Characterization of the prothrombotic phenotype of AT-deficient and AT-infused mice. Mice were treated with control siRNA or AT siRNA followed by infusion of saline, AT-WT, AT-4Mut, or AT-R425del at 24 and 48 hours of siRNA treatment as shown in Figure 1. Mice were euthanized after 72 hours of siRNA treatment and perfused with PBS. (A) Perfused liver cryosections were fixed, permeabilized, and incubated with anti-fibrin(ogen) (rabbit) and anti-CD31 (goat) antibodies followed by Alexa Fluor 555–conjugated anti-rabbit and Alexa Fluor 488–conjugated anti-goat antibodies. DAPI (4′,6-diamidino-2-phenylindole) was used to stain the nucleus. The arrows indicate intravascular thrombosis. Inset boxes from each group are magnified. Scale bar, 50 μm. (B) Relative mean fluorescence intensity (MFI) of fibrinogen stain in (panel A) is presented. (C-D) The perfused left lobe of the liver was collected in the tissue lysis buffer and immunoblotted for fibrin(ogen). β-actin was used as a loading control. (E-F) Densitometric analysis of fibrin(ogen) deposition in the liver tissue sample is presented. All data are presented as mean ± SEM. n ≥ 3. Statistical analysis was performed using Graph Pad Prism 10. P values are determined by 1-way ANOVA, followed by Bonferroni multiple comparison test. ∗P < .05; ∗∗P < .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001. MFI, mean fluorescence density; ns, not significant.