Detection of PN-modified fibrin(ogen) in the APAP-injured liver and effect of PN modification on fibrin polymerization and cross-linking. WT mice were challenged with 300 mg/kg APAP, and livers were collected 6 or 24 hours later to prepare homogenates (n = 5 mice/group). (A) Fibrin(ogen) detected by western blot using a rabbit anti-fibrinogen polyclonal antibody in whole-liver homogenates. (B-C) Immunoprecipitation of 3-NT adducted proteins from whole-liver homogenates and detection of fibrin(ogen) by western blot using a rabbit anti-fibrinogen polyclonal antibody. (B) 3-NT adducts in purified human fibrinogen modified by PN detected by western blotting (C). (D-E) Polymerization of purified human fibrinogen after PN modification was measured by turbidity (D) and modified Clauss assay (E). (F) Fibrin(ogen) cross-linking was assessed in clots prepared from vehicle- or PN-modified human fibrinogen incubated with thrombin for the indicated time points. (G) Human fibrinogen was reacted with the indicated concentration of PN in 3 independent reactions and then incubated with thrombin (0.25 U/mL) for 15 minutes. Quantitation of Fibγ (i) and γ-γ dimer (ii) in a Coomassie-stained gel (as shown in supplemental Figure 5) is expressed as a ratio to Fibβ compared with vehicle-modified fibrinogen (expressed as dashed line set at 1). *P < .05 compared with vehicle-modified fibrinogen. (H) Human fibrinogen was reacted with the indicated concentration of PN and observed using SEM. Fibers are visible beneath a fibrin film (white arrowhead). Representative photomicrographs are shown (×10 000 magnification).