Neutrophil Extracellular Traps (NETs) Thrombogenicity and Endothelial Cell Toxicity Are Ameliorated By Platelet Factor 4 (PF4): Implications to the Treatment of Sepsis

Sepsis, the dysregulated response to infection, is a leading cause of mortality worldwide for which there are no targeted treatments. Neutrophils play a crucial role in sepsis by releasing NETs, webs of DNA complexed with histones and antimicrobial proteins that capture pathogens. However, when NETs are degraded by circulating nucleases, they release NET-degradation products (NDPs) including cell-free (cf) DNA, histones and myeloperoxidase, which trigger thrombosis, induce complement activation and cause oxidative vascular damage. We propose a novel NET-directed therapy in sepsis, in which NETs are stabilized by the platelet chemokine PF4 (CXCL4), which enhances NET resistance to DNase degradation, promoting NDP sequestration and bacterial capture, improving sepsis outcomes.

As NETs are traditionally considered to be prothrombotic, we investigated whether PF4-stabilization alters the thrombogenicity of NETs. We examined the effect of PF4 on both NET- and DNA-mediated thrombus formation and endothelial cell injury using NET and DNA fragments of different lengths. Human neutrophils were stimulated to release NETs and treated with mild DNase I (4U/mL) to liberate high-molecular-weight (hmw) NETs (>50kb), and then digested with restriction enzymes to generate low-molecular-weight (lmw) fragments of ~4kb and ~250bp. We also studied single-stranded (ss) DNA, generated by heating double stranded (ds) DNA fragments to 100°C. We assessed the effects of PF4 on NETs/dsDNA/ssDNA-induced thrombin and fibrin generation in normal and in factor (F) XII- and FXI-depleted plasma. PF4 significantly reduced plasma thrombin and fibrin generation induced by digested NET and DNA fragments and by ssDNA without impairing thrombolysis. PF4 did not exert any effects on DNA-induced fibrin generation in FXII- or FXI-depleted plasma, while supplementation with purified FXII or FXI restored the anticoagulant effects of PF4, suggesting that PF4 binding reduces the ability of cfDNA and NETs to activate the contact pathway of coagulation.

We next studied how PF4 alters the ability of DNA to induce a prothrombotic phenotype in human umbilical vein endothelial cells (HUVECs) incubated with dsDNA or ssDNA fragments by measuring von Willebrand factor (VWF) release with immunofluorescence and by quantifying tissue factor (TF) expression with a chromogenic substrate assay. To study the mechanism of endothelial activation, HUVECs were treated with inhibitors of toll-like receptor (TLR) 9, a pattern recognition receptor that binds to dsDNA. Unlike intact NETs or hmwDNA, both digested dsDNA and ssDNA upregulated VWF release and TF expression by endothelial cells via a TLR9-dependent mechanism, which was prevented by the addition of PF4.

To study these observations in vivo, hmwDNA was administered intravenously to wildtype (WT) and Cxcl4^-/- mice and plasma thrombin-anti-thrombin (TAT) levels were measured 4h post infusion. Cxcl4^-/- mice exhibited higher TAT levels as compared to WT mice. We also studied two distinct sepsis-like states: (1) WT mice exposed to lipopolysaccharide (LPS, IP; 35mg/kg) ± human (h)PF4 (IV; 40mg/kg), where plasma cfDNA and TAT levels were measured at 6h, and (2) the cecal slurry model of polymicrobial infection in endothelial-specific TLR9^-/- mice, where plasma TAT levels were measured at 4h. In the LPS endotoxemia model (1), TAT and cfDNA levels were elevated at 6h post LPS challenge, which were prevented with the administration of hPF4. In the cecal slurry model (2), endothelial TLR9^-/- mice exhibited lower TAT levels at 4h post challenge as compared to WT controls.

In summary, while intact NETs help to contain infection, NET degradation liberates lmwDNA and ssDNA that activate the contact pathway and induce a prothrombotic phenotype in endothelial cells via activation of TLR9. PF4-stabilization of NETs decreases the risk of NET-mediated toxicity by (1) inhibiting DNase cleavage of intact NETs and (2) neutralizing released lmwDNA and ssDNA. NET stabilization by PF4 tips the balance, limiting NET- and NDP-induced thrombosis and vascular toxicity, while enhancing NET's ability to fight infection by microbial entrapment. These studies add support to our hypothesis that PF4 stabilization of NETs is protective in sepsis. Strategies that augment the beneficial effects of PF4 merit further investigation.

Rauova:Astra Zeneca: Research Funding; Rigel Pharmaceuticals Inc: Research Funding. Poncz:AstraZeneca: Research Funding; Johnson n Johnson: Consultancy.