Project 1. Role of extracellular thiol-modifying machinery in arterial thrombosis Mounting evidence indicates that oxidation or reduction of allosteric disulfide bonds in plasma proteins and cell surface molecules induces conformational changes and alters their functions in the initiation and progression of cardiovascular disease. Protein disulfide isomerase (PDI) and PDI family member oxidoreductases catalyze the modification of allosteric disulfide bonds. We demonstrated that platelet-released PDI directly interacts with cell surface molecules, such as β3 integrin and glycoprotein Ibα (GPIbα), during cell activation and promotes platelet adhesive function in arterial thrombosis and thromboinflammation (Kim et al. Blood 2013; Li et al. Circulation 2019). While these results demonstrate the essential role of PDI in the disease process, it is unknown how intravascular PDI activity is regulated under thrombotic conditions. We have found that endoplasmic reticulum oxidoreductin 1α (ERO1α), a key oxidase of PDI in the ER, is released from activated intravascular cells such as platelets and plays a critical role in arteriolar thrombosis but not hemostasis. Using ERO1α knockout (KO) and conditional KO (CKO) mice, blocking antibodies, and small-molecule inhibitors which my lab has recently developed, we will identify cell surface molecules targeted by extracellular ERO1α using proteomics, unravel the role of ERO1α in modifying allosteric disulfide bonds in cell surface molecules, and determine the presence and function of extracellular ERO1α-thiol isomerase (e.g., PDI) redox-relay signaling in arterial thrombosis. There is evidence that hypoxia stimulates platelet activity and contributes to the pathophysiology of cerebral and arterial thrombosis. Because hypoxia up-regulates the expression of ERO1α, our long-term goal is to elucidate novel roles of ERO1α and thiol isomerases in ischemia/reperfusion-induced injuries, such as stroke and myocardial infarction. Project 2. Mechanisms regulating neutrophil recruitment to sites of vascular inflammation Neutrophils are essential for innate immunity, yet excessive recruitment to sites of inflammation causes severe tissue damage. Furthermore, adherent neutrophils support adhesion of other blood cells such as platelets and red blood cells, leading to cell-cell aggregation and vascular occlusion. Hence, a better understanding of the mechanisms regulating the interactions of neutrophils with endothelial cells and other blood cells is of great importance for designing effective therapies to attenuate inflammatory conditions without impairing host defense. We demonstrated that the AKT2-NOX2 signaling axis promotes the ligand-binding function of neutrophil αMβ2 integrin and platelet GPIbα to induce cell-cell interactions under thromboinflammatory conditions, including sterile inflammation and ischemia/reperfusion-induced injury (Li et al. J Clin Invest 2014; Kim et al. Blood 2015). In particular, our animal studies revealed that intravascular neutrophil-platelet interactions directly contribute to vaso-occlusion in sickle cell disease (SCD), which is a trigger for pain crisis and acute chest syndrome in patients (Barazia et al. Blood 2015; Kim et al. Haematologica 2017). In our latest studies, we demonstrated that blocking intravascular PDI activity attenuates these cell-cell interactions and vaso-occlusion in SCD mice (Li et al. Circulation 2019). We have identified a crucial role of AKT2 in promoting calcium release and influx during neutrophil activation. Aided by proteomics and mass spectrometric analysis, we will discover AKT2-regulated target molecules controlling calcium mobilization in neutrophils under inflammatory conditions and to identify the AKT2 phosphorylation sites on the candidate molecules. Since NOX2-generated reactive oxygen species alter the local redox environment, our long-term project conducted in relation to though independent of Project 1 is to explore how the AKT2-NOX2 signaling axis influences the activities of extracellular ERO1α and thiol isomerases in inflammatory disease. Furthermore, we have found that in addition to PDI, extracellular ERO1α and other thiol isomerases contribute to vaso-occlusion in patients with SCD. We will determine the role of intravascular ERO1α and thiol isomerases in regulating neutrophil adhesive function in SCD. Importantly, we will test the ability of novel ERO1α blocking antibodies and small-molecule inhibitors to attenuate vaso-occlusion-mediated events in SCD. Our studies will be of substantial clinical importance by providing insight into the design of novel therapeutic strategies for SCD patients. Project 3. Role of transcription regulators in platelet activation and thrombosis Anucleate platelets can synthesize proteins using mRNAs and microRNAs under disease conditions. Intriguingly, platelets contain transcriptional regulators, which could be transferred from megakaryocytes during platelet production. Our recent studies provide the first evidence that the mRNA and protein of Downstream Regulatory Element Antagonist Modulator (DREAM, a transcriptional repressor and a positive regulator of NF-κB) are present in platelets and that platelet DREAM promotes the activity of phosphoinositide-3-kinase during platelet activation and arterial thrombosis (Kim et al. Blood 2017). Our work has identified DREAM as a novel target for anti-thrombotic agents. Furthermore, platelets express toll-like receptors (TLRs) such as TLR4 and their downstream signaling molecules, such as NF-κB. However, their roles in platelet function remain poorly understood. Therefore, we will investigate the role of TLR and NF-κB signaling in platelet activation and arterial thrombosis.

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As a principal investigator who was originally from Seoul, South Korea, I am aware of the importance of training a diverse range of people. Particularly, I understand that training minority and women scientists are essential for our scientific community. I have trained five female postdoctoral fellows. Two of them are tenure-track faculty members in academia. Also, I trained one female M.S. degree African American student and had three medical school students who are Hispanic or African American for their summer research. Further, I had several summer high school students who were Asian, Indian, and Hispanic and prepared for a pre-med in college. I have enjoyed a diverse group of my trainees very much.