

2, 1985

BIOCHEMICAL

thrombin

was

Tryptase

and its

synthetic

during

complex

substrate.

mixture

of

for

increase

in

period

for

about

mixture of

additional activity

the

that

tryptase

that

trypstatin

1.

This

of

in

the

free

for

from

completely

buffer

rather

occurred

after

in

a lag to

and the

scarecely

slight

any

decrease These

in

results

had prothrombinase the

the

prothrombin

trypstatin

minute.

inhibited

after

in

resulted

trypstatin

in

of

with

another

reaction

increase

addition

but

incubation

basal

tryptase

activity,

on the

the

thrombin

increase

buffer

12 hr.

and time-dependent

the

contrast,

complex

thrombin after

over

up to

activity to

a significant

In

thrombin

of

COhlMUNlCATlONS

for

showed

substrate

Fig.

20 s.

of

substrate

in

37'C

of prothrombin

activity

shown

RESEARCH

at

trypstatin

45 s produced

as

activity,

with

and the

thrombin

BIOPHYSICAL

incubation

Addition

tryptase

incubation

the

detected

AND

show

activity

activation

of

and

prothrombin

by tryptase. To measure for

activation

tions

of

the of

kinetic

prothrombin,

prothrombin in

the

tryptase

in

50 mM Tris-HCl

presence

determined

difference

as the the

prothrombin with

prothrombin.

prothrombin measured

in under

of Xa but

the

conditions

in

activity

(Km) the

tryptase employed.

of

of

for

apparent 616

bovine

I).

The

of

than

Km value

were tryp-

without mixture

factor

Xa for

Va were

also

apparent was

proteolytic

higher

thrombin

reaction

prothrombin

of

1.4-fold

in

higher

efficiency that of

nM

100 mM NaCl

activity

and factor

(Table

were and 6.1

mixture

of

phospholipid

tryptase

The

tryptase

generated basal

tryptase

concentra-

containing

observed

coefficient

was

of

reaction

constants

same conditions

than

of

the

the

constant

(k,,t/Km)

between

absence

Michaelis that

newly

the

of

various

activity

of

The kinetic

of

pH 8.0,

substrate total

activity

mM Boc-Val-Pro-Arg-MCA

activities

synthetic and the

0.6

the

effects

buffer,

The

of

uM) on the of

and I.0 mM CaC12.

for

the

(2-50

examined

tase

constants

of Xa under tryptase

for

BIOCHEMICAL

Vol. 132, No. 2, 1985

AND BIOPHYSICAL

Table

Kinetic Kinetic

constants

for

I

activation

constant

of prothrombin

Tryptase

Km (PM) k,,t

RESEARCH COMMUNICATIONS

Xa

2.3

(s-l)

kcatlKm (5-l

PM-')

0.74

46.3

10.3

20.1

13.9

The rate of conversion of prothrombin to cc-thrombin was calculated by subtracting the basal activity of tryptase or Xa with Boc-Val-Pro-Arg-MCA as substrate from the total Km and kcat values were activity, as described in the text. determined as described in the text. prothrombin from rat process

was relatively mast cells

of

fibrosis

The time

course

low (2.3

PM),

suggesting

contributes

to blood

coagulation

in tissues,

such as pulmonary

of prothrombin

Xa was examined

by analysis

electrophoresis

(Fig.

2).

activation

of the

ABCDEFG

II

I

tryptase

or the fibrosis.

by tryptase

activation

The weight

that

products

ratio

or by

on SDS-gel

of prothrombin

to

JitLM

”

r*)

-3GK

-84K

Fig. the

2: SDS-gel electrophoresis

reaction

of

bovine

prothrombin

of products with

-28

K

-14

K

at various

tryptase

or with

stages in factor

Xa.

Prothrombin (4 ug) and factor Xa (40 ng) were incubated in 20 ul of 50 mMTris-HCl buffer, pH 7.5, containing 100 mMNaCl and 10 mM CaC12 for 0 min (A), 45 min (B), 2 hr (C), 4 hr (D), 6 hr (E) and 8 hr (F). Prothrombin (4 ug) and tryptase (1.5 ng) were incubated in 20 ul of buffer containing 100 mMNaCl and 10 mMCaC12 for 0 hr (H), 4 hr (I), 8 hr (J), 12 hr (K) and 16 hr (L). The reaction was stopped by boiling the mixtures and electrophoresis was done in 8% polyacrylamide gel with reduction. Lanes G and M: 6 ug and 2 ug of purified bovine thrombin, respectively. 617

Vol.

132,

No. 2, 1985

factor

BIOCHEMICAL

Xa was

1OO:l

and that

At zero

time

a single

band

(+2,000)

was

observed

on the

tryptase,

intermediates

daltons

were

mediate

of

8 hr,

67,000

whereas

that 12 hr

thrombin

2 (11)

of

thrombin

of daltons

daltons of

activation

39,000

of

mediate

of

67,000

pathway

of

prothrombin

the

result

of

prothrombin formed

by the

daltons,

activation tryptase

concomitant

Thus,

further

products are

thrombin-catalyzed

due to

was

as

is

whereas

1)

named are

studies formed

the

(fl,OOO)

the

other

daltons

Fig.

process that

formed

2.

The

reported

to

and u-thrombin

are

products,

as

such

1 (prothrombin

be required activation

tryptase-catalyzed

to of

proteolysis,

be

(20-23):

by thrombin-catalyzed

will

of

inter-

and an intermediate

prethrombin

during

26,000

that

2,

ct2 of

to

been

con-

fragment

by Xa and thrombin

prethrombin

1 o f 28,000

of

similar

in

for

of pre-

This

shown

by Xa has

(111,

a maximum

of

except

proteolyses

of Xa,

which fragment

formed

activation

fragment

prothrombin lysis.

was

incubation

incubation.

by tryptase

daltons

action

the

by Xa (18,19)

fragment-l.2

prothrombin 57,000

during

1 (11)

by

inter-

B-chain

prothrombin

fragment

increased

the

73,000

(?2,000)

The

to

of

2667:l.

prothrombin

The amounts

daltons,

daltons,

wt

further

increased

(fl,OOO)

prothrombin

4 hr.

disappeared.

(+l,OOO)

a mol of

during

daltons

was

and 57,000 for

disappeared

of prothrombin

activation

(+2,000)

COMMUNICATIONS

tryptase

with

incubation

and prothrombin

gradually

to

On activation

67,000

57,000

RESEARCH

prothrombin

gel.

and then

34,000

BIOPHYSICAL

prothrombin

after

of

at

14,000

of

daltons

centration

of

of

separated

AND

of minus proteo-

determine

prothrombin

which by

and which

to

proteolysis.

ACKNOWLEDGMENTS:We thank Ms. E. Inai for expert secretarial assistance. work was supported in part by a Grant-in-Aid (56880028) from the Ministry Education, Science and Culture of Japan.

This of

REFERENCES 1. Katunuma, N., Kominami, E., Kobayashi, K., Banno, Y., Suzuki, K Chichibu, K., Hamaguchi, Y., and Katsunuma, T. (1975) E;;. J. Biochem. 52: 37-50. 618

Vol.

132,

No. 2, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

2. Kido, H., Fukusen, N., and Katunuma, N. (1985) Arch. Biochem. Biophys. 239: 436-443. 3. Sanada, Y., Yasogawa, N., and Katunuma, N. (1978) Biochem. Biophys. Res. Commun. 82: 108-113. 4. Yurt, R.W., and Austen, K.F. (1977) J. Exp. Med. 146: 1405-1419. 5. Schwartz, L.B., Lewis, R.A., and Austen, K.F. (1981) J. Biol. Chem. 256: 11939-11943. 6. Kido, H., Fukusen, N., and Katunuma, N. (1985) Biochem. International 10: 863-871. 7. Maier, M., Spragg, J., and Schwartz, L.B. (1983) J. Immunol. 130: 2352-2356. 8. Schwartz, L.B., Maier, M., and Spragg, J. (1985) Adv. Exp. Med. Biol. in press. 9. Schwartz, L.B., Bradford, T., and Griffin, J.H. (1985) Biochem.Biophys. Res. Commun. 129: 76-81. 10. Iwanaga, S., Morita, T., Kato, H., Harada, T., Adachi, N., Sugo, T., Maruyama, I., Takada, K., Kimura, T., and Sakakibara, S. (1979) in Kinins-II: Biochemistry, and Clinical Aspects (Fujii, S., Moriya, H., and Suzuki, T., eds) pp. 147-163, Plenum, New York. 11. Jackson, C.M., and Nemerson, Y. (1980) Annu. Rev. Biochem. 49: 765-811. 12. Izzo, P., and Gantt, R. (1977) Biochemistry 16: 3576-3581. 13. Hashimoto, N., Morita, T., and Iwanaga, S. (1985) J. Biochem. 97: 1347-1355. 14. Morita, T., and Jackson, C.M. (1980) in Vitamin K Metabolism and Vitamin K-Dependent Proteins (Suttie, J.W., ed) pp. 120123, University Park Press, Baltimore. 15. Laemmli, U.K. (1970) Nature (London) 227: 680-685. 16. Ames, G.F-L.(1974) J. Biol. Chem. 249: 634-644. 17. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193: 265-275. 18. Morita, T., Nishibe, H., Iwanaga, S., and Suzuki, T. (1974) J. Biochem. 76: 1031-1048. 19. Owen, W.G., Esmon, C.T., and Jackson, C.M. (1974) J. Biol. Chem. 249 : 594-605. 20. Stenn, K.S., and Blout, E.R. (1972) Biochemistry 11: 4502-4515. 21. Esmon, C.T., Owen, W.G., and Jackson, C.M. (1974) J. Biol. Chem. 249: 606-611. 22. Kisiel, W., and Hanahan, D.J. (1974) Biochem. Biophys. Res. Commun. 59: 570-577. 23. Downing, M.R., Butkowski, R.J., Clark, M.M., and Mann, K.G. (1975) J. Biol. Chem. 250: 8897-8906.

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