MINIREVIEW 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. FEBS Journal 277 (2010) 2223–2229 ª 2010 The Author Journal compilation ª 2010 FEBS 2223 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 (K i ) of 6.1 nm and 6.3 nm, respectively [26]. The second-order rate constants (m )1 Æs )1 ) of the reaction between 34 kDa HGFA and PCI in the pres- ence or absence of heparin (10 unitsÆmL )1 ) were 6.6 · 10 4 and 6.3 · 10 4 , 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 K i of 14 nm and second-order rate constants (m )1 Æs )1 ) of 1.3 · 10 4 in the absence of hepa- rin and 6.5 · 10 5 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: HGFA: its regulation by protein C inhibitor K. Suzuki 2224 FEBS Journal 277 (2010) 2223–2229 ª 2010 The Author Journal compilation ª 2010 FEBS 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) R362 R362 K270 R269 K270 R269 on on H-helix on H-helix K38 I 35 G36 K37 K39 D37 R362 R362 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]. K. Suzuki HGFA: its regulation by protein C inhibitor FEBS Journal 277 (2010) 2223–2229 ª 2010 The Author Journal compilation ª 2010 FEBS 2225 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, suggesting 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, G 1 -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 HGFA: its regulation by protein C inhibitor K. Suzuki 2226 FEBS Journal 277 (2010) 2223–2229 ª 2010 The Author Journal compilation ª 2010 FEBS 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 K. Suzuki HGFA: its regulation by protein C inhibitor FEBS Journal 277 (2010) 2223–2229 ª 2010 The Author Journal compilation ª 2010 FEBS 2227 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. 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