Fibrinogen alpha C chain 5.9 kDa fragment (FIC5.9), a new

biomarker for chronic hepatitis, reflects a minute change of

fibrinolysis and coagulation factors

（新規慢性肝疾患マーカーFIC5.9は微小な凝固線溶因子の変

動を反映する）

千葉大学大学院医学薬学府

先進医療科学専攻

（主任： 野村 文夫教授）

菊地 渉

ABSTRACT

Fibrinogen alpha C chain 5.9 kDa fragment (FIC5.9) is a new serum biomarker for chronic hepatitis

that was discovered by proteomics analysis. FIC5.9 is derived from the C-terminal region of fibrinogen

alpha chain. The serum levels of FIC5.9 decrease in chronic hepatitis. Previous studies have suggested that

FIC5.9 cannot be detected in the systemic circulation and is only synthesized during blood clotting.

However, the mechanism of FIC5.9 synthesis is unclear. Based on our previous work, I formulated a

hypothesis that FIC5.9 is synthesized by enzymes that are activated post-blood collection and may be

coagulation and fibrinolysis factors. In this study, I analyzed the mechanisms of FIC5.9 synthesis in

healthy blood. Our analysis showed that thrombin acts as an initiator for FIC5.9 synthesis, and that plasmin

and neutrophil elastase cleaves FIC5.9 from fibrinogen. The results indicate that FIC5.9 reflects and

amplifies minute changes in coagulation and fibrinolysis factors associated with pathological conditions.

INTRODUCTION

Many biomarkers have been discovered by proteomics analysis, but fewer have been developed for

clinical use (1, 2). Most reported biomarkers involve posttranslational modification or degradation, and it

may be unclear why the level of the biomarker changes in disease. Thus, there is a need to establish links

between the synthetic mechanism of the biomarker and disease conditions for practical use in clinical

diagnosis (3).

Fibrinogen alpha C chain 5.9 kDa fragment (FIC5.9) is a new serum marker for chronic hepatitis that

was discovered in samples from cases of alcoholic liver disease using SELDI-TOF MS (4). FIC5.9 is

derived from the C-terminal region of fibrinogen alpha chain and has a molecular weight is 5890 Da. The

level of FIC5.9 is high in healthy people and decreases with onset of hepatitis (5-7). I developed an ELISA

system for FIC5.9 and evaluated the clinical utility of FIC5.9 in various kinds of chronic hepatitis (8). This

analysis showed that the serum level of FIC5.9 is significantly decreased in the early stage of liver fibrosis

and that this is a strong indicator for liver fibrosis. The results also indicated that FIC5.9 is not synthesized

in the circulation, but in the blood collection tube during the process of blood coagulation. That is, FIC5.9

is synthesized from blood collected from healthy subjects and in vitro products in the collection tube. Since

our initial identification of FIC5.9, the same peptide or an unidentified peptide with the same nominal

mass has been reported to be of diagnostic value in a variety of pathological conditions including cancers

and inflammatory disorders (8-14). However, the mechanism of FIC5.9 production is unknown.

Based on these results, I formulated a hypothesis that FIC5.9 is synthesized from fibrinogen or fibrin by

an enzyme that is activated post-blood collection, with this synthesis involving coagulation and fibrinolysis

factors. The FIC5.9 sequence includes amino acids 576-629 of the fibrinogen alpha chain (Fig. 1) and

FIC5.9 appears to be synthesized by cleavage at RGK/SSS (N-terminal region) and RPV/RGI (C-terminal

region) (4). Trypsin cleaves to the carboxyl side of lysine, but plasmin and thrombin are the major enzymes

that work with fibrinogen or fibrin (15). There are fewer reports of enzymes in blood that cleave to the

carboxyl side of valine, but neutrophil elastase is one such example (16). In this study, as a first step

toward understanding the mechanisms of FIC5.9 alterations in various pathologies, I tested the hypothesis

that FIC5.9 is synthesized by these enzymes in apparently healthy subjects.

EXPERIMENTAL PROCEDURES

FIC5.9 synthesis during blood clotting

To confirm the conclusion of our previous study (8) regarding the roles of coagulation factors in

synthesis of FIC5.9, the coagulation cascade was reactivated by adding Thrombocheck APTT-SLA

(Sysmex Corp., Hyogo, Japan) to coagulation-deficient plasma (factor II, V, VII, VIII, IX, X, XI, XII;

Sysmex). Control plasma was collected with Insepack II (sodium citrate type, Sekisui Medical Co., Tokyo,

Japan). After a 1-hour incubation at 25°C, samples were centrifuged at 3000 rpm for 20 min. The

supernatant was purified with C18/WCX cartridges (4). FIC5.9 levels in samples were measured by

MALDI-TOF MS on a Bruker AUTOFlex® mass spectrometer, using stable isotope-labeled FIC5.9 as an

internal standard (4).

In vitro degradation of purified fibrinogen

Purified fibrinogen (Wako Pure Chemical Industries, Tokyo, Japan; 70 µg) in PBS buffer was incubated

with thrombin (Wako; final conc. 2 U/mL), plasmin (Wako; 1 U/mL) or neutrophil elastase (Sigma-Aldrich,

St. Louis, MO, USA; 1 U/mL) for 2 h at 25°C. The reaction was stopped by adding EDTA (pH 8.0, 10

mM) and aprotinin (Wako; 1 U/mL).

LC-MS/MS analysis of degradation products of fibrinogen

StageTips C18 (Thermo Fisher Scientific, Waltham, MA, USA) was used for desalting the degradation

products of fibrinogen (17). Obtained peptides were identified by LC/MS/MS analysis (18).

Analysis and time course of FIC5.9 synthesis in blood collection tubes

Blood collection tubes (Insepack II, Sekisui Medical Co.) with added thrombin (Wako; 20 U/mL (19)),

hirudin (Thermo Fisher; 1 U/mL (19)), plasmin (Wako; 0.8 U/mL (20)), tranexamic acid (Wako; 10 mM

(20)), sivelestat sodium (Cosmo Bio Co.; Tokyo Japan; 80 µM (21)) or the same volume of saline were

used to collect blood samples from six healthy volunteers. The collected blood was clotted for 0, 5, 30, 60,

90 min at 25°C. After blood clotting, serum was obtained by centrifugation at 3000 rpm for 10 min at 4°C.

The level of FIC5.9 was measured using a FIC5.9 ELISA kit (6). All volunteers gave informed consent and

the study was approved by the ethics committee of our institute.

Statistical analysis

Statistical analysis was performed by Mann-Whitney U-test with SPSS software, version 18.0 (SPSS

Inc., Chicago, IL, USA). A P value less than 0.05 was considered significant.

RESULTS

LC-MS/MS analysis of degradation products of fibrinogen in vitro

FIC5.9 is synthesized in the blood collection tube during blood clotting (8). Confirmatory experiments

in the current study showed that a deficiency of factors II, V, VIII and X decreased the synthesis of FIC5.9

(Fig. 2), and that factor II (thrombin) had a particularly marked effect on the synthesis of FIC5.9 (8). To

determine the coagulation and fibrinolysis factors that produce FIC5.9, I narrowed down the candidate

enzymes based on these results, well-known enzymes that catalyze fibrinogen, and enzymes that cleave at

sites consistent with synthesis of FIC5.9. Information on enzyme cleavage sites was obtained from the

Peptidase Database (MEROPS: http://merops.sanger.ac.uk). Thrombin, plasmin and neutrophil elastase

were selected. Next, the sequences of degradation products obtained from the reaction of fibrinogen and

these enzymes were analyzed by LC-MS/MS (Fig. 3, and Table). The results suggested that the N-terminal

region of FIC5.9 (RGK/SSS) is cleaved by thrombin or plasmin, and that the C-terminal region of FIC5.9

(RPV/RGI) is cleaved by neutrophil elastase.

Analysis and time course of FIC5.9 synthesis in blood collection tubes

To determine if thrombin, plasmin and neutrophil elastase can synthesize FIC5.9 in blood, I analyzed

the synthesis and time course of FIC5.9 in blood collection tubes containing thrombin, hirudin (a thrombin

inhibitor), plasmin, tranexamic acid (a plasmin inhibitor), or sivelestat sodium (a neutrophil elastase

inhibitor). I first examined FIC5.9 synthesis and clotting time in plain and silica-coated tubes by

measurement of FIC5.9 in serum after 0, 5, 30, 60, 90 min of clotting and centrifugation. The rate of

synthesis of FIC5.9 in a silica-coated tube was significantly faster than that in a plain tube (Fig. 4), but the

final FIC5.9 levels did not differ significantly between these tubes. All further analyses (including clinical

evaluation of FIC5.9 as a biomarker in chronic hepatitis) were performed in silica-coated tubes

In all experiments, FIC5.9 was detected at 0 min of clotting. However, there is a time lag between

blood collection and dispensing into the collection tube. The rate of synthesis of FIC5.9 in tubes with

added thrombin was significantly faster than that in silica-coated tubes. Synthesis of FIC5.9 reached a

plateau after 30 min clotting and the same amount of FIC5.9 was synthesized in the silica-coated and plain

tubes (Fig. 5A). Addition of hirudin (thrombin inhibitor) significantly delayed the synthesis of FIC5.9, but

prolonged synthesis to after 90 minutes of clotting. With plasmin, the synthesis rate and total amount of

FIC5.9 were significantly increased, and addition of tranexamic (plasmin inhibitor) markedly decreased

the synthesis of FIC5.9 (Fig. 5B). Addition of sivelestat sodium (neutrophil elastase inhibitor) completely

inhibited synthesis of FIC5.9 (Fig. 5C). These results suggest that plasmin and neutrophil elastase

synthesize FIC5.9 directly.

DISCUSSION

In this study, I formulated a hypothesis that FIC5.9 is synthesized from fibrinogen by coagulation and

fibrinolysis factors. This hypothesis was tested by digesting purified fibrinogen with thrombin, plasmin

and neutrophil elastase in vitro and analysis of fragments of fibrinogen by LC-MS/MS. The results showed

that the N-terminal end of FIC5.9 (RGK/SSS) is cleaved by thrombin or plasmin, and the C-terminal end

of FIC5.9 (RPV/RGI) is cleaved by neutrophil elastase (Fig. 3) in vitro. To confirm that these enzymes can

synthesize FIC5.9 during blood coagulation, the synthesis and time course of FIC5.9 were analyzed in

blood collection tubes spiked with thrombin, plasmin, neutrophil elastase, or inhibitors of these enzymes.

Addition of thrombin or plasmin accelerated the synthesis of FIC5.9, and inhibition of all three enzymes

significantly decreased the synthesis of FIC5.9 (Fig. 5). These results show that the mechanism of FIC5.9

synthesis in healthy people is strongly related to coagulation and fibrinolysis factors.

Both thrombin and plasmin can cleave the N-terminal end of FIC5.9 (RGK/SSS), but addition of

thrombin into blood collection tube resulted in only acceleration of FIC5.9 synthesis and no significant

difference did not alter the final amount of FIC5.9 synthesized, whereas addition of plasmin markedly

increased the final amount of FIC5.9. The different results for thrombin or plasmin are of interest.

Inhibition of thrombin (adding hirudin) delayed the synthesis of FIC5.9, but there was no significant

difference at 90 minutes clotting. These results indicate that thrombin is essential for initiation of FIC5.9

synthesis, but is less important with regard to the amount of FIC5.9 produced. Plasmin is the significant

enzyme that cleaves the N-terminal region of FIC5.9 and affects the amount of FIC5.9. In nature,

activation of plasminogen to plasmin occurs after activation of coagulation factors (22, 23). These reports

also support the role of thrombin as the initiator of FIC5.9 synthesis.

Our results suggests why FIC5.9 is a marker of alcoholic liver disease and liver fibrosis (4)-(8).

Neutrophil elastase cleaves the C-terminal site of FIC5.9, but only a few reports indicate that the level of

neutrophil elastase (or the neutrophil count) changes in the early stage of chronic hepatitis (24, 25).

Therefore, the C-terminal site of FIC5.9 is likely to be cleaved at a similar level in healthy people and

patients with chronic hepatitis. However, an extreme increase or decrease in neutrophils might affect the

synthesis of FIC5.9. A spike test of neutrophil elastase into the collection tube could not be performed, but

the results of LC-MS/MS indicated that neutrophil elastase can cleave the C-terminal region of FIC5.9.

This requires confirmation in a further study.

Thrombin (the initiator of FIC5.9 synthesis) and plasmin (which significantly cleaves the N-terminal

site of FIC5.9) are molecular markers of liver disease, partly because both enzymes are secreted from liver

(26-30). Our study suggests that the decrease in FIC5.9 levels in chronic hepatitis is caused by dysfunction

of coagulation and fibrinolysis factors, since some levels of these factors change in chronic liver disease

(31, 32). The marked change in the level of FIC5.9 in the early stage of liver fibrosis (8) might occur

because changes in all of these factors affect FIC5.9 synthesis synergistically. The presence of other factors

related to FIC5.9 synthesis also requires consideration. Recently, Marfa et al. reported that TGF-β reduces

the expression level of fibrinogen alpha chain mRNA (7). The level of TGF-β is closely related to liver

fibrosis and hepatitis (33, 34). Plasma fibrinogen is also a marker for chronic liver disease (35). These

reports are important for investigation of the mechanisms of FIC5.9 synthesis.

Measurement of degradation products in blood circulation is commonly used in clinical tests (36, 37).

Most coagulation and fibrinolysis factors are unstable for measurement of activity (38)-(40), as

exemplified by the activated partial thromboplastin time (APTT) and the prothrombin time (PT). FIC5.9 is

also a degradation product from fibrinogen alpha chain that is synthesized by coagulation and fibrinolysis

factors, and reflects a minute change in these factors. Thrombin, plasmin and neutrophil elastase are

involved in the synthetic mechanism of FIC5.9 in clotting of normal blood. This provides the basis for

understanding the decrease in FIC 5.9 in clotting of blood from patients with chronic hepatitis. Further

analysis may show similar effects in blood from patients with other diseases.

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FIGURE LEGENDS

FIGURE 1. Sequences of fibrinogen alpha chain and FIC5.9. The mature alpha chain is indicated by a grey

shadow. The FIC5.9 sequence (amino acids 576-629) is underlined.

FIGURE 2. Analysis of FIC5.9 synthesis in coagulation factor-deficient plasma. (A) Mass spectrum of

coagulation-depleted plasma reactivated using an APTT reagent. Synthesized FIC5.9 is indicated with a

red line and SI-labeled FIC5.9 peptide is indicated with a blue line. (B) Quantification of FIC5.9

synthesized by coagulation reactivation. The relative intensity of FIC5.9 was calculated by comparing with

the intensity of the internal standard (SI-FIC5.9).

FIGURE 3. Cleavage site mapping of thrombin, plasmin and neutrophil elastase in FIC5.9 and surrounding

regions. Cleavage sites are indicated with scissors, depending on the number of peptides identified by

LC-MS/MS analysis. Identified peptide sequence information is shown in Supplementary Table 1.

TABLE Identified peptides cleaved by thrombin, plasmin and neutrophil elastase in FIC5.9 and

surrounding regions by LC-MS/MS analysis. (O) indicates “oxidized”, and duplication is eliminated.

FIGURE 4. Analysis of synthesis and time course of FIC5.9 in plain and silica-coated tubes. The relative

amount of FIC5.9 was measured by FIC5.9 ELISA. Statistical analysis was performed as described in the

Materials and Methods.

FIGURE 5. Analysis of synthesis and time course of FIC5.9 with thrombin spiked/inhibited (A), plasmin

spiked/inhibited (B), or neutrophil elastase inhibited (C) blood collection tubes. The relative amount of

FIC5.9 was measured by FIC5.9 ELISA. Statistical comparison of silica-coated tube vs. enzyme-spiked

tube (*), or inhibitor spiked tube (†) was performed as described in the Materials and Methods.

FIGURE 1

FIGURE 2

FIGURE 3

TABLE

Identified peptides cleaved by thrombin, plasmin and neutrophil elastase in FIC5.9 and surrounding

regions by LC-MS/MS analysis.

Enzyme Confirmed sequences

Thrombin

(7 Peptides)

574- GKSSSYSKQFTSSTSYNRGDSTFESKSYKMADEA -604

574- GKSSSYSKQFTSSTSYNRGDSTFESKSYKM -603

576- SSSYSKQFTSSTSYNRGDSTFESKSYKMA -604

576- SSSYSKQFTSSTSYNRGDSTFESKSYKM -603

576- SSSYSKQFTSSTSYNRGDSTFESKS -600

576- SSSYSKQFTSSTSYNRGDSTFESK -599

576- SSSYSKQFTSSTSYNRGDSTFES -598

Plasmin

(26 Peptides)

528- TFPGFFSPMLGEFVSETESRGSESGIFTNTKESSSHHPGIAEFPSRGK -575

528- TFPGFFSPMLGEFVSETESRGSESGIFTNTKESSSHHPGIAEFPSRG -574

528- TFPGFFSPMLGEFVSETESRGSESGIFTNTKESSSHHPGIAEFPSR -573

528- TFPGFFSPMLGEFVSETESRGSESGIFTNTKESSSHHPGIAEFPS -572

540- FVSETESRGSESGIFTNTKESSSHHPGIAEFPSRGK -575

541- VSETESRGSESGIFTNTKESSSHHPGIAEFPSRGK -575

548- GSESGIFTNTKESSSHHPGIAEFPSRGK -575

548- GSESGIFTNTKESSSHHPGIAEFPSRGKSSSYS -580

559- ESRGSESGIFTNTKESSSHHPGIAEFPSRGKSSSYSK -582

559- ESRGSESGIFTNTKESSSHHPGIAEFPSRGK -575

559- ESRGSESGIFTNTKESSSHHPGIAEFPSRG -574

559- ESSSHHPGIAEFPSRGKSSSYS -580

576- SSSYSKQFTSSTSYNRGDSTFESKSYK -602

576- SSSYSKQFTSSTSYNRGDSTFESKS -600

576- SSSYSKQFTSSTSYNRGDSTFESK -599

576- SSSYSKQFTSSTSYNRGDSTFES -598

576- SSSYSKQFTSSTSYNRGDST -595

576- SSSYSKQFTSSTSYNRG -592

576- SSSYSKQFTSSTSYN -590

576- SSSYSKQFTSSTSY -589

576- SSSYSKQFTSSTS -588

582- QFTSSTSYNRGDSTFESKSYK -602

582- QFTSSTSYNRGDSTFESKS -600

603- MADEAGSEADHEGTHSTKRGHAK -625

603- MADEAGSEADHEGTHSTKRGHA -624

603- MADEAGSEADHEGTHSTK -620

Neutrophil

Elastase

(39 peptides)

557- NTKESSSHHPGIAEFPSRGKSS -577

557- NTKESSSHHPGIAEFPSRGKS -576

557- NTKESSSHHPGIAEFPSRGK -575

558- TKESSHHPGIAEFPS -572

561- SSHHPGIAEFPSRGKS -576

562- SHHPGIAEFPSRG -574

562- SHHPGIAEFPSR -573

568- AEFPSRGKSSSYSKQFT -584

569- EFPSRGKSSSYSKQFT -584

585- SSTSYNRGDSTFESKSYKMADEAGSEADHEGTHSTKRGHAKSRPV -629

585- SSTSYNRGDSTFESKSYKMADEAGSEADHEGTHSTK -620

585- SSTSYNRGDSTFESKSYKMA -604

585- SSTSYNRGDSTFESKSYKM -603

585- TSYNRGDSTFESKSYKMADEAGSEADHEGTHSTKRGHAKSRPV -629

587- TSYNRGDSTFESKSYKMA -604

587- TSYNRGDSTFESKSYKM -603

588- SYNRGDSTFESKSYKMADEAGSEADHEGTHSTKRGHAKSRPV -629

588- SYNRGDSTFESKSYKMADEAGSEADHEGTHSTKRGHAKSRPV -629(O)

588- SYNRGDSTFESKSYKMADEAGSEADHEGTHSTKRGHAK -625

588- SYNRGDSTFESKSYKMADEAGSEADHEGTHSTK -620

588- SYNRGDSTFESKSYKMADEAGSEADHEGTH -617

588- SYNRGDSTFESKSYKMADEA -607

588- SYNRGDSTFESKSYKMA -604

588- SYNRGDSTFESKSYKM -603

588- SYNRGDSTFESKSYK -602

596- FESKSYKMADEAGSEADHEGTHSTKRGHAKSRPV -629

596- FESKSYKMADEAGSEADHEGTHSTKRGHAK -625

597- ESKSYKMADEAGSEADHEGTHSTKRGHAKSRPV -629

599- KSYKMADEAGSEADHEGTHSTKRGHAKSRPV -629

599- KSYKMADEAGSEADHEGTHSTKRGHAKSRPV -629(O)

599- KSYKMADEAGSEADHEGTHSTKRGHAKSR -627

599- KSYKMADEAGSEADHEGTHSTK -620

599- KSYKMADEAGSEADHEGTHST -619

599- KSYKMADEAGSEADHEGT -616

601- YKMADEAGSEADHEGTHSTKRGHAKSRPV -629

601- YKMADEAGSEADHEGTHSTKRGHAKSRPV -629(O)

601- YKMADEAGSEADHEGTHSTKRGHAKSRP -628

601- YKMADEAGSEADHEGTHSTKRGHAK -625

603- ADEAGSEADHEGTHSTKRGHAKSRPV -629

None N.D.

(O) indicates “oxidized”, and duplication is eliminated.

FIGURE 4

FIGURE 5

Proteomics

平成２７年１月９日 投稿中