Cell-Free DNA Modulates Clot Structure and Impairs Fibrinolysis in Sepsis

Abstract

Objectives-Sepsis is characterized by systemic activation of inflammation and coagulation in response to infection. In sepsis, activated neutrophils extrude neutrophil extracellular traps composed of cell-free DNA (CFDNA) that not only trap pathogens but also provide a stimulus for clot formation. Although the effect of CFDNA on coagulation has been extensively studied, much less is known about the impact of CFDNA on fibrinolysis. To address this, we (1) investigated the relationship between CFDNA levels and fibrinolytic activity in sepsis and (2) determined the mechanisms by which CFDNA modulates fibrinolysis. Approach and Results-Plasma was collected from healthy and septic individuals, and CFDNA was quantified. Clot lysis assays were performed in plasma and purified systems, and lysis times were determined by monitoring absorbance. Clot morphology was assessed using scanning electron microscopy. Clots formed in plasma from septic patients containing >5 µg/mL CFDNA were dense in structure and resistant to fibrinolysis, a phenomenon overcome by deoxyribonuclease addition. These effects were recapitulated in control plasma supplemented with CFDNA. In a purified system, CFDNA delayed fibrinolysis but did not alter tissue-type plasminogen activator-induced plasmin generation. Using surface plasmon resonance, CFDNA bound plasmin with a K d value of 4.2±0.3 µmol/L, and increasing concentrations of CFDNA impaired plasmin-mediated degradation of fibrin clots via the formation of a nonproductive ternary complex between plasmin, CFDNA, and fibrin. Conclusions-Our studies suggest that the increased levels of CFDNA in sepsis impair fibrinolysis by inhibiting plasmin-mediated fibrin degradation, thereby identifying CFDNA as a potential therapeutic target for sepsis treatment. Gould et al DNA Impairs Fibrinolysis in Sepsis 2545patients increase thrombin generation by activating the intrinsic pathway of blood coagulation, 26 whereas DNA-histone complexes trigger platelet activation and aggregation by signaling through toll-like receptor-2 and toll-like receptor-4. [26][27][28] In addition, recent evidence suggests that fibrin, along with von Willebrand factor and chromatin, form a colocalized network within the thrombus that provides a scaffold for localized coagulation activation coupled with platelet and red blood cell adhesion, thereby promoting thrombus formation. 16,20,29 Although the contributions of CFDNA to coagulation activation and thrombus formation have been well characterized, studies on the influence of CFDNA on the fibrinolytic system are limited. NETs have previously been shown to intercalate with fibrin to form a structural network that is resistant to lysis by tissue-type plasminogen activator (tPA) or degradation by DNase. 20 Conversely, CFDNA has been shown to facilitate the recruitment of profibrinolytic enzymes, such as tPA, urokinase plasminogen activator, plasminogen, and plasmin, and their endogenous inhibitors, plasminogen activator inhibitor-1 (PAI-1) and α2-antiplasmin.30 Thus, although CFDNA may...

Acute Fibrinolysis Shutdown after Injury Occurs Frequently and Increases Mortality: A Multicenter Evaluation of 2,540 Severely Injured Patients

“…However, it remains to be identified what drives the hypecoaguability and fibrinolysis shutdown after trauma. Recent work in sepsis indicated that cell free DNA contributes to fibrinolysis resistance(27). These DNA fragments, which co-localize to histones, have been proposed to be drivers of microvascular thrombosis in septic shock(28).…”

Elevated levels of circulating cell-free DNA and neutrophil proteins are associated with neonatal sepsis and necrotizing enterocolitis in immature mice, pigs and infants

“…[20][21][22] Increased NET formation and accumulation may occur before, or close to the disease onset without proper clearance mechanisms, as shown for traumatized or septic adults. 39,40 NET formation in the intestine with released DNA and neutrophil-derived calprotectin has been shown to be associated with NEC, 26 but it was not clear if circulating NETs may play a role. In the present study, elevated circulating cfDNA levels 1-6 d before NEC onset in preterm infants suggest that sub-clinical systemic inflammation at an early stage of NEC may stimulate neutrophils to release DNA and antimicrobial proteins in the circulation, which may add further inflammatory insults that could advance NEC progression.…”

The role of leukocytes in thrombosis

“…147 NETosis may also influence fibrinolysis and thrombus stability, as the addition of CF-DNA and histones to clotting plasma results in the formation of thicker fibers with greater mechanical stability. 148 In patients with sepsis and elevated CF-DNA levels, clot lysis times are attenuated compared with controls 149 ; this effect can be replicated by the addition of histone-DNA complex 148 or in the presence of NETing neutrophils. 53 The presence of CF-DNA within the clot has been shown to impair plasminogen activation by tPA, 53 and the binding of plasmin to fibrin.…”