Fibrinogen α-Chain as a Serum Marker of Liver Disease
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Liver fibrosis is the hepatic response to an insult characterized by an accumulation of extracellular matrix proteins. If the underlying cause is not treated or eliminated, the disease can progress and may lead to several clinical complications including hepatocellular carcinoma or even death. Thus, detection, staging, and follow-up of liver fibrosis are the main issues in the prognosis and treatment of patients with chronic liver disease. In recent years, new advances in mass spectrometry-based proteomics technology and protein fractionation techniques have improved protein identification as well as protein quantification in many different samples and diseases including liver fibrosis. In particular, the fibrinogen α chain and more specifically the serum levels of the 5.9 kDa fragment of fibrinogen α C-chain have shown to be altered in several hepatic etiologies. In fact, these results have been reproduced by different laboratories, and recently a marked downregulation of this protein fragment has also been described in the initial stages of liver fibrosis. In this chapter, we have described the potential role of fibrinogen α chain and particularly the 5.9 kDa fragment of fibrinogen α C-chain as a circulating marker of liver fibrosis.
KeywordsBiomarker Liver Early detection Fibrinogen Fibrinogen alpha chain Fibrosis Mass spectrometry Proteomics
List of Abbreviations
Alcoholic liver disease
Enhanced liver fibrosis
Hepatitis B virus
Hepatitis C virus
- m/z ratio
- MALDI-TOF/TOF MS
Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry
Amino-terminal peptide of type III procollagen
Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry
Stable-isotope dilution mass spectrometry
Transforming growth factor beta
Tissue inhibitor of matrix metalloproteinase-1
Time of flight
Key Facts of Liver Function and Structure
The liver is the largest organ in the body and is located in the upper-right region of the abdominal cavity.
It is comprised of four different lobes.
It is the only organ that has a dual blood supply: the hepatic artery and the portal vein.
While the hepatic artery provides the oxygen necessary to achieve the different vital functions, the portal vein, which comes from the intestines, is detoxified in the organ. Both blood flows converge and form the hepatic vein which finally drains into the inferior vena cava.
Several cell types compose this organ, and they can be classified as parenchymal and non-parenchymal cells.
Hepatic stellate cells (HSC), Kupffer cells, and hepatocytes are the most relevant cell types. The latter, however, are the principal cell types in the liver and occupy around 80 % of its volume.
The liver, but generally hepatocytes, executes many different functions including protein synthesis, bile synthesis and its secretion, toxin metabolism, etc.
Key Facts of Mass Spectrometry (MS)
It was invented by J. J. Thomson.
Its function is to separate, identify, and even quantify molecules based on their mass-to-charge ratio (m/z). However, molecules must be firstly converted into gaseous ions.
- A mass spectrometer consists of three basic elements:
Ion source: this is where the sample is ionized. Examples of this component are electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI), and surface-enhanced laser desorption/ionization (SELDI).
Mass analyzer: this is where molecules are separated according to their mass-to-charge ratio (m/z). Examples of this component are time of flight (TOF) and quadrupole ion trap, among others.
Ion detector: this is used to detect and record the relative abundance of each charged molecule.
Definitions of Words and Terms
- Alternative splicing
This is a biological process by which multiple mRNAs (and subsequently proteins) are generated from the same gene. Basically, DNA is transcribed into a pre-mRNA, which contains both introns and exons. Subsequently, mature mRNA is formed by the total or partial retention of exons, creating a diverse panel of mRNA from a single pre-mRNA. This phenomenon enhances proteome diversity and the functional capacity of each gene.
This is a physiological process by which fibrinogen is converted to fibrin by the action of thrombin. This cleavage leads to the appearance of a fibrin clot which turns liquid blood into a thickened mass.
This is the mechanism by which fibrin clots are broken down by the action of plasmin. Its physiological function is to prevent the growth of blood clots and reduce their potential side effects.
Hyaluronic acid is one of the components of extracellular matrix (ECM). Its serum levels have proven to have a good correlation with the severity of liver disease. In fact, it is an excellent tool to identify late stages of liver fibrogenesis. However, it is not as useful for the detection of early fibrotic stages.
Hepatitis C virus is one of the main causes of liver fibrosis and cirrhosis worldwide and the main indication for liver transplantation. However, recent advances in drug development have allowed the obtaining of better and highly effective drugs that will predictably reduce all the clinical complications caused by this virus.
- Liver biopsy
Until recently, liver biopsy has been the gold standard method to stage liver fibrosis. This invasive procedure involves the removal of a small sample of liver tissue to be further histologically examined to determine the underlying cause and the severity of the liver disease.
- m/z ratio
The mass-to-charge ratio is the relation between the mass (m) of a molecule and its charge number (z). In this chapter, the molecules analyzed by MS are proteins. In addition, since many of the ions carry a charge of +1, the m/z ratio is often considered to be the molecular weight of the compound.
- MALDI-TOF/TOF MS
This is a proteomic approach that allows the analysis, identification, and even quantification of several biomolecules including proteins. It can be used for biomarker discovery.
Nonalcoholic steatohepatitis is one of the most advanced consequences of nonalcoholic fatty liver diseases (NAFLD). Subsequently, NASH can lead to cirrhosis, liver failure, or even HCC. In terms of prevalence, this phenomenon has increased in Western societies during the last decades due to sedentary habits, unhealthy diet, and the metabolic syndrome. Basically, it is characterized by fat accumulation in the liver which causes inflammation and damage.
- Posttranslational modifications
These are biological mechanisms that enhance proteome diversity. In addition, they play a pivotal role in several cellular processes. Posttranslational modifications (PTM) include all the modifications generated during or after protein synthesis. Phosphorylation, methylation, and proteolysis are just some examples of the wide variety of these modifications.
- Scheuer classification
This is one of the most commonly used classifications for the assessment of chronic hepatitis in liver biopsy specimens. Basically, liver fibrosis can be staged from zero to four. No fibrosis corresponds to fibrosis stage 0 (F0); minimal portal fibrosis would be staged as F1, periportal fibrosis (F2), fibrosis beyond the portal tract making septums (F3), and, finally, cirrhosis (F4).
It is a high-throughput proteomic approach similar to MALDI-TOF, which is also used for biomarker discovery. However, it incorporates solid-phase chromatography that reduces the complexity of the biological sample protein and allows better selectivity of the proteins of interest.
Transforming growth factor beta is mainly produced by Kupffer cells and is the most important cytokine that promotes wound healing and repair. TGF-β induces apoptosis of hepatocytes and activates HSC.
Tissue inhibitor of metalloproteinase 1 is produced by several types of hepatic cells and inhibits the degradation of newly formed collagen fibrils by MMPs. It is also highly upregulated during fibrogenic processes.
Liver fibrosis is a dynamic and reversible wound-healing process characterized by the accumulation of extracellular matrix proteins (ECM) in response to chronic injury (Bataller and Brenner 2005). Despite the remarkable regenerative capacity of the liver, if the underlying cause is not treated and eliminated, chronic liver injury can lead to cirrhosis, hepatocellular carcinoma, liver failure, or even death (Bataller and Brenner 2005; Lee and Friedman 2011; Forner et al. 2012; Schuppan and Kim 2013; Tsochatzis et al. 2014). Many different causes can contribute to liver fibrosis. However, the main etiologies in developed countries are alcoholic liver disease (ALD), cholestasis, chronic hepatitis B virus (HBV) and C virus (HCV) infections, and, recently, nonalcoholic steatohepatitis (NASH) (Schuppan and Kim 2013; Friedman 2010). Knowledge of the extent of hepatic fibrosis is critical to define and establish its prognosis and progression as well as make treatment decisions. However, in clinical practice, early detection of liver fibrosis is, in many cases, relatively complex since the detection and clinical symptoms of hepatic decompensation can occur within weeks, months, or even years after the onset of injury (Hernandez-Gea and Friedman 2011). In fact, some studies have pointed out that about 40 % of patients with liver disease are asymptomatic for 10–15 years before any clinical complication appears (Heidelbaugh and Bruderly 2006). Thus, prompt detection of hepatic fibrogenesis is of major importance in the prognosis and treatment of patients with chronic liver disease.
At present, liver biopsy remains the gold standard method to stage liver fibrosis (Fernandez-Varo and Jimenez 2011). Nevertheless, this procedure must be performed by a trained physician due to its invasiveness. In addition, it may involve several clinical complications including pain, hemorrhage, potential morbidity, or even death. Furthermore, the small amount of specimen obtained in the liver biopsy might not represent the fibrosis stage of the whole organ due to the heterogeneity of fibrosis. Moreover, interobserver variability should also be taken into account, and liver biopsy does not allow fibrosis progression/reversion follow-up (Castera and Pinzani 2010; Bedossa et al. 2003; Fernandez-Varo and Jimenez 2011). For these reasons, many efforts have been made during the last decade to find new noninvasive procedures as alternatives to liver biopsy. These efforts have essentially been focused on developing imaging techniques and identifying new circulating biomarkers (Carrión et al. 2010; Castera et al. 2008; Crespo et al. 2012). Despite both options having several important advantages including their noninvasiveness and easy sequential follow-up of the disease, the latter allows better accessibility (Alrawashdeh and Crnogorac-Jurcevic 2011).
Ideally, a marker of liver fibrosis should be specific and highly sensitive when discriminating the different stages of liver fibrosis of any etiology. It should also be easy to measure and economical as well as reproducible. In addition, it should not vary with inflammation or with any metabolic disorder and should be useful to monitor fibrosis progression/regression (Baranova et al. 2011). Finally, a biomarker should also be able to discriminate the underlying causes of liver disease. Nonetheless, there is currently no marker that fulfills all these criteria (Watkins 2009). However, the advances made in this field have allowed the identification of new potential and very promising biological markers. Additionally, the combination of several serum markers appears to be another interesting strategy in discriminating different stages of liver fibrosis (Sebastiani et al. 2006; Castera 2012; Crespo et al. 2012). In fact, one of the most well-known algorithms is enhanced liver fibrosis (ELF), which combines serum levels of three different direct biological markers: HA, PIIINP, and TIMP-1. This algorithm was developed in an international multicenter study including a cohort of 1,021 patients with different hepatic etiologies (Rosenberg et al. 2004). Age was initially included in the algorithm but was finally excluded on validation of the ELF tool. In fact, ELF is highly accurate for detecting patients with significant fibrosis and cirrhosis (Martinez et al. 2011; Crespo et al. 2012). However, several studies have reported lower diagnostic capacity in early and mild stages of hepatic fibrosis in which the values obtained from fibrotic patients are similar to those of healthy subjects (Martinez et al. 2011; Lichtinghagen et al. 2013). In fact, this is a common trend in many other noninvasive markers and algorithms. Thus, discriminating early stages of liver fibrosis has remained a challenge in this disease.
In view of the fact that hepatocytes, the predominant cell type in the liver, are responsible for the synthesis of most intrahepatic and extrahepatic proteins including plasma coagulation proteins such as prothrombin or fibrinogen (Tennent et al. 2007), it is feasible to think that even subtle changes in liver architecture can incur abnormalities in protein synthesis and be further detected in the bloodstream. Thus, circulating proteins have been one of the most studied areas of research. Recent advances in mass spectrometry -based proteomics technology and protein fractionation techniques have improved protein identification as well as protein quantification in many different samples and diseases (Aebersold and Mann 2003; Camerini and Mauri 2015). In particular, thanks to these technical advances, not only changes in many abundant and non-abundant proteins can now be detected but also in protein fragments released into the circulation (Hortin 2006). In this context, some studies have demonstrated alterations in circulating serum fibrinogen fragments.
Human fibrinogen is one of the most abundant proteins in blood and plays a pivotal role in the coagulation cascade (Mosesson et al. 2001; Davalos and Akassoglou 2012). Structurally, this 340 kDa acute phase glycoprotein synthesized in the liver is comprised of two symmetric sets of three different chains known as Aα, Bβ, and γ. The predominant Aα chain is the longest polypeptide chain, containing 610 residues and weighs 63.5 kDa. The Bβ (56 kDa) and γ (48 kDa) chains consist of 461 and 411 amino acids, respectively. However, all the fibrinogen chains are very heterogeneous as a result of alternative splicing, posttranslational modification, and even proteolytic degradation (Herrick et al. 1999). The N-terminus of each chain is bound by disulfide knots, forming its central E domain. In this region, the Aα and Bβ chains present a small peptide sequence known as fibrinopeptide A and B, respectively, which are crucial for preventing the conversion of fibrinogen to fibrin. At the opposite end, the C-terminus fragments of the Bβ and γ chains together with a portion of the Aα chain constitute both outer D domains. The end peptide sequence of the Aα chain is called the αC domain and forms “free-swimming appendages” (Doolittle 1973) which can interact not only with each other but with the central region of the molecule or even with other αC domains (intermolecular interactions) in fibrin polymerization. In addition, this region is susceptible to being cleaved from fibrinogen by several proteases including plasmin and being released into the systemic circulation where it is further degraded (Cesarman-Maus and Hajjar 2005). However, some fragments from this region can be detected before their dissolution and can be used as potential biomarkers of many diseases including liver fibrosis.
Coagulation and Fibrinolysis
Under physiological conditions, both coagulation and fibrinolysis mechanisms are precisely regulated by many different elements (Cesarman-Maus and Hajjar 2005). Due to different stimuli, fibrinogen is converted to fibrin by thrombin. Afterward, the fibrin clot is broken down in order to prevent excessive clot formation and subsequently blood vessel blockage. Several factors are involved in this complex process, although plasmin is basically the enzyme that lyses the fibrin mesh at different locations, generating fibrinogen fragments of different sizes (Cesarman-Maus and Hajjar 2005). The resulting fragments are released into the circulation and are subsequently degraded by other proteases. However, this process is accelerated during liver injury (Takahashi et al. 1990). Furthermore, it has also been well established that these breakdown fragments can exert direct effects on tissue repair (Herrick et al. 1999). Therefore, it is possible that the soluble fragments released into the bloodstream or even the molecules related to coagulation or fibrinolysis processes could be differentially expressed in liver diseases and be used as biomarkers of hepatic fibrosis. Nevertheless, it seems that depending on the etiology, the mechanisms of degradation and even the cleavage sites could be different. In fact, some studies have reported that conformational changes occur in fibrinogen molecule after moderate alcohol consumption (Gorinstein et al. 2003). It is also well known that there is a lower concentration of serum fibrinogen levels in patients with moderate alcohol consumption (Sierksma et al. 2002). Additional investigations have suggested that moderate consumption entails no changes in fibrinolysis (van Golde et al. 2002), despite other studies describing the presence of clear changes in this mechanism (Dimmitt et al. 1998).
Fibrinogen as a Marker of Liver Cirrhosis
It is well established that serum fibrinogen levels remain unaltered in patients with stable liver diseases. However, this behavior differs in patients with more advanced hepatic fibrosis (de Maat et al. 1995). In fact, several studies have reported that fibrinogen levels are usually diminished in these patients, even though some cirrhotic patients may present fibrinogen serum concentrations within the normal range (Pluta et al. 2010). It is of note that Child-Pugh C cirrhotic patients show the lowest serum levels, probably due to an overall reduction of protein synthesis in this stage. Furthermore, dysfibrinogenemia is also often observed in patients with acute liver failure (Mistry and Jain 2011). Therefore, serum fibrinogen alterations seem to mainly be found in advanced liver disease, and only in very specific cases such as acute liver failure are early alterations observed.
Early stages of liver fibrosis are associated with a clear inflammatory process. In this context, the analysis of fragmentome (also called peptidome) may play a pivotal role for its detection. The fragmentome approach is the analysis of the entire set of tiny peptide components most of which are the fruit of the degradation of the abundant proteins in serum or plasma (Hortin 2006). In fact, it has been reported that fragmentome modifications may be better indicators of inflammation and even organ injury than intact proteins. With regard to fibrinogen, several molecular fragments of the α, β, and γ chains are cleaved and released into the bloodstream due to several stimuli. A more in-depth discussion of β and γ chain fragments is outside the scope of this chapter. However, α chain fragments are particularly interesting due to their rapid alteration in early stages in liver fibrosis (Marfà et al. 2014). Indeed, recent studies using MS technology have identified altered fragments of the fibrinogen α chain with a 5.9 kDa C-terminal fragment of the fibrinogen α chain being one the most frequently described.
5.9 kDa Fibrinogen Alpha C-Chain Fragment as a Marker of ALD
At present, determination of blood ethanol levels is performed for recent alcohol intake. On the other hand, there are several biochemical markers for the diagnosis of ALD. Among them, carbohydrate-deficient transferrin (CDT) and gamma-glutamyltransferase (GGT) are the most commonly used (Anton et al. 2002). However, in some heavy drinkers, no alterations are observed in blood, with GGT serum concentrations being within the standard range (Matsuda et al. 1993).
Early Serum 5.9 kDA Fibrinogen Alpha C-Chain Fragment Alterations in HCV Patients
5.9 kDA Fibrinogen Alpha C-Chain Fragment Behavior in Other Hepatic Etiologies
Cell Regulation and Serum Stability of the 5.9 kDA Fibrinogen Alpha C-Chain Fragment
Potential Applications to Prognosis, Other Diseases, or Conditions
At present there is no study specifically addressed at estimating the strength of the 5.9 kDa fibrinogen α C-chain fragment as a prognostic or diagnostic tool in liver disease. On the other hand, taking this biomarker from the research lab to the daily routine in the clinical diagnostic lab depends not only on accurately defining the sensitivity, specificity, positive predictive value, and negative predictive value as indicators of early fibrogenic activity but also determining the availability of high-throughput methods, such as automatic immunoassays, to measure its concentration in biological fluids.
Finally, neither is it known whether the 5.9 kDa fragment is a specific biomarker for liver fibrosis. In this regard, a study performed by Pang et al. (2006) showed that the MS spectra of patients with severe acute respiratory syndrome (SARS) during the outbreak period displayed a diminution in the intensity of the 5.9 kDa fragment in comparison to non-SARS patients.
The SELDI-TOF-MS proteomic technique has also been widely used to investigate altered expression of the 5.9 kDa fragment in other pathologies including different types of neoplasia. Several experimental data have suggested that this fragment could be a biomarker in this condition. For instance, a diminution in the same m/z intensity of the fragment has been described in breast, ovarian, prostate, and non-small cell lung cancers (Engwegen et al. 2006; Belluco et al. 2007). In contrast, increased intensities were found in colorectal cancer and pancreatic adenocarcinoma (Koopmann et al. 2004; Engwegen et al. 2006). It should be noted that none of these investigations identified the amino acid sequence of the protein peak. However, it is presumably the same 5.9 kDa fragment of fibrinogen α C-chain since the sample processing and MS methods were very similar to those investigations in which the amino acid sequence identification was performed. Therefore, it is quite plausible that the 5.9 kDa fragment of fibrinogen α C-chain can also be useful as a biomarker in some types of neoplastic diseases.
Until the last decade, invasive techniques were the only option to stage liver fibrosis.
Recent technological advances have allowed the development of new imaging techniques and the identification of novel circulating protein markers to detect and monitor the evolution of liver diseases.
Although many circulating biomarkers are appropriate for the identification and follow-up of the severe stages of liver fibrosis, their diagnostic capacity is lower in early and mild stages.
The 5.9 kDa fragment of the fibrinogen α C-chain detected in serum is able to identify patients in early stages of liver fibrosis.
No differences were observed between fibrotic patients in terms of age, gender, etiology, or fibrosis stage when the 5.9 kDa fragment of the fibrinogen α C-chain was evaluated.
TGF-β seems to be one of the potential stimuli that affect the release of this 5.9 kDa fibrinogen peptide.
Part of the work described in this chapter has been supported by grants from Dirección General de Investigación Científica y Técnica (SAF 2009-08839 and 2012-35979 to W. Jiménez and BES-2010-035452 to S. Marfà) and co-financed by the European Union through the European Regional Development Fund (ERDF) “A way of making Europe.” CIBEREHD is funded by the Instituto de Salud Carlos III.
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