SESSION TITLE: Biomarkers in Severe Sepsis and Septic Shock
SESSION TYPE: Original Investigation Slide
PRESENTED ON: Wednesday, October 28, 2015 at 02:45 PM - 04:15 PM
PURPOSE: Neutrophil extracellular traps (NETs; webs of DNA coated in anti-microbial proteins expelled from activated neutrophils) are released within the microvasculature during sepsis and aid in host defense, but also contribute to organ damage. Various components of NETs (including neutrophil proteases and histones) have been shown in vitro to modulate the coagulation cascade. We hypothesized that the release of NETs within the vasculature activates intravascular coagulation, leading to microvascular hypoperfusion and organ dysfunction.
METHODS: Multi-color confocal intravital microscopy (IVM) was used in mouse models of sepsis (endotoxemia and E. coli peritonitis) to visualize and quantify neutrophil trafficking, NETs release, intravascular coagulation (using a novel method of in vivo zymography to quantify intravascular thrombin activity, and in vivo immunofluorescene for fibrin deposition), and real time microvascular perfusion analysis. Serum lactate, ALT, and thrombin-antithrombin complex (TAT) levels were determined using commercial assays.
RESULTS: In vivo imaging of intravascular thrombin activity within the liver microcirculation of septic mice revealed spatial co-localization between NETs (released from infiltrating neutrophils), intravascular coagulation (thrombin activity and fibrin deposition), and concomitant microvascular occlusion. Inhibition of intravascular NETs in septic mice using intravenous DNase infusion (to digest NETs), PAD4-deficient mice (that are unable to generate NETs), or a novel PAD4 inhibitor (GSK199) resulted in a significant reduction in microvascular thrombin activity, and subsequent fibrin deposition. Real-time assessment of microvascular perfusion revealed that DNase infusion or PAD4 inhibition (or deficiency) improved microvascular perfusion. In mice with Gram-negative sepsis, treatment with i.v. DNase or a PAD4-inhibitor reduced markers of disseminated intravascular coagulation (TAT complex levels), as well as reduced serum lactate and ALT levels compared to vehicle-treated control mice.
CONCLUSIONS: Neutrophil extracellular traps activate thrombin leading to disseminated intravascular coagulation during sepsis. Inhibition of NETs in septic animals prevents coagulation, improves microvascular perfusion, and attenuates end-organ damage.
CLINICAL IMPLICATIONS: Inhibition of NETs (i.v. DNase or PAD4 inhibitor) represents a novel therapeutic strategy to prevent disseminated intravascular coagulation and organ dysfunction in severe sepsis.
DISCLOSURE: Huw Lewis: Employee: GlaxoSmithKline Employee. Involved in development of GSK199, a product used in this study, for which a salary was paid as an employee. The following authors have nothing to disclose: Braedon McDonald, Rachelle Davis, Paul Kubes, Craig Jenne
This presentation includes information about a small molecule PAD4-inhibitor called "GSK199", designed by GlaxoSmithKline, that has not been approved for any purpose.