The novel tethered ligand P3R peptide improves endothelial barrier functions. Endothelial barrier function in vitro was determined by the permeability of an endothelial cell layer to Evans blue-albumin complexes. (A) Contribution of PAR1 and PAR3 using the cleavage-blocking antibodies ATAP-2 (25 μg/mL) against PAR1 or H103 (25 μg/mL) against PAR3 to thrombin-induced endothelial permeability. (B-D) Modulation of thrombin-induced endothelial permeability by PAR3 peptides (50 μM) derived from cleavage at Lys38 (P3K) or Arg41 (P3R) of various lengths (supplemental Figure 4) compared with APC (20 nM). Shown are data for (B) short peptides (P3K_S and P3R_S) comprised of 6 amino acids, (C) medium-length peptides ending at Ser54 (P3K_M and P3R_M), and (D) long peptides ending at Thr65 (P3K_L and P3R_L). (E) Protection of endothelial barrier function by PAR3 peptides (50 μM) derived from cleavage at Lys38 (P3K_M) or Arg41 (P3R_M) against permeability induced by histones. (F) Modulation of endothelial barrier function of near confluent endothelial by PAR3 peptides (50 μM) derived from cleavage at Lys38 (P3K_M) or Arg41 (P3R_M) in the absence (gray bars) and presence (dark bars) of the PAR1 antagonist SCH79797. Data points represent the mean ± SEM (n ≥ 3 independent inserts). Asterisk denotes a statistically significant difference (P < .05).