Hepatitis B virus (HBV) infection is the most common etiology of liver cirrhosis (1). Portal venous system thrombosis (PVST) is one of major complications of liver cirrhosis (2). Its prevalence ranges from 1 to 26%, which increases with the severity of liver disease (3, 4). In addition, PVST can increase the risk of long-term death, portal hypertension-related bleeding, ascites, and acute kidney injury in cirrhotic patients (5). Therefore, it is necessary to explore the mechanism regarding the development of PVST in HBV-related liver cirrhosis.

C-type lectin-like receptor 2 (CLEC-2) is a type II transmembrane receptor composed of an extracellular ligand-binding C-type lectin-like domain, stalk region, single transmembrane helix, and short cytoplasmic tail (6). It is encoded by the gene CLEC1B located on 12p13.31 with a molecular weight of 32-40 kDa (7). It is highly expressed in megakaryocytes and platelets and slightly expressed in myeloid cell subsets (8, 9). It has been shown that CLEC-2 mediates the activation and aggregation of platelets (10). Moreover, deep venous thrombosis is significantly inhibited in CLEC-2-deficient mice, suggesting that CLEC-2, especially platelet CLEC-2, should be critical for the development of venous thrombosis (11).

Galectin-1 is the firstly discovered member of the galectin family and belongs to the prototype galectin containing a carbohydrate-binding domain, which can exist as monomers or dimers (12). It is a 14.5 kDa protein encoded by the gene LSGALS1 located on 22q12 (13). Due to the lack of signal peptides, galectin-1 is secreted through a non-classical secretion pathway that translocate directly across the plasma membrane, which performs important biological functions both inside and outside cells (14). Galectin-1 knockout mice have a significantly longer median tail bleeding time than wild-type mice (15). Furthermore, galectin-1-deficient platelets exhibit impairment in fibrinogen adhesion and clot retraction, suggesting that galectin-1 should contribute to hemostasis and involve in thrombosis (15).

Some animal studies have demonstrated a relationship of CLEC-2 and galectin-1 with venous thrombosis, but human studies are lacking (10, 15–17). On the other hand, their association with PVST has never been explored yet. For this reason, this cross-sectional study aimed to evaluate the relationship between CLEC-2/galectin-1 and PVST in patients with HBV-related liver cirrhosis, and further explore the impact of degree and location of PVST on their relationship.

Some major findings of this study were as follows: (1) serum CLEC-2/galectin-1 level was positively associated with the presence of PVST in HBV-related cirrhotic patients; (2) patients with partial/complete PVST or fibrotic cord, but not those with mural PVST, had significantly higher serum CLEC-2 and galectin-1 levels than those without PVST; (3) patients with MPV or SMV thrombosis had a significantly higher serum CLEC-2 level than those without PVST; and (4) patients with SMV thrombosis, but not those with MPV thrombosis, had a significantly higher serum galectin-1 level than those without PVST.

Platelets are denucleated blood cells from megakaryocytes in the bone marrow (22). Thrombocytopenia, a common complication of liver cirrhosis, is mainly due to decreased synthesis of thrombopoietin in the liver and increased breakdown of platelets in the spleen (23). However, the coagulation system can be often compensated in people with liver disease (24). A reduction in the number of platelets can induce an increase in von Willebrand factor and a decrease in plasma metalloproteinase ADAMTS13, compensatively promoting platelets function (24). On the other hand, platelets adhesion, dissemination, and aggregation are essential for venous thrombosis (25). They also express phosphatidylserine, which provides procoagulant surface for prothrombinase complexes to activate coagulation cascade, and exert pro-inflammatory effects by inducing neutrophil extracellular trap formation, enhancing leukocyte recruitment, and secreting granular contents to activate coagulation system, which further promote the development of venous thrombosis (26, 27). Taken together, it should be reasonable to speculate that the change of platelets function in cirrhotic patients contributes to the development of PVST.

CLEC-2 is an important platelet-activating receptor (9). Its ligand, podoplanin, expresses near the lumen side of endothelial cell in the vascular wall, and the expression of podoplanin is increased in the presence of inflammation and blood flow obstruction (11). Systemic and local inflammation caused by viral hepatitis and increased portal pressure can damage vascular endothelial cells, allowing circulating platelet CLEC-2 to interact with subendothelial podoplanin and possibly with some other unidentified ligands (11, 28, 29). CLEC-2 binds to its ligand to induce phosphorylation of tyrosine residues in a single YXXL motif in the CLEC-2 intracellular domain (30, 31). Splenic tyrosine kinase is activated by binding its tandem Src homologous 2 domain to two tyrosine-phosphorylated YXXL motifs, subsequently triggering downstream signaling pathways that ultimately promote platelets activation and aggregation (7, 17, 32).

Extracellular galectin-1 can induce platelets activation by binding to different surface receptors on platelets (15). As potent platelets agonist, it activates the “inside-out” signal transduction pathway to promote a conformational change in the integrin α_IIbβ₃ on the platelets surface, changing from a low-affinity/resting state to a high-affinity/active state, exposing the high-affinity binding sites of fibrinogen (15, 33–35). At the same time, galectin-1 can also directly bind to platelets surface integrin α_IIbβ₃ through α_IIb subunit, trigger the “outside-in” signal transduction pathway, and then promote platelets activation and aggregation (15, 34). Moreover, platelets can also express galectin-1, and galectin-1 contained in platelets may play a role in platelets activation, which requires further studies (15, 35).

Our study has for the first time demonstrated a relationship between CLEC-2/galectin-1 level and PVST in cirrhotic patients, which may be dependent upon the degree of PVST. Similarly, an animal study also showed that the progression to stable large thrombosis in CLEC-2-deficient mice was almost completely eliminated compared to the control group, suggesting the critical role of CLEC-2 in pathologically occlusive thrombosis (10). At present, both the Chinese consensus (5) and Baveno VII consensus (36) indicate that the timing of anticoagulation should be related to the degree of PVST. Therefore, both CLEC-2 and galectin-1 levels may be valuable for deciding the initiation of anticoagulant therapy.

Our study also demonstrated that serum galectin-1 level was higher in patients with MPV thrombosis than those without PVST, but this difference was not statistically significant. There were two possible explanations for this unexpected phenomenon. First, Xu et al. (37) showed that galectin-1 level in the peripheral blood of mice decreased with its age. Indeed, the mean age was significantly higher in our patients with MPV thrombosis than those without PVST, suggesting that age might weaken the difference in serum galectin-1 level between the two groups. Second, the sample size of this study was relatively insufficient, thereby compromising the statistical power.

There were some limitations in our study. First, the number of patients included was limited, thus large-scale studies are needed to further verify our results. Second, only patients with HBV-related liver cirrhosis were selected, thus further validation is needed in liver cirrhosis secondary to other etiology.

Our current findings support a relationship between CLEC-2/galectin-1 and PVST in patients with HBV-related liver cirrhosis. Thus, CLEC-2/galectin-1 may predict the risk of more severe PVST, and they should be considered as potential targets for the prevention and treatment of PVST. In future, the mechanism regarding how CLEC-2/galectin-1 influences the development of PVST needs to be explored.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

This study followed the Declaration of Helsinki and was approved by the Medical Ethical Committee of the General Hospital of Northern Theater Command (approval number: Y2023-008).

XQ: conception and design, administrative support. YZ, XX, SX, JC, and XQ: provision of study materials or patients. YZ, XX, SX, and JC: collection and assembly of data. YZ and XQ: data analysis and interpretation. YZ, XZ, XX, XG, SX, SM, JC, and XQ: manuscript writing. YZ, XZ, XX, XG, SX, SM, JC, and XQ: final approval of manuscript. All authors contributed to the article and approved the submitted version.