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. Approach and 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.

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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 K d 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. Gould et al DNA Impairs Fibrinolysis in Sepsis 2545patients increase thrombin generation by activating the intrinsic pathway of blood coagulation, 26 whereas DNA-histone complexes trigger platelet activation and aggregation by signaling through toll-like receptor-2 and toll-like receptor-4. [26][27][28] In addition, recent evidence suggests that fibrin, along with von Willebrand factor and chromatin, form a colocalized network within the thrombus that provides a scaffold for localized coagulation activation coupled with platelet and red blood cell adhesion, thereby promoting thrombus formation. 16,20,29 Although the contributions of CFDNA to coagulation activation and thrombus formation have been well characterized, studies on the influence of CFDNA on the fibrinolytic system are limited. NETs have previously been shown to intercalate with fibrin to form a structural network that is resistant to lysis by tissue-type plasminogen activator (tPA) or degradation by DNase. 20 Conversely, CFDNA has been shown to facilitate the recruitment of profibrinolytic enzymes, such as tPA, urokinase plasminogen activator, plasminogen, and plasmin, and their endogenous inhibitors, plasminogen activator inhibitor-1 (PAI-1) and α2-antiplasmin.30 Thus, although CFDNA may...

SummaryThere is mounting evidence that zinc, the second most abundant transition metal in blood, is an important mediator of haemostasis and thrombosis. Prompted by the observation that zinc deficiency is associated with bleeding and clotting abnormalities, there now is evidence that zinc serves as an effector of coagulation, anticoagulation and fibrinolysis. Zinc binds numerous plasma proteins and modulates their structure and function. Because activated platelets secrete zinc into the local microenvironment, the concentration of zinc increases in the vicinity of a thrombus. Consequently, the role of zinc varies depending on the microenvironment; a feature that endows zinc with the capacity to spatially and temporally regulate haemostasis and thrombosis. This paper reviews the mechanisms by which zinc regulates coagulation, platelet aggregation, anticoagulation and fibrinolysis and outlines how zinc serves as a ubiquitous modulator of haemostasis and thrombosis.

Histidine-rich glycoprotein (HRG) circulates in plasma at a concentration of 2M and binds plasminogen, fibrinogen, and thrombospondin. Despite these interactions, the physiologic role of HRG is unknown. Previous studies have shown that mice and humans deficient in HRG have shortened plasma clotting times. To better understand this phenomenon, we examined the effect of HRG on clotting tests. HRG prolongs the activated partial thromboplastin time in a concentrationdependent fashion but has no effect on tissue factor-induced clotting, localizing its effect to the contact pathway. Plasma immunodepleted of HRG exhibits a shortened activated partial thromboplastin time that is restored to baseline with HRG replenishment. To explore how HRG affects the contact pathway, we examined its binding to factors XII, XIIa, XI, and XIa. HRG binds factor XIIa with high affinity, an interaction that is enhanced in the presence of Zn 2؉ , but does not bind factors XII, XI, or XIa. In addition, HRG inhibits autoactivation of factor XII and factor XIIa-mediated activation of factor XI. These results suggest that, by binding to factor XIIa, HRG modulates the intrinsic pathway of coagulation, particularly in the vicinity of a thrombus where platelet release of HRG and Zn 2؉ will promote this interaction.(Blood. 2011;117(15): 4134-4141) IntroductionDespite the capacity of the intrinsic pathway to enhance thrombin generation, patients deficient in factor XII (FXII) do not bleed. 1 Even patients with FXI deficiency rarely have hemorrhagic complications, except with surgery or major trauma. 2 Because of these observations, it is well accepted that the contact system plays little part in hemostasis; and, by extension, it was also thought to be unimportant for thrombosis. However, a number of recent studies challenge this thinking. First, mice with deficiencies of highmolecular-weight kininogen (HK), bradykinin B2 receptor, FXII, or FXI are protected against injury-induced thrombosis, and an antibody against FXI inhibits thrombus formation in a baboon arteriovenous shunt model. [3][4][5][6][7] These observations raise the possibility that the contact pathway contributes to thrombogenesis. 8 Second, potential physiologic activators of FXII have been identified, which could initiate the contact system at sites of vascular injury. In addition to glycosaminoglycans and collagen, novel activators include polyphosphates and RNA. 9,10 Polyphosphates, which are released from the dense granules of platelets activated at sites of injury, trigger coagulation in a FXII-dependent fashion. Likewise, RNA released from the damaged vessel wall also can activate FXII, and RNAase administration to animals attenuates thrombosis at sites of injury, observations that have sparked a renewed interest in the contact pathway. 10 Consequently, it is important to better understand this pathway and how it is regulated.Histidine-rich glycoprotein (HRG) is an abundant plasma protein whose role is largely unknown. 11 HRG circulates at a concentration of approximately 2M. I...

Genome-scale metabolic models have proven useful for answering fundamental questions about metabolic capabilities of a variety of microorganisms, as well as informing their metabolic engineering. However, only a few models are available for oxygenic photosynthetic microorganisms, particularly in cyanobacteria in which photosynthetic and respiratory electron transport chains (ETC) share components. We addressed the complexity of cyanobacterial ETC by developing a genome-scale model for the diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142. The resulting metabolic reconstruction, iCce806, consists of 806 genes associated with 667 metabolic reactions and includes a detailed representation of the ETC and a biomass equation based on experimental measurements. Both computational and experimental approaches were used to investigate light-driven metabolism in Cyanothece sp. ATCC 51142, with a particular focus on reductant production and partitioning within the ETC. The simulation results suggest that growth and metabolic flux distributions are substantially impacted by the relative amounts of light going into the individual photosystems. When growth is limited by the flux through photosystem I, terminal respiratory oxidases are predicted to be an important mechanism for removing excess reductant. Similarly, under photosystem II flux limitation, excess electron carriers must be removed via cyclic electron transport. Furthermore, in silico calculations were in good quantitative agreement with the measured growth rates whereas predictions of reaction usage were qualitatively consistent with protein and mRNA expression data, which we used to further improve the resolution of intracellular flux values.

Key Points Mice deficient in HRG have normal hemostasis, but demonstrate accelerated thrombosis via the contact system. HRG abrogates nucleic acid–driven coagulation and serves as a novel modulator of the contact system in vivo.

Cyanobacteria are ideal metabolic engineering platforms for carbon-neutral biotechnology because they directly convert CO2 to a range of valuable products. In this study, we present a computational assessment of biochemical production in Synechococcus sp. PCC 7002 (Synechococcus 7002), a fast growing cyanobacterium whose genome has been sequenced, and for which genetic modification methods have been developed. We evaluated the maximum theoretical yields (mol product per mol CO2 or mol photon) of producing various chemicals under photoautotrophic and dark conditions using a genome-scale metabolic model of Synechococcus 7002. We found that the yields were lower under dark conditions, compared to photoautotrophic conditions, due to the limited amount of energy and reductant generated from glycogen. We also examined the effects of photon and CO2 limitations on chemical production under photoautotrophic conditions. In addition, using various computational methods such as minimization of metabolic adjustment (MOMA), relative metabolic change (RELATCH), and OptORF, we identified gene-knockout mutants that are predicted to improve chemical production under photoautotrophic and/or dark anoxic conditions. These computational results are useful for metabolic engineering of cyanobacteria to synthesize value-added products.