Abstract Number: PB2029
Meeting: ISTH 2020 Congress
Background: Inflammasomes are intracellular multiprotein complexes of the innate immune system that can provide NLRP3-dependent activation of caspase-1 (casp-1) which enables release of proinflammatory cytokines, including interleukin (IL)-1β which is implicated in immunothrombosis. Activated protein C (APC) suppresses NLRP3-inflammasome activity in vivo, in part by signaling via protease activated receptor (PAR)1.
Aims: To determine the anti-inflammatory effects of APC and of PAR1-derived peptide (residues 47-66(P1-47)) and/or PAR3-derived peptide (residues 42-65(P3-42)) on phorbol myristate acetate-differentiated human monocytes that are stimulated to induce casp-1 activity and IL-1β, markers of inflammasome activity.
Methods: Monocytes (THP-1 cells) were differentiated into macrophages, then pretreated with APC or PAR1/PAR3 peptides prior to stimulation with LPS and ATP to induce casp-1 whose activity was measured using a luminescent substrate.
Results: APC, P1-47 or P3-42 each dose-dependently reduced NLRP3-dependent casp-1 activity and IL-1β release in differentiated, stimulated THP-1 cells. The P1-47 and P3-42 peptides at low concentrations where either peptide alone had little inhibitory activity synergistically inhibited generation of casp-1 and IL-1β. Studies showed that the following variations of PAR1/PAR3 peptides retained anti-inflammatory activity (e.g., reduction of casp-1 activity in stimulated THP-1 cells): shortened P1-47 variants with ≥8 amino acids; P1-47 variant with the N-terminal Asn47Gln replacement; or a shortened P3-42 of medium length. Moreover, combinations of low doses of a PAR1-derived variant peptide and a PAR3-derived variant peptide synergistically inhibited casp-1 activity generation.
Conclusions: APC or a non-canonical PAR1-agonist peptide or PAR3-agonist peptide are anti-inflammatory by reducing NLRP3-driven casp-1 activity in human THP-1 cells. Remarkably, combinations of low doses of P1-47 and P3-42 peptides, or of their variants, act synergistically to provide anti-inflammatory activity. The synergy observed for simultaneous agonism of PAR1 and PAR3 likely reflects the previously suggested existence of PAR1-PAR3 heterodimers. Our results suggest that combinations of PAR1- and PAR3-derived peptides have significant translational implications.
To cite this abstract in AMA style:Healy L, Fernandez J, Mosnier L, Griffin J. PAR1-Derived and PAR3-Derived Peptides and their Variants Synergistically Inhibit Human Macrophage NLRP3-Dependent Inflammasome Activity [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/par1-derived-and-par3-derived-peptides-and-their-variants-synergistically-inhibit-human-macrophage-nlrp3-dependent-inflammasome-activity/. Accessed August 15, 2022.
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