OC 72.4 - Toxicity of neutrophil extracellular traps (NETs) and the effects of platelet factor 4 (PF4) on NET thrombogenicity: Therapeutic implications

Wednesday, July 13, 2022

11:00 AM – 11:15 AM

Location: ExCel Center, ICC Capital Suite Room 7&12

Presentation Format: In-Person

Presenting Author(s)

Anh T. Ngo, PhD

Postdoctoral Fellow
Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States

Co-Author(s)

CF

Conroy Field

Graduate Student
University of Pennsylvania
Philadelphia, Pennsylvania, United States
AS

Amrita Sarkar

Research Associate
Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
IY

Irene Yarovoi

Research Assistant
Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
GZ

Guohua Zhao

Research Technician
Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
LR

Lubica Rauova

Research Associate Professor of Pediatrics
Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
MK

M. Anna Kowalska

Scientist III
Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
MP

Mortimer Poncz

Division Chief
Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
KG

Kandace Gollomp

Assistant Professor
Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States

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.