Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver injury worldwide. NAFLD is highly associated with obesity and the metabolic syndrome (MetS). It might be accompanied by liver inflammation and signs of hepatocyte damage, leading to nonalcoholic steatohepatitis (NASH). NASH can lead to progressive fibrosis and cirrhosis.
In recent years, a possible role of NAFLD in the development of cardiovascular disease (CVD) has been suggested and this relationship seems to be independent from obesity and the MetS.[3, 4] However, the exact mechanisms linking NAFLD to CVD are still poorly understood. Different pathways could be involved, one of which is overproduction of prothrombotic factors in patients with fatty liver.
Prothrombotic factors have also been identified in patients with cirrhosis and portal vein thrombosis (PVT) and might be implicated in the pathogenesis of several liver diseases. Recently, Villa et al. reported a lower rate of complications in patients with cirrhosis treated with low-molecular-weight heparins, suggesting again the role of local thrombotic events in the progression of liver disease.
Different human studies have assessed the relationship between liver steatosis and prothrombotic factors. Although most of these studies report several alterations in different coagulation factors, results are inconsistent. This might, in part, be explained by methodological issues. Several studies used elevated serum liver tests,[9, 10] ultrasonography,[9, 11] or magnetic resonance spectroscopy (MRS)[12, 13] for the diagnosis of liver dysfunction. Only a few studies used data obtained from liver biopsy.[11, 14-16] Low patient numbers and retrospectivity might further account for the discrepant results.
The aim of the present study was therefore to assess the relation between an extended set of coagulation factors and the histological subtypes and severity grades of NAFLD in patients with obesity, independent of metabolic risk factors (including visceral fat measurement by computed tomography [CT]). Therefore, NAFLD and coagulation status were assessed in a large group of thoroughly characterized, consecutively recruited subjects who were overweight or obese, using liver biopsy as the gold standard for diagnosis and staging of NAFLD/NASH.
The aim of the present study was to investigate whether NAFLD and its different subtypes in obese subjects were associated with a prothrombotic state and whether this link existed independently of metabolic risk factors. Therefore, we studied a large group of overweight or obese subjects, using liver biopsy as the gold standard for the definition and classification of NAFLD. From an extended panel of prothrombotic factors, only PAI-1 showed a significant increase in PAI-1 levels throughout the different stages of the histological features of NAFLD, with the highest levels in patients with more severe steatohepatitis. No consistent differences were found in other prothrombotic factors. Multiple regression showed C-peptide, steatosis, waist circumference, and levels of GGT, a marker of liver damage, as independent determinants of PAI-1 levels, together explaining 21% of its variance.
Obesity and the MetS are known to be associated with an increased risk of thromboembolic events. An increase in PAI-1, fibrinogen, FVIII, and vWF and a decrease in antithrombin III are most frequently reported. Also, in our large series of overweight and obese patients, the increase in PAI-1, fibrinogen, FVIII, and vWF and decrease in antithrombin III in relation to metabolic factors is confirmed, whereas the other coagulation factors were not significantly altered.
Because the liver is an important source of the majority of coagulation factors and NAFLD and NASH are closely linked to the features of the MetS, the specific contribution of NAFLD and NASH to the observed alterations needs to be assessed. Furthermore, NAFLD has been identified as a cardiovascular risk factor independent of frequently associated metabolic risk factors. Therefore, it can be hypothesized that alterations in the hepatic production of coagulant factors might, in part, explain both the prothrombotic state in obesity and the role of NAFLD in increasing the risk of cardiovascular events. Furthermore, prothrombotic factors have been implicated in the pathogenesis of cirrhosis and in PVT. Recently, anticoagulant therapy has shown to beneficially influence the natural history of cirrhosis. Therefore, prothrombotic factors may play an important role in disease progression in several liver diseases.
A first conclusion of our study is that most of the alterations observed in obesity are unrelated to liver status. The correlations between alterations in fibrinogen or vWF and liver histology are insignificant when corrected for metabolic factors, such as BMI or VAT, which implies that the observed alterations are related to the common underlying metabolic features, but not to a specific contribution, of the fatty or inflamed liver. For many other factors studied, no consistent correlation with liver histology was observed, except for PAI-1. We can therefore conclude that in NAFLD and NASH, at least in obese patients, most of the prothrombotic factors are unaltered.
Few studies have assessed a large panel of coagulation factors in patients with NAFLD. Kotronen et al. found an increase in FVIII, FIX, FXI, and XII in a retrospective analysis of 54 patients with NAFLD, mostly diagnosed by MRS, compared to 44 controls. The differences remained significant after adjustment for metabolic factors. Assy et al. found an increase in protein C in relation to the degree of fibrosis in 15 patients with biopsy-proven NAFLD and 15 with NAS,H compared to chronic hepatitis C or healthy individuals. There was no correction for metabolic factors. Papatheodoridis et al. studied 60 biopsy-proven NAFLD patients, compared them to 90 chronic viral hepatitis patients, and found an increase in fibrinogen, protein S, and protein C in NAFLD. There was no correction for metabolic factors. They also showed a relation between NAFLD and the number of thrombotic risk factors, with the highest numbers in patients with NASH and fibrosis. Targher et al. studied 100 apparently healthy volunteers and found no differences between 35 NAFLD patients (based on US) and the no-NAFLD group after correction for VAT. Hence, most of the studies have low patient numbers or lack histological diagnosis.
In contrast to these studies, our study consecutively recruited a large series of patients with a wide range of BMI and other metabolic factors in whom there was no a priori suspicion of liver disease. Liver biopsy was performed in all patients who underwent bariatric surgery, and in patients not going to surgery, liver biopsy was proposed if there was any suspicion of NAFLD. This resulted in a large series of histologically characterized patients presenting the whole spectrum from normal liver to NASH and cirrhosis. The patients who ultimately appeared to have a histologically normal liver were considered to be an internal control group, avoiding the need to compose a matched control group (in which histological data are usually lacking). These methodological considerations strengthen the conclusion that, besides PAI-1, the liver is not contributing to the prothrombotic state associated with obesity. It might be worthwhile, in future studies, to include another type of control group consisting of nonobese patients, such as liver donor patients or patients undergoing elective abdominal surgery.
A second conclusion of our study is that NAFLD is independently contributing to the increase in PAI-1, observed in obese patients, and that the increase in PAI-1 is related to histological severity, especially steatosis and NASH.
Literature data on PAI-1 are mostly, but not all, consistent with an increase in PAI-1 in relation to NAFLD. In the study of Targher et al., the link between NAFLD and PAI-1 disappeared after correction for VAT, suggesting that not the liver, but VAT was the main contributor to thrombophilia in the MetS. Also, in the study of Sookoian et al., studying 113 biopsy-proven NAFLD patients, compared to 102 age- and gender-matched controls without biopsy, and of Cigolini et al. (studying 31 NAFLD patients, based on US, with 32 controls) differences in PAI-1 disappeared upon correction for metabolic parameters. Not all studies corrected the associations for metabolic features. In the studies by Kotronen et al., Barbato et al. and Bruckert et al., the association of increased PAI-1 levels with NAFLD (based on MRS, US, or liver enzymes, respectively) remained significant after adjusting for different metabolic parameters. By contrast, Espino et al. found no differences in PAI-1 serum levels according to the presence or absence of NAFLD. In the latter study, histology was, however, only available in a small group of 50 morbidly obese patients and comparison was only made between patients with NAFLD versus patients without NAFLD.
As outlined before, some of the methodological shortcomings of other reported studies were avoided in our study. Therefore, we estimate that we can reliably conclude that, in the case of obesity, the liver independently contributes to the observed increase in PAI-1 and that this increase is correlated with the severity of steatosis and steatohepatitis, with the highest values in the most severe NASH.
Although the grade of steatosis does seem to have a higher effect on PAI-1 levels, compared to inflammation, our data clearly show increasing levels of PAI-1 with increasing grades of inflammation or severity of steatohepatitis, as expressed by the NAS. We also found highly significantly increased levels of PAI-1 in patients with NASH, compared to patients with simple steatosis. Furthermore, PAI-1 gene expression was also significantly higher in patients with NASH, compared to those with simple steatosis, further substantiating the role of steatohepatitis. This is probably not restricted to NASH, because PAI-1, being an acute-phase reactant, has shown to be increased in different types of both acute and chronic hepatic inflammation.
Previous studies have also shown a link between the prothrombotic state and hepatic fibrosis. In multiple regression analysis entering only histological features, we found fibrosis to be independently correlated with PAI-1 levels. However, steatosis was the most important determinant, showing that even patients with simple steatosis had increased levels of PAI-1. When metabolic factors were included in multiple regression, fibrosis was no longer an independent determinant of PAI-1. Our population comprises low numbers of patients with advanced fibrosis, probably resulting in insufficient statistical power to show an independent correlation between fibrosis and PAI-1.
Different mechanisms could explain the link between NAFLD/NASH and increased PAI-1 levels. One of the possible mechanisms is that increased liver fat could directly stimulate hepatocytes to secrete PAI-1. Sookoian et al. found higher circulating levels of PAI-1 in patients with NAFLD and also higher scores of PAI-1 expression in the liver, compared to controls. In our series, we confirm the higher expression of PAI-1 in livers of patients with NASH. Patients with normal livers or patients with simple steatosis have lower levels, again pointing toward an effect of the inflammatory component of the disease in the increase in PAI-1 levels.
The true clinical significance of the increase in PAI-1 can, of course, not be assessed by a cross-sectional analysis. In plasma, PAI-1 inhibits degradation of fibrin clots, contributing to an increased atherothrombotic risk. Large epidemiological studies have shown elevated plasma levels of PAI-1 as a predictor of myocardial infarction. PAI-1 levels are related to the severity of vessel wall damage and are good predictors of subsequent development of a first acute myocardial infarction. One can hypothesize that the increase in PAI-1 levels by the steatotic and inflamed liver, at least in part, explains the observed link between NAFLD and increased cardiovascular risk. Furthermore, local thrombotic events might contribute to fibrogenesis and liver disease progression. Antithrombotic treatment has recently been proven to beneficially affect disease evolution in cirrhosis. However, large longitudinal studies are needed to assess the role of PAI-1 in NAFLD-associated cardiovascular risk and fibrosis progression and the potential therapeutic implications of these findings.
The association found between a prothrombotic status and NAFLD, independent of obesity, might be of relevance for the treatment paradigm of NAFLD and NASH. As more effective treatments become available in the future, it might be considered to treat patients with NAFLD and NASH, regardless of the degree of fibrosis, in an attempt to reduce the associated cardiovascular risks.
In conclusion, our results show that NAFLD and NASH independently contribute to the prothrombotic state in obesity by an increase in PAI-1, whereas other prothrombotic factors are unaffected by liver status.