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

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.摘要 背景 骚扰锥蝽（Triatoma infestans，半翅目：猎蝽科）是一种吸血昆虫，同时是克氏锥虫（Trypanosoma cruzi，动基体目：锥虫科）的主要传播媒介。本研究中，作者探究了骚扰锥蝽唾液中的丝氨酸蛋白酶（serine protease）活性是否参与血管运动调节，并通过切割并激活蛋白酶激活受体（protease-activated receptors, PARs）来影响该昆虫的吸血行为。 方法 参照此前用于纯化triapsin（一种丝氨酸蛋白酶）的报道方法，对骚扰锥蝽唾液进行色谱层析。基于荧光共振能量转移（FRET, Fluorescence Resonance Energy Transfer）技术，探究triapsin对PAR肽的切割活性。采用质谱分析法分析triapsin切割PAR-2肽的位点。使用DAN测定法（2,3-二氨基萘，2,3-diaminonapthalene）进行一氧化氮（NO, Nitric Oxide）水平检测。在预先用苯肾上腺素（phenylephrine，3 μM）预收缩的、具备或不具备功能完整内皮的血管标本中，检测triapsin的血管舒张活性。采用活体显微镜成像技术评估triapsin对小鼠皮肤微循环的影响。 结果 triapsin可诱导PAR肽的水解反应，且对PAR-2肽的切割偏好性更高。质谱分析证实其存在唯一切割位点，该位点对应PAR-2受体的激活位点。triapsin可在培养的人脐静脉内皮细胞（HUVECs, human umbilical vein endothelial cells）中诱导剂量依赖性的NO释放，在17.58 nM浓度时达到最大效应。将经凝胶过滤层析纯化的triapsin（10^-16 至 10^-9 M）以累积给药方式施加于小鼠肠系膜动脉环，结果显示其具备强效的内皮依赖性血管舒张效应（EC30 = 10^-12 M）。一氧化氮似乎部分参与了该血管舒张效应，因为一氧化氮合酶抑制剂L-NAME（L-NG-硝基精氨酸甲酯，L-NG-nitroarginine methyl ester，300 μM）并未完全阻断triapsin介导的血管舒张反应。抗PAR-2抗体可完全抑制triapsin活性所介导的血管舒张。triapsin活性还可诱导小鼠耳静脉管径增大。 结论 本研究数据表明，由triapsin介导的PAR-2激活与骚扰锥蝽唾液所致的血管舒张之间存在潜在关联。