Acetaminophen overdose reveals protease activated receptor 4 as a low expressing but potent receptor on the hepatic endothelium in mice

There is a paucity of data on how protease-activated receptors (PARs) regulate transcriptional reprogramming. We sought to determine how the expression of two members of the PAR family (PAR1 and PAR4) on hepatic endothelial cells (ECs) influence transcription in the liver after acetaminophen (APAP) overdose. We combined EC-specific translating ribosome affinity purification with next-generation sequencing (EC-TRAPseq) in endothelial-specific Par1 and Par4 knockout mice. This allowed us to gain an in vivo snapshot of how EC gene expression profiles change with APAP overdose and how the loss of endothelial PARs impacts transcriptional reprogramming in hepatic ECs. This approach also provided a high degree of sensitivity for detecting low-expressing transcripts. We found distinct transcriptional changes in APAP-overdosed ECs between endothelial Par1 and Par4 knockout mice. Overall design: Total tissue lysate and EC-TRAP RNASeq data were collected from livers of 11-15 week-old male littermate control mice (RiboTag;Par1fl/+;iCdh5(PAC)-CreERT2) and experimental endothelial Par1 knockout mice (RiboTag;Par1fl/fl;iCdh5(PAC)-CreERT2) treated with saline or overdosed with 300 mg/kg APAP (n=3 for each genotype and treatment). Comparable samples were collected from control and endothelial Par4 knockout mice. This resulted in liver lysates and EC-TRAP samples from 24 mice (i.e., 48 distinct samples).