Abstract Number: OC 72.4
Meeting: ISTH 2022 Congress
Background: Sepsis is a life-threatening response to infection. Neutrophils prevent sepsis by releasing NETs to entrap bacteria; yet most experimental NET-directed therapies are designed to accelerate NET degradation. We hypothesize that NET degradation products induce greater harm than intact NETs, and propose the alternative strategy of NET-stabilization with PF4.
Aims: We compared coagulopathy and endothelial dysfunction induced by intact or degraded NETs and cell-free (cf)DNA, and effects of PF4 on those properties.
Methods: Human neutrophils were stimulated to release NETs and treated with mild DNase to liberate high-molecular-weight (hmw) NETs. High and low molecular weight (lmw) cfDNA and single-stranded (ss) DNA were also studied. We assessed effects of PF4 on DNA/NET/ssDNA-induced thrombin and fibrin generation in normal, factor (F) XII- and FXI-depleted plasma. Effects of PF4 on DNA-induced endothelial dysfunction were studied using human umbilical vein endothelial cells (HUVECs) exposed to DNA/ssDNA and stained for von Willebrand factor (vWF) release. HmwDNA was administered intravenously to wildtype (WT) and PF4-/- mice. Cremaster venous thrombosis and plasma thrombin-anti-thrombin (TAT) were assessed.
Results: HmwNETs/DNA were less thrombogenic than lmwNETs/DNA or ssDNA. PF4 delayed thrombin and fibrin generation induced by NET/DNA fragments of any length. PF4 did not affect DNA-induced fibrin generation in FXII- or FXI-depleted plasma, but supplementation with FXII or FXI rescued PF4 anticoagulant effects. LmwDNA and ssDNA induced vWF release by HUVECs, which was prevented by addition of PF4. Infused cfDNA markedly enhanced fibrin accumulation and neutrophil recruitment into thrombi in PF4-/- compared to WT mice, corresponding to higher TAT levels in PF4-/- mice.
Conclusion(s): NET-stabilization with PF4 decreases thrombosis by (1) inhibiting DNase digestion of intact NETs and subsequent liberation of prothrombotic cfDNA, and (2) preventing further digestion of circulating cfDNA into shorter and more prothrombotic fragments. These studies further support our hypothesis that NET-stabilization with PF4 is protective in sepsis, meriting further translational studies.
To cite this abstract in AMA style:Ngo A, Field C, Sarkar A, Yarovoi I, Zhao G, Rauova L, Kowalska M, Poncz M, Gollomp K. Toxicity of neutrophil extracellular traps (NETs) and the effects of platelet factor 4 (PF4) on NET thrombogenicity: Therapeutic implications [abstract]. https://abstracts.isth.org/abstract/toxicity-of-neutrophil-extracellular-traps-nets-and-the-effects-of-platelet-factor-4-pf4-on-net-thrombogenicity-therapeutic-implications/. Accessed September 26, 2022.
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