Platelet Factor 4 (PF4) Modulates the Prothrombotic Nature of Neutrophil-Extracellular Traps (NETs): Therapeutic Implications of a NET-Stabilization Strategy

Sepsis is a dysregulated response to infection leading to life-threating organ damage. Although it remains one of the most common causes of mortality worldwide, it lacks targeted treatments. Neutrophils play a crucial role in sepsis by releasing NETs, webs of DNA complexed with histones and antimicrobial proteins that capture pathogens and prevent bacterial dissemination. However, when NETs are degraded by circulating nucleases they release NET-degradation products (NDPs) including cell-free (cf) DNA, histones and myeloperoxidase, which trigger coagulation, induce complement activation, and cause oxidative tissue damage. We proposed a novel NET-directed therapy in sepsis, in which NETs are stabilized by the platelet chemokine PF4. Binding of PF4 enhances NET DNase-resistance, promotes NDP sequestration and increases bacterial capture, improving survival in murine sepsis. As NETs are considered prothrombotic, we were concerned that NET stabilization may increase the risk of clot formation. We therefore sought to determine the effect of PF4-NET stabilization on the thrombogenicity of NETs to learn if this strategy is safe for clinical application. To that end, we examined the effect of PF4 on the thrombotic potential of DNA and NET fragments at different states of nuclease digestion. High molecular weight (hmw) genomic DNA (hmwDNA, >50kbp) was isolated from human whole blood. hmwDNA was digested with restriction enzymes (EcoRI and AluI) for 15min to generate DNA fragments of ~4kbp and ~250bp, respectively. Neutrophils were also isolated from human blood and stimulated with 100 nM PMA to produce neutrophil-adherent NETs, which were cleaved from cell bodies by treatment with 4U/mL DNase I for 20 minutes, releasing NETs >50kbp (hmwNETs). Additional incubation of hmwNETs with DNase I yielded smaller NET fragments. We assessed in vitro activation of coagulation by DNA and NETs by measuring thrombin generation and fibrin formation in platelet-poor plasma using fluorogenic substrate and turbidity assays. Neutrophil-adherent NETs induced far less thrombin generation and fibrin formation in plasma than hmwDNA and hmwNETs. PF4 significantly increased lag time and reduced peak thrombin formation induced by both hmwDNA and hmwNETs. Binding of PF4 also delayed clot initiation time and reduced the rate of fibrin generation. Digestion of hmwDNA and hmwNETs to smaller fragments markedly enhanced thrombogenicity. We posited that shorter DNA fragments are more thrombogenic be