Effets anti-inflammatoires de l'inhibiteur dépendant de la protéine Z : intérêt potentiel comme traitement adjuvant du sepsis

Abstract : Septic shock is an acute circulatory failure caused by an infectious agent, resulting in hemodynamic, metabolic and visceral disorders, in particular due to pro-inflammatory cytokines. The mortality rate is high (about 40 %). Progression of severe sepsis is often complicated by thrombotic events, in part because of a direct coagulation activation by bacteria, but also because of Neutrophil Extracellular Traps (NETs) released by polymorphonuclear neutrophils. Disseminated intravascular coagulation (DIC) is frequently present in these patients, characterized by an unstable equilibrium, where thrombotic and bleeding risks coexist, due to the consumption of coagulation factors.Several studies suggested that administration of coagulation inhibitors, such as antithrombin (AT) or activated Protein C, decreased mortality, not only by preventing the activation of coagulation, but also through their cytoprotective and anti-inflammatory effects, independent of their anticoagulant activity. However, the cytoprotective effects require the administration at very high doses, leading to a bleeding tendency.The protein Z-dependent protease inhibitor (ZPI) belongs to the serpin superfamily, as AT, but in contrast to AT, inhibits only factors Xa (FXa) and XIa (FXIa) of coagulation. FXa inhibition by ZPI is potentiated by Protein Z (PZ), a vitamin K-dependent factor, which circulates in plasma in a complex with ZPI. In a whole blood model, I observed that ZPI exerts an inhibitory effect on lipopolysaccharide (LPS)-induced pro-inflammatory cytokines production (IL-6 and TNF-α). At high concentration (4 times physiological concentration), ZPI anti-inflammatory effect (AIE) is not modified by PZ or unfractionned heparin, which increase ZPI anticoagulant activity. Moreover, this AIE is still present using a reactive center loop variant of ZPI (ZPI Y387A), suggesting that the AIE of ZPI is independent of its anticoagulant activity. In vitro, in whole blood, ZPI induced an early increased of CCL-5, a chemokine with anti-inflammatory properties. These data are confirmed in vivo in a murine model of endotoxin shock where LPS is injected intraperitoneally. Simultaneously injection of recombinant human ZPI (rhZPI) with LPS led to lower plasma levels of IL-6 and TNF-α than in control mice, whereas higher CCL-5 levels were observed in peritoneal lavages.In addition, using purified proteins, we have shown that neutrophil elastase, an enzyme which decorates NETs, induces several cleavages of rhZPI. A quick and first cleavage is observed on the reactive centre loop of ZPI, inducing a loss of inhibitory activity towards FXa and FXIa. PZ does not protect ZPI from elastase degradation. ZPI proteolysis induced by NETs could contribute to their procoagulant activity.Lastly, in collaboration with the intensive care unit of Strasbourg Hospital, we studied plasma levels of PZ and ZPI in 100 patients with severe sepsis. During the first 24 hours, there was a significant decrease of plasma PZ levels, compared to levels of healthy subjects, whereas an approximately 2.5 times increase was observed for ZPI levels. These high levels of ZPI were still present at D3 and D7, whereas PZ levels regularly increased. Variations of PZ and ZPI levels were not predictive of the 30-day mortality rate, and not associated with DIC development.These results suggest that elevated concentrations of ZPI (4 times physiological concentration) could be an adjuvant therapy to septic shock, by decreasing pro-inflammatory cytokines production, but devoid of bleeding risk, since FXIa inhibition has antithrombotic activity without inducing haemorrhages.
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Mahita Razanakolona. Effets anti-inflammatoires de l'inhibiteur dépendant de la protéine Z : intérêt potentiel comme traitement adjuvant du sepsis. Biotechnologie. Université Paris-Saclay, 2018. Français. ⟨NNT : 2018SACLS564⟩. ⟨tel-02158060⟩

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