Project Summary Preclinical and clinical studies have provided compelling evidence that adherent neutrophils on activated vascular endothelium contribute to cell-cell aggregation and vaso-occlusion (VOC) in sickle cell disease (SCD). VOC positively correlates with severe pain crisis and acute chest syndrome, the common complication and cause of death in SCD patients. Currently, the effective treatment options for VOC remain limited. Thus, a better understanding of the regulatory mechanisms of neutrophil adhesive function will help develop novel therapies for treating VOC in SCD. We previously discovered that neutrophil surface-bound protein disulfide isomerase (PDI) is crucial for the ligand-binding activity of αMβ2 integrin and neutrophil-endothelial cell interactions during vascular inflammation. These findings have raised several fundamental questions regarding how the activity of cell surface-bound PDI is controlled and whether other thiol isomerases also regulate the function of neutrophil surface receptors. Our preliminary data demonstrate that endoplasmic reticulum protein 57 (ERp57, a thiol isomerase with structural similarity with PDI) and ER oxidoreductin 1α (ERO1α, a key oxidase of thiol isomerases in the ER) are detected on the surface of activated neutrophils and play overlapping and distinct roles in regulating neutrophil adhesive function under inflammatory conditions. Using biochemical, cellular, and animal studies with novel mouse models, blocking antibodies and cell-impermeable peptides, we will test the hypothesis that extracellular ERp57 and PDI in concert with ERO1α target different surface receptors and regulate the ligand-binding function, contributing to intravascular cell-cell interactions and VOC in SCD. In Aim 1, using a series of biochemical and cell biological studies, we will determine the molecular basis of neutrophil thiol isomerases and ERO1α in regulating neutrophil adhesive function. In Aim 2, using in vivo imaging techniques, we will test whether inhibition of extracellular ERp57, PDI and ERO1α attenuates intravascular cell-cell aggregation and VOC in peripheral and pulmonary vessels of SCD mice. In Aim 3, using blood samples from SCD patients under different VOC conditions, we will test whether extracellular thiol isomerases and ERO1α contribute to cell-cell aggregation and correlate with acute VOC conditions in SCD patients. Our studies will provide insights into novel molecular mechanisms mediating intravascular cell-cell interactions, which can be used to design safe and effective strategies to treat VOC-mediated conditions in SCD.