Oliveira Karla A, Torquato Ricardo J S, Lustosa Daniela C G Garcia, Ribeiro Tales, Nascimento Bruno W L, de Oliveira Lilian C G, Juliano Maria A, Paschoalin Thaysa, Lemos Virginia S, Araujo Ricardo N, Pereira Marcos H, Tanaka Aparecida S
Department of Biochemistry and Pharmacology, Federal University of Piauí, Teresina, PI, Brazil.
Department of Biochemistry, Federal University of São Paulo (Unifesp), São Paulo, SP, Brazil.
J Venom Anim Toxins Incl Trop Dis. 2021 Mar 8;27:e20200098. doi: 10.1590/1678-9199-JVATITD-2020-0098.
(Hemiptera: Reduviidae) is a hematophagous insect and the main vector of (Kinetoplastida: Trypanosomatidae). In the present study, the authors investigated whether a serine protease activity from the saliva of has a role in vasomotor modulation, and in the insect-blood feeding by cleaving and activating protease-activated receptors (PARs).
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.
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 to 10 M) was applied cumulatively to mouse mesenteric artery rings and showed a potent endothelium-dependent vasodilator effect (EC = 10 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.
Data from this study suggest a plausible association between triapsin activity mediated PAR-2 activation and vasodilation caused by saliva.
（半翅目：猎蝽科）是一种吸血昆虫，也是（动质体目：锥虫科）的主要传播媒介。在本研究中，作者调查了该昆虫唾液中的一种丝氨酸蛋白酶活性是否在血管舒缩调节以及通过裂解和激活蛋白酶激活受体（PARs）来促进昆虫取食血液过程中发挥作用。
按照先前报道的用于纯化丝氨酸蛋白酶三肽酶的方法对该昆虫唾液进行色谱分析。基于荧光共振能量转移（FRET）技术研究三肽酶对PAR肽的裂解活性。采用质谱分析法分析三肽酶裂解PAR - 2肽的位点。使用DAN检测法（2,3 - 二氨基萘）进行一氧化氮（NO）测定。在预先用去氧肾上腺素（3 μM）预收缩的有或无功能性内皮的血管中测量三肽酶的血管舒张活性。利用活体显微镜评估三肽酶对小鼠皮肤微循环的影响。
三肽酶能够诱导PAR肽的水解，并且对PAR - 2肽的裂解表现出更高的偏好性。质谱分析证实了一个单一的裂解位点，该位点对应于PAR - 2受体的激活位点。三肽酶在培养的人脐静脉内皮细胞（HUVECs）中诱导剂量依赖性的NO释放，在17.58 nM时达到最大效应。通过凝胶过滤色谱法纯化的三肽酶（10至10 M）累积应用于小鼠肠系膜动脉环，显示出强大的内皮依赖性血管舒张作用（EC = 10 M）。一氧化氮似乎部分介导了这种血管舒张作用，因为一氧化氮合酶抑制剂L - NAME（L - NG - 硝基精氨酸甲酯300 μM）并未消除三肽酶激活的血管舒张。抗PAR - 2抗体完全抑制了在三肽酶活性存在下观察到的血管舒张。三肽酶活性还导致小鼠耳静脉直径增加。
本研究数据表明，三肽酶活性介导的PAR - 2激活与该昆虫唾液引起的血管舒张之间存在合理的关联。
