Extracellular Histones Inhibit Fibrinolysis through Noncovalent and Covalent Interactions with Fibrin

Abstract

Histones released into circulation as neutrophil extracellular traps are causally implicated in the pathogenesis of arterial, venous, and microvascular thrombosis by promoting coagulation and enhancing clot stability. Histones induce structural changes in fibrin rendering it stronger and resistant to fibrinolysis. The current study extends these observations by defining the antifibrinolytic mechanisms of histones in purified, plasma, and whole blood systems. Although histones stimulated plasminogen activation in solution, they inhibited plasmin as competitive substrates. Protection of fibrin from plasmin digestion is enhanced by covalent incorporation of histones into fibrin, catalyzed by activated transglutaminase, coagulation factor FXIII (FXIIIa). All histone subtypes (H1, H2A, H2B, H3, and H4) were crosslinked to fibrin. A distinct, noncovalent mechanism explains histone-accelerated lateral aggregation of fibrin protofibrils, resulting in thicker fibers with higher mass-to-length ratios and in turn hampered fibrinolysis. However, histones were less effective at delaying fibrinolysis in the absence of FXIIIa activity. Therapeutic doses of low-molecular-weight heparin (LMWH) prevented covalent but not noncovalent histone–fibrin interactions and neutralized the effects of histones on fibrinolysis. This suggests an additional antithrombotic mechanism for LMWH beyond anticoagulation. In conclusion, for the first time we report that histones are crosslinked to fibrin by FXIIIa and promote fibrinolytic resistance which can be overcome by FXIIIa inhibitors and histone-binding heparinoids. These findings provide a rationale for targeting the FXIII–histone–fibrin axis to destabilize fibrin and prevent potentially thrombotic fibrin networks.

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

“…In general, denser clots with smaller pores and more closely packed fibers are more resistant against fibrinolysis; however, several fibrin structure modifiers exert direct effects on the efficiency of the plasminogen/plasmin system, which could add up or perhaps override the anticipated effects based on the structural changes. Histones favor lateral aggregation of protofibrils and are covalently cross‐linked to fibrin by FXIIIa resulting in a more plasmin‐resistant clot structure; moreover, they compete for plasmin as alternative substrates 36 . DNA interacts directly with plasmin and fibrin forming an unproductive ternary plasmin‐DNA‐fibrin complex, whereas polyphosphates may shield positively charged plasminogen binding sites in fibrin leading to hampered plasminogen activation 8,20 …”

“…Histones favor lateral aggregation of protofibrils and are covalently cross-linked to fibrin by FXIIIa resulting in a more plasmin-resistant clot structure; moreover, they compete for plasmin as alternative substrates. 36 DNA interacts directly with plasmin and fibrin forming an unproductive ternary plasmin-DNA-fibrin complex, whereas polyphosphates may shield positively charged plasminogen binding sites in fibrin leading to hampered plasminogen activation. 8,20 Despite the size-, charge-, and chemical nature-dependent structural and biomechanical effects discussed above, most polyanions showed a rather uniform, but charge-dependent, prolongation of fibrinolysis with plasmin incorporated in fibrin-histone clots.…”

Circulating Histones in Sepsis: Potential Outcome Predictors and Therapeutic Targets

“…It appears that the controversial effects of histones on the fibrinolytic system are relevant for sepsis-induced DIC. Early studies have consistently revealed that histones could accelerate fibrin polymerization and enhance clot structure rendering it stronger and resistant to fibrinolysis, which could be neutralized by histone-binding low-molecular-weight heparin, or FXIIIa inhibitors (70)(71)(72). On one hand, noncovalent histone-fibrin interactions induce lateral aggregation of fibrin protofibrils and increase fibrin-fiber thickness, giving the fibrin network increased mechanical stability.…”

“…On one hand, noncovalent histone–fibrin interactions induce lateral aggregation of fibrin protofibrils and increase fibrin-fiber thickness, giving the fibrin network increased mechanical stability. On the other hand, histones also inhibit plasmin as competitive substrates to delay fibrinolysis, and protect fibrin from plasmin digestion through their covalent interactions with fibrin, catalyzed by activated coagulation factor FXIII (FXIIIa) ( 70 ). On the contrary, Fabrizio et al argued that histones strongly accelerated single-chain urokinase-type plasminogen activator (scu-PA)-driven clot lysis through the enhancement of scu-PA to urokinase-type plasminogen activator conversion, mediated by factor seven activating protease ( 73 ).…”

A Modified in vitro Clot Lysis Assay Predicts Outcomes in Non-traumatic Intracerebral Hemorrhage Stroke Patients—The IRONHEART Study

“…It must be emphasized, that our primary goal was to find assay conditions where fibrinolysis kinetics are optimally influenced by the addition of cfDNA and histones. The mechanisms behind the observed effect of cfDNA and histones resulting in prolongation of clot lysis are complex (14,37). Among others, the addition of cfDNA promotes the formation of densely packed networks of thick fibers less susceptible to plasmin digestion, while the addition of histones competitively inhibits plasmin and delays fibrinolysis.…”