Thrombomodulin favors leukocyte microvesicle fibrinolytic activity, reduces NETosis and prevents septic shock-induced coagulopathy in rats

Abstract

BackgroundSeptic shock-induced disseminated intravascular coagulation is responsible for increased occurrence of multiple organ dysfunction and mortality. Immunothrombosis-induced coagulopathy may contribute to hypercoagulability. We aimed at determining whether recombinant human thrombomodulin (rhTM) could control exaggerated immunothrombosis by studying procoagulant responses, fibrinolysis activity borne by microvesicles (MVs) and NETosis in septic shock.MethodsIn a septic shock model after a cecal ligation and puncture-induced peritonitis (H0), rats were treated with rhTM or a placebo at H18, resuscitated and monitored during 4 h. At H22, blood was sampled to perform coagulation tests, to characterize MVs and to detect neutrophils extracellular traps (NETs). Lungs were stained with hematoxylin–eosin for inflammatory injury assessment.ResultsCoagulopathy was attenuated in rhTM-treated septic rats compared to placebo-treated rats, as attested by a significant decrease in procoagulant annexin A5+-MVs and plasma procoagulant activity of phospholipids and by a significant increase in antithrombin levels (84 ± 8 vs. 64 ± 6%, p < 0.05), platelet count (582 ± 157 vs. 319 ± 91 × 109/L, p < 0.05) and fibrinolysis activity borne by MVs (2.9 ± 0.26 vs. 0.48 ± 0.29 U/mL urokinase, p < 0.05). Lung histological injury score showed significantly less leukocyte infiltration. Decreased procoagulant activity and lung injury were concomitant with decreased leukocyte activation as attested by plasma leukocyte-derived MVs and NETosis reduction after rhTM treatment (neutrophil elastase/DNA: 93 ± 33 vs. 227 ± 48 and citrullinated histones H3/DNA: 96 ± 16 vs. 242 ± 180, mOD for 109 neutrophils/L, p < 0.05).ConclusionThrombomodulin limits procoagulant responses and NETosis and at least partly restores hemostasis control during immunothrombosis. Neutrophils might thus stand as a promising therapeutic target in septic shock-induced coagulopathy.Electronic supplementary materialThe online version of this article (10.1186/s13613-017-0340-z) contains supplementary material, which is available to authorized users.

A Review of Neutrophil Extracellular Traps (NETs) in Disease: Potential Anti-NETs Therapeutics

“…Helms et al did a study where they looked into the use of a recombinant human thrombomodulin (rhTM) and found out that it can limit procoagulant responses. rhTM was also shown to fully inhibit NETosis in neutrophils cultured with platelets and in the presence of LPS [ 138 ]. There is not much research in effect of rhTM, but this provides a starting point for further research into its potential use to inhibit NETs.…”

Neutrophil-Extracellular Traps, Cell-Free DNA, and Immunothrombosis in Companion Animals: A Review

“…69 Similarly, thrombomodulin attenuates histone-induced thrombin generation and endothelial cell death in vitro, likely through its ability to activate protein C. 154 Thrombomodulin also reduces NETosis and blunts the prothrombotic state resulting from cecal-ligation and puncture-induced peritonitis in rats. 72 Recombinant human thrombomodulin is used successfully in humans with sepsis-induced DIC in Japan, 68,228 and Phase III clinical trials are ongoing in the USA (NCT01598831, NCT03517501). If these prove successful, then some of the benefits may derive from inhibition of immunothrombosis.…”

Thrombomodulin as a Physiological Modulator of Intravascular Injury

“…In a mouse histone-induced septic model, pretreatment with rTM reduced mortality rates by neutralizing histones ( 20 ). In a rat sepsis/peritonitis model ( 33 ) and a murine LPS-induced septic model ( 34 ), rTM controlled sepsis-related immunothrombosis by limiting abnormal hemostasis and NET formation.…”

“…In particular, autoimmune ANCA vasculitis, which is characterized by immune dysregulation and intravascular injury, might be a candidate for rTM treatment. However, the dosage of rTM in many experimental situations ( 15 , 33 , 41 ) is 15–50 times of therapeutic dosage in patients with DIC and the effective concentration as an anti-inflammatory and immune-regulatory property remains unclear. Thus, in the future the indications of rTM therapy and the suitable dosage with no serious complications such as bleeding tendency should be carefully addressed.…”