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.
Arterioscler Thromb Vasc Biol. 2015 Dec;35(12):2544-53. doi: 10.1161/ATVBAHA.115.306035. Epub 2015 Oct 22.
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.
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.
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.
脓毒症的特征是感染导致全身炎症和凝血激活。在脓毒症中，活化的中性粒细胞挤出由无细胞 DNA（cfDNA）组成的中性粒细胞细胞外陷阱，这些陷阱不仅可以捕获病原体，还可以为血栓形成提供刺激。尽管 cfDNA 对凝血的影响已被广泛研究，但对 cfDNA 对纤溶的影响知之甚少。为了解决这个问题，我们（1）研究了脓毒症中 cfDNA 水平与纤溶活性之间的关系，（2）确定了 cfDNA 调节纤溶的机制。
从健康和脓毒症个体中采集血浆，并定量 cfDNA。在血浆和纯化系统中进行了血块溶解测定，并通过监测吸光度来确定溶解时间。使用扫描电子显微镜评估了血块形态。在含有>5μg/ml cfDNA 的脓毒症患者血浆中形成的血块结构致密，不易溶解，脱氧核糖核酸酶的添加可克服这一现象。在补充 cfDNA 的对照血浆中也重现了这些效应。在纯化系统中，cfDNA 延迟纤溶，但不改变组织型纤溶酶原激活物诱导的纤溶酶生成。使用表面等离子体共振，cfDNA 与纤溶酶的结合 Kd 值为 4.2±0.3μmol/L，并且随着 cfDNA 浓度的增加，cfDNA 通过形成纤溶酶、cfDNA 和纤维蛋白之间的非生产性三元复合物，损害纤溶酶介导的纤维蛋白血块降解。
我们的研究表明，脓毒症中 cfDNA 水平的升高通过抑制纤溶酶介导的纤维蛋白降解来损害纤溶，从而确定 cfDNA 是脓毒症治疗的潜在治疗靶点。
