title: Neutrophil extracellular traps promote thrombin generation through platelet-dependent and platelet-independent mechanisms.
abstract: OBJECTIVE
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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.
authors.forename: Travis J
authors.initials: TJ
authors.lastname: Gould
authors.affiliation: 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.
authors.forename: Trang T
authors.initials: TT
authors.lastname: Vu
authors.affiliation: 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.
authors.forename: Laura L
authors.initials: LL
authors.lastname: Swystun
authors.affiliation: 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.
authors.forename: Dhruva J
authors.initials: DJ
authors.lastname: Dwivedi
authors.affiliation: 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.
authors.forename: Safiah H C
authors.initials: SH
authors.lastname: Mai
authors.affiliation: 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.
authors.forename: Jeffrey I
authors.initials: JI
authors.lastname: Weitz
authors.affiliation: 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. patricia.liaw@taari.ca.
authors.forename: Patricia C
authors.initials: PC
authors.lastname: Liaw
mesh_terms: D001777:Blood Coagulation / Q000502:physiology*; D001792:Blood Platelets / Q000502:physiology*; D002474:Cell-Free System; D004247:DNA / Q000097:blood / Q000494:pharmacology; D005109:Extracellular Matrix / Q000502:physiology*; D006657:Histones / Q000097:blood / Q000494:pharmacology; D006801:Humans; D018375:Neutrophil Activation / Q000187:drug effects / Q000502:physiology*; D053657:Platelet-Rich Plasma; D018805:Sepsis / Q000097:blood*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000096:biosynthesis* / Q000235:genetics; D051195:Toll-Like Receptor 2 / Q000502:physiology; D051197:Toll-Like Receptor 4 / Q000502:physiology
chemical_list: D006657:Histones; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D004247:DNA; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate
keywords: blood platelets; neutrophils; sepsis