Donor sex and platelet storage change the therapeutic effects of platelet-derived extracellular vesicles on endothelium

Female-derived PEVs from resting platelets offer superior endothelial barrier protection versus male-derived and stored-platelet PEVs. Platelet-derived extracellular vesicles (PEVs) play an active role in vascular protection and repair and are being explored as a viable alternative to platelet therapy. Because platelet function and stability are shaped by donor sex and storage conditions, these same factors are likely to influence the PEVs they release. Understanding these influences is key to developing PEVs into a safe and dependable therapeutic option. In this study, we investigated how donor sex and platelet storage affect the therapeutic properties of PEVs. To address this, PEVs were isolated from platelets of healthy male and female donors. Platelets were either processed immediately after blood collection to represent a resting state or stored overnight at room temperature on a rocker to mimic platelet storage conditions. PEVs isolated from these preparations displayed similar size, morphology, and cellular uptake across groups, but their biological effects diverged. Female PEVs, particularly from resting platelets, provided the strongest protection against thrombin-induced endothelial barrier disruption, stabilized junctional proteins, and reduced oxidative stress. Male PEVs showed weaker barrier protection compared to female-derived PEVs but more pronounced modulation of certain inflammatory mediators. In addition, PEVs derived from resting platelets consistently showed stronger protective effects than those from stored platelets, regardless of donor sex. These results highlight that donor sex and platelet storage influence PEVs function and underscore the need to account for both when developing PEV-based therapies.