Proteolytic activity of Triatoma infestans saliva associated with PAR-2 activation and vasodilation

Karla A Oliveira; Ricardo J S Torquato; Daniela C G Garcia Lustosa; Tales Ribeiro; Bruno W L Nascimento; Lilian C G de Oliveira; Maria A Juliano; Thaysa Paschoalin; Virginia S Lemos; Ricardo N Araujo; Marcos H Pereira; Aparecida S Tanaka

doi:10.1590/1678-9199-JVATITD-2020-0098

Proteolytic activity of Triatoma infestans saliva associated with PAR-2 activation and vasodilation

J Venom Anim Toxins Incl Trop Dis. 2021 Mar 8:27:e20200098. doi: 10.1590/1678-9199-JVATITD-2020-0098.

Authors

Affiliations

¹ Department of Biochemistry and Pharmacology, Federal University of Piauí, Teresina, PI, Brazil.
² Department of Biochemistry, Federal University of São Paulo (Unifesp), São Paulo, SP, Brazil.
³ Department of Pharmacology, Institute of Biomedical Sciences, Federal University of Minas Gerais (UFMG), Belo Horizonte, MG, Brazil.
⁴ Department of Parasitology, Institute of Biomedical Sciences, Federal University of Minas Gerais (UFMG), Belo Horizonte, MG, Brazil.
⁵ Department of Biophysics, National Institute of Pharmacology and Molecular Biology (INFAR), Federal University of São Paulo (Unifesp), São Paulo, SP, Brazil.
⁶ Department of Physiology and Biophysics, Institute of Biomedical Sciences, Federal University of Minas Gerais (UFMG), Belo Horizonte, MG, Brazil.
⁷ National Institute of Science and Technology in Molecular Entomology (INCT-EM), Rio de Janeiro, RJ, Brazil.

PMID: 33747067
PMCID: PMC7939238
DOI: 10.1590/1678-9199-JVATITD-2020-0098

Abstract

Background: Triatoma infestans (Hemiptera: Reduviidae) is a hematophagous insect and the main vector of Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae). In the present study, the authors investigated whether a serine protease activity from the saliva of T. infestans has a role in vasomotor modulation, and in the insect-blood feeding by cleaving and activating protease-activated receptors (PARs).

Methods: T. infestans saliva was chromatographed as previously reported for purification of triapsin, a serine protease. The cleavage activity of triapsin on PAR peptides was investigated based on FRET technology. Mass spectrometry was used to analyze the sites of PAR-2 peptide cleaved by triapsin. NO measurements were performed using the DAN assay (2,3-diaminonapthalene). The vasorelaxant activity of triapsin was measured in vessels with or without functional endothelium pre-contracted with phenylephrine (3 µM). Intravital microscopy was used to assess the effect of triapsin on mouse skin microcirculation.

Results: Triapsin was able to induce hydrolysis of PAR peptides and showed a higher preference for cleavage of the PAR-2 peptide. Analysis by mass spectrometry confirmed a single cleavage site, which corresponds to the activation site of the PAR-2 receptor. Triapsin induced dose-dependent NO release in cultured human umbilical vein endothelial cells (HUVECs), reaching a maximum effect at 17.58 nM. Triapsin purified by gel-filtration chromatography (10^-16 to 10^-9 M) was applied cumulatively to mouse mesenteric artery rings and showed a potent endothelium-dependent vasodilator effect (EC₃₀ = 10^-12 M). Nitric oxide seems to be partially responsible for this vasodilator effect because L-NAME (L-NG-nitroarginine methyl ester 300 µM), a nitric oxide synthetase inhibitor, did not abrogate the vasodilation activated by triapsin. Anti-PAR-2 antibody completely inhibited vasodilation observed in the presence of triapsin activity. Triapsin activity also induced an increase in the mouse ear venular diameter.

Conclusion: Data from this study suggest a plausible association between triapsin activity mediated PAR-2 activation and vasodilation caused by T. infestans saliva.

Keywords: Endothelial cells; Nitric oxide; PAR-2 receptor; Triapsin; Vasodilation.