An orthosteric/allosteric bivalent peptide agonist comprising covalently linked protease activated receptor-derived peptides mimics in vitro and in vivo activities of activated protein C

Background Activated protein C (APC) has anticoagulant and cytoprotective cell signaling activities which often require protease-activated receptor (PAR)1 and PAR3 and PAR cleavages at noncanonical sites (R46-N47 and R41-G42, respectively). Some PAR1-derived peptides(P1) and PAR3-derived peptides(P3), e.g., P1-47-66 and P3-42-65, mimic APC’s cell signaling. In anti-inflammatory assays, these two peptides at low concentrations synergistically attenuate cellular inflammation. Objective To determine whether a P1 peptide covalently linked to a P3 peptide mimics APC’s anti-inflammatory and endothelial barrier stabilization activities. Methods Anti-inflammatory assays employed stimulated THP-1 cells and caspase-1 measurements. Cultured human EAhy926 or murine aortic endothelial cells (EC) exposed to thrombin were monitored for transendothelial electrical resistance (TEER). Bivalent covalently-linked P1:P3 peptides were studied for APC-like activities. Results In anti-inflammatory assays, P1-47-55 was as active as P1-47-66 and some P3 peptides (e.g., P3-44-54 and P3-51-65) were as active as P3-42-65. The bivalent P1:P3 peptide comprising P1-47-55-[Gly(10 residues)]-P3-51-65 (designated “G10 peptide”) was more potently anti-inflammatory than the P1 or P3 peptide alone. In TEER studies of thrombin-challenged EC’s, P1-47-55 and the G10 peptide mimicked APC’s protective actions. In dose-response studies, the G10 peptide was more potent than the P1-47-55 peptide. In murine EC studies, the murine PAR-sequence-derived G10 peptide mimicked murine APC’s activity. Anti-PAR1 and anti-PAR3 antibodies, but not anti-EPCR antibodies, abated G10’s cytoprotection, showing G10’s actions involve PAR1:PAR3. G10 significantly increased survival in murine endotoxemia. Conclusions The PAR-sequence-derived G10 peptide is a bivalent agonist that mimics APC’s cytoprotective anti-inflammatory and endothelial barrier stabilizing actions and APC’s protection against endotoxemic mortality.