Kyungho Kim1, Jing Li1, Alan Tseng1, Robert K. Andrews2, and Jaehyung Cho1*
1 Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL, United States;
2 Australian Centre for Blood Diseases, Monash University, Melbourne, VIC, Australia
* Corresponding author; Jaehyung Cho
Platelet-leukocyte interactions on activated endothelial cells play an important role during microvascular occlusion under oxidative stress conditions. However, it remains poorly understood how neutrophil-platelet interactions are regulated during vascular inflammation. Using intravital microscopy with mice lacking NADPH oxidase 2 (NOX2) and their bone marrow chimera, we demonstrated that both hematopoietic and endothelial cell NOX2 are crucial for neutrophil-platelet interactions during TNF-α-induced venular inflammation. Platelet NOX2-produced reactive oxygen species (ROS) regulated P-selectin exposure upon agonist stimulation and the ligand-binding function of glycoprotein Ibα. Furthermore, neutrophil NOX2-generated ROS enhanced the activation and ligand-binding activity of αMβ2 integrin following fMLF stimulation. Studies with isolated cells and a mouse model of hepatic ischemia/reperfusion injury revealed that both platelet and neutrophil NOX2 are required for cell-cell interactions, which contribute to the pathology of hepatic ischemia/reperfusion injury. Platelet NOX2 modulated intracellular Ca2+ release but not store-operated Ca2+ entry (SOCE), whereas neutrophil NOX2 was crucial for SOCE but not intracellular Ca2+ release. Different regulation of Ca2+ signaling by platelet and neutrophil NOX2 correlated with differences in the phosphorylation of AKT, ERK, and p38MAPK. Our results indicate that platelet and neutrophil NOX2-produced ROS are critical for the function of surface receptors essential for neutrophil-platelet interactions during vascular inflammation.