Platelet aggregation elicits FasL expression and hepatocyte apoptosis in sinusoidal obstruction syndrome

Abstract

Sinusoidal obstruction syndrome (SOS) is a fatal liver condition resulting from sinusoidal endothelial cell injury and hepatocyte death, following liver or hematopoietic stem cell transplantation as well as chemotherapy. We showed evidence of platelet displacement and aggregation within the space of Disse in SOS. However, the relationship between platelets and hepatocyte death remains unclear. Using a mouse SOS model by intraperitoneal monocrotaline (270 mg/kg; a pyrrolizidine alkaloide plant toxin) administration, we observed positive stains for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase-3, which are markers for apoptosis, in the liver by immunohistochemistry. At 48 h of the SOS liver, aggregated platelets and hepatocytes around zone 3 were found to express Fas ligand (FasL) and Fas, respectively. Human peripheral blood platelets, when aggregated, could induce expression of FASL on themselves and then lead to apoptosis in co-cultured HepG2 cells. Treatment of recombinant soluble thrombomodulin (rTM), an anticoagulant and vascular endothelium-protective drug, prevented the hepatocyte death in the SOS mice. These findings suggest that the prevention of platelet aggregation is a potential therapeutic intervention against hepatocyte death and severe liver damage in SOS.

Fig. 1

Immunostaining for SE-1, a LSEC marker, and CD42b, a platelet marker, in the liver of MCT-injected SOS model mice. (a) SE-1 staining for LSEC detection at 0 and 48 h after MCT injection. (b, c) HE (b) or CD42b (c) staining at 0, 12, 24, and 48 h after MCT injection.

Fig. 2

Immunostaining for cleaved caspase-3 and TUNEL assay in the liver of MCT-injected SOS model mice. (a) Cleaved caspase-3 staining at 0 and 48 h after MCT injection. +rTM, pretreatment of recombinant human soluble thrombomodulin (4 mg/kg). (b) TUNEL assay at 0 and 48 h after MCT injection.

Fig. 3

(a) Immunostaining for Fas in the liver at 0, 24 and 48 h after MCT injection. (b) The quantitative evaluation of the Fas-positive area. Data are expressed as mean ± SE (n = 3). **, p < 0.01. *, p < 0.05.

Fig. 4

Immunostaining for FasL in the liver of MCT-injected SOS model mice. (a) FasL staining at 0, 24 and 48 h after MCT injection. (b) Higher magnification images at 48 h after MCT injection. Red arrows indicate positive stain. (c) FasL staining at 48 h after MCT injection in combination with pretreatment of recombinant human soluble thrombomodulin (4 mg/kg). (d) The quantitative evaluation of the FasL-positive area. Data are expressed as mean ± SE (n = 3). *, p < 0.05. (e) Immunofluorescent staining for FasL and CD42b.

Fig. 5

FasL appearance in activated and aggregated platelets. (a) Western blotting for mouse FasL protein in platelets isolated from the peripheral blood and the liver of MCT-injected SOS model mice (SOS model) or non-injected controls (Control). (b) Immunofluorescent study for the detection of CD62P and FasL using human platelets with or without their activation and coagulation [Agg(+) or Agg(-), respectively]. Normal IgG used as a negative control. (c) The quantitative data of CD62P and FasL fluorescence intensity. Data are expressed as mean ± SE (n = 3). **, p < 0.01. (d) Western blotting for human FasL protein in platelets with or without their activation and coagulation [Agg(+) or Agg(-), respectively]. (e) The quantitative data of the band intensity of FASL and GAPDH. Data are expressed as mean ± SE (n = 3). **, p < 0.01. (f) qPCR analyses for FasL mRNA in human platelets with or without their activation and coagulation [Agg(+) or Agg(-), respectively]. CACO-2 cells, colorectal cancer cell line. PBMC, peripheral blood mononuclear cells as a positive control.

Fig. 6

Immunofluorescent study for the detection of cleaved caspase-3 in HepG2 cells (a-d) or primary mouse hepatocytes (f-i) at 2–24 h after co-incubation of activated and coagulated human or mouse platelets with or without the treatment of 300 µM of Kp7-6, a Fas/FasL antagonist. (e and j) The quantitative data of cleaved caspase-3 fluorescence intensity. Data are expressed as mean ± SE (n = 3). **, p < 0.01.

Fig. 7

A proposed model of hepatocyte apoptosis after the interaction between FasL on activated and coagulated platelets and Fas-expressed hepatocytes in the pathogenesis of SOS.