Travis J Gould1, Trang T Vu1, Alan R Stafford1, Dhruva J Dwivedi1, Paul Y Kim1, Alison E Fox-Robichaud1, Jeffrey I Weitz1, Patricia C Liaw2. 1. From the Department of Medical Sciences (T.J.G., T.T.V.), Thrombosis and Atherosclerosis Research Institute (T.J.G., T.T.V., A.R.S., D.J.D., P.Y.K., A.E.F.-R., J.I.W., P.C.L.), and Department of Medicine (A.R.S., P.Y.K., A.E.F.-R., J.I.W., P.C.L.), McMaster University, Hamilton, Ontario, Canada. 2. From the Department of Medical Sciences (T.J.G., T.T.V.), Thrombosis and Atherosclerosis Research Institute (T.J.G., T.T.V., A.R.S., D.J.D., P.Y.K., A.E.F.-R., J.I.W., P.C.L.), and Department of Medicine (A.R.S., P.Y.K., A.E.F.-R., J.I.W., P.C.L.), McMaster University, Hamilton, Ontario, Canada. patricia.liaw@taari.ca.
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 Kd 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.
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 Kd 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.
Authors: Hunter B Moore; Ernest E Moore; Ioannis N Liras; Eduardo Gonzalez; John A Harvin; John B Holcomb; Angela Sauaia; Bryan A Cotton Journal: J Am Coll Surg Date: 2016-01-22 Impact factor: 6.113
Authors: Emma G Bouck; Frederik Denorme; Alisa S Wolberg; Robert A Campbell; Lori A Holle; Elizabeth A Middelton; Antoinette M Blair; Bas de Laat; Joshua D Schiffman; Christian Con Yost; Matthew T Rondina Journal: Arterioscler Thromb Vasc Biol Date: 2020-11-16 Impact factor: 8.311