Size‐ and charge‐dependent modulation of the lytic susceptibility and mechanical stability of fibrin‐histone clots by heparin and polyphosphate variants

Abstract

Background: Neutrophil extracellular traps (NETs) containing DNA and histones are expelled from neutrophils in infection and thrombosis. Heparins, anticoagulant polyanions, can neutralize histones with a potential therapeutic advantage in sepsis. Polyphosphates, procoagulant polyanions, are released by platelets and microorganisms.Objectives: To characterize the combined effects of NET components and polyanions on clot structure, mechanical properties and lytic susceptibility.Methods: Scanning electron microscopy, pressure-driven permeation, turbidimetry, and oscillation rheometry were used for the characterization of the structure, viscoelasticity, and kinetics of formation and lysis of fibrin and plasma clots containing histones+/-DNA in combination with unfractionated heparin, its desulfated derivatives, low molecular weight heparin (LMWH), pentasaccharide, and polyphosphates of different sizes.Results: Histones and DNA inhibited fibrin lysis by plasmin, but this behavior was not neutralized by negatively charged heparins or short polyphosphates. Rather, fibrin lysis was further inhibited by added polyanions. Histones inhibited plasma clot lysis by tissue plasminogen activator and the response to added heparin was size dependent. Unfractionated heparin, LMWH, and pentasaccharide had no effect, exacerbated, or reversed histone inhibition, respectively. Histones increased the mechanical strength of fibrin, which was exacerbated by smaller heparin and polyphosphate molecules.Histones increased fibrin diameter and pore size of fibrin clots and this effect was neutralized by all heparin variants but enhanced by polyphosphates. Conclusions:Despite their common polyanionic character, heparins and polyphosphates exert distinct effects on fibrin mechanical and fibrinolytic stability. Antifibrinolytic effects of histones were more often enhanced by polyanions not counteracted. Careful selection of anti-histone strategies is required if they are to be combined with thrombolytic therapy.This is an open access article under the terms of the Creat ive Commo ns Attri butio n-NonCo mmercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Polyphosphate nanoparticles enhance the fibrin stabilization by histones more efficiently than linear polyphosphates

“…As for PolyP, it was shown earlier by others, that the linear form incorporates into polymerizing fibrin clots [ 33 ], which may be the reason for its fiber thickening effect. We have recently shown that linear polyphosphates enhance the fiber-thickening effect of histones [ 5 ], and our current results provide evidence that in the form of nanoparticles they are even stronger structural modifiers.…”

“…However, if the cause of changes in fibrin architecture is incorporation of modifier molecules, the thicker fibers are more resistant to lysis. Because the structure of the major fibrin-degrading protease, plasmin is optimal for the inter-protofibril distances within modifier-free fibrin fibers (reviewed in [ 23 ]), looser packaging of the protofibrils in the fibers would decrease its catalytic efficiency, as we have previously described for the breakdown of fibrin containing histones [ 2 ] or combination of histone and linear polyphosphate [ 5 ]. This interpretation is supported by the current result that the nanoparticulate PolyP with the strongest steric effect on the fiber geometry enhances stronger the anti-fibrinolytic action of histones than the linear variants.…”

“…Our previous studies on the effects of NET components on structural and lytic stability of fibrin and plasma clots revealed that DNA, histones and their combinations increase the mechanical stability of clots and hamper tissue-type plasminogen activator (tPA) mediated clot lysis [ 2 , 3 ]. Furthermore, we found, that linear PolyPs stabilize fibrin via size-dependent modulation of its structure and kringle-dependent inhibition of plasmin-mediated fibrinolysis [ 4 ], while in combination with NET components, linear PolyPs enhanced the clot stabilizing effects of DNA and histones [ 5 ]. In addition to the linear form, nanoparticulate polyphosphates (PolyP-NPs) have also been shown to localize at the surface of activated platelets and activate the intrinsic pathway of coagulation [ 6 ], but in contrast to the linear PolyP effects, little is known about their contribution to the stabilization of thrombi by NETs.…”

The Emerging Role of Neutrophils in the Pathogenesis of Thrombosis in COVID-19

“…It is well-documented that neutrophils are key factors in the cytokine storm and thrombotic complications experienced by COVID-19 patients and that therapeutic targeting of neutrophils might ameliorate hyperinflammatory syndrome in COVID-19. Therefore, several studies proposed that NETs could be a therapeutic target in COVID-19 patients (Table 3) [90][91][92][93].…”

Neutrophils: As a Key Bridge between Inflammation and Thrombosis

“…During NET formation, histones are also released into the plasma in large quantities, which cross-link with fibrin and induce changes in fibrin conformation, accelerating the aggregation of fibrinogenic fibrils and causing them to form thicker fibrin to prevent their lysis by fibrinolytic enzymes [ 62 ]. It has also been shown that heparin and DNA, as anionic substances, can also inhibit fibrinolysis and increase the mechanical stability of fibrin [ 63 , 64 ]. Ultimately, activated neutrophils compromise the body's anticoagulant and fibrinolytic systems through this series of pathways, ultimately leading to the formation of immunothrombosis.…”