Tài liệu Báo cáo khoa học: Tissue factor pathway inhibitor is highly susceptible to chymase-mediated proteolysis pptx

Tissue factor pathway inhibitor is highly susceptible
to chymase-mediated proteolysis
Tsutomu Hamuro1, Hiroshi Kido2, Yujiro Asada3, Kinta Hatakeyama3, Yuushi Okumura2,
Youichi Kunori4, Takashi Kamimura4, Sadaaki Iwanaga1 and Shintaro Kamei1
1
2
3
4

Therapeutic Protein Products Research Department, The Chemo-Sero-Therapeutic Research Institute, Kaketsuken, Japan
Division of Enzyme Chemistry, Institute for Enzyme Research, University of Tokushima, Japan
Department of Pathology, Faculty of Medicine, University of Miyazaki, Japan
Institute for Biomedical Research, Teijin Pharma Limited, Japan

Keywords
chymase; inflammation; protease inhibitor;
serine proteinase; tissue factor pathway
inhibitor (TFPI)
Correspondence
T. Hamuro, Therapeutic Protein Products
Research Department, The Chemo-SeroTherapeutic Research Institute, Kaketsuken,
1-6-1 Okubo, Kumamoto, 860-8568, Japan
Fax: +81 96 3449234
Tel: +81 96 3442189
E-mail:
(Received 20 December 2006, revised 12
March 2007, accepted 17 April 2007)
doi:10.1111/j.1742-4658.2007.05833.x

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor that primarily inhibits the extrinsic pathway of blood coagulation. It is synthesized by various cells and its expression level increases in
inflammatory environments. Mast cells and neutrophils accumulate at sites

of inflammation and vascular disease where they release proteinases as well
as chemical mediators of these conditions. In this study, the interactions
between TFPI and serine proteinases secreted from human mast cells and
neutrophils were examined. TFPI inactivated human lung tryptase, and its
inhibitory activity was stronger than that of antithrombin. In contrast,
mast cell chymase rapidly cleaved TFPI even at an enzyme to substrate
molar ratio of 1 : 500, resulting in markedly decreased TFPI anticoagulant
and anti-(factor Xa) activities. N-Terminal amino-acid sequencing and MS
analyses of the proteolytic fragments revealed that chymase preferentially
cleaved TFPI at Tyr159-Gly160, Phe181-Glu182, Leu89-Gln90, and
Tyr268-Glu269, in that order, resulting in the separation of the three individual Kunitz domains. Neutrophil-derived proteinase 3 also cleaved TFPI,
but the reaction was much slower than the chymase reaction. In contrast,
a-chymotrypsin, which shows similar substrate specificities to those of chymase, resulted in a markedly lower level of TFPI degradation. These data
indicate that TFPI is a novel and highly susceptible substrate of chymase.
We propose that chymase-mediated proteolysis of TFPI may induce a
thrombosis-prone state at inflammatory sites.

Tissue factor pathway inhibitor (TFPI) is the main
inhibitor of tissue factor-induced blood coagulation.
Human TFPI contains 276 amino acids that comprise
an acidic N-terminal domain followed by three tandem
Kunitz-type trypsin inhibitor domains and a C-terminal basic amino-acid cluster region [1]. The first
Kunitz domain is necessary for the inhibition of the
factor VIIa–tissue factor complex, which forms a tertiary complex with factor Xa, whereas the second
Kunitz domain inhibits factor Xa. TFPI also inhibits a

variety of serine proteases, such as trypsin, a-chymotrypsin, plasmin, and cathepsin G, demonstrating that
this inhibitor has a relatively broad spectrum of inhibition [2]. The third Kunitz domain as well as the C-terminal basic region is important for binding to heparin
[3–5]; however, whether this Kunitz domain possesses
an inhibitory activity is still unknown. TFPI is mainly

synthesized and secreted from endothelial cells [6]. In
addition, it is expressed in smooth muscle cells, fibroblasts, monocytes, and cardiomyocytes in response to

Abbreviations
pNA, p-nitroanilide; TFPI, tissue factor pathway inhibitor; TFPI-C, TFPI with truncated C-terminal basic-amino-acid region.

FEBS Journal 274 (2007) 3065–3077 ª 2007 The Authors Journal compilation ª 2007 FEBS

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Chymase-mediated proteolysis of TFPI

T. Hamuro et al.

various stimuli produced in inflammatory states [6–9].
It is well known that neutrophils and mast cells are
important effector cells at sites of vascular perturbation. These cells release secretory granules that contain
a variety of biologically active substances. In particular, neutrophil-derived proteolytic enzymes participate in the destruction of inflamed regions through the
degradation and inactivation of matrix proteins and
various protease inhibitors, including antithrombin, C1
inhibitor, heparin cofactor II, and a2-antiplasmin [10].
Previous reports have described the interactions of
TFPI with inflammatory cell-derived proteinases, such
as neutrophil elastase [11,12], cathepsin G [12], and
matrix metalloproteinases [13,14]. The experimental
conditions used in these studies, however, did not precisely mimic physiological conditions. Furthermore, little is known about the interactions between TFPI and
serine proteinases derived from the secretory granules
of inflammatory cells.
To investigate the functional role of TFPI in inflammation, we examined the interactions between TFPI

and several serine proteinases derived from mast cells
and neutrophils. Here, we demonstrate that TFPI
inhibited human lung tryptase. In contrast, the activity
of chymase was not inhibited by TFPI, and chymase
rapidly cleaved TFPI even at a low enzyme to substrate molar ratio, resulting in its inactivation. In addition, we identified the cleavage sites in TFPI, and
determined the apparent kinetic constant for its proteolysis by chymase. Neutrophil-derived proteinase 3
also cleaved TFPI, but the reaction rate was much
slower than that of chymase. These data identify TFPI
as a novel, highly susceptible substrate of chymase.
Thus, chymase-mediated degradation and inactivation
of TFPI may induce a thrombosis-prone state at
inflammatory sites.

Results
Inhibitory properties of TFPI on proteases
To investigate the inhibitory properties of TFPI against
human mast cell-derived and neutrophil-derived proteinases, inhibition assays were performed using appropriate synthetic substrates. For the mast cell-derived
proteinases, TFPI inhibited the activity of tryptase with
a 50% inhibitory concentration (IC50) of  10 lm,
whereas it did not inhibit the activity of chymase
(Fig. 1A,B). Next, we tested the effects of TFPI on the
amidolytic activities of elastase, cathepsin G, and neutrophil-derived proteinase 3. As shown in Fig. 1C,D,
TFPI inhibited the amidolytic activities of elastase
(IC50 ¼ 1.4 lm) and cathepsin G (IC50 ¼ 0.13 lm),
3066

which agrees with a previous report [12]. In contrast,
TFPI produced only weak inhibition of the amidolytic
activity of proteinase 3 (Fig. 1E), even though this protein is structurally similar to elastase and cathepsin G.
Inhibitory properties of TFPI derivatives

on tryptase
It is well known that the conversion of tryptase into
an active tetrameric form requires sulfated polysaccharides such as heparin [15,16]. On the other hand, TFPI
strongly binds to heparin via its C-terminal basic
amino-acid cluster region and the third Kunitz domain
[3–5]. Therefore, we tested the specificity of the inhibition of tryptase by TFPI using TFPI and a TFPI
derivative. In the presence of a relatively low concentration of heparin (0.5 lgỈmL)1), TFPI strongly inhibited tryptase activity throughout a 60 min incubation
(Fig. 2A), whereas TFPI-C, which lacked the C-terminal basic region and ended at Lys249, showed no
inhibitory activity (Fig. 2B). In addition, antithrombin
inhibited tryptase activity with an IC50 of 6.5 lm
(Fig. 2C); however, its inhibitory activity was weaker
than that of TFPI (IC50 ¼ 1.7 lm). In the presence of
excess heparin (500 lgỈmL)1), the activity of tryptase
was not affected by TFPI, TFPI-C, or antithrombin
(Fig. 2A–C). These results strongly suggest that TFPI
converted tryptase into an inactive monomer by
removing the ‘essential heparin’, which was necessary
for the tetramerization of tryptase, whereas an excess
of free heparin prevented TFPI from accessing the
‘essential heparin’. These results were consistent with a
previous report that used heparin antagonists [17].
Proteolysis of TFPI by chymase and other
proteinases
To examine whether TFPI is degraded by mast cellderived and neutrophil-derived proteinases, each proteinase was incubated with TFPI at a molar ratio of
1 : 500. Surprisingly, chymase rapidly cleaved TFPI,
even at a low enzyme to substrate ratio. As shown in
Fig. 3A, TFPI was cleaved by chymase within 15 min,
as evidenced by the two smeared bands (bands 1 and
2) with molecular masses of 20–30 kDa observed on
SDS ⁄ PAGE. Subsequently, the level of the approximately 15-kDa band increased, and TFPI (43 kDa) as

well as bands 1 and 2 disappeared entirely after incubation for 20 h. These results indicate that TFPI was
completely converted into fragments with molecular
masses of  15 kDa by chymase. Chymostatin, an
inhibitor of chymotrypsin-type serine proteinases, completely blocked the chymase-mediated proteolysis of

FEBS Journal 274 (2007) 3065–3077 ª 2007 The Authors Journal compilation ª 2007 FEBS


B

120
100
80
60
40
20

120
100

0

1
0.1
TFPI (µM)

60
40
20

10

100
80
60
40
20

100
80
60
40
20
0

0.1
1
TFPI (µM)

10

0

E

120

0
0

0.1
1
TFPI (µM)

10

0

0.1
1
TFPI (µM)

10

120

Proteinase 3 activity
(% of control)

Cathepsin G activity
(% of control)

80

120

0

0

D

C
Elastase activity
(% of control)

Chymase-mediated proteolysis of TFPI

Tryptase activity
(% of control)

A
Chymase activity
(% of control)

T. Hamuro et al.

100
80
60
40
20
0

0

0.1
1
TFPI (µM)

10

Fig. 1. Inhibition of mast cell-derived and neutrophil-derived proteinases by TFPI. Each proteinase was incubated with various concentrations
of TFPI at 37 °C, and the residual proteinase activity was measured. (A) Chymase (7 nM) was assayed using Suc-Ala-Ala-Pro-Phe-pNA
(5 mM). (B) Tryptase (14 nM) was assayed using H-D-Ile-Pro-Arg-pNA (0.15 mM). (C) Neutrophil elastase (40 nM) was assayed using MeOSuc-Ala-Ala-Pro-Val-pNA (0.6 mM). (D) Cathepsin G (167 nM) was assayed using Suc-Ala-Ala-Pro-Phe-pNA (0.6 mM). (E) Neutrophil-derived
proteinase 3 (100 nM) was assayed using MeO-Suc-Ala-Ala-Pro-Val-pNA (5 mM). Data are presented as the mean ± SD from three independent experiments.

B

120

100

100
80
60
40
20
0

Tryptase activity
(% of control)

Fig. 2. Inhibition of tryptase by TFPI derivatives and antithrombin. TFPI (A), TFPI-C (B),
and antithrombin (C) were incubated with
tryptase in the presence of 0.5 lgỈmL)1 (d)
or 500 lgỈmL)1 heparin (s) for 60 min. A
chromogenic substrate was then added, and
the amidolytic activity of tryptase was
measured. Data are presented as the

mean ± SD from three independent experiments.

80
60
40
20
0

0

C

120

Tryptase activity
(% of control)

Tryptase activity
(% of control)

A

1
TFPI (µM)

10

0

1

Antithrombin (µM)

10

0

1
TFPI (µM)

10

120
100
80
60
40
20
0

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Chymase-mediated proteolysis of TFPI

A

T. Hamuro et al.

min
Mr 0 15 30 60 120 180 1200

B

min
Mr 0 15 30 60 120 180

C

kDa

kDa

kDa

97
66
45

97
66
45

97
66
45

30

Band 1 30

30

20.1

Band 2 20.1

20.1

14.4

14.4

min
Mr 0 15 30 60 120 180

14.4

D

min
Mr 0 15 30 60 120 180

E

min
Mr 0 15 30 60 120 180

F

kDa

kDa

kDa

97
66

97
66

97
66

45

45

45

30

30

30

20.1

20.1

20.1

14.4

14.4

min
Mr 0 15 30 60 120 180

14.4

Fig. 3. Cleavage of TFPI by mast cell-derived and neutrophil-derived serine proteinases. Chymase (A), tryptase (B), a-chymotrypsin (C), neutrophil elastase (D), cathepsin G (E), or proteinase 3 (F) at a concentration of 7 nM was incubated with TFPI (3.5 lM) for the indicated times
at 37 °C. Proteins were separated on 15–25% polyacrylamide gels under reducing conditions and the gels were stained with Coomassie Brilliant Blue. Intermediate degradation products were designated as bands 1 and 2 (A). Mr, Low molecular mass marker.

TFPI (data not shown). In addition, neither human
mast cell tryptase (Fig. 3B) nor a-chymotrypsin
(Fig. 3C) degraded TFPI. In the same analysis, elastase
cleaved TFPI, resulting in the appearance of three new
bands on SDS ⁄ PAGE which were estimated to be
38 kDa, 12 kDa, and 10 kDa (Fig. 3D). This result
agrees with a previous report from Higuchi et al. [11].
Petersen et al. [12] previously reported that TFPI was
degraded by cathepsin G; we, however, did not detect
any degradation products after incubating TFPI with
cathepsin G (Fig. 3E) at a similar enzyme to inhibitor
molar ratio (1 : 20, data not shown). This discrepancy
may be due to a difference in the experimental materials. In addition to these results, we found that proteinase 3 produced a similar digestion pattern on SDS ⁄
PAGE to that of elastase, although the digestion was

less efficient (Fig. 3F). N-Terminal sequencing of the
3068

reaction products after a 20-h incubation demonstrated
that proteinase 3 primarily cleaved TFPI between
Thr87 and Thr88, which was the cleavage site targeted
by elastase. In addition, trace amounts of peptide
sequences starting with Leu90, Asp157, or Asp194
were found. Of the proteinases examined in this study,
chymase most rapidly and specifically processed TFPI.
In the light of this finding, we further characterized the
chymase-mediated proteolysis of TFPI.
Characterization of the chymase-mediated
proteolysis of TFPI
To characterize the proteolysis of TFPI by chymase,
we digested TFPI with chymase for 20 h and separated
the resulting peptides using RP-HPLC. As shown in
Fig. 4A, six major peptide peaks were separated using

FEBS Journal 274 (2007) 3065–3077 ª 2007 The Authors Journal compilation ª 2007 FEBS


Chymase-mediated proteolysis of TFPI

A

B

Mr

Pe
ak
Pe I I I
ak
Pe I V
ak
Pe V
ak
VI

T. Hamuro et al.

kDa
97
66
45

Peak V Peak VI

2

Peak IV
40

Peak I Peak II

1

20
0

0
10

20

30

40

50

60

30
CH3 CN (%)

Absorbance at 214nm

Peak III

3

20.1

14.4

70

Elution time (min)

Fig. 4. Separation of TFPI degradation products produced by treatment with chymase. (A) TFPI digested with chymase for 20 h was applied
to a Vydac C8 column that had been equilibrated with 0.1% trifluoroacetic acid. The products were eluted with 0.1% trifluoroacetic acid containing a linear concentration gradient of acetonitrile from 0% to 50%. (B) SDS ⁄ PAGE of the peak III, IV, V, and VI fractions after RP-HPLC.
Mr, Low molecular mass marker.

this type of chromatography. Four of them, designated
peaks III, IV, V, and VI, all migrated at  15 kDa
during SDS ⁄ PAGE (Fig. 4B). The broad peak IV was
also visualized as a smeared band on the gel, even
though only a single N-terminal residue was detected
for this fragment. Because recombinant TFPI contains
a variety of carbohydrate chains, this fragment may
have included N-linked carbohydrate chains [18]. To
identify the chymase cleavage sites in TFPI, the fragments that resulted in these six peaks were analyzed to
determine their amino-acid compositions, N-terminal
sequences, and mass spectra. The results of these analyses are summarized in Table 1. Peaks III and IV corresponded to the third and second Kunitz domains of
TFPI, respectively. Multiple MS signals were observed
for the peak IV fragment, supporting the idea that this
fragment was glycosylated. Peaks V and VI both corresponded to the first Kunitz domain of TFPI; only
peak VI, however, was consistent with the calculated

MS value of this domain. We assumed that the peak V
fragment had an O-linked carbohydrate on Thr14 for
two reasons: (1) N-terminal sequencing of this fragment did not produce a signal corresponding to Thr14,
although such a signal was observed for the peak VI
fragment; (2) the difference between the MS values for
these two fragments (948 Da) perfectly matched the
mass of a ubiquitous O-linked carbohydrate chain,
Gal1–3GalNAc, with two N-acetylneuraminic acid residues. On the basis of these observations, we believe
that the recombinant TFPI carried an O-linked carbohydrate chain on Thr14. Moreover, the peak heights

and the areas under the peaks suggested that about
half of the TFPI carried the O-linked carbohydrate
chain on Thr14 (Fig. 4A). Peak II corresponded to the
third Kunitz domain without the C-terminal basic
region (Table 1), and presumably resulted from a trace
amount of contaminating TFPI-C [4]. In addition,
peak patterns obtained using RP-HPLC after 48 h of

Table 1. N-Terminal amino-acid sequences and mass spectra of degradation products. The individual fragments designated in Figs 2 and 3
were separated by RP-HPLC, and the amino-acid sequences and mass spectra were determined.
Fragment

Sequence

Cleavage site

Observed mass (m ⁄ z)

Calculated mass (m ⁄ z)

Band 1a

DSEEDEE

–

–

–

a

GTQLNAV

Tyr159-Gly160

–

–

Peak I

EEIFVKN

Tyr268-Glu269

1010.61

1010.17

Peak II

EFHGPSW

Phe181-Glu182

7778.76

7778.72

Peak III

EFHGPSW

Phe181-Glu182

10031.02

Band 2

Deduced structure

10032.50
b

Peak IV

QQEKPDF

Leu89-Gln90

9705–12656

–

Peak V

DSEEDEE

–

11334.61

–

Peak VI

DSEEDEE

–

10386.94

10386.63

a

Intermediate degradation products;

b

multiple signals were observed.

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Chymase-mediated proteolysis of TFPI

T. Hamuro et al.

Anticoagulant activity of the TFPI degradation
products produced by chymase treatment
The effects of the chymase-mediated degradation on
TFPI function were evaluated by testing the residual
anticoagulant activities of the fragments and also by
determining the amount of residual TFPI antigen
using an ELISA. Figure 5 shows the time course of
the decrease in anticoagulant and anti-(factor Xa)
activities determined under conditions in which 70 nm
TFPI was incubated at 37 °C with 7 nm chymase. As
shown in Fig. 5A, TFPI antigen promptly disappeared; the two fragments generated by cleavage at
Tyr159-Gly160 were not detected with this ELISA system. The anticoagulant and anti-(factor Xa) activities
also decreased in a time-dependent manner; after 5 min,
however, these activities decreased at relatively slow
rates (Fig. 5B,C). In particular, 20% of the anti(factor Xa) activity was observed after 120 min, suggesting that the digested TFPI was still able to inhibit
the protease. Petersen et al. [2] reported that a single
Kunitz domain can act as a protease inhibitor, although
its activity was weaker than that of the full-length
protein. These results indicate that chymase rapidly
reduces, but does not completely eliminate, the anticoagulant activity of TFPI.
3070

TFPI antigen
(% of control)

A

100

80
60
40
20
0
0

30

60

90

120

0

30

60

90

120

0

30
60
90

Incubation time (min)

120

Anticoagulant activity
(% of control)

B
100
80
60
40
20
0
C

Anti-factor Xa activity
(% of control)

incubation were essentially identical with those
obtained with a 20-h incubation (data not shown),
implying that chymase acted on specific cleavage sites
in TFPI. To clarify the order in which chymase processed these sites in TFPI, we next separated the intermediate fragments (Fig. 3A, bands 1 and 2) using
RP-HPLC after incubation with the enzyme for 6 h.
N-Terminal sequencing analyses identified bands 1 and
2 as the first and second Kunitz domains and the third
Kunitz domain, respectively (Table 1). The sample
obtained after a 3-h incubation was also subjected to
N-terminal sequencing. In addition to the original
N-terminal sequence of native TFPI, four unique

sequences starting with Gln90, Gly160, Glu182, and
Glu269 were observed. These sequences were present
at an approximate molar ratio of 2 : 5 : 4 : 1, respectively. These data revealed that chymase first cleaved
TFPI at Tyr159-Gly160 to generate two molecules
(bands 1 and 2), which was followed by cleavage at
Phe181-Glu182 and Leu89-Gln90 to generate the fragments corresponding to peaks IV, V, and VI. Finally,
chymase-mediated cleavage at Tyr268-Glu269 generated the peaks I and III. Taken together, these data
indicate that chymase selectively cleaved TFPI into five
fragments that were not disulfide linked, three of
which contained individual Kunitz domains.

100
80
60
40
20
0

Fig. 5. Effects of chymase on the anticoagulant and anti-(factor Xa)
activities of TFPI. TFPI (70 nM) was incubated with chymase (7 nM)
at 37 °C. After various incubation times, the reaction was terminated with chymostatin, and aliquots of the sample were subjected
to ELISA, a dilute tissue factor clotting assay, and anti-(factor Xa)
assay (see Experimental procedures). (A) Residual TFPI antigen. (B)
Residual anticoagulant activity of TFPI. (C) Residual anti-(factor Xa)
activity of TFPI. Data are presented as the mean ± SD from three
independent experiments.

Kinetic analyses of the degradation of TFPI
by inflammatory proteinases
To quantify the abilities of chymase, elastase, and proteinase 3 to proteolytically cleave TFPI, we performed

kinetic analyses using ELISAs. With this method,
kinetic constants were calculated as apparent values,

FEBS Journal 274 (2007) 3065–3077 ª 2007 The Authors Journal compilation ª 2007 FEBS


T. Hamuro et al.

Chymase-mediated proteolysis of TFPI

chymase occurred in the presence of every polysaccharide tested. Unfractionated heparin and low-molecularweight heparin slightly delayed the chymase-mediated
proteolysis of TFPI over the first 60 min; after 180 min,
however, the levels of residual TFPI antigen in these
samples were the same as observed in the control
sample (Fig. 6B). These results suggest that mast cellderived heparin and cell-surface heparan sulfate do not
prevent the proteolysis of TFPI by chymase.

Table 2. Apparent kinetic constants for the proteolytic cleavage of
TFPI by chymase, neutrophil elastase, and proteinase 3. The velocity of TFPI degradation was measured using an ELISA as described in Experimental procedures. Kinetic constants were
calculated from a Lineweaver–Burk plot. Values are expressed as
the mean ± SD from three independent experiments.

Enzyme

Km
Substrate (lM)

Chymase
TFPI
Elastase

TFPI
Proteinase 3 TFPI

kcat
(min)1)

kcat ⁄ Km
(lM)1Ỉmin)1)

5.01 ± 0.84 23.16 ± 1.98 4.62
2.00 ± 0.06 10.20 ± 0.71 5.10
17.47 ± 4.46 8.10 ± 1.11 0.46

Discussion

because the efficiency of enzymatic proteolysis was
estimated on the basis of the amount of remaining
TFPI antigen. As shown in Table 2, the apparent catalytic efficiency (kcat ⁄ Km) of chymase was almost equivalent to that of elastase. On the other hand, although
the enzymatic properties of proteinase 3 are similar to
those of elastase, this proteinase showed lower activity
toward TFPI.

Mast cells, which reside mainly in connective tissue
matrices, lung, heart, and epithelial surfaces, are
effector cells that participate in innate and acquired
immunity [23–26]. In pathological conditions, such as
inflammation, fibrosis, and malignancy, mast cells as
well as neutrophils and macrophages accumulate at
the affected sites. Recent studies indicate that mast
cells also accumulate at sites of atrial appendages

[27], deep venous thrombosis [28], periprostate vein
thrombosis [29], and atherosclerotic plaques [30–33].
These findings imply that mast cells are involved in
thrombosis and fibrinolysis. In fact, mast cells express
tissue-type plasminogen activator and urokinase-type
plasminogen activator receptor [34]. Little, however,
is known about the functional roles of proteinases
released from mast cell granules during thrombosis
and other pathological states. In this report, we
focused on the reactivity of TFPI with serine proteinases released from mast cells.

Effects of sulfated polysaccharides

kDa
97

Mr

TF
PI
Bu on
ffe ly
UF r
H

A

LM
W
He H

pa
Ch ran
su
o
De ndr lfa
rm oit te
Hy at in
al an sul
u
f
De ron sulf ate
xt
i a
ra c a te
ci
n
su d
lfa
te

TFPI is thought to localize on various cell surfaces,
especially the surfaces of endothelial cells, by binding to
sulfated proteoglycans such as heparan sulfate [19,20].
In addition, sulfated polysaccharides bind to TFPI and
enhance its anticoagulant activity in vitro [21,22].
Therefore, we investigated whether sulfated polysaccharides affected the chymase-mediated degradation of
TFPI. As shown in Fig. 6A, cleavage of TFPI by

B

100

45

30

20.1

TFPI antigen
(% of control)

66

80
60
40
20
0

14.4

0

60
120
180
Incubation time (min)

Fig. 6. Effects of various polysaccharides on chymase-mediated proteolysis of TFPI. (A) TFPI (3.5 lM) was incubated with chymase (7 nM)
for 60 min at 37 °C in the presence of each polysaccharide (100 lgỈmL)1). Proteins were separated on 15–25% polyacrylamide gels under

reducing conditions, and the gels were stained with Coomassie Brilliant Blue. The arrow indicates TFPI. (B) TFPI (70 nM) was incubated with
chymase (7 nM) in the presence or absence of heparin (100 lgỈmL)1) for up to 180 min. After various incubation times, the reaction was terminated with chymostatin, and the level of TFPI that remained was measured using an ELISA. (d) Control incubation; (j) incubation with
low molecular weight heparin (LMWH); (h) incubation with unfractionated heparin (UFH). Mr, Low molecular mass marker.

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T. Hamuro et al.

We first found that TFPI inactivates the amidolytic
activity of tryptase, presumably by removing heparin;
heparin or acidic polysaccharides allow tryptase to
form a stabilized noncovalent tetramer and are indispensable for tryptase activity [15,16]. Owing to the
loss of heparin, tetrameric tryptase rapidly and irreversibly dissociates into inactive monomers [35]. For
example, polybrene and protamine convert tetrameric
tryptase into monomers, resulting in the loss of tryptase activity [17]. The conformational structure of
b-tryptase suggests that the active site of each tryptase
monomer is largely inaccessible to macromolecular
inhibitors [16], which probably explains why tryptase
is resistant to endogenous proteinase inhibitors, such
as a1-proteinase inhibitor and antithrombin [36,37].
Therefore, this inactivating process has been proposed to be a control system that regulates tryptase
activity in vivo. In this study, TFPI, but not TFPI-C,
inactivated the amidolytic activity of tryptase, suggesting that the domain responsible for the inactivation was the C-terminal basic-amino-acid cluster
region. A synthetic peptide representing Lys254 to
Met276, however, was rapidly degraded by tryptase

(data not shown). This synthetic peptide probably
did not mimic the native structure of the C-terminal
region of TFPI, because TFPI was not cleaved by
tryptase in this study (Fig. 3B). The mechanism by
which TFPI inactivates tryptase requires further
investigation.
Secondly, we found that chymase efficiently cleaved
TFPI, even at a very low enzyme to substrate molar
ratio (1 : 500). As shown in Fig. 7, TFPI is known to
be degraded by several proteinases, including thrombin, plasmin, factor Xa, matrix metalloproteinases,
and neutrophil elastase [11–14,38–40]. Those results,
however, were obtained from reactions performed at
high enzyme to substrate molar ratios, or after long
incubations. The present study revealed that chymase
selectively cleaves TFPI at four peptide bonds
(Tyr159-Gly160, Phe181-Glu182, Leu89-Gln90, and
Tyr268-Glu269 in that order), which separated the
three individual Kunitz inhibitor domains and abolished the anticoagulant activity of TFPI. The previously reported natural substrates of human chymase
include angiotensin I [41], bradykinin [42], C1-inhibitor
[43], interleukin-1b [44], neurotensin [45], interstitial
procollagenase (proMMP-1) [46], kit ligand [47], big
endothelins [48], type-I procollagen [49], a2-macroglobulin [50], profilin [51], albumin [52], and connective
tissue-activating peptide III [53]. The cleaving sites of
these natural substrates are summarized in Table 3.
Using a combinatorial peptide screening method, Raymond et al. [52] demonstrated that chymase preferen3072

IIa, Pm
MMPs

T14

Xa, Pm
MMPs

L89-Q90
Y159-G160

F181-E182

Pm
IIa

Y268-E269

Fig. 7. Schematic structure of TFPI and cleavage sites by chymase.
The cleavage sites in TFPI are summarized in this figure. Data
obtained in this study including the four chymase cleavage sites
are shown below the TFPI structure, whereas previously determined data are shown above the TFPI structure. The thick arrows
indicate the locations of the sites cleaved by chymase, which
include the amino acids and residue numbers. The open circles and
branches indicate O-linked glycosylation sites and N-linked glycosylation sites, respectively. Our findings suggest that the threonine
residue at amino-acid position 14 carried an O-linked carbohydrate
in half of the TFPI molecules used here. The solid bar indicates a
‘hot region’, which contains cleavage sites for thrombin (IIa), plasmin (Pm), factor Xa (Xa), neutrophil elastase, proteinase 3, and
chymase. The thin arrows indicate the cleavage sites for each proteolytic enzyme. MMP, Matrix metalloproteinase.

tially acts at sites with Tyr or Phe as the P1 residue,
which is supported by the presence of these residues at
the P1 positions in the natural substrates (Table 3). In
agreement with those results, we found that three of

the four chymase cleavage sites in TFPI have either
Tyr or Phe as the P1 residue. At the fourth site,
chymase cleaved TFPI between Leu89 and Gln90.
Interestingly, the region containing Lys86-Thr87Thr88-Leu89-Gln90 appears to be a ‘hot region’,
because it contains cleavage sites for thrombin, plasmin, factor Xa, and elastase in addition to chymase
Table 3. Sites of hydrolysis of natural substrates of human chymase.
P1 site

P4–P1_P1¢

Substrate

Reference

Tyr

NEAY_V
RRPY_I
VVPY_G
VGFY_E
RETY_G
VDNY_G
KIAY_E
IHPF_H
FSPF_R
KMLF_V
TKPF_M
RVGF_Y
PSLF_E
QFVL_T

LKSL_S
KTTL_Q
PSVW_A

Interleukin 1b
Neurotensin
Endothelin-1
a2-Macroglobulin
Albumin
TFPI
TFPI
Angiotensin I
Bradykinin
C1 inhibitor
Kit ligand
a2-Macroglobulin
TFPI
Procollagenase
Procollagen 1a
TFPI
Profilin

[44]
[45]
[48]
[50]
[52]
This
This
[41]

[42]
[43]
[47]
[50]
This
[46]
[49]
This
[51]

Phe

Leu

Trp

work
work

work

work

FEBS Journal 274 (2007) 3065–3077 ª 2007 The Authors Journal compilation ª 2007 FEBS


T. Hamuro et al.

(Fig. 7). Presumably, this region has a distinct conformation and is exposed on the surface of the molecule,
making it highly susceptible to attack by these proteinases. It was also reported that the P2 and P3 subsite

preferences of chymase were Thr ⁄ Pro and Thr ⁄ Glu ⁄
Ser, respectively [52]. This could explain why TFPI
was cleaved by chymase at Leu89-Gln90, because both
of the P2 and P3 subsites are Thr.
TFPI-b is an alternatively spliced form of TFPI
that lacks the third Kunitz domain and the C-terminal portion of TFPI and instead contains a glycosylphosphatidylinositol anchor [54]. It is thought that
TFPI-b binds and localizes to the cell surface via its
glycosylphosphatidylinositol anchor domain. Because
the N-terminal 181 amino acids of TFPI-b are identical with those of TFPI, TFPI-b has at least two
chymase cleavage sites, and chymase might be able
to release TFPI-b from the cell surface. The interaction between chymase and TFPI-b requires further
elucidation.
In addition, we investigated the effects of sulfated
polysaccharides on the interaction between TFPI and
chymase, because both of these proteins bind to heparin [55,56]. Heparin, which is produced and secreted
by mast cells, did not inhibit the cleavage of TFPI by
chymase. Moreover, heparan sulfate did not influence
the proteolysis of TFPI. It was reported that heparin
has no affect on the amidolytic activity of chymase
for a chromogenic substrate, whereas it inhibited the
chymase-mediated proteolysis of casein and angiotensin I [56,57], suggesting that the regulation of chymase activity by heparin is dependent on the
substrate. Therefore, cell-surface TFPI, which is bound
to proteoglycans, could be cleaved by chymase.
Although Valentin & Schousboe [58] reported that
TFPI interacts with acidic phospholipids such as phosphatidylserine in vitro, we found that phosphatidylserine did not affect the cleavage of TFPI (data not
shown).
The human gastrointestinal tract contains numerous
mast cells, which are located primarily in the lamina
propria mucosa. We confirmed that a large number
of chymase-positive mast cells are located around

microvessels in the lamina propria mucosa, and TFPI
was detected on the intraluminal surface of these
microvessels (K. Hatakeyama and Y. Asada, unpublished data). It was previously reported that human
intestinal mast cells produce and release tumor necrosis factor-a in response to Gram-negative bacteria
such as Escherichia coli [59]. Furthermore, tumor necrosis factor-a induces the expression of tissue factor
on vascular endothelial cells [60]. Clot formation
resulting from tissue factor induction and chymase-

Chymase-mediated proteolysis of TFPI

mediated proteolysis of TFPI might be a protective
function of intestinal mast cells against bacterial invasion into the bloodstream.
In conclusion, the present study suggests that the
presence of mast cells and the associated release of
chymase may accentuate local thrombosis due to the
local inactivation of TFPI at inflammatory sites.

Experimental procedures
Materials
S-2288 (H-d-Ile-Pro-Arg-pNHCl where pNA is p-nitroanilide) and S-2222 [Bz-Ile-Glu(GlucOMe)-Gly-Arg-pNHCl]
were obtained from Chromogenix AB (Stockholm, Sweden).
Succinyl-Ala-Ala-Pro-Phe-pNA, methoxysuccinyl-Ala-AlaPro-Val-pNA, unfractionated heparin, and low molecular
weight heparin (average molecular mass 3000 Da) were
purchased from Sigma-Aldrich (St Louis, MO, USA).
Chymostatin was purchased from Peptide Institute, Inc.
(Osaka, Japan). HemolianceÒ human control plasma was
obtained from Instrumentation Laboratory (Lexington,
MA, USA). Dad thromboplastinỈC plus was purchased
from Dade International Inc. (Miami, FL, USA), and heparan sulfate, chondroitin sulfate A, dermatan sulfate, and
hyaluronic acid were obtained from Seikagaku kogyo Co.

(Tokyo, Japan). Sodium dextran sulfate was purchased
from ICN Biochemicals, Inc. (Aurora, OH, USA). All
other chemicals were of analytical grade or of the highest
quality commercially available.

Proteins
Human lung tryptase, human neutrophil elastase, and cathepsin G were obtained from Calbiochem (La Jolla, CA,
USA). Human neutrophil proteinase 3 was purchased from
Athens Research and Technology (Athens, GA, USA).
Bovine a-chymotrypsin was obtained from Worthington
Biochemical Corp. (Lakewood, NJ, USA). Recombinant
human chymase was expressed in Trichoplusia ni insect cells
using a baculovirus expression system and purified from the
culture medium as described previously [61]. Activated
human factor X (factor Xa) was prepared by incubating
purified factor X with Russell’s viper venom factor X activator (Haematologic Technologies, Essex Junction, VT, USA)
and then separating factor Xa by gel filtration on a column
of Sephacryl S-200 (Amersham Biosciences, Piscataway,
NJ, USA) as described in a previous paper [62]. Human
antithrombin was purified from human plasma using a
procedure based on heparin affinity chromatography [63].
Recombinant human TFPI was expressed in Chinese hamster ovary (CHO) cells and purified from the culture medium
as described previously [4]. TFPI-C, which lacked the C-terminal basic region and ended at Lys249, was separated from

FEBS Journal 274 (2007) 3065–3077 ª 2007 The Authors Journal compilation ª 2007 FEBS

3073


Chymase-mediated proteolysis of TFPI

T. Hamuro et al.

full-length TFPI [4]. TFPI expressed in CHO cells had
N-linked carbohydrate chains at Asn117 and Asn167, and
O-linked carbohydrate chains at Ser174 and Thr175 [18].

Inhibition assay of TFPI
All inhibition experiments were performed in Tris ⁄ NaCl
buffer (50 mm Tris ⁄ HCl containing 150 mm NaCl, pH 7.5)
at 37 °C in 96-well microtiter plates. TFPI was incubated
with each proteinase and synthetic substrate, and the
velocities of the initial reactions were measured with a
THERMOmax microplate spectrometer (Molecular Devices,
Sunnyvale, CA, USA) as the linear increase in A405 over
1 min. The amount of residual active proteinase was determined by comparing the result to a standard curve constructed using known amounts of the proteinase. Chymase
(7 nm) was assayed using Suc-Ala-Ala-Pro-Phe-pNA
(5 mm). Tryptase (14 nm) was assayed using H-d-Ile-ProArg-pNA (0.15 mm) in the presence of 0.5 lgỈmL)1 heparin. Neutrophil-derived proteinase 3 (100 nm) was assayed
using MeO-Suc-Ala-Ala-Pro-Val-pNA (5 mm). Neutrophil
elastase (40 nm) was assayed using MeO-Suc-Ala-Ala-ProVal-pNA (0.6 mm). Cathepsin G (167 nm) was assayed
using Suc-Ala-Ala-Pro-Phe-pNA (0.6 mm).

Inhibition of human lung tryptase by TFPI
derivatives
Tryptase (2 nm) in Tris ⁄ NaCl containing a low or high concentration of heparin was incubated with various concentrations of TFPI, TFPI-C, or antithrombin for 60 min at
37 °C. S-2288 was then added to a concentration of 5 mm,
and the initial rate of hydrolysis was measured.

Proteolysis of TFPI by proteinases
Reactions containing 7 nm chymase, tryptase, a-chymotrypsin, elastase, cathepsin G, or proteinase 3 and 3.5 lm TFPI

(molar ratio of 1 : 500) were incubated for the designated
times (15, 30, 60, 120, and 180 min) at 37 °C. Experiments
using chymase, a-chymotrypsin, elastase, cathepsin G, and
proteinase 3 were carried out in Tris ⁄ NaCl. Reactions using
tryptase were carried out in Tris ⁄ NaCl containing
500 lgỈmL)1 unfractionated heparin. At the indicated time
points, samples were taken from the reaction mixture and
subjected to SDS ⁄ PAGE under reducing conditions. Protein bands were visualized by staining with Coomassie Brilliant Blue R-250.

RP-HPLC
RP-HPLC was carried out on a Vydac 208TP54 C8-300
column (Cypress International Ltd, Tokyo, Japan). After
the sample was injected, the column was washed with

3074

a solution of 0.1% trifluoroacetic acid for 10 min. TFPI
fragments were eluted with a linear gradient of this
solution containing 24–37% acetonitrile at a flow rate of
1.0 mLỈmin)1. Each peak fraction was pooled, lyophilized,
and dissolved in water for further analyses.

Amino-acid composition analysis, N-terminal
sequencing, and MS analysis
The amino-acid compositions of the fragments were analyzed using an AccQTagTM system (Waters, Milford, MA,
USA) according to the manufacturer’s protocol. Automated Edman degradation was carried out using an Applied
Biosystems 492 protein sequencer and standard methods.
MS analysis was performed using matrix-assisted laser
desorption ⁄ ionization time-of-flight MS and a Voyager-DE
STR workstation (Applied Biosystems, Foster City, CA,

USA).

Measurement of the level of TFPI antigen using
an ELISA
TFPI antigen was detected with a sandwich ELISA method
using two different monoclonal antibodies against TFPI.
One monoclonal antibody (designated K9), which was
immobilized on the microtiter plate, recognized the third
Kunitz domain of TFPI [64], whereas the other monoclonal
antibody (designated K270), which was conjugated with
horseradish peroxidase, recognized the region between the
first and second Kunitz domains of TFPI [62]. Only TFPI
antigen consisting of all three Kunitz domains was detected
with this ELISA. In this procedure, each sample and horseradish peroxidase-conjugated K270 (110 ngỈmL)1) were premixed and incubated in a K9-coated microtiter well for 2 h
at 4 °C in a reaction volume of 200 lL. Then, the plate
was washed five times with Tris ⁄ NaCl ⁄ 0.05% Tween 20
buffer, and mixed with 200 lL 3,3¢,5,5¢-tetramethylbenzidine solution (2 mm EDTA containing 350 lgặmL)1
3,3Â,5,5Â-tetramethylbenzidine and 0.015% H2O2). After a
30-min incubation, development was terminated by the
addition of 100 lL 0.5 m H2SO4, and the absorbance at
405 nm was measured using a THERMOmax microplate
spectrometer. The concentration of TFPI was calculated
from a standard curve prepared with known amounts of
TFPI.

Dilute tissue factor clotting assay
TFPI (70 nm) was incubated with chymase (7 nm) at 37 °C
in Tris ⁄ NaCl. After various incubation times, the reaction
was terminated with 100 lm chymostatin, and a 15-lL aliquot of the sample was added to 135 lL human control
plasma and mixed for 2 min at 37 °C. Then, 150 lL thromboplastin [diluted 1 : 40 in 50 mm Tris ⁄ HCl (pH 7.0)

FEBS Journal 274 (2007) 3065–3077 ª 2007 The Authors Journal compilation ª 2007 FEBS


T. Hamuro et al.

containing 100 mm NaCl, 30 mm CaCl2, and 0.2% BSA]
was added to the sample ⁄ plasma mixture, and the clotting
time was measured using a Fibrintimer coagulometer (Dade
Behring Ltd, Tokyo, Japan). The concentration of the diluted thromboplastin was adjusted to yield clotting times
of about 30 s in the absence of TFPI. The clotting time
was related to the anticoagulant activity (expressed as the
percentage of the control sample) using a reference curve
constructed with known amounts of TFPI.

Measurement of the inhibitory activity of TFPI
toward factor Xa
The inhibition of factor Xa by TFPI was measured using
S-2222 as a substrate. First 20 lL 2 mm substrate and
75 lL sample were mixed with 75 lL Tris ⁄ NaCl ⁄ 0.2%
BSA and preincubated for 5 min at 37 °C. The reaction
was initiated by the addition of 20 lL 70 nm factor Xa,
and the change in A405 was monitored by means of a
THERMOmax microplate spectrometer. The anti-(factor Xa) activity of TFPI (expressed as the percentage of the
control sample) was calculated using a reference curve constructed with known amounts of TFPI.

Chymase-mediated proteolysis of TFPI

3

4

5

6

7

Determination of Km and kcat values

8

Chymase (7 nm) was mixed with various concentrations of
TFPI (0.31, 0.68, 1.25, 2.5, 5, and 10 lm) in Tris ⁄ NaCl.
After incubation for 30 min at 37 °C, p-amidinophenylmethanesulfonyl fluoride hydrochloride was added at a
concentration of 20 mm to terminate the reaction. The
remaining substrate was measured using an ELISA, and the
Km and kcat values were calculated from a Lineweaver–
Burk plot.

9

10

Acknowledgements
This work was supported through Special Coordination Funds of the Ministry of Education, Culture,
Sports, Science and Technology, the Japanese Government (to TH and SK). We thank Dr Yu-ichi Kamikubo for valuable advice and helpful discussion. We
also thank Kumiko Arita, Rumiko Onitsuka and
Michiko Kihara for technical assistance.

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