Fibrinogen superfamily proteins: Key regulators in hepatic disorders

Abstract

Liver diseases including hepatic injury, hepatitis, metabolic-associated fatty liver disease, and hepatocellular carcinoma have emerged as critical public health challenges globally. The fibrinogen (FG) superfamily proteins, primarily comprising FG, FG-like protein (FGL) 1, and FGL2, have been demonstrated to exert regulatory effects on hepatic tissue regeneration, lipid metabolism homeostasis, and oncogenic processes in hepatocytes. This review systematically examines the pathophysiological correlations between FG superfamily members and major hepatic disorders. Physiologically, FG superfamily proteins function as hepatoprotective mediators through their intrinsic capacity to enhance hepatic parenchymal regeneration and extracellular matrix remodeling. However, emerging evidence reveals their dual regulatory properties, whereby overactivation of these hepatokines paradoxically induces a pathological shift characterized by pro-inflammatory cascades, metabolic derangement potentiation, and tumor invasion and metastasis promotion. Specifically, we elucidate the molecular mechanisms underlying their involvement in hepatitis progression, metabolic-associated fatty liver disease pathogenesis, and hepatocellular carcinoma tumorigenesis. Furthermore, we highlight their therapeutic potential in hepatic disease management, particularly emphasizing that targeting the FGL1/lymphocyte activation gene 3 immune checkpoint axis represents a novel paradigm in precision cancer immunotherapy.

Key Words: Fibrinogen superfamily; Liver injury; Hepatitis; Metabolic associated fatty liver disease; Hepatocellular carcinoma

Core Tip: In this article, we introduce three fibrinogen superfamily proteins: Fibrinogen, FG-like protein 1, and FG-like protein 2. We focus on their roles in liver injury, metabolic associated fatty liver disease, and hepatocellular carcinoma, as well as potential therapeutic approaches. This review aims to deepen the understanding of the relationship between the fibrinogen superfamily and liver diseases, providing new insights for the treatment of hepatic disorders.

INTRODUCTION

Liver diseases, mainly including liver injury, hepatitis, metabolic associated fatty liver disease (MAFLD), and hepatocellular carcinoma (HCC), are huge killers of human health globally. More than 2 million people die every year worldwide due to these kinds of diseases[1]. Therefore, finding potential mechanisms of action and relevant therapeutic targets are critical to establish effective diagnostic protocols and treatment strategies for these kinds of liver diseases.

The fibrinogen (FG) superfamily includes FG, fibrinogen-like protein (FGL) 1, FGL2, and other fibrinogen-related proteins[2-4]. Many studies have shown that the FG superfamily proteins play protective roles in self-repair of damaged livers, whereas overactivation of these hepatokines paradoxically induces a pathological shift characterized by pro-inflammatory cascades, metabolic derangement potentiation, as well as tumor invasion and metastasis promotion[2]. In the current review, the origin, structure, and function of FG, FGL1, and FGL2 proteins in the FG superfamily are introduced, and their roles in liver diseases are discussed. This review would be helpful to further understand the relationship between FG superfamily proteins and liver diseases, providing a new idea for rational targeted treatment of these disorders.

LIVER DISEASES

Emerging research has demonstrated the crucial involvement of FG superfamily proteins in three major hepatic pathologies: Acute/chronic liver injury progression, MAFLD pathogenesis, and HCC tumorigenesis[2]. Thus, herein we mainly introduce these three pathological conditions. Liver injury refers to the structural and functional impairment of the liver resulting from diverse etiological factors, which can be categorized into acute and chronic forms based on the duration and progression of the damage[5]. Acute hepatic damage can be categorized into several types, including inflammation-mediated injury triggered by viral infections, hepatocyte necrosis resulting from exposure to carbon tetrachloride or acetaminophen, and physical trauma caused by partial hepatectomy or radiation exposure[5,6]. On the other hand, chronic liver injury could result from fibrin deposition and cholestatic liver injury induced by drugs[5,7]. Such hepatic injuries not only disrupt the organ’s physiological functions but also lead to severe complications, including inflammatory responses, fibrotic scarring, and potentially HCC. Furthermore, the liver’s ability to self-repair and regenerate are also impaired when it is injured[8].

MAFLD is a chronic hepatic condition characterized by abnormal fat deposition in the liver and metabolic disturbances, including obesity, overweight, and insulin resistance. Previously termed nonalcoholic fatty liver disease, this condition has been redefined to reflect its metabolic origins[9]. Unlike nonalcoholic fatty liver disease, the diagnostic framework for MAFLD does not necessitate the exclusion of other liver disorders, such as viral hepatitis or alcohol-related liver damage[10]. The spectrum of MAFLD encompasses metabolic fatty liver disease, metabolic dysfunction-associated steatohepatitis (MASH), and their potential progression to chronic hepatic injury, cirrhosis, and HCC[11]. Moreover, MAFLD is frequently associated with a range of metabolic comorbidities, including type 2 diabetes mellitus, cardiovascular disorders, hyperuricemia, and obesity[12].

HCC ranks as the third most lethal malignancy globally. The tumorigenesis of HCC involves multifaceted interactions among: (1) Demographic determinants (age, sex, and ethnic predisposition); (2) Disease progression parameters (fibrosis stage, hepatic inflammatory activity, and therapeutic interventions); (3) Metabolic dysregulation (diabetes mellitus and obesity spectrum disorders); and (4) Behavioral risk factors (chronic alcohol consumption and tobacco use). Approximately 90% of HCC cases typically arise from preexisting chronic liver conditions, including hepatitis B/C viral infections, alcoholic steatohepatitis, and MAFLD[13]. Notably, while the incidence of viral hepatitis-related HCC has shown a declining trend, emerging epidemiological data reveal a progressive increase in MAFLD-associated HCC cases[14,15].

FG SUPERFAMILY PROTEINS

In this section, we are primarily focused on presenting the structure and characteristics of FG, FGL1, and FGL2. FG is a 340 kDa soluble glycoprotein primarily synthesized in hepatocytes, which is composed of three different polypeptide chains encoded by three independent genes: Aα (FGA, 52.0 kDa), Bβ (FGB, 52.0 kDa), and Gγ (FGG, 46.5 kDa)[16-18]. FG is initially formed in the endoplasmic reticulum through interactions to create Aα-γ and Bβ-γ complexes, which are then connected by disulfide bonds to form a symmetrical dimeric structure[19]. The secretion of FG is subject to a strict quality control mechanism, ensuring that only fully assembled FG is secreted into the bloodstream[20,21]. In addition to liver cells, FG synthesis also occurs in monkey kidney cells (cercopithecus aethiops SV40 transformed cells), hamster kidney fibroblasts, lung epithelial cells, and fibroblast-like cell lines of human breast cancer epithelial cells, indicating that FG assembly is not liver cell-specific (Figure 1)[22-25].

Figure 1 Structure of fibrinogen superfamily proteins. A: Fibrinogen (FG) is a soluble glycoprotein with a molecular weight of 340 kDa, comprising three distinct subunits: Aα, Bβ, and Gγ chains; B: The monomeric form of FG-like protein (FGL) 1 contains an N-terminal C-terminal FG-like domain and a C-terminal FG-related domain. Two identical 34 kDa subunits assemble into a functional dimer via disulfide linkages; C: Membrane-bound FGL2 is classified as a type II transmembrane glycoprotein, featuring an intracellular domain at its N-terminus, a transmembrane segment, and an extracellular region at the C-terminus; D: Soluble FGL2 exhibits structural polymorphism, existing as both monomeric and tetrameric forms. Initially, monomers dimerize through disulfide bonds, followed by further association into tetramers via additional covalent bridges. FG: Fibrinogen; FGL1: Fibrinogen-like protein 1; mFGL2: Membrane-bound fibrinogen-like protein 2; sFGL2: Soluble Fibrinogen-like protein 2.

FGL1, also known as hepatocyte-derived fibrinogen-like protein-1 or hepassocin, is primarily synthesized and secreted by hepatocytes and represents a novel hepatokine that regulates hepatic metabolism[3]. FGL1 is a 68 kD homodimeric protein, which is composed of two 34 kD subunits linked by disulfide bonds and contains approximately 312 amino acid residues[26]. It shares high homology with fibrinogen, featuring a hydrophobic signal peptide and characteristic amino acids at the carboxyl terminus, with β and γ subunits[3]. However, due to the absence of platelet binding sites and thrombin-sensitive sites, FGL1 does not participate in coagulation functions (Figure 1)[27].

FGL2, known as prothrombin, is an evolutionarily highly conserved protein that has a 36% homology to the FG beta and gamma chains and 40% homology to the fibrinogen-related domain of cytotactin[28,29]. Till now, two distinct subtypes of FGL2, membrane-associated FGL2 (mFGL2) and soluble FGL2 (sFGL2), have been identified[30]. mFGL2 is a 70 kDa transmembrane protein that is mainly expressed in epithelial cells, endothelial cells, macrophages, and dendritic cells[30,31]. mFGL2 could activate prothrombin and transform it into thrombin that plays an important role in innate immunity[30]. sFGL2 with a size of 50 kDa is highly expressed in regulatory T cells (Tregs) rather than helper T lymphocytes and B lymphocytes. When acting as an immunosuppressor, it can restrain the proliferation of T cells and the maturation of bone marrow dendritic cells (Figure 1)[30].

RELATIONSHIPS OF FG SUPERFAMILY PROTEINS WITH LIVER DISEASES

Role of FG in liver diseases

FG plays a crucial role in liver injury and affects several specific types of damage[2]. Luyendyk et al[31] found that during cholestasis-induced liver injury, the expression of FG is enhanced, leading to the recruitment and activation of inflammatory cells and local hypoxia in the liver. Additionally, FG can limit the release of bile from the damaged liver, thereby inhibiting the severity of liver injury[32]. Liu et al[33] discovered that a derivative peptide of the FGβ-chain (Bβ15-42) inhibits hepatic inflammation by preventing the release of high mobility group box 1 protein and the activation of mitogen-activated protein kinase (MAPK), thereby inhibiting the activation of c-Jun N-terminal kinase to slow down the necrosis and apoptosis of hepatocytes. Poole et al[34] indicated that altering the structure of the FG γ-chain can affect the fibrin polymerization reaction induced by thrombin, thereby exacerbating the severity of acute liver injury. Kopec et al[35] demonstrated that during acetaminophen-induced liver injury repair, FG interacts with the leukocyte integrin αMβ2, upregulates the expression of Mmp12, promotes hepatocyte proliferation, and repairs damaged liver tissue. In summary, FG and its chains could play roles in hemostasis, reduce cell necrosis, and initiate targeted repair during liver injury[36].

Additionally, FG is a biomarker for pro-inflammatory responses and thrombosis in metabolic syndrome[27]. The deposition of FG in tissues can promote obesity and disrupt related metabolic balance. Hur et al[37] reported that the polymerization and cross-linking of fibrin can induce the formation of binding motifs for leukocyte integrin receptor αMβ2, thereby promoting the development of obesity, MAFLD, and hypercholesterolemia. Therefore, selectively eliminating the polymerization and cross-linking of fibrin could prevent related diseases.

FG also plays a significant role in the invasion, growth, and metastasis of tumor cells[35,36]. It deposits around tumor cells and forms platelet-fibrin microthrombi, which act as a physical barrier to hinder the clearance of tumor cells by natural killer cells, allowing them to evade recognition by the innate immune system[35,37]. Additionally, tumor cells secrete pro-coagulant factors that convert FG into fibrin, further exacerbating the hypercoagulable state of the blood and enhancing the growth, proliferation, and invasive capacity of tumor cells[38]. FG can bind to various cell growth factors, including transforming growth factor-β, vascular endothelial growth factor, platelet-derived growth factor, and the fibroblast growth factor family, thereby promoting the proliferation of tumor cells[39,40]. Yan et al[41] found that in patients with liver cancer, tumor growth can compress tissue vessels, induce the aggregation and adhesion of platelets and tumor cells, inhibit the transcription of thrombomodulin, and lead to elevated FG levels. Therefore, FG could serve as a biomarker for the detection of HCC and be used as a prognostic factor for liver cancer.

Role of FGL1 in liver diseases

FGL1 serves as a hepatic protective factor, which promotes the self-repair of damaged livers. Han et al[42] found that the expression level of FGL1 is associated with the degree of radiation-induced liver injury. Hara et al[26] discovered that human FGL1 obtained through cloning can stimulate cellular uptake of 3H-thymidine and further increase DNA synthesis in primary hepatocytes. Additionally, FGL1 binds to the membrane-specific receptor of the L02 hepatocyte line, inducing cell proliferation through the epidermal growth factor receptor/extracellular signal-regulated kinase/Src family of tyrosine kinases pathway[43,44].

FGL1 is involved in the regulation of adipogenesis, gluconeogenesis, and insulin resistance[45,46]. Carbohydrates and unsaturated fatty acids enhance the transcription of FGL1 by activating signal transducer and activator of transcription 3[46-48], which in turn promotes the phosphorylation of extracellular signal-regulated kinase 1/2 and induces adipogenesis (Figure 2)[45].

Figure 2 Role of fibrinogen-like protein 1 in liver diseases. In liver injury, fibrinogen-like protein (FGL) 1 exerts its effects by stimulating the Src family of tyrosine kinases/epidermal growth factor receptor/extracellular signal-regulated kinase 1/2 cascade through autocrine signaling, thereby modulating hepatocyte proliferation and apoptotic processes. Under conditions of metabolic-associated fatty liver disease, elevated levels of palmitate and monounsaturated fatty acids upregulate FGL1 expression in hepatocytes via activation of the interleukin-6/signal transducer and activator of transcription 3 pathway. This upregulation subsequently triggers lipid deposition and inflammatory responses through mechanisms dependent on extracellular signal-regulated kinase 1/2 signaling. In the context of hepatocellular carcinoma, the assembly of the hepatocyte nuclear factor 1 homeobox α/high mobility group box 1 /cyclic adenosine monophosphate-response element binding transcriptional complex boosts FGL1 transcription. Additionally, FGL1 suppresses tumor cell growth by interfering with the cell cycle and inhibiting the protein kinase B/mammalian target of rapamycin signaling axis. Furthermore, FGL1 interacts with lymphocyte activation gene 3 receptors expressed on CD4⁺ T cells, CD8⁺ T cells, and natural killer cells, leading to the suppression of T cell-mediated antitumor immune responses. FGL: Fibrinogen-like protein; Src: Src family of tyrosine kinases; ER: Endoplasmic reticulum; EGFR: Epidermal growth factor receptor; ERK: Extracellular signal-regulated kinase; FAS: Fatty acid synthase; ACC: Acetyl-CoA carboxylase; STAT3: Signal transducer and activator of transcription 3 ; HCC: Hepatocellular carcinoma; LAG: Lymphocyte activation gene; CREB: Cyclic adenosine monophosphate response element-binding protein; HNF1α: Hepatocyte nuclear factor 1 α; HMGB1: High mobility group box 1 protein; IL: Interleukin; mTOR: Mammalian target of rapamycin; AKT: Protein kinase B; MAFLD: Metabolic-associated fatty liver disease.

FGL1 plays paradoxical roles in normal liver cells and liver cancer cells. Cao et al[43] found that FGL1 acts as a positive regulator in non-tumor cells through autocrine signaling, whereas it inhibits cell growth through the endocrine pathway in the HCC cell line HepG2. Yu et al[49] demonstrated that hepatocyte nuclear factor-1α is an important liver-specific cis-acting element of FGL1, which activates the FGL1 promoter and promotes its transcription by binding to Box-1 protein (high mobility group box 1 protein) and cyclic adenosine monophosphate response element binding protein in the progression of HCC. Moreover, FGL1 inhibits the proliferation of liver cancer cells by suppressing the protein kinase B/mammalian target of rapamycin pathway (Figure 2)[50].

Strikingly, FGL1 is reported to impair CD8⁺ T-cell function through FGL1-lymphocyte activation gene 3 (LAG3) interaction, ultimately fostering a pro-tumorigenic microenvironment. LAG3 is a crucial negative regulator of T cell activation and function[51], which is expressed on the surface of lymphocytes in various tumors and exerts immunosuppressive effects by binding to its ligand[52]. FGL1 can inhibit T cell responses by binding to the functional ligand LAG3 Located on the surface of cells such as CD4⁺ T cells, CD8⁺ T cells, and natural killer cells, allowing tumor cells to evade the body’s immune tracking and promoting tumor growth by altering the immune microenvironment. Thus, blocking the interaction between LAG-3 and FGL1 can enhance T cell responses to tumors and lead to a reduction in tumor size[53]. Therefore, the FGL1/LAG3 axis has become a new target for cancer immunotherapy. Clinically, the use of anti-LAG3 monoclonal antibodies or drugs that block the interaction between LAG3 and FGL1 could enhance antitumor immunity[54]. Additionally, the use of bispecific antibodies to block the interaction between programmed death-ligand 1 and LAG3 enhances the activity of immune cells and increases the number of CD4⁺ and CD8⁺ T cells, thereby improving antitumor efficacy[54]. Currently, monoclonal antibodies targeting LAG3 are undergoing clinical trials and have shown promising antitumor potential. However, monoclonal antibodies targeting FGL1 have not yet entered the clinical trial phase (Figure 2).

Role of FGL2 in liver diseases

FGL2 plays multifaceted roles in regulating the progression and outcomes of both acute and chronic hepatitis through its immunomodulatory functions and signaling pathway regulation. In acute liver injury settings, FGL2 demonstrates protective effects by suppressing p38 MAPK signaling to reduce drug-induced hepatotoxicity[55]. In viral hepatitis, sustained FGL2 expression contributes to hepatic stellate cell activation through both coagulation-dependent and -independent mechanisms, ultimately driving collagen deposition and fibrotic progression[30]. Additionally, FGL2 may facilitate viral immune evasion by enhancing Treg activity or suppressing cytotoxic T lymphocyte responses, thereby promoting chronic infection. Clinical evidence suggests a positive correlation between elevated FGL2 Levels and increased hepatitis B virus load. In chronic hepatitis conditions, FGL2 is an important immunosuppressive effector of Tregs. When Tregs accumulate at the inflammatory site, the expression of FGL2 will be enhanced and bound to the inhibitory Fc fragment of IgG low-affinity IIb receptor on the upper surface of antigen-presenting cells[56]. According to the study of Liu et al[57], mesenchymal stem cells overexpressing sFGL2 can promote the differentiation of Treg cells, T helper 17 cells, and T helper 1 cells by enhancing the phosphorylation of Src homology 2 domain-containing protein tyrosine phosphatase 2 and mothers against decapentaplegic homolog 2/3 that can alleviate liver damage caused by autoimmune hepatitis. While the immunosuppressive properties of FGL2 could be beneficial in certain contexts, such as mitigating excessive immune responses in autoimmune hepatitis, its dual role in fibrosis promotion and anti-inflammatory regulation necessitates careful therapeutic consideration.

FGL2 also plays an important role in metabolic disorders. Hu et al[58] indicated that in alcoholic fatty liver disease, FGL2 accelerates alcoholic liver injury by mediating the dependence of pyruvate kinase M2 subtype in pro-inflammatory macrophages on the aerobic glycolysis pathway. FGL2 deficiency alleviates hepatitis injury in MASH mice[59]. Plasma FGL2 Levels in MALFD patients are higher than those in healthy controls[60]. Hu et al[59] suggested that FGL2 overgenerates pro-inflammatory cytokines and reactive oxygen species through up-regulation of nuclear factor kappa B and p38-MAPK signaling pathways and production of nucleotide-binding domain, leucine-rich repeat, and PYD-containing protein 3 inflammasome that result in liver lipid metabolism disorder and severe liver injury in MASH. Therefore, FGL2 may be a potential biomarker and therapeutic target for MASH in the future.

FGL2 is highly expressed in liver cancer, whose expression is positively correlated with the expression levels of CD11b, CD33, and surface markers of myeloid-derived suppressor cells[61]. Knocking out Fgl2 can significantly inhibit the growth of liver tumors and retard the development of liver cancer[62]. FGL2 facilitates the development of HCC by promoting the differentiation of macrophages and the proliferation of Tregs in the tumor microenvironment[63]. In addition, FGL2 promotes the development of liver cancer by affecting the proliferation of CD8⁺ T cells and the activation of dendritic cells[64]. Wu et al[65] evidenced that FGL2 regulates the differentiation of myeloid-derived suppressor cells by activating the X-box binding protein 1 signal to generate reactive oxygen species, thus promoting the development of liver cancer. FGL2 is also involved in the regulation of the FGL2-Fc fragment of IgG low-affinity IIb receptor pathway during the development of HCC[66]. Knocking down Fgl2 would hinder the activation of PAR2 and impair downstream c-Jun N-terminal kinase phosphorylation, which gives rise to the arrest of the tumor cell cycle, thus inhibiting tumor proliferation and angiogenesis[62,67]. In conclusion, FGL2 serves as a new target for tumor immunotherapy.

CONCLUSION

The liver serves as the central hub for systemic energy regulation, orchestrating metabolic processes through hepatokines, a class of liver-derived signaling proteins including FG, FGL1, and FGL2[68]. These pleiotropic mediators significantly influence hepatic pathophysiology and interorgan communication. Emerging evidence positions hepatokine research as a pivotal frontier in understanding liver-systemic crosstalk, with particular focus on their dual roles in tissue repair and disease progression[69,70]. FG acts as an acute-phase protein, participating in the inflammatory process and the activation and migration of leukocytes. In the liver tumor microenvironment, it is involved in the regulation of the cell cycle and the expression of metabolism-related genes, promoting liver tumor growth and inhibiting tumor cell senescence[2,27]. Additionally, FG levels are associated with obesity, insulin resistance, and pro-thrombotic and inflammatory conditions in MAFLD[71]. FGL1 functions as a protective hepatokine in tissues that could promote cell growth[72]. Under high-fat conditions, elevated levels of hepatic FGL1 can lead to lipid accumulation and trigger inflammatory responses, thereby accelerating the progression of metabolic fatty liver disease. FGL1 can also act as a ligand to promote the proliferation of tumor cells and accelerate the transformation of liver cancer[53]. FGL2 functions as a prothrombin and exerts pro-coagulant effects. Its immunomodulatory functions play a vital role in combating hepatitis, immune rejection in organ transplantation, and cancer progression[73].

In practical applications, FGL1 acts as a ligand for LAG3, and their interaction can stimulate the activation and proliferation of T cells in the tumor microenvironment. The discovery of immunotherapy through the LAG3/FGL1 axis represents a milestone in cancer treatment, which also prompts us to consider whether FGL1 regulates tumor progression through other mechanisms as well[74,75]. Monitoring FGL2 Levels is crucial for assessing the severity of viral hepatitis. In liver transplantation, FGL2 exhibits immunosuppressive functions, protecting both the host and the graft. Dynamic monitoring of FGL2 Levels has been proven to be beneficial for evaluating transplant tolerance. In cases of MAFLD, it has been found that Fgl2 depletion could improve ethanol-induced hepatic steatosis, oxidative damage, and pro-inflammatory cytokines[58]. Targeting FGL2 in hepatic macrophages may hold promise as an intervention for MAFLD and alcoholic hepatitis. Therefore, elucidation of these mechanisms would yield the following significant implications: (1) Uncovering shared regulatory checkpoints in the liver inflammation-fibrosis transition; and (2) Establishing a novel paradigm for developing multi-target interventions based on the FGL molecular network. Additionally, their potential as biomarkers and therapeutic targets is of significant value in detecting liver diseases and identifying new treatment strategies.