Abstract 569: Neutrophil Extracellular Traps Promote Thrombin Generation Through Platelet-Dependent and -Independent Mechanisms

Abstract

Objective: Activation of neutrophils by microbial or inflammatory stimuli results in the release of neutrophil extracellular traps (NETs) which are comprised 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 septic patients. Approach and Results: NETs released from phorbyl myristate (PMA) activated neutrophils enhance thrombin generation in platelet-poor plasma (PPP). This effect was DNA-dependent (confirmed by DNase treatment) and occurred via the intrinsic pathway of coagulation (confirmed with FXII- and FXI-depleted plasma). In platelet-rich plasma treated with corn-trypsin inhibitor, addition of PMA-activated neutrophils increased thrombin generation and shortened the lag time in a TLR-2- and TLR-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 PPP plasma samples from septic patients, we found a positive correlation between endogenous CFDNA and thrombin generation, and addition of DNase attenuated thrombin generation. Conclusions: These studies are the first to 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.