Mechanisms of the stimulatory effect of neutrophil extracellular traps on blood clot retraction

Introduction. Immunothrombosis significantly exacerbates the course and outcomes of inflammatory diseases. Immune thrombi contain leukocytes, primarily neutrophils, that modulate their properties such as resistance to lysis, mechanical stability (embologenicity), and others. We have previously shown that activated neutrophils enhance clot contraction (retraction); however, the pathways and pathogenetic consequences of the influence of inflammatory cells on clot retraction remain unknown. Aim: to establish the mechanisms underlying the stimulatory effect of activated neutrophils on the rate and extent of blood clot retraction mediated by platelet contraction. Materials and methods. Isolated human neutrophils were activated with phorbol 12-myristate 13-acetate (PMA). Thrombin-induced clots were formed in whole blood or platelet-rich plasma with the addition of non-activated or PMA-activated neutrophils forming neutrophil extracellular traps (NETs). Clot retraction kinetics was tracked optically, and the viscoelastic properties of clots were measured using thromboelastography. Results. PMA-activated neutrophils significantly increased the rate and the final extent of clot retraction. These effects were associated with the presence of NETs detected in the clots and their degradation which eliminated the stimulatory effect of neutrophils. The experimentally identified mechanisms of how NETs influence blood clot retraction included the hyperstimulation of platelets by endogenous thrombin and/or alterations in the mechanical properties of clots. Specifically, rivaroxaban, a factor Xa inhibitor, neutralized the stimulatory effect of NETs on clot and plasma retraction// on clot retraction both in whole blood and platelet-reach plasma, indicating the important role of active thrombin generation and secondary platelet hyperactivation in the process of retraction. Furthermore, according to the results of thromboelastography, in the presence of NETs clots were less stiff and, consequently, more susceptible to compressive deformation. Conclusion. PMA-activated neutrophils within blood clots form NETs that incorporate into a fibrin network and enhance clot retraction via two independent mechanisms: the generation of endogenous thrombin causing platelet hyperactivation, and an increase in clot susceptibility to mechanical compression. Understanding these mechanisms may facilitate the development of novel treatments for immunothrombosis based on modulation of NETs formation, as well as on their biological and mechanical properties that influence the retraction of inflammatory blood clots and thrombi.