PROJECT SUMMARY/ABSTRACT This proposed renewal of “Purinergic modulation of the autoimmune vascular phenotype” builds upon 4 years of discovery by an early stage investigator in the areas of lupus, COVID-19, and antiphospholipid syndrome (APS). Despite the routine use of traditional anticoagulants in the latter disease, 1 in 5 APS patients is still expected to experience a breakthrough thrombotic event. Furthermore, anticoagulants do little to mitigate the chronic occlusive microangiopathy that damages APS organs over time. How to combat anticoagulant- resistant manifestations of APS is unknown. This project now endeavors to use a deeply-phenotyped patient cohort, selected in vitro systems, and the most relevant animal models to identify adjuvant therapeutic approaches for the APS clinic. Its successful completion will shed additional light on neutrophil phenotypes in APS and will provide a new understanding of the role that purinergic signaling, neutrophil-platelet interactions, and neutrophil metabolism play in thrombotic events. The goal is that by the next cycle of this grant, we will have identified the 1-2 most promising drug candidates for repurposing in our APS clinic. APS is a leading acquired cause of both thrombosis and late-term pregnancy loss. In pursuit of a mechanistic understanding of immunothrombosis in APS, our group was the first to show that neutrophil extracellular traps (NETs, tangles of chromatin and microbicidal proteins expelled from activated neutrophils via “NETosis”) are required for APS-associated thrombosis. Since our last competitive submission, we have found that extracellular adenosine generated by the ectonucleotidase CD73 restrains NET release by activating surface adenosine A2A receptors (A2AR) and thereby boosting intracellular cAMP. Key preliminary data that inform this renewal demonstrate (i) restraint of platelet-mediated neutrophil activation by the CD73-A2AR axis; (ii) exaggerated thrombosis in myeloid lineage-specific A2AR knockout mice; (iii) hyperactive glucose metabolism in APS neutrophils that normalizes with A2AR agonists; and (iv) mitigation of thrombosis in APS mice by metabolism-focused interventions. The hypothesis is that manipulation of the CD73-A2AR-cAMP axis will restore neutrophil homeostasis in APS. Specific Aim 1 will define mechanisms by which purinergic signaling influences neutrophil and platelet function in APS. This Aim will define for the first time the purinergic landscape of a thrombophilic disorder, elucidate mechanisms by which purinergic signaling regulates neutrophil-platelet communication, and potentially identify the subset of APS patients most likely to benefit from antiplatelet and/or adenosine receptor-modulating therapies. Specific Aim 2 will determine the extent to which purinergic signaling can be leveraged to normalize neutrophil metabolism in APS. This Aim is expected to provide a new understanding of the metabolic requirements of NETosis, elucidate strategies for ma...