Real-time fluorescence intravital microscopy has provided compelling evidence that platelet-neutrophil interactions on the activated endothelium are a major determinant of microvascular occlusion during vascular inflammation. Previous studies demonstrated that neutrophil Mac-1 (?M?2 integrin) plays a critical role in platelet-neutrophil interactions under inflammatory conditions. Further, we found that neutrophil AKT2 is a critical regulator for the membrane translocation and activation of Mac-1 during cell activation. However, the detailed mechanisms by which AKT2 regulates Mac-1 function remain unclear. Our preliminary studies showed that neutrophil AKT2 phosphorylates stromal interaction molecule 1 (STIM1), a Ca2+ sensor essential for store- operated Ca2+ entry (SOCE). Importantly, peptides mimicking AKT2 phosphorylation sites on STIM1 significantly inhibited SOCE during neutrophil activation, suggesting the important role of AKT2-phosphorylated STIM1 in inducing SOCE. Another key result is that neutrophil NADPH oxidase 2 (NOX2) is important for mediating Mac- 1 activation but not membrane translocation. In this proposal, we will test the novel hypothesis that neutrophil AKT2-induced SOCE stimulates reactive oxygen species (ROS) generation and ligand-binding activity of Mac- 1, thereby promoting the deleterious neutrophil-platelet interaction and microvascular occlusion under inflammatory conditions. We propose the following specific aims to test this hypothesis:
AIM 1 : Determine the role of neutrophil AKT2 in stimulating STIM1 and Mac-1 function. We will define the role of AKT2 in mediating SOCE through STIM1 phosphorylation. We will also address how AKT2-induced SOCE stimulates ligand-binding activity of Mac-1.
AIM 2 : Define the role of neutrophil AKT2-induced SOCE in activating NOX2 and thiol-disulfide exposure in Mac-1. We will determine the role of AKT2-induced SOCE in activating NOX2 and investigate whether AKT2-NOX2 signaling induces thiol exchange in the extracellular domain of Mac-1 and stimulates its ligand-binding function through cell surface PDI activity. We will also identify Cys residues in allosteric disulfide bonds of Mac-1.
AIM 3 : Investigate the pathophysiologic role of AKT2-induced SOCE in mediating neutrophil-platelet interaction and microvascular occlusion as a central mechanism of vascular inflammation. We will determine the role of AKT2-induced SOCE in neutrophil recruitment, neutrophil-platelet interactions, and vascular occlusion under inflammatory conditions. Further, we will also examine the role of AKT2-induced SOCE signaling in heterotypic cell-cell interaction and aggregation during vaso-occlusive events in sickle cell disease (SCD) patients and mice.
The goal of the proposed work is to elucidate the detailed mechanism(s) by which neutrophil AKT2 signaling regulates calcium influx and platelet-neutrophil interaction during vascular occlusion in inflammatory diseases. This project will define the pathophysiologic role of AKT2-induced calcium influx in enhancing the thiol exposure and ligand-binding activity of Mac-1 and mediating platelet-neutrophil interaction during vascular inflammation. Our studies will provide novel insight into a potential druggable target(s) to treat thromboinflammatory disease.
