Heterobivalent Peptide Agonists Comprising Covalently Linked Protease Activated Receptor 1 and Protease Activated Receptor 3 Peptides Mimic the Cytoprotective Cell Signaling Activities of Activated Protein C

Introduction: Activated protein C (APC) exhibits both anticoagulant and cytoprotective cell signaling actions, including anti-inflammatory and anti-apoptotic activities and endothelial barrier stabilization. Extensive in vitro and in vivo data for APC's cell signaling benefits identified requirements for two G-protein coupled receptors (GPCR), protease-activated receptor (PAR) PAR1 and PAR3 which are cleaved by APC at the noncanonical sites (R46 and R41, respectively). Agonism of PARs following proteolysis of the N-terminal tail is driven by the newly revealed N-terminus and related synthetic peptides, e.g., for APC, PAR1 peptides (P1) beginning with N47 or PAR3 peptides (P3) beginning with G42. We found that the non-canonical P1-47-66 and P3-42-65 peptides mimic APC in that each is anti-inflammatory by reducing NLRP3-driven caspase-1 activity in human THP-1 cells (Healy LD et al, J Thromb Haemost 2021;19:269-280). Remarkably, these two peptides act synergistically to attenuate activity of caspase-1 at low concentrations where either peptide alone has little activity. Thus, we undertook studies of P1 peptides that were covalently linked to P3 peptides for ability to mimic APC's anti-inflammatory and endothelial barrier stabilization actions.

Methods: Monocytes (THP-1 cells) were differentiated into macrophages using PMA, then pretreated with APC or peptides prior to stimulation with lipopolysaccharide and adenosine triphosphate to induce caspase-1 whose activity was measured using a luminescent caspase-1 substrate. Endothelial barrier stability was monitored for transendothelial electrical resistance (TEER) using an electric cell-substrate sensing system (iCelligence apparatus) when cultured human EAhy926 or murine aortic endothelial cells (EC) were exposed to thrombin treatment (0.25 nM) plus or minus treatment with P1 and/or P3 peptides or G10-related peptides. Synthetic P1 and P3 sequence-derived peptides of various compositions were made, and bivalent P1:P3 peptides were made using oligo-Gly-peptide linkers containing from 4 to 14 Gly residues.

Results: Using THP-1 cell anti-inflammatory assays for various peptides, studies showed that P1-47-55 was as potent as P1-47-66 and that the N-terminal N47 was essential. In anti-inflammatory activity assays, some P3 peptides shorter than P3-42-65 (e.g., P3-44-54, P3-51-65, and P3-51-59) were as active as P3-42-65. Remarkably, this is the first example for any PAR-agonist peptide that does not require a native-like sequence with an intact free alpha-amino N-terminus to agonize the PAR response. For bivalent P1:P3 studies, we covalently linked P3-47-55 to P3-51-65 using oligo-Gly linkers with 4-14 Gly residues. In anti-inflammatory activity assays, the maximal activity was seen for a 10-Gly linker. The bivalent peptide comprising P1-47-55-[Gly(10 residues)]-P3-51-65, designated as the G10 peptide, was much more potent than any P1 peptide or P3 peptide in anti-inflammatory THP-1 cell assays in dose-response studies. In TEER studies of thrombin-challenged EAhy926 EC where APC markedly attenuates thrombin-induced endothelial barrier dysfunction, P1-47-55 and the G10 peptide mimicked APC's protective actions. In dose-response studies, the G10 peptide was much more potent than the P1-47-55 peptide or any combination of P1-47-55 with a P3 peptide. The P3 peptides alone were very weak. In TEER studies of thrombin-challenged murine aortic EC where murine APC markedly attenuates thrombin-induced endothelial barrier dysfunction, the G10 peptide comprising murine PAR sequences mimicked murine APC's protective actions.

Conclusions: Covalent linkage of a PAR1-derived peptide agonist to a PAR3-derived peptide agonist using a 10-Gly-residue linker provided the first heterobivalent PAR peptide GPCR agonist. This heterobivalent G10 peptide potently mimicked APC's cytoprotective activities of anti-inflammation and endothelial barrier stabilization. Molecular mechanisms for G10 likely involve both orthosteric and allosteric effects for PAR1 and PAR3 which can form heterodimers. Given APC's beneficial cell signaling effects in many preclinical injury models and given the increasing usage of peptide-based drugs, studies of the G10 peptide for beneficial APC-mimicking effects may help to identify important directions for future translational research.