1612 Contribution of activated beta3 integrin in the PDI release from endothelial cells Halszka Ponamarczuk 1 , Marcin Popielarski 1 , Marta Stasiak 1 , Radosław Bednarek 1 , Maciej Studzian 2 , Lukasz Pulaski 2,3 , Anna Babinska 4 , Maria Swiatkowska 1 1 Department of Cytobiology and Proteomics, Medical University of Lodz, Lodz, Poland, 2 Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland, 3 Laboratory of Transcriptional Regulation, Institute of Medical Biology PAS, Lodz, Poland, 4 Department of Medicine, SUNY Downstate Medical Center, Brooklyn, New York, USA TABLE OF CONTENTS 1. Abstract 2. Introduction 3. Material and methods 3.1. Reagents and antibodies 3.2. EA.hy926 cell culture 3.3. Secretion of PDI from endothelial cells 3.4. Cell adhesion assay 3.5. Wound healing migration assay 3.6. Coimmunoprecipitation and immunoblotting 3.7. Coimmunoprecipitation beta1/PDI 3.8. Confocal microscopy 3.9. Sulfhydryl group labeling 3.10. In vitro angiogenesis assay - endothelial cell spheroids 3.11. Statistical analysis 4. Results 4.1. Secretion of PDI from endothelial cells 4.2. Association of PDI with alphaVbeta3 integrin on endothelial cells during adhesion 4.3. Infuence of PDI inhibitors and thiol group blockers on the functions of endothelial cells 5. Discussion 6. Acknowledgment 7. References [Frontiers In Bioscience, Landmark, 23, 1612-1627, March 1, 2018] 1. ABSTRACT Protein disulfde isomerase (PDI) is an abundant reticulum endoplasmic protein but also acts as an important functional regulator of some extracellular surface proteins. Recent studies suggest that PDI plays a role in integrin activation and thrombus formation. The aim of this study was to examine whether activation of integrin is the frst stage leading to release of PDI from the subcellular compartments of endothelial cells to extracellular space. Our results show that endothelial cells which adhere to fbronectin or fbrinogen release signifcantly more PDI than those which adhere to poly-L-lysine. Cells treated with RGD peptide, Src and FAK kinase inhibitors and anti alphaVbeta3 antibody display lower PDI secretion. The destruction of the actin cytoskeleton of endothelial cells by cytochalasin D inhibits PDI release. When the endothelial cells adhere to fbrinogen or fbronectin, PDI and alphaVbeta3 gain free thiol groups. Our data suggest that upon activation of integrins, PDI is released from endothelial cells and forms a disulfde bond complex with alphaVbeta3 integrin. 2. INTRODUCTION PDI, a member of the thiol-disulfde oxidoreductase family, displays thiol isomerase, oxidase and reductase activity. PDI is found on the surface of several types of cells, including endothelial cells, hepatocytes, pancreatic cells, neutrophils and cancer cells (1). It has also been found on the surface of platelets, where it plays an important role in platelet