Báo cáo khoa học: Hepatocyte growth factor activator (HGFA): its regulation by protein C inhibitor ppt

Hepatocyte growth factor activator HGFA: its regulationby protein C inhibitor Koji Suzuki Department of Molecular Pathobiology, Mie University Graduate School of Medicine, Japan Introduc

Hepatocyte growth factor activator (HGFA): its regulation

by protein C inhibitor

Koji Suzuki

Department of Molecular Pathobiology, Mie University Graduate School of Medicine, Japan

Introduction

Protein C inhibitor (PCI; SERPINA5), a member of

the plasma serine protease inhibitor (serpin) family, was

isolated from human plasma as an inhibitor of activated

protein C (APC), the major protease of the

anticoagu-lant protein C pathway, which regulates thrombosis

and hemostasis [1] PCI, an irreversible inhibitor, forms

an acyl-bonded complex with APC, and complex

for-mation is enhanced in the presence of heparin [2] In

humans, the liver is the main source of plasma PCI [3],

and PCI synthesis also occurs in the kidneys [3], lungs,

pancreas, spleen, megakaryocytes, platelets [4], and

reproductive organs, including testis, epididymis,

prostate, and seminal vesicles [5] Because of its broad

tissue distribution, human PCI may regulate several

physiological and pathological events in which serine

proteases are involved, and its molecular targets may include APC and thrombomodulin–thrombin [6], which play roles in the anticoagulant protein C pathway, thrombin and plasma kallikrein [7], which play roles in the blood coagulation pathway, plasminogen activators released during matrix invasion by tumor cells [8], and acrosin in the fertilization system [9] Thus, PCI plays many physiological and pathological roles beyond thrombosis and hemostasis in humans [10] However, in rodents, including rats and mice, PCI is detected only in the testes and ovaries, and not in the liver or plasma [11,12] Therefore, to study the physiological and path-ological roles of PCI in experimental disease animal models, we established human PCI-transgenic (hPCI-Tg) mice that mimic PCI expression in humans; in this

Keywords

activated protein C; hepatocyte growth

factor (HGF); HGF activator; liver

regeneration; protein C inhibitor

Correspondence

K Suzuki, Department of Molecular

Pathobiology, Mie University Graduate

School of Medicine, Edobashi 2-174,

Tsu-city, Mie 514-8507, Japan

Fax: +81 59 231 9797

Tel: +81 59 231 9702

E-mail: suzuki@doc.medic.mie-u.ac.jp

(Received 30 November 2009, revised

29 January 2010, accepted 26 February

2010)

doi:10.1111/j.1742-4658.2010.07639.x

Protein C inhibitor (PCI; SERPINA5) is a plasma serine protease inhibitor, and a potent inhibitor of activated protein C (APC), which plays a critical role in the anticoagulant protein C pathway Recently, PCI was also found

to form a complex with the serine protease hepatocyte growth factor activator (HGFA), inhibiting the HGFA-catalyzed activation of the single-chain hepatocyte growth factor precursor In vivo studies using human PCI-transgenic (hPCI-Tg) mice, which mimic PCI expression in humans, showed that the regeneration rate of the liver after partial hepatectomy was significantly impaired as compared with wild-type mice The decreased liver regeneration in hPCI-Tg mice was restored by pretreatment with anti-body against human PCI Furthermore, APC protected hepatic nonparen-chymal cells from thrombin-induced inflammation in vitro, suggesting that plasma PCI may inhibit the cytoprotective action of APC on hepatic cells

in hPCI-Tg mice It was shown that the levels of HGFA–PCI are increased

in plasma of patients who have been subjected to hepatectomy, as com-pared with complex levels in the plasma of normal individuals Thus, PCI may play a role as a potent inhibitor of HGFA and APC in plasma and⁄ or

at the sites of tissue injury in the regulation of tissue regeneration

Abbreviations

APC, activated protein C; BrdU, bromodeoxyuridine; HAI, hepatocyte growth factor activator inhibitor; HGF, hepatocyte growth factor; HGFA, hepatocyte growth factor activator; hPCI-Tg, human protein C inhibitor-transgenic; PCI, protein C inhibitor; PDB, Protein Data Bank.

model, high levels of human PCI are detected in plasma

and in selected organs, including the liver [13]

Hepatocyte growth factor (HGF) plays a critical role

in the regeneration of various tissues, including the

liver, by stimulating the proliferation and motility of

various types of cell, including epithelial and

endothe-lial cells [14] HGF is synthesized and secreted as an

inactive single-chain precursor from liver sinusoidal

endothelial cells and megakaryocytes (platelets) [15]

Limited proteolytic activation of this precursor is

required for the biological activity of HGF [16] The

most potent activator of HGF precursor is HGF

acti-vator (HGFA), a serine protease homologous to

coag-ulation factor XIIa [17,18] HGFA is synthesized in

hepatocytes, and its zymogen, pro-HGFA, circulates in

blood as a 98 kDa single-chain form [18,19] Thrombin

generated at sites of tissue injury activates pro-HGFA

by limited proteolysis, generating an activated 98 kDa

two-chain form of HGFA that contains a

disulfide-linked 65 kDa heavy chain and a 31 kDa light chain

[18,20,21] The HGFA heavy chain is then further

cleaved in the systemic circulation by plasma

kallik-rein, releasing the 34 kDa mature form of HGFA

[20,21] Recently, cell membrane-bound Kunitz-type

HGFA inhibitors (HAIs) were isolated [22,23], and it

was determined that HAI-1 acts as an inhibitor and a

receptor of HGFA on the cell surface [24,25]

We recently found that PCI inhibits HGFA by

form-ing HGFA–PCI in the absence of heparin, and inhibits

HGFA-catalyzed activation of HGF precursor in vitro

[26] To determine the pathophysiological significance

of PCI inhibition of HGFA, we investigated the

influ-ence of PCI on liver regeneration, using hPCI-Tg mice,

and found that PCI decreased the regeneration rate of

the liver after hepatectomy by forming HGFA–PCI

Furthermore, PCI aggravated hepatic nonparenchymal

cell injury by inhibiting the cytoprotective effects of

APC [27] The levels of HGFA–PCI in the plasma of

patients who had been subjected to hepatectomy were

found to be significantly increased as compared with

levels in the plasma of normal individuals [26,27]

In this review, I describe the mechanism of regulation

of HGFA by PCI in vitro, a possible role of PCI in liver

regeneration triggered by HGFA in the mouse model,

and clinical data related to the regulation of HGFA

by PCI in patients and normal individuals after

hepatectomy

PCI inhibits HGFA by forming

HGFA–PCI independently of heparin

Thrombin-catalyzed activation of pro-HGFA is

enhanced in the presence of heparin, and this

pro-HGFA activation by thrombin appears to be sig-nificantly inhibited by PCI Furthermore, PCI was found to inhibit activated HGFA directly and potently

in the absence of heparin [26] PCI inhibits the 34 and

98 kDa forms of HGFA equally, with apparent inhibi-tion constants (Ki) of 6.1 nm and 6.3 nm, respectively [26] The second-order rate constants (m)1Æs)1) of the reaction between 34 kDa HGFA and PCI in the pres-ence or abspres-ence of heparin (10 unitsÆmL)1) were 6.6· 104 and 6.3· 104, respectively The residual amidolytic activity of HGFA decreased concomitantly with increased HGFA–PCI formation, as determined

by SDS⁄ PAGE and western blotting, with an inverse relationship being observed between HGFA inhibition

by PCI and HGFA–PCI formation HGFA–PCI for-mation was also observed in plasma in vitro When HGFA was added to human plasma, the concentration

of HGFA–PCI increased in a time-dependent manner PCI also inhibits the HGFA-catalyzed activation of HGF precursor [26] In the absence of PCI, almost all

of the single-chain HGF precursor was activated by HGFA, releasing the 98 kDa disulfide-linked active form of HGF In contrast, in the presence of PCI-pre-treated HGFA, the single-chain HGF precursor was minimally converted into the two-chain form

HGFA–PCI formation was competitively inhibited

by APC in the presence of heparin PCI inhibits APC with an apparent Ki of 14 nm and second-order rate constants (m)1Æs)1) of 1.3· 104 in the absence of hepa-rin and 6.5· 105 in the presence of heparin [1,2] To evaluate whether APC and HGFA are competitive tar-gets of PCI in plasma, the effect of APC on HGFA– PCI formation was examined in the presence or absence

of heparin APC competitively inhibited HGFA–PCI formation in the presence of heparin, but exhibited only

a weak inhibitory effect in the absence of heparin;

in this latter condition, PCI effectively inhibits HGFA [26] Plasma kallikrein competitively inhibited HGFA–PCI formation in the presence and absence of heparin

Structural analysis of APC–PCI and HGFA–PCI formation

To study the different effects of heparin on APC–PCI and HGFA–PCI formation, three-dimensional models

of both complexes were constructed by using the three-dimensional structure of the trypsin–serpin Michaelis complex [Protein Data Bank (PDB) accession number: 1K9O] for the reference template, as described previ-ously [26] The sequences of human pro-HGFA prote-ase domain (Swiss-Prot accession number: Q04756) and of the human PCI domain (PIR accession number:

A39339) were compared with the PDB structures of

trypsin and serpin, respectively, and this was followed

by standard homology modeling and energy

minimiza-tion procedures Figure 1A shows three-dimensional

structures with docking models of PCI and APC or

HGFA In these models, the estimated heparin-binding

sites are Arg269-Lys270 of the PCI H-helix [28] and

the 37-loop structure of APC, containing

Lys37-Lys38-Lys39 [29] Figure 1B shows the detailed structures of

the docking models of PCI and APC or HGFA In the

PCI–APC model, PCI Arg362, which is the first

resi-due of the s1C strand after the reactive center loop, is

estimated to be the nearest residue to the 37-loop structure of APC, and the distances from Arg362 of PCI to Lys37 and Lys39 are estimated to be 6.8 and 5.6 A˚, respectively These values are small enough for positive charge repulsion to exist between PCI and APC in the absence of heparin This repulsion can be neutralized by heparin, as Lys37 and Lys39 from APC interact with heparin On the other hand, the 37-loop structure in APC is replaced by Ile35-Gly36-Asp37 in HGFA The negatively charged Asp37 of HGFA is located very near (7.6 A˚) to Arg362 of PCI As Asp37

of HGFA is able to interact strongly with Arg362 of

37-loop

A

B

(K37-K38-K39)

(I35-G36-D37)

K270 R269

K270 R269 on

on H-helix

on H-helix

K38

I 35

G36

D37

Fig 1 (A) Spatial representation of molecular homology between APC–PCI (left) and HGFA–PCI (right) APC (green), HGFA (red–orange) and PCI (gray) are shown as ribbon models In each complex, acidic and basic amino acids are in red and blue, respectively There is no basic residue in the loop structure (Ile35-Gly36-Asp37) of HGFA that corresponds with the 37-loop structure of APC Heparin is estimated to bind

to Lys269-Lys270 on the H-helix of PCI [28] and the 37-loop structure of APC containing Lys37-Lys38-Lys39 [29] (B) Comparative molecular modeling of the reactive center loop region of PCI with the 37-loop of APC (left) and HGFA (right) APC (green), HGFA (red–orange) and PCI (gray) are shown as ribbon models The reactive center loop structure of PCI is gold-colored The space occupied by Lys37, Lys38, and Lys39, forming the 37-loop structure of APC, is occupied by Ile35, Gly36 and Asp37 in the homology model of HGFA Hydrogen atoms are not displayed for clarity The distances from Arg362 of PCI to Lys37 and Lys39 of APC in APC–PCI are estimated to be 6.8 and 5.6 A ˚ , respectively, in the absence of heparin The blue dotted arrows indicate repulsion between Arg362 of PCI and Lys37 or Lys39 of APC The distance from Arg362 of PCI to Asp37 of HGFA in HGFA–PCI is estimated to be 7.6 A ˚ in the absence of heparin The red dotted arrow indi-cates an interaction between Arg362 of PCI and Asp37 of HGFA These models are in part modified from Fig 10 of our previous article [26].

PCI, we hypothesize that heparin does not affect PCI

inhibition of HGFA

To confirm this hypothesis, we compared the

inhibi-tion of HGFA by recombinant mutated PCI

(R362A-PCI; Arg362 replaced by Ala) and that by wild-type

PCI in the presence or absence of heparin The data

showed that the inhibitory activity of R362A-PCI

against HGFA in the absence of heparin was markedly

decreased as compared with wild-type PCI, but it was

accelerated in the presence of heparin [26] On the

other hand, the inhibition of APC by R362A-PCI was

relatively increased in the absence of heparin as

com-pared with wild-type PCI, and it was also accelerated

by heparin These findings suggest that Arg362 of PCI

is important for HGFA inhibition

Recently, Li et al [30] determined a crystallographic

structure of the Michaelis complex of PCI, thrombin

and heparin to 1.6 A˚ resolution, and found that

thrombin interacts with PCI, depending on the length

of PCI’s reactive center loop to align the

heparin-bind-ing sites of the two proteins, suggestheparin-bind-ing that the

cofac-tor activity of heparin depends on the formation of a

heparin-bridged Michaelis complex and

substrate-induced exosite contacts

PCI regulates HGFA-mediated liver

regeneration in the mouse model

To investigate the influence of HGFA inhibition by

PCI in vivo, we compared liver regeneration after 70%

hepatectomy in wild-type and hPCI-Tg mice, which

mimic human PCI expression [27] All procedures were

conducted according to the National Institutes of

Health guidelines for animal experiments, and the Mie

University Review Board approved the experimental

protocol for the animal investigation, as previously

described [27] The livers of both wild-type and

hPCI-Tg mice started to regenerate by postoperative

day 5, and the liver weight in wild-type mice recovered

to preoperative levels by postoperative day 9

However, in hPCI-Tg mice, the liver weight was below

normal on postoperative day 5 (about 80%), and took

up to 13 days to recover to the normal weight shown

in wild-type mice To test liver regenerative ability,

bromodeoxyuridine (BrdU) incorporation and

expres-sion of cell proliferation markers, G1-phase cyclin D1

and S-phase cyclin A, were assessed in the remnant

livers The BrdU labeling index peaked 48 h after

hepatectomy in hPCI-Tg and wild-type mice, but the

numbers of BrdU-positive cells were significantly

decreased in hPCI-Tg mouse livers at each time point

after hepatectomy (19.2 ± 2.5% in wild-type mice;

4.9 ± 0.1% in hPCI-Tg mice) The expression levels of

cyclin D1 and cyclin A were significantly decreased in hPCI-Tg mice 48 h after hepatectomy These findings suggest that impaired liver regeneration in hPCI-Tg mice was due to decreased hepatocyte proliferation

To investigate the mechanism of impaired liver regeneration in hPCI-Tg mice, the dynamics of HGFA, PCI, HGFA–PCI and HGF were determined

at early stages after hepatectomy Pro-HGFA mRNA levels were the same in wild-type and hPCI-Tg naı¨ve livers, and were upregulated to similar levels after hep-atectomy [27] The levels of active 34 kDa HGFA protein in plasma of wild-type mice increased 10-fold from preoperative levels 6 h after hepatectomy How-ever, they did not increase to the same levels in hPCI-Tg mice; the highest active HGFA protein level (five-fold of the preoperative stage) was observed at 12 h The plasma PCI levels in hPCI-Tg mice also decreased rapidly after partial hepatectomy; the lowest PCI level was observed 12 h postoperation, and it recovered at

48 h Plasma HGFA–PCI was detected 12 h after hep-atectomy in hPCI-Tg mice The generation of activated HGF was lower in hPCI-Tg mice than in wild-type mice, and the activation rate of HGF in hPCI-Tg mice was substantially lower than in wild-type mice (63.1% ± 4.1% in wild-type mice; 43.0% ± 3.1% in hPCI-Tg mice) These data suggest that HGF precur-sor activation is impaired in the remnant liver of hPCI-Tg mice because of HGFA–PCI formation

Plasma PCI in hPCI-Tg mice inhibits the cytoprotective activity of APC

The anticoagulant protease APC has marked cytopro-tective and anti-inflammatory activities [31,32], and it

is generated from its precursor protein C by activation with thrombin bound to thrombomodulin on the vascular endothelial cells [33,34] As PCI is a potent inhibitor of both APC and thrombomodulin–throm-bin, we hypothesized that hepatic nonparenchymal cells are aggravated in hPCI-Tg mice after partial hep-atectomy because of human PCI-mediated inhibition

of APC generation from protein C by thrombomodu-lin–thrombin and the resulting loss of APC cytoprotec-tive effects Twenty-four hours after hepatectomy, histological evaluation showed vacuolized hepatocytes

in the remnant livers of both wild-type and hPCI-Tg mice [27] However, sinusoidal congestion and bleeding were detected focally in hPCI-Tg livers, and more sinu-soidal fibrin deposition was observed in hPCI-Tg livers than in wild-type livers at this time point The plasma hyaluronic acid concentration was higher in hPCI-Tg mice than in wild-type mice at each time point after hepatectomy, suggesting that severe sinusoidal

dysfunction occurs in hPCI-Tg mice In vitro studies

showed that thrombin-induced interleukin-6

produc-tion by cultured nonparenchymal cells isolated from

wild-type mice was similar to that by cells from

hPCI-Tg mice, and addition of exogenous APC

decreased thrombin-stimulated interleukin-6

produc-tion in the nonparenchymal cells of both wild-type and

hPCI-Tg mice equally [27] These findings suggest that

APC protects hepatic nonparenchymal cells from

thrombin-induced inflammation, and that plasma PCI

in hPCI-Tg mice inhibits the cytoprotective action of

APC and may also inhibit

thrombomodulin–thrombin-mediated APC generation

To investigate the inhibitory effect of PCI in

HGFA-mediated liver regeneration and the

cytoprotec-tive activity of APC on hepatic nonparenchymal cells,

we evaluated the effect of treatment with antibody

against human PCI on hPCI-Tg mice [27] The plasma

levels of snake venom (Protac)-activated APC activity

in hPCI-Tg mice were 35.8% ± 4.3% (P < 0.01) of

the plasma levels observed in wild-type mice However,

after administration of antibody against human PCI

through the tail vein 12 h before hepatectomy and

sub-sequently every 72 h, APC activity in hPCI-Tg mice

increased to levels similar to those observed in

wild-type mice The antibody showed no effect on liver

weight in wild-type mice; however, the antibody

signifi-cantly (P < 0.01) restored the impaired liver

regenera-tion in hPCI-Tg mice as compared with hPCI-Tg mice

treated with saline The BrdU labeling index at 48 h in

the regenerated liver of the antibody-treated hPCI-Tg

mice increased significantly (12.1% ± 1.2%,

P< 0.01) as compared with hPCI-Tg mice treated

with saline and also significantly (P < 0.05) as

com-pared with wild-type mice These data suggest that the

antibody against human PCI significantly improves

impaired liver regeneration in hPCI-Tg mice

HGFA–PCI formation in human plasma

The concentrations of HGFA–PCI, pro-HGFA and

PCI in peripheral plasma obtained from normal

sub-jects and from patients with hepatitis or hepatocellular

carcinoma have been determined [26] The plasma

con-centrations of pro-HGFA (40.2 ± 5.4 nm) and PCI

(115.4 ± 10.5 nm) in normal subjects were significantly

(P < 0.05) higher than in patients with hepatocellular

carcinoma (22.5 ± 4.5 and 55.7 ± 6.5 nm,

respec-tively) In addition, the plasma concentration of

HGFA–PCI was significantly (P < 0.01) higher in

hepatocellular carcinoma patients (60 ± 20 pm) than

in normal subjects (27 ± 10 pm) On the other hand,

the plasma concentrations of pro-HGFA and PCI were

not significantly different between hepatitis patients and normal subjects, but the plasma levels of HGFA– PCI were significantly (P < 0.01) higher in patients with hepatitis (112 ± 50 pm) than in normal subjects The concentrations of PCI, pro-HGFA and HGFA– PCI in plasma of normal individuals (n = 6; 114.5 ± 18.4, 45.7 ± 6.2, and 30 ± 5 pm, respec-tively, before hepatectomy) were also determined after hepatectomy [27] The plasma PCI level in human liver donors rapidly decreased after hepatectomy Concomi-tantly with the decrease in plasma PCI level, pro-HGFA and HGFA–PCI levels were significantly increased, reaching peak levels 12 h after surgery (a three-fold increase in pro-HGFA level and a 1.5-fold increase in HGFA–PCI level as compared with preoperative levels) Thereafter, the pro-HGFA and HGFA–PCI levels gradually decreased These findings suggest that thrombin generated at the site of tissue injury stimulates the liver cells, resulting in increased HGFA levels and HGFA–PCI formation, similar to that observed in partially hepatectomized hPCI-Tg mice

Conclusions

The data obtained in in vitro studies using isolated PCI, HGFA and HGF precursor suggest that PCI inhibits HGFA directly and potently in the absence of heparin

by forming HGFA–PCI; this inhibition of HGFA regu-lates the catalytic activation of HGF precursor The inhibition of HGFA by PCI is competitively impaired

by APC in the presence of heparin Figure 2 shows a possible mechanism of PCI-mediated regulation of HGFA and regulation of the protein C pathway PCI may regulate HGFA in solution, resulting in HGFA-mediated activation of HGF precursor In the protein C pathway, PCI regulates APC generation from protein C

by inhibition of thrombomodulin–thrombin, and also regulates the anticoagulant and anti-inflammatory activ-ities of APC in the presence of heparin-like proteogly-can In vivo studies using hPCI-Tg mice suggest that the liver regeneration rate after partial hepatectomy is regulated by plasma PCI One of the mechanisms of PCI-mediated regulation of liver regeneration may result from PCI inhibition of the cytoprotective activity

of APC following thrombin-induced injury, because the decreased liver regeneration in hPCI-Tg mice was restored by pretreatment with antibody against human PCI The regulation of HGFA by PCI was also shown

in humans, as PCI levels were observed to decrease and levels of HGFA–PCI to increase in plasma of human donors after hepatectomy, in a manner similar to that observed in hPCI-Tg mice Treatment with antibody against human PCI therefore had a beneficial effect on

liver regeneration, and thus may become valuable

therapy for liver regeneration in the future

Acknowledgements

The author thanks J Nishioka, T Hayashi, T Hamada,

H Kamada and T Okamoto in the Department of

Molecular Pathobiology, Mie University Graduate

School of Medicine, Tsu-city, Mie, who have worked

together on the characterization of HGFA, PCI, and

HGFA–PCI, and studied liver regeneration using

hPCI-Tg mice The author also thanks T Kobayashi,

Discovery Platform Technology Department,

Kamak-ura Research Laboratories, Chugai Pharmaceutical

Co., Kamakura, Kanagawa, for his excellent work on

homology modeling of PCI and APC or HGFA This

study was supported in part by a Grant-in-Aid for

Scientific Research from the Ministry of Education,

Culture, Sports, Science and Technology of Japan

(18659280, 19390262, and 21390292) and the Mie

University COE-A project

References

1 Suzuki K, Nishioka J & Hashimoto S (1983) Protein C

inhibitor Purification from human plasma and

charac-terization J Biol Chem 258, 163–168

2 Suzuki K, Nishioka J, Kusumoto H & Hashimoto S

(1984) Mechanism of inhibition of activated protein C

by protein C inhibitor J Biochem (Tokyo) 95, 187–195

3 Suzuki K, Deyashiki Y, Nishioka J & Toma K (1989)

Protein C inhibitor: structure and function Thromb

Haemost 61, 331–342

4 Nishioka J, Ning M, Hayashi T & Suzuki K (1998) Protein C inhibitor secreted from activated platelets efficiently inhibits activated protein C on phosphatidyl-ethanolamine of platelet membrane and microvesicles

J Biol Chem 273, 11281–11287

5 Laurell M, Christensson A, Abrahamsson PA, Stenflo

J & Lilja H (1992) Protein C inhibitor in human body fluids Seminal plasma is rich in inhibitor antigen deriving from cells throughout the male reproductive system J Clin Invest 89, 1094–1101

6 Rezaie AR, Cooper ST, Church FC & Esmon CT (1995) Protein C inhibitor is a potent inhibitor of the thrombin–thrombomodulin complex J Biol Chem 270, 25336–25339

7 Meijers JCM, Kanters DH, Vlooswijk RA, van Erp

HE, Hessing M & Bouma BN (1988) Inactivation of human plasma kallikrein and factor XIa by protein C inhibitor Biochemistry 27, 4231–4237

8 Wakita T, Hayashi T, Tamaru H, Nishioka J, Akita

N, Asanuma K, Kamada H, Gabazza EC, Ido M, Kawamura J et al (2004) Regulation of carcinoma cell invasion by protein C inhibitor whose expression is decreased in renal cell carcinoma Int J Cancer 108, 516–523

9 Hermans JM, Jones R & Stone SR (1994) Rapid inhibition of the sperm protease acrosin by protein C inhibitor Biochemistry 33, 5440–5444

10 Suzuki K (2008) The multi-functional serpin, protein C inhibitor: beyond thrombosis and hemostasis J Thromb Haemost 6, 2017–2026

11 Wakita T, Hayashi T, Yuasa H, Nishioka J, Kawamura

J & Suzuki K (1998) Molecular cloning, tissue distribu-tion and androgen reguladistribu-tion of rat protein C inhibitor FEBS Lett 429, 263–268

APC

HGF

PCI

Tissue injury

Tissue regeneration Protein C

HGFA

Blood

coagulation

TM

precursor HAI

Pro-HGFA

Sp1 AP2

PKC

Pro-HGFA gene

S AP2

g

Hepatocytes

Fig 2 A possible mechanism of PCI-medi-ated regulation of HGFA and regulation of the protein C pathway PCI regulates HGFA

in the absence of heparin (solution phase), resulting in HGFA-mediated activation of HGF precursor In addition, PCI regulates APC generation from protein C by inhibition

of the thrombomodulin (TM)–thrombin com-plex, and inhibits APC in the presence of heparin-like proteoglycan in the protein C pathway PKC, protein kinase C.

12 Zechmeister-Machhart M, Hufnagl P, Uhrin P,

Korschineck I, Binder BR & Geiger M (1997)

Molecular cloning and sequence analysis of the mouse

protein C inhibitor gene Gene 186, 61–66

13 Hayashi T, Nishioka J, Kamada H, Asanuma K,

Kondo H, Gabazza EC, Ido M & Suzuki K (2004)

Characterization of a novel human protein C inhibitor

(PCI) gene transgenic mouse useful for studying the role

of PCI in physiological and pathological conditions

J Thromb Haemost 2, 949–961

14 Kan M, Zhang G, Zarnegar R, Michalopoulos G,

Myoken Y, McKeehan WL & Stevens JI (1991)

Hepatocyte growth factor⁄ hepatopoietin A stimulates

the growth of rat kidney proximal tubule epithelial cells

(RPTE), rat nonparenchymal liver cells, human

mela-noma cells, mouse keratinocytes and stimulates

anchor-age-independent growth of SV-40 transformed RPTE

Biochem Biophys Res Commun 17, 331–337

15 Nakamura Y, Morishita R, Higaki J, Kida I, Aoki M,

Moriguchi A, Yamada K, Hayashi S, Yo Y,

Matsumoto K et al (1995) Expression of local

hepatocyte growth factor system in vascular tissues

Biochem Biophys Res Commun 215, 483–488

16 Naka D, Ishii T, Yoshiyama Y, Miyazawa K, Hara H,

Hishida T & Kitamura N (1992) Activation of

hepato-cyte growth factor by proteolytic conversion of a single

chain form to a heterodimer J Biol Chem 267, 20114–

20119

17 Shimomura T, Miyazawa K, Komiyama Y, Hiraoka

H, Naka D, Morimoto Y & Kitamura M (1995)

Acti-vation of hepatocyte growth factor by two

homolo-gous proteases, blood-coagulation factor XIIa and

hepatocyte growth factor activator Eur J Biochem

229, 257–261

18 Miyazawa K (2010) Hepatocyte growth factor activator

(HGFA): a serine protease that links tissue injury to

activation of hepatocyte growth factor FEBS J 277,

2208–2214

19 Miyazawa K, Shimomura T, Kitamura A, Kondo J,

Morimoto Y & Kitamura N (1993) Molecular cloning

and sequence analysis of the cDNA for a human serine

protease responsible for activation of hepatocyte growth

factor Structural similarity of the protease precursor to

blood coagulation factor XII J Biol Chem 268, 10024–

10028

20 Shimomura T, Kondo J, Ochiai M, Naka D, Miyazawa

K, Morimoto Y & Kitamura N (1993) Activation of

the zymogen of hepatocyte growth factor activator by

thrombin J Biol Chem 268, 22927–22932

21 Kataoka H & Kawaguchi M (2010) Hepatocyte growth

factor activator (HGFA): pathophysiological functions

in vivo FEBS J 277, 2230–2237

22 Shimomura T, Denda K, Kitamura A, Kawaguchi T,

Kito M, Kondo J, Kagaya T, Qin L, Takata H,

Miyaz-awa K et al (1997) Hepatocyte growth factor activator

inhibitor, a novel Kunitz-type serine protease inhibitor

J Biol Chem 272, 6370–6376

23 Kawaguchi T, Qin L, Shimomura T, Kondo J, Matsumoto K, Denda K & Kitamura N (1997) Purification and cloning of hepatocyte growth factor activator inhibitor type 2, a Kunitz-type serine protease inhibitor J Biol Chem 272, 27558–27564

24 Kataoka H, Shimomura T, Kawaguchi T, Hamasuna

R, Itoh H, Kitamura N, Miyazawa K & Koono M (2000) Hepatocyte growth factor activator inhibitor type 1 is a specific cell surface binding protein of hepa-tocyte growth factor activator (HGFA) and regulates HGFA activity in the pericellular microenvironment

J Biol Chem 275, 40453–40462

25 Eigenbrot C, Ganesan R & Kirchhofer D (2010) Hepa-tocyte growth factor activator (HGFA): molecular structure and interactions with HAI-1 FEBS J 277, 2215–2222

26 Hayashi T, Nishioka J, Nakagawa N, Gabazza EC, Kamada H, Kobayashi T, Hattori A & Suzuki K (2007) Protein C inhibitor directly and potently inhibits hepatocyte growth factor activator J Thromb Haemost

5, 1477–1485

27 Hamada T, Kamada H, Hayashi T, Nishioka J, Gabazza

EC, Isaji S, Uemoto S & Suzuki K (2008) Protein C inhibitor regulates hepatocyte growth factor activator-mediated liver regeneration in mice Gut 57, 365–373

28 Shirk RA, Elisen MG, Meijers JC & Church FC (1994) Role of the H helix in heparin binding to protein C inhibitor J Biol Chem 269, 28690–28695

29 Glasscock LN, Gerlitz B, Cooper ST, Grinnell BW & Church FC (2003) Basic residues in the 37-loop of activated protein C modulate inhibition by protein C inhibitor but not by alpha(1)-antitrypsin Biochim Biophys Acta 1649, 106–117

30 Li W, Adams TE, Nangalia J, Esmon CT & Hunting-ton JA (2008) Molecular basis of thrombin recognition

by protein C inhibitor revealed by the 1.6-A structure

of the heparin-bridged complex Proc Natl Acad Sci USA 105, 4661–4666

31 Shimizu S, Gabazza EC, Taguchi O, Yasui H, Taguchi

Y, Hayashi T, Ido M, Shimizu T, Nakagaki T, Kobayashi H et al (2003) Activated protein C inhibits the expression of platelet-derived growth factor in the lung Am J Respir Crit Care Med 167, 1416–1426

32 Esmon CT (2006) Inflammation and the activated protein C anticoagulant pathway Semin Thromb Hemost 32(Suppl 1), 49–60

33 Esmon CT (1987) The regulation of natural anticoagulant pathways Science 235, 1348–1352

34 Suzuki K, Kusumoto H, Deyashiki Y, Nishioka J, Hashimoto S, Maruyama I, Yamamoto S & Horiuchi S (1987) Structure and expression of human thrombomod-ulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation EMBO J 6, 1891–1897