DNAse-dependent, NET-independent pathway of thrombus formation in vivo

Abstract

The contribution of NETs (neutrophil extracellular traps) to thrombus formation has been intensively documented in both arterial and venous thrombosis in mice. We previously demonstrated that adenosine triphosphate (ATP)–activated neutrophils play a key role in initiating the tissue factor–dependent activation of the coagulation cascade, leading to thrombus formation following laser-induced injury. Here, we investigated the contribution of NETs to thrombus formation in a laser-induced injury model. In vivo, treatment of mice with DNase-I significantly inhibited the accumulation of polymorphonuclear neutrophils at the site of injury, neutrophil elastase secretion, and platelet thrombus formation within seconds following injury. Surprisingly, electron microscopy of the thrombus revealed that neutrophils present at the site of laser-induced injury did not form NETs. In vitro, ATP, the main neutrophil agonist present at the site of laser-induced injury, induced the overexpression of PAD4 and CitH3 but not NETosis. However, compared to no treatment, the addition of DNase-I was sufficient to cleave ATP and adenosine diphosphate (ADP) in adenosine. Human and mouse platelet aggregation by ADP and neutrophil activation by ATP were also significantly reduced in the presence of DNase-I. We conclude that following laser-induced injury, neutrophils but not NETs are involved in thrombus formation. Treatment with DNase-I induces the hydrolysis of ATP and ADP, leading to the generation of adenosine and the inhibition of thrombus formation in vivo.

Role of Neutrophils and NETs in Animal Models of Thrombosis

“…The authors also showed the importance of adenosine triphosphate (ATP), which plays an indispensable role in the recruitment and activation of neutrophils in the vascular endothelium, and the activated endothelium is a potential physiological source of ATP [2]. Finally, in 2021, our team demonstrated the involvement of NETs in this laser thrombosis model [24]. Indeed, intravital microscopy experiments showed that the injection of DNAse I (Figure 4), as well as the injection of apyrase, a known ATPase, into the general circulation of WT mice induced a strong decrease in the size of the thrombus after laser beam injury, thus suggesting a possible involvement of NETs in this model.…”

“…Morphologically, electron microscopy experiments in particular allow the characterization of neutrophils entering in NETosis with disrupted nuclear and plasma membranes, a clear cytoplasm with granules approaching the center of the cell close to the nucleus and finally extracellular DNA fibers [24].…”

“…Regarding the protein profile of these structures, both in vitro [24] and in vivo [8] studies show that NETs express several proteins originally contained in neutrophilic granules such as serine proteases NE, MPO, and cathepsin G. In an original way, NETs show a significant citrullination of its histones (2, 3, and 4). CitH3 was used as a NETs reference marker.…”

“…In addition, the use of a calcium mobilization reporter compound, Fluo-4-AM, revealed that neutrophils accumulated at the site of endothelial injury were activated. They release granular contents such as serine protease NE [24] by exocytosis and express tissue factor on their surface either by de novo production or by acquisition via monocytes or TF+ microparticles. The procoagulant TF+ activity of these neutrophils is still under debate.…”

Inflammatory Mediators of Platelet Activation: Focus on Atherosclerosis and COVID-19

“…Li et al noted a decrease in inflammatory cytokines paired with attenuation of platelet activation and thrombus formation following NET degradation with the inhibitor DNAse [168]. In another study, DNAse lessened ATP and ADP-induced thrombus formation in vivo, although it did not affect NET formation [169]. The DNAse inhibitor dornase alfa has recently gained attention towards COVID-19 treatment, with improvement in patient oxygenation being noted following its use in vitro and in vivo [170][171][172], while it remains to be explored whether these agents can influence clinical outcomes by halting platelet activation among other pathways.…”