Mast cells are significantly involved in IgE-mediated allergic reactions; however, their roles in health and disease are incompletely understood.,We aimed to define the proteome contained in mast cell releasates on activation to better understand the factors secreted by mast cells that are relevant to the contribution of mast cells in diseases.,Bone marrow-derived cultured mast cells (BMCMCs) and peritoneal cell-derived mast cells were used as "surrogates" for mucosal and connective tissue mast cells, respectively, and their releasate proteomes were analyzed by mass spectrometry.,Our studies showed that BMCMCs and peritoneal cell-derived mast cells produced substantially different releasates following IgE-mediated activation. Moreover, we observed that the transglutaminase coagulation factor XIIIA (FXIIIA) was one of the most abundant proteins contained in the BMCMC releasates. Mast cell-deficient mice exhibited increased FXIIIA plasma and activity levels as well as reduced bleeding times, indicating that mast cells are more efficient in their ability to downregulate FXIIIA than in contributing to its amounts and functions in homeostatic conditions. We found that human chymase and mouse mast cell protease-4 (the mouse homologue of human chymase) had the ability to reduce FXIIIA levels and function via proteolytic degradation. Moreover, we found that chymase deficiency led to increased FXIIIA amounts and activity, as well as reduced bleeding times in homeostatic conditions and during sepsis.,Our study indicates that the mast cell protease content can shape its releasate proteome. Moreover, we found that chymase plays an important role in the regulation of FXIIIA via proteolytic degradation.,Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.