APC causes biased signaling via PAR1 cleavage at Arg46. PAR1 is a 7-transmembrane GPCR receptor capable of many conformational states. (A) The extracellular N-terminus contains the intramolecular ligands, which become exposed after proteolysis by APC or thrombin. PAR1 cleavage by thrombin at Arg41 exposes the canonical N-terminal tethered agonist that begins with residue Ser42 (SFLLRN-), whereas noncanonical cleavage by APC at Arg46 results in a different N-terminal tethered agonist that begins with residue Asn47 (NPNDKY-). (B) Synthetic agonist peptides with the N-terminal tethered-ligand sequences beginning with residue 42 (TRAP) or residue 47 (TR47) cause thrombin-like or APC-like effects on cells, respectively. (C) Activation of PAR1 by thrombin or TRAP stabilizes PAR1 conformers, whose intracellular loops provide surfaces that are highly favorable for interactions with G proteins, resulting in G-protein–dependent signaling. These PAR1 conformers are termed “G-protein biased.” In contrast, activation of PAR1 by APC or TR47 stabilizes different PAR1 conformers that preferentially interact with β-arrestin-2, resulting in β-arrestin-2–dependent signaling. Such PAR1 conformers are termed “β-arrestin biased.” Thus, the agonist bias is directly related to the cleavage site used to activate PAR1 because the cleavage determines which tethered ligand is exposed and which subsets of PAR1 conformations are stabilized. Biased agonism results in the induction of uniquely different signaling repertoires, such as the activation of ERK1/2 and RhoA, etc, resulting in vascular leakage by thrombin or the activation of PI3K, Akt, and Rac1, etc, by APC resulting in neuroprotection. (D-E) APC-mediated neuroprotection in ischemic stroke requires PAR1-dependent biased signaling due to cleavage at Arg46 in PAR1. To assess PAR1-biased signaling, studies employed homozygous QQ41-PAR1 mice, homozygous QQ46-PAR1 mice, and wt control mice. At 4 hours after a 60-minute MCAO, 3K3A-APC (0.04 mg/kg) or placebo was given IV, and then at 24 hours after occlusion, various parameters were measured. Treatment of mice with 3K3A-APC or vehicle is indicated by plus or negative signs under panels D and E. Data are shown for infarct volume (D) and for degenerating neurons as determined by Fluoro-Jade C stain (E) for each mouse group. For panels D and E, bars indicate mean ± standard deviation, n = 4-6 mice per group. (For details regarding panels D and E, see Sinha et al²² ).