Ticks obtain a blood meal by lacerating small blood vessels and ingesting the blood that flows to the feeding site, which triggers various host responses. However, ticks face the challenge of wound healing, a process involving hemostasis, inflammation, cell proliferation and migration, and remodeling, hindering blood acquisition. To overcome these obstacles, tick salivary glands produce an array of bioactive molecules. Here, we characterize ixochymostatin, an Ixodes scapularis protein belonging to the trypsin inhibitor-like (TIL) family. It is expressed in multiple developmental stages and in tick salivary glands and acts as a slow and tight-binding inhibitor of chymase, cathepsin G, and chymotrypsin. Predictions for the tertiary structure complex between ixochymostatin and chymase suggest a direct interaction between the inhibitor reactive site loop and protease active sites. In vitro, ixochymostatin protects the endothelial cell barrier against chymase degrading action, decreasing cell permeability. In vivo, it reduces vascular permeability induced by chymase and compound 48/80, a mast cell degranulator agonist, in a mouse model. Additionally, ixochymostatin inhibits the chymase-dependent generation of vasoconstrictor peptides. Antibodies against ixochymostatin neutralize its inhibitory properties, with epitope mapping identifying potential neutralization regions. Ixochymostatin emerges as a novel tick protein modulating host responses against tick feeding, facilitating blood acquisition.

Protein Chemistry Section Rocky Mountain Laboratories Division of Intramural Research National Institute of Allergy and Infectious Diseases National Institutes of Health Hamilton MT USA

Rocky Mountain Veterinary Branch Rocky Mountain Laboratories Division of Intramural Research National Institute of Allergy and Infectious Diseases National Institutes of Health Hamilton MT USA

Tick Pathogen Transmission Unit Laboratory of Bacteriology Division of Intramural Research National Institute of Allergy and Infectious Diseases Hamilton MT USA

Tick Pathogen Transmission Unit Laboratory of Bacteriology Division of Intramural Research National Institute of Allergy and Infectious Diseases Hamilton MT USA; Centro de Pesquisa Experimental Hospital de Clínicas de Porto Alegre Porto Alegre RS Brazil

Tick Pathogen Transmission Unit Laboratory of Bacteriology Division of Intramural Research National Institute of Allergy and Infectious Diseases Hamilton MT USA; Department of Biochemistry and Molecular Biology University of Nevada Reno NV USA

Tick Pathogen Transmission Unit Laboratory of Bacteriology Division of Intramural Research National Institute of Allergy and Infectious Diseases Hamilton MT USA; Department of Medical Biology Faculty of Science University of South Bohemia in České Budějovice Budweis Czech Republic

Vector Biology Section Laboratory of Malaria and Vector Research Division of Intramural Research National Institute of Allergy and Infectious Diseases Rockville MD USA

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