Evaluation of the Hemostatic Profile in Mice with Chronic Liver Disease: Effects of Protein S and Combined Gas6 and Protein S Deficiencies

Introduction

Advanced chronic liver disease (ACLD) is characterized by changes in the coagulation system that embrace both hypo- and hypercoagulability. In this rebalanced situation, hypercoagulability can contribute to fibrosis progression and cirrhosis complications.

Common hemostasis tests fail to reflect bleeding and thrombotic tendencies in ACLD patients. Therefore, a better understanding of the underlying mechanisms is mandatory for setting-up new assays and treatments.

Here, we assessed the hemostatic changes by means of routine laboratory tests and thrombin generation assay (TGA) in two murine hepatotoxin-induced ACLD models.

To better evaluate hemostatic changes, wild-type mice, mice carrying a partial protein S (Pros1) deficiency -thrombotic risk factor- and with a complete deficiency in Gas6, protective factor for fibrosis progression, have been investigated.

Methods

Adult C57Bl6/129 Pros1^+/+ and Pros1^+/- mice received 1ml/kg carbon tetrachloride (CCl₄) i.p. and 0.3g/L phenobarbital (PB) in drinking water 2x/week for 11 weeks. Similarly, Pros1^+/+, Pros1^+/-, Gas6^-/-Pros1^+/+, Gas6^-/-Pros1^+/- mice received 200mg/kg of thioacetamide (TAA) i.p. 3x/week for 12 weeks.

At the end of the treatment, body, liver and spleen were weighed. Liver sections were stained with Sirius Red to score fibrosis according to METAVIR system. Plasma was analyzed for prothombin time (PT), fibrinogen (Fb), factor V (FV), factor VIII coagulant (FVIII:c) and thrombin and antithrombin complexes (TAT). TGA was performed in presence/absence of activated protein C (APC) or thrombomodulin (TM).

Results

After 11 weeks of CCl₄/PB treatment, only 45% mice survived and developed an advanced CLD independently of their genotype (Pros1^+/+ vs Pros1^+/-). In particular, 42% Pros1^+/+ (n=9)and 58% Pros1^+/-(n=7) mice developed a severe cirrhosis (METAVIR: F4), 48% Pros1^+/+ (n=10)and 42% Pros1^+/-(n=5) an advanced fibrosis stage (F3) and only 10% Pros1^+/+ (n=2) a modest fibrotic stage (F2). Treated mice presented hepatomegaly with darkish discoloration and nodular surface. The liver to body weight ratio was significantly increased compared to untreated controls (Pros1^+/+=9.7% and Pros1^+/-= 10.8% vs Pros1^+/+ untreated =5.3%, P<0.05 and P<0.001, respectively).

No variations were observed on PT, Fb, FV and platelets. Differently, increased FVIII:c (Pros1^+/+=340±69 and Pros1^+/-=378±103 vs Pros1^+/+ untreated 212±62% vs hFVIII, P<0.05 and P<0.001, respectively) and TAT (Pros1^+/+=30.2 and Pros1^+/-=41.8±36.7 vs Pros1^+/+ untreated 8.4±2.2 ug/L) were measured independently of disease severity (F3 vs F4).

TAA treatment showed a reduced mortality paralleled by the development of less severe fibrotic stages in comparison to CCl₄/PB treatment. In particular, 11% Pros1^+/+ (n=4)and 31% Pros1^+/-(n=4) mice were found at F4, 50% Pros1^+/+ (n=12)and 54% Pros1^+/-(n=7) at F3 while 39% Pros1^+/+ (n=10)and 15% Pros1^+/-(n=3) showed a moderate fibrotic disease (F2). In line with published data, preliminary data indicate a frequency reduction of the severe/advanced fibrotic stages in Gas6^-/-Pros1^+/+ mice: 37% (n=3) at F3 and 63% (n=5) at F2. Interestingly, this trend was reversed in Gas6^-/-Pros1^+/- mice: 54% (n=6) at F3 and 46% (n=5) at F2.

No variations in body, liver and spleen weight and morphology were observed. The hemostatic profile did not show relevant variations of PT, Fb, FV, FVIII and platelets. TAT was increased in Pros1^+/+ and Pros1^+/-mice while levels in Gas6^-/-Pros1^+/+mice were comparable to the untreated group (Pros1^+/+=50.2±34.8 and Pros1^+/-=47.7±57.9 vs Gas6^-/-Pros1^+/+=10.8±5.5 Gas6^-/-Pros1^+/-=13.9±17.9). Preliminary data on TGA showed a lack of APC resistance in all the treated groups (nAPCsr: Pros1^+/+=1.0±0.3, Pros1^+/-=1.3±0.7, Gas6^-/-Pros1^+/+=1.3±0.3and Gas6^-/-Pros1^+/-=1.1) but an acquired resistance to TM in Pros1^+/+ and Pros1^+/-mice (nTMsr: Pros1^+/+=2.1±0.4, Pros1^+/-=1.9±0.5, Gas6^-/-Pros1^+/+=1.2±0.4and Gas6^-/-Pros1^+/-=1.1). Primary results suggest a positive correlation between nTMsr and the METAVIR score for Pros1^+/+ mice (r=0.46, n=6).

Conclusion

These data provide the first evaluation of the hemostatic profile in murine ACLD at different stages. Preliminary data suggest that TM resistance might be discriminatory for the severity of the disease. Further TGA analysis and evaluation of the thrombotic risk on treated and control mice are needed.