Fibrinogen alpha C chain 5.9 kDa fragment (FIC5.9), a biomarker for various pathological conditions, is produced in post-blood collection by fibrinolysis and coagulation factors
© The Author(s) 2016
Received: 21 January 2016
Accepted: 23 September 2016
Published: 7 October 2016
Fibrinogen alpha C chain 5.9 kDa fragment (FIC5.9) is a new serum biomarker for chronic hepatitis that was discovered by proteomics analysis. Previous studies have shown that FIC5.9 is derived from the C-terminal region of fibrinogen alpha chain and the serum levels of FIC5.9 decrease in chronic hepatitis. It also have been reported that FIC5.9 cannot be detected in the blood stream of the systemic circulation and it is released from fibrinogen during blood clotting in collecting tube. However, the mechanism of FIC5.9 releasing from fibrinogen is unclear.
We formulated a hypothesis that FIC5.9 is released by enzymes that are activated by post-blood collection and may be coagulation and fibrinolysis factors. In this study, we analyzed the mechanisms of FIC5.9 releasing from fibrinogen in healthy blood.
Our analysis showed that thrombin acts as an initiator for FIC5.9 releasing, and that mainly plasmin cleaves N-terminal end of FIC5.9 and neutrophil elastase cleave C-terminal end of FIC5.9.
FIC5.9 reflects minute changes in coagulation and fibrinolysis factors and may be associated with pathological conditions.
KeywordsBiomarker Fibrinogen Plasmin Thrombin Fibrinogen alpha C chain 5.9 kDa fragment (FIC5.9) Coagulation Hepatitis
Many biomarkers have been discovered by proteomics analysis, but fewer have been developed for clinical use [1, 2]. Most of the reported biomarkers involve posttranslational modification or degradation, and they are unclear why the level of biomarker changes in disease. Thus, there is a need to establish the links between synthetic mechanism of the biomarker and disease conditions for practical use in clinical diagnosis .
FIC5.9 synthesis during blood clotting
To confirm the conclusion of our previous study  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 1-h incubation at 25 °C, samples were centrifuged at 1500g for 20 min. The supernatant was purified with C18/WCX cartridges . 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 . Each experiment was done in triplicate.
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). Under these conditions, purified fibrinogen is extensively degraded by thrombin, plasmin and neutrophil elastase [17–19].
LC–MS/MS analysis of degradation products of fibrinogen
Analysis and time course of FIC5.9 synthesis in serum collection tubes
Serum collection tubes (tubes are evacuated; Insepack II, Sekisui Medical Co.) with added thrombin (Wako; 20 U/mL), hirudin (Thermo Fisher; 1 U/mL ), plasmin (Wako; 0.8 U/mL), tranexamic acid (Wako; 10 mM ), sivelestat sodium (Cosmo Bio Co.; Tokyo Japan; 80 µM ) or the same volume of saline were used to collect blood samples from eleven healthy volunteers with an evacuated by Safetouch™ Blood Collection system (NIPRO Co., Osaka Japan). The collected blood was clotted for 0, 5, 30, 60, 90 min at 25 °C. After blood clotting, serum was obtained by centrifugation at 1500g for 10 min at 4 °C. The level of FIC5.9 was measured using a FIC5.9 ELISA kit described in . Written informed consent was obtained prior to the sample collection, and the study was approved by the research ethics committee of the graduate school of medicine, Chiba University (Approval no. 677).
Statistical analysis was performed by Mann–Whitney U-test with SPSS software, version 18.0 (SPSS Inc., Chicago, IL, USA). A P value <0.05 were considered significant.
LC–MS/MS analysis of degradation products of fibrinogen in vitro
Analysis and time course of FIC5.9 releasing in serum collection tubes
To determine if thrombin, plasmin and neutrophil elastase can release FIC5.9 from fibrinogen in blood, we analyzed the time course of FIC5.9 releasing in an evacuated blood collection system. We firstly examined the time course of FIC5.9 releasing in plain and silica-coated tubes by measuring the levels of FIC5.9 in serum after 0, 5, 30, 60, 90 min of clotting. The rate of FIC5.9 releasing in a silica-coated tube was significantly faster than that in a plain tube (Additional file 3), but the final FIC5.9 levels did not differ significantly. All further analyses were performed in silica-coated tubes.
In this study, we formulated a hypothesis that FIC5.9 is released from fibrinogen by coagulation and fibrinolysis factors. We firstly analyzed in vitro released fragments from fibrinogen by digestion with thrombin, plasmin or neutrophil elastase using LC–MS/MS (Fig. 3). Successively we analyzed the time course of FIC5.9 release was determined in serum collection tubes spiked with thrombin (and its inhibitor, hirudin), plasmin (and its inhibitor, tranexamic acid) and sivelestat sodium (neutrophil elastase inhibitor) (Fig. 4). We concluded our analysis showed that thrombin acts as an initiator for FIC5.9 releasing, and that mainly plasmin cleaves N-terminal end of FIC5.9 and neutrophil elastase cleave C-terminal end of FIC5.9 from fibrinogen.
LC–MS/MS analysis showed that N-terminal end of FIC5.9 (RGK/SSS) is cleaved by thrombin or plasmin, and C-terminal end of FIC5.9 (RPV/RGI) is cleaved by neutrophil elastase (Fig. 3). To confirm that these enzymes can release FIC5.9 during blood coagulation, we analyzed the time course of FIC5.9 release was determined in serum collection tubes spiked with thrombin (and its inhibitor, hirudin), plasmin (and its inhibitor, tranexamic acid) and sivelestat sodium (neutrophil elastase inhibitor) (Fig. 4). Addition of thrombin (or their inhibitor, hirudin) accelerated (or delayed) the releasing of FIC5.9 from fibrinogen, but the amounts of FIC5.9 at 90 min clotting had no significance. It is clear that blood clotting initiates FIC5.9 releasing, and FIC5.9 is negligible in the blood circulation, but found in serum collection tubes. However, these results do not demonstrate thrombin plays a main role in FIC5.9 releasing when once blood clotting process is started in serum collection tubes. We conclude thrombin play a role of initiation element of FIC5.9 releasing by starting blood clotting with its activity.
On the other hands, addition of plasmin sufficiently accelerated the releasing of FIC5.9 and increased substantially the amount of FIC5.9. While thinking thrombin is an initiation element of FIC5.9 releasing, these results indicate that plasmin is the major 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 including thrombin [22, 23]. These reports also support the role of thrombin as the initiator of FIC5.9 synthesis. Inhibition of plasmin activity significantly suppressed FIC5.9 releasing from fibrinogen (Fig. 4). The suppression of plasmin by tranexamic acid was significant, but not completely. It may be due to other enzymes which can cleave the N-terminal end of FIC5.9; in fact, our in vitro digestion experiments (Fig. 3) show thrombin can cleave N-terminal end of FIC5.9. Taken together, plasmin seems to play a main role in cleaving N-terminal end of FIC5.9 and releasing FIC5.9.
Up to here, we discussed enzymes cleaving N-terminal end of FIC5.9. Our results also indicate neutrophil elastase play an important role in cleaving C-terminal end of FIC5.9 (Figs. 3, 4). Results of the in vitro digestion experiments (Fig. 3) showed that C-terminal end of FIC5.9 (RPV/RGI) is cleaved by neutrophil elastase, while inhibition of its activity strongly suppress FIC5.9 releasing in serum collection tube (Fig. 4). The suppression of neutrophil elastase completely inhibited the releasing of FIC5.9 from fibrinogen (Fig. 4c), which implies neutrophil elastase plays a major role in cleaving the C-terminal end and releasing of FIC5.9 from fibrinogen.
In the experiments of serum collection tubes, FIC5.9 was detected at 0 min of clotting or inhibitor added tubes (Fig. 4). We estimate this phenomenon is caused by blood collection process; by the moment blood reaches serum collection tubes, blood passes blood collection needle and a thin tube connecting between the needle and a serum collection tube, clearly blood coagulation process have already started before blood reaches serum collection tubes; and centrifugation duration is 10 min.
Our results showed that the mechanism of FIC5.9 releasing in healthy people is strongly related to coagulation and fibrinolysis factors. And our results suggest 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 FIC5.9 releasing. A spike test of neutrophil elastase into the collection tube could not be performed because we could not obtain enough amount of elastase. Indeed, significant volume of enzyme was needed for experiment with blood collection tubes. However, 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 (seems to be an 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 [26–29].
The presence of other factors related to FIC5.9 releasing should also be considered. Recently, Marfa et al. reported that TGF-β reduces the expression level of fibrinogen alpha chain mRNA , which is of note because the level of TGF-β is related to liver fibrosis and hepatitis [31, 32]. In addition, plasma fibrinogen has been proposed as a marker for chronic liver disease . As these reports show, the low level of FIC5.9 releasing in chronic hepatitis is not simply due to enzymatic reactions, but also to a decrease in fibrinogen synthesis (a decrease in the precursor to FIC5.9). Thus, it seems that several factors are involved in determining the level of FIC5.9.
Measurement of degradation products in blood circulation is commonly used in clinical tests [34, 35]. Most coagulation and fibrinolysis factors are unstable for measurement of activity [36–38], 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 released by coagulation and fibrinolysis factors, and reflects a minute change in these factors. Thrombin, plasmin and neutrophil elastase are involved in the key mechanism of FIC5.9 releasing in clotting of normal blood. This provides the basis for understanding the decrease in FIC5.9 in clotting of blood from patients with chronic hepatitis. Further analysis may show similar effects in blood from patients with several diseases.
fibrinogen alpha C chain 5.9 kDa fragment
- SELDI-TOF MS:
surface-enhanced laser desorption/ionization-time of flight mass spectrometry
enzyme-linked immunosorbent assay
- MALDI-TOF MS:
matrix assisted laser desorption/ionization-time of flight mass spectrometry
activated partial thromboplastin time
WK performed and designed research, analyzed data, and wrote the manuscript; MN conceived and designed the experiments, analyzed data, and wrote the manuscript; TK performed and designed research, analyzed data; ST contributed the experiments; TS provided technical input on the mass-spectrometry and chromatography; MS contributed the experiments; KN, contributed reagents, analysis; YK provided technical input on the mass-spectrometry and chromatography; TT contributed reagents/materials/analysis tools; FN conceived and designed the experiments, contributed reagents/materials/analysis. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
In all studies, written informed consent was obtained prior to the sample collection, and the study was approved by the research ethics committee of the graduate school of medicine, Chiba University (Approval no. 677).
This work was supported by JSPS KAKENHI (Grant-in-Aid for Scientific Research (C)) Grant No. 15K08611.
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