From the Department of Medical Sciences (T.J.G., T.T.V., L.L.S., S.H.C.M.), Thrombosis and Atherosclerosis Research Institute (T.J.G., T.T.V., L.L.S., D.J.D., S.H.C.M., J.I.W., P.C.L.), and Department of Medicine (J.I.W., P.C.L.), McMaster University, Hamilton, Ontario, Canada.
Arterioscler Thromb Vasc Biol. 2014 Sep;34(9):1977-84. doi: 10.1161/ATVBAHA.114.304114. Epub 2014 Jul 10.
Activation of neutrophils by microbial or inflammatory stimuli results in the release of neutrophil extracellular traps (NETs) that are composed of DNA, histones, and antimicrobial proteins. In purified systems, cell-free DNA (CFDNA) activates the intrinsic pathway of coagulation, whereas histones promote thrombin generation through platelet-dependent mechanisms. However, the overall procoagulant effects of CFDNA/histone complexes as part of intact NETs are unknown. In this study, we examined the procoagulant potential of intact NETs released from activated neutrophils. We also determined the relative contribution of CFDNA and histones to thrombin generation in plasmas from patients with sepsis.
NETs released from phorbyl myristate-activated neutrophils enhance thrombin generation in platelet-poor plasma. This effect was DNA dependent (confirmed by DNase treatment) and occurred via the intrinsic pathway of coagulation (confirmed with coagulation factor XII- and coagulation factor XI-depleted plasma). In platelet-rich plasma treated with corn trypsin inhibitor, addition of phorbyl myristate-activated neutrophils increased thrombin generation and shortened the lag time in a toll-like receptor-2- and toll-like receptor-4-dependent mechanism. Addition of DNase further augmented thrombin generation, suggesting that dismantling of the NET scaffold increases histone-mediated, platelet-dependent thrombin generation. In platelet-poor plasma samples from patients with sepsis, we found a positive correlation between endogenous CFDNA and thrombin generation, and addition of DNase attenuated thrombin generation.
These studies examine the procoagulant activities of CFDNA and histones in the context of NETs. Our studies also implicate a role for the intrinsic pathway of coagulation in sepsis pathogenesis.
微生物或炎症刺激可激活中性粒细胞，导致中性粒细胞胞外诱捕网（NETs）的释放，NETs 由 DNA、组蛋白和抗微生物蛋白组成。在无细胞体系中，游离 DNA（cfDNA）激活凝血的内在途径，而组蛋白通过血小板依赖的机制促进凝血酶生成。然而，作为完整 NETs 的一部分，cfDNA/组蛋白复合物的总体促凝作用尚不清楚。在这项研究中，我们研究了激活中性粒细胞释放的完整 NETs 的促凝潜力。我们还确定了 cfDNA 和组蛋白在脓毒症患者血浆中生成凝血酶中的相对贡献。
佛波醇肉豆蔻酸激活的中性粒细胞释放的 NETs 增强了血小板缺乏血浆中的凝血酶生成。这种效应依赖于 DNA（经 DNA 酶处理证实），并通过凝血的内在途径发生（用凝血因子 XII 和 XI 耗尽的血浆证实）。在用玉米胰蛋白酶抑制剂处理的富含血小板的血浆中，添加佛波醇肉豆蔻酸激活的中性粒细胞可增加凝血酶生成，并通过 Toll 样受体 2 和 Toll 样受体 4 依赖性机制缩短延迟时间。添加 DNA 酶进一步增强了凝血酶生成，表明 NET 支架的解体增加了组蛋白介导的、血小板依赖的凝血酶生成。在脓毒症患者的血小板缺乏血浆样本中，我们发现内源性 cfDNA 与凝血酶生成呈正相关，添加 DNA 酶可减弱凝血酶生成。
这些研究在 NETs 的背景下研究了 cfDNA 和组蛋白的促凝活性。我们的研究还表明，凝血的内在途径在脓毒症发病机制中起作用。
