Endogenous mechanisms regulating the host response during inflammation resolution are critical in ensuring disposal of noxious stimuli and return to homeostasis. In this article, we engineered novel Annexin A1 (AnxA1)–based peptides, AnxA12–50, that displayed specific binding to the AnxA1 receptor (formyl peptide receptor 2/Lipoxin A4 receptor [FPR2/ALX]; IC50 ∼4 nM). Intravenous administration of AnxA12–50 markedly reduced (>60%) leukocyte adhesion to postcapillary venules in wild type and Fpr1−/−, but not Fpr2/Alx−/−, mice. Generation of a metabolically stable form of this peptide (CR-AnxA12–50), engineered by substituting a cleavage site shared by human proteinase 3 and neutrophil elastase, yielded an agonist that was resistant to neutrophil-mediated cleavage and displayed enhanced proresolving actions: accelerated resolution of self-limited inflammation and enhanced macrophage efferocytosis after sterile injury, when compared with AnxA12–50. These actions were retained with human primary leukocytes where CR-AnxA12–50 decreased neutrophil–endothelial interactions (∼25–45%), and stimulated neutrophil apoptosis and macrophage efferocytosis (∼45%). In murine cardiac ischemia/reperfusion injury, CR-AnxA12–50 elicited tissue-protective actions reducing infarct size (∼60%) and incidence of 24-h death. These results identify AnxA12–50 and CR-AnxA12–50 as FPR2/ALX agonists that harness the proresolving actions of AnxA1, and thus may represent therapeutic tools for treatment of inflammatory conditions.

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The biosynthesis of C-terminal alpha-amidated peptides from their corresponding C-terminal glycine-extended precursors is catalyzed by peptidylglycine alpha-amidating enzyme (alpha-AE) in a reaction that requires copper, ascorbate, and molecular oxygen. Using bifunctional type A rat alpha-AE, we have shown that O2 is the source of the alpha-carbonyl oxygen of pyruvate produced during the amidation of dansyl-Tyr-Val-[alpha-13C]-D-Ala, as demonstrated by the 18O isotopic shift in the 13C NMR spectrum of [alpha-13C]lactate generated from [alpha-13C]pyruvate in the presence of lactate dehydrogenase and NADH. In addition, one-to-one stoichiometries have been determined for glyoxylate formed/dansyl-Tyr-Val-Gly consumed, pyruvate formed/dansyl-Tyr-Val-D-Ala consumed, dansyl-Tyr-Val-NH2 formed/ascorbate oxidized, and dansyl-Tyr-Val-NH2 formed/O2 consumed. Quantitative coupling of NADH oxidation to dansyl-Tyr-Val-NH2 production using Neurospora crassa semidehydroascorbate reductase showed that two one-electron reductions by ascorbate occurred per alpha-AE turnover. The stoichiometry of approximately 1.0 dansyl-Tyr-Val-NH2 produced/ascorbate oxidized observed in the absence of a semidehydroascorbate trap resulted from the disproportionation of two semidehydroascorbate molecules to ascorbate and dehydroascorbate.

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