DNA‐bound elastase of neutrophil extracellular traps degrades plasminogen, reduces plasmin formation, and decreases fibrinolysis: proof of concept in septic shock plasma

Abstract

Activation of platelets and neutrophils in septic shock results in the formation of microvascular clots containing an intricate scaffold of fibrin with neutrophil extracellular traps (NETs) DNA. NETs contain multiple components that might impact endogenous fibrinolysis, resulting in failure to lyse clots in the microcirculation and residual systemic microthrombosis. We propose herein that the reservoir of human neutrophil elastase (HNE) on NETs may directly interfere with the fibrinolytic mechanism via a plasminogen proteolytic pathway. To investigate this mechanism, we constructed fibrin-NETs matrices by seeding and activating neutrophils onto a fibrin surface and monitored plasminogen activation or degradation. We demonstrate that the elastase activity of HNE-DNA complexes is protected from inhibition by plasma antiproteases and sustains its ability to degrade plasminogen. Using mass spectrometry proteomic analysis, we identified plasminogen fragments composed of kringle (K) domains (K 1+2+3 , k 1+2+3+4 ) and the serine protease (SP) region (K 5 -SP). We further demonstrate that patients with septic shock with disseminated intravascular coagulation have circulating HNE-DNA complexes, HNE-derived plasminogen fragments, a low plasminogen concentration, and a reduced capacity to generate plasmin onto fibrin. In conclusion, we show that NETs bearing active HNE-DNA complexes reduce plasminogen into fragments, thus impairing fibrinolysis by decreasing the local plasminogen concentration, plasminogen binding to fibrin, and localized plasmin formation.

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“…With regard to the interaction of plasmin with NETs, the serine proteases, thrombin and plasmin, were also found to interact with DNA, and bound to NETs in vitro (1,9). In this respect, it is interesting to note that thrombin and plasmin, like NE, belong to the vast family of S1 peptidases, which share an overall similar structure and folding (1). Thrombin can induce neutrophil chemotaxis and aggregation at submicromolar concentrations.…”

“…In addition to this, NET components [DNA, histone, and granule proteins such as myeloperoxidase and elastase (NE)] also contribute to the triggering of an inflammatory process. The dysregulation of NET formation and the consecutive release of NET byproducts is involved in thrombosis and fibrinolysis disorders in autoimmune diseases, as well as non-autoimmune diseases, in particular viral infection (1,5,10).…”

“…In this way, NE highly degrades plasminogen without generating plasmin, which in turn leads to the production of antifibrinolytic plasminogen fragments. NETs can therefore serve as a platform for NE-mediated activation of intravascular coagulation in vivo (1,10).…”

“…Platelet-neutrophil interactions at the site of deep vein thrombosis formation were found to induce NETosis, and to be of substantial relevance for thrombogenesis in the context of deep vein thrombosis in general. Barbosa da Cruz et al ( 1 ) revealed that NE/DNA complexes in NETs play a central role in a mechanism that results in severe fibrinolytic failure. NE forms a tight complex with DNA that strongly impairs its inhibition by the α1-proteinase inhibitor (α1-PI) ( 1 ).…”

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“…In addition to its proteolytic effect, NE is also known to induce pro-inflammatory cytokines in epithelial cells, thus exacerbating inflammatory damage ( 37 , 38 ). Moreover, the NETs’ DNA-backbone has been demonstrated to enhance the activity of NE by protecting it from inhibition by antiproteases ( 39 ). Considering the pathogenic role of the interaction between neutrophils and endothelium in AOSD ( 40 ), NE-DNA complex may mediate vascular damage, leading to serositis and interstitial lung infiltration in AOSD.…”