Platelet Factor 4 Modulates the Bactericidal Ability of Neutrophil Extracellular Traps

Introduction: Neutrophils play a crucial role in sepsis by releasing neutrophil extracellular traps (NETs), webs of DNA complexed with histones and antimicrobial proteins that capture pathogens and prevent bacterial dissemination. However, whether or not NETs are capable of killing bacteria remains controversial. Our group has preciously shown that the positively charged, platelet-specific chemokine, platelet factor 4 (PF4, CXCL4), binds to NETs, enhancing NET resistance to nuclease digestion and markedly increasing their ability to capture bacteria. In this study, we investigate the heterogeneity of in vitro NET bactericidal ability between donors, and examined whether PF4-NET binding modulates NET antimicrobial functions. Methods: Neutrophils (2-3x10 7/mL) were purified from healthy donors (n=4) and treated with phorbol-2-myristate-13-acetate (100nM) for 4 hours at 37°C to produce neutrophil-adherent NETs, which were cleaved from cell bodies by treatment with a low dose of DNase I (4 U/mL), yielding NETs >50 kilobase pairs (bp) long. Additional treatment of NETs with DNase I (100U/ml) for 1h at 37°C generated smaller NET fragments (