PROJECT SUMMARY/ABSTRACT The threat of nuclear accidents or attacks makes it critical to develop medical countermeasures. Preclinical studies have shown a relation between vascular dysfunction and chronic organ radiation damage, but little is known about the underlying protective mechanisms. Hence, to identify targets for mitigation, research is needed to elucidate pathways involved in radiation-induced vascular dysfunction and vascular protection. Radiation-induced endothelial dysfunction is associated with detrimental alterations in the protein C pathway. Loss of endothelial surface thrombomodulin (Thbd) leads to reduced levels of activated protein C (APC), a critical component in plasma that has anticoagulant and anti-inflammatory properties and that enhances endothelial cell survival. We have previously shown that recombinant APC is an effective mitigator of acute radiation injury when administered 24 h after total-body irradiation in a mouse model. We will further explore the paradigm that the protein C pathway plays a central role in radiation-induced vascular dysfunction and that APC is an effective mitigator of both acute and late radiation toxicity in multiple organs. In vitro studies with wild-type and recombinant APC using irradiated human endothelial cells in culture will determine which structural features of APC and which endothelial APC receptors are critical for enhancing post-radiation endothelial function. In vivo studies with wild-type mice, Thbd-deficient mice, and mice with enhanced vascular responses to radiation in the small intestine, heart and brain?three organ systems critical in the endogenous levels of APC will determine the role of the Thbd?protein C pathway in both the acute and the late delayed response to radiation. Gene expression profiling focused on endothelial cells extracted from mice will identify radiation-induced changes in the translatome and the effects of APC on those gene expression profiles. Plasma samples from the same mice will be used to identify metabolite profiles indicative of radiation injury and reflective of how APC alters host responses. Such metabolic data may lead to novel biomarkers, as well as enlightening us about how radiation and radiomitigation affect various metabolic pathways. In summary, these studies will provide novel insights into mechanisms by which the Thbd?protein C pathway components, i.e., APC and its endothelial receptors, achieve endothelial radiomitigation. Studies of endothelial gene expression profiles will provide insights into which endothelial regulatory systems are significantly altered by radiation and rescued by APC. Basic knowledge from this project will provide key data required for thoughtful development of countermeasures addressing radiation-induced endovascular injury.