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Extracellular vesicles (EVs), including exosomes and microvesicles, are membranous vesicles released from nearly all cellular types. They contain various bioactive molecules, and their molecular composition varies depending on their cellular origin. As research into venomous animals has progressed, EVs have been discovered in the venom of snakes and parasitic wasps. Although vesicle secretion in spider venom glands has been observed, these secretory vesicles' origin and biological properties are unknown. In this study, the origin of the EVs from Ornithoctonus hainana venom was observed using transmission electron microscopy (TEM). The Ornithoctonus hainana venom extracellular vesicles (HN-EVs) were isolated and purified by density gradient centrifugation. HN-EVs possess classic membranous vesicles with a size distribution ranging from 50 to 150 nm and express the arthropod EV marker Tsp29Fb. The LC-MS/MS analysis identified a total of 150 proteins, which were divided into three groups according to their potential function: conservative vesicle transport-related proteins, virulence-related proteins, and other proteins of unknown function. Functionally, HN-EVs have hyaluronidase activity and inhibit the proliferation of human umbilical vein endothelial cells (HUVECs) by affecting the cytoskeleton and cell cycle. Overall, this study investigates the biological characteristics of HN-EVs for the first time and sheds new light on the envenomation process of spider venom.

Origin and Characterization of Extracellular Vesicles Present in the Spider Venom of Ornithoctonus hainana