Trang T. Vu

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

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.

Cell-Free DNA Modulates Clot Structure and Impairs Fibrinolysis in Sepsis

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 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. 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.

Histidine-rich glycoprotein binds factor XIIa with high affinity and inhibits contact-initiated coagulation

Histidine-rich glycoprotein (HRG) circulates in plasma at a concentration of 2μM 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 concentration-dependent 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 Zn²(+), 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 Zn²(+) will promote this interaction.

Zinc: An important cofactor in haemostasis and thrombosis

There 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 Binds Fibrin(ogen) with High Affinity and Competes with Thrombin for Binding to the γ′-Chain

Histidine-rich glycoprotein (HRG) is an abundant protein that binds fibrinogen and other plasma proteins in a Zn2+-dependent fashion but whose function is unclear. HRG has antimicrobial activity, and its incorporation into fibrin clots facilitates bacterial entrapment and killing and promotes inflammation. Although these findings suggest that HRG contributes to innate immunity and inflammation, little is known about the HRG-fibrin(ogen) interaction. By immunoassay, HRG-fibrinogen complexes were detected in Zn2+-supplemented human plasma, a finding consistent with a high affinity interaction. Surface plasmon resonance determinations support this concept and show that in the presence of Zn2+, HRG binds the predominant γA/γA-fibrinogen and the γ-chain elongated isoform, γA/γ′-fibrinogen, with Kd values of 9 nm. Likewise, 125I-labeled HRG binds γA/γA- or γA/γ′-fibrin clots with similar Kd values when Zn2+ is present. There are multiple HRG binding sites on fibrin(ogen) because HRG binds immobilized fibrinogen fragment D or E and γ′-peptide, an analog of the COOH terminus of the γ′-chain that mediates the high affinity interaction of thrombin with γA/γ′-fibrin. Thrombin competes with HRG for γ′-peptide binding and displaces 125I-HRG from γA/γ′-fibrin clots and vice versa. Taken together, these data suggest that (a) HRG circulates in complex with fibrinogen and that the complex persists upon fibrin formation, and (b) by competing with thrombin for γA/γ′-fibrin binding, HRG may modulate coagulation. Therefore, the HRG-fibrin interaction may provide a novel link between coagulation, innate immunity, and inflammation.

Batroxobin Binds Fibrin with Higher Affinity and Promotes Clot Expansion to a Greater Extent than Thrombin

Background: Snake venom protease batroxobin clots fibrinogen in a manner distinct from thrombin. Results: Batroxobin binds fibrin(ogen) with higher affinity than thrombin and promotes greater clot expansion. Conclusion: Batroxobins distinctive interaction with fibrin(ogen) may contribute to its unique pattern of fibrinopeptide release. Significance: Clinically, batroxobin is used as a defibrinogenating agent, but its capacity to promote clot expansion may promote microvascular thrombosis. Batroxobin is a thrombin-like serine protease from the venom of Bothrops atrox moojeni that clots fibrinogen. In contrast to thrombin, which releases fibrinopeptide A and B from the NH2-terminal domains of the Aα- and Bβ-chains of fibrinogen, respectively, batroxobin only releases fibrinopeptide A. Because the mechanism responsible for these differences is unknown, we compared the interactions of batroxobin and thrombin with the predominant γA/γA isoform of fibrin(ogen) and the γA/γ′ variant with an extended γ-chain. Thrombin binds to the γ′-chain and forms a higher affinity interaction with γA/γ′-fibrin(ogen) than γA/γA-fibrin(ogen). In contrast, batroxobin binds both fibrin(ogen) isoforms with similar high affinity (Kd values of about 0.5 μm) even though it does not interact with the γ′-chain. The batroxobin-binding sites on fibrin(ogen) only partially overlap with those of thrombin because thrombin attenuates, but does not abrogate, the interaction of γA/γA-fibrinogen with batroxobin. Furthermore, although both thrombin and batroxobin bind to the central E-region of fibrinogen with a Kd value of 2–5 μm, the α(17–51) and Bβ(1–42) regions bind thrombin but not batroxobin. Once bound to fibrin, the capacity of batroxobin to promote fibrin accretion is 18-fold greater than that of thrombin, a finding that may explain the microvascular thrombosis that complicates envenomation by B. atrox moojeni. Therefore, batroxobin binds fibrin(ogen) in a manner distinct from thrombin, which may contribute to its higher affinity interaction, selective fibrinopeptide A release, and prothrombotic properties.

Histidine-rich glycoprotein binds DNA and RNA and attenuates their capacity to activate the intrinsic coagulation pathway

When triggered by factor (F) XII and nucleic acids, we showed that thrombosis in HRG-deficient mice is accelerated compared with that in wild-type mice. In this study, we set out to identify the mechanisms by which nucleic acids promote contact activation, and to determine whether HRG attenuates their effects. DNA or RNA addition to human plasma enhances thrombin generation via the intrinsic pathway and shortens the clotting time. Their effect on the clotting time is seven- to 14-fold greater in HRG-deficient plasma than in control plasma. Investigations into the mechanisms of activation reveal that nucleic acids a) promote FXII activation in the presence of prekallikrein- and high molecular weight kininogen (HK), and b) enhance thrombin-mediated FXI activation by 10- to 12-fold. Surface plasmon resonance studies show that DNA and RNA bind FXII, FXIIa, HK, FXI, FXIa and thrombin with high affinity. HRG attenuates DNA- and RNA-mediated FXII activation, and FXI activation by FXIIa or by thrombin, suggesting that HRG down regulates the capacity of DNA and RNA to activate the intrinsic pathway. Therefore, HRG attenuates the procoagulant activity of nucleic acids at multiple levels.

Arterial thrombosis is accelerated in mice deficient in histidine-rich glycoprotein

Factor (F) XII, a key component of the contact system, triggers clotting via the intrinsic pathway, and is implicated in propagating thrombosis. Although nucleic acids are potent activators, it is unclear how the contact system is regulated to prevent uncontrolled clotting. Previously, we showed that histidine-rich glycoprotein (HRG) binds FXIIa and attenuates its capacity to trigger coagulation. To investigate the role of HRG as a regulator of the intrinsic pathway, we compared RNA- and DNA-induced thrombin generation in plasma from HRG-deficient and wild-type mice. Thrombin generation was enhanced in plasma from HRG-deficient mice, and accelerated clotting was restored to normal with HRG reconstitution. Although blood loss after tail tip amputation was similar in HRG-deficient and wild-type mice, carotid artery occlusion after FeCl3 injury was accelerated in HRG-deficient mice, and HRG administration abrogated this effect. To confirm that HRG modulates the contact system, we used DNase, RNase, and antisense oligonucleotides to characterize the FeCl3 model. Whereas DNase or FVII knockdown had no effect, carotid occlusion was abrogated with RNase or FXII knockdown, confirming that FeCl3-induced thrombosis is triggered by RNA in a FXII-dependent fashion. Therefore, in a nucleic acid-driven model, HRG inhibits thrombosis by modulating the intrinsic pathway of coagulation.

Reduced Plasminogen Binding and Delayed Activation Render γ′-Fibrin More Resistant to Lysis than γA-Fibrin

Background: When compared with degradation of the predominant γA-fibrin, lysis of variant γ′-fibrin is delayed. Results: Thrombin-mediated fibrinopeptide B release is slower from γ′-fibrinogen than from γA-fibrinogen, resulting in delayed binding and activation of plasminogen. Conclusion: Delayed plasmin generation renders γ′-fibrin resistant to lysis. Significance: The association between slower clotting and delayed lysis highlights the links between coagulation and fibrinolysis. Fibrin (Fn) clots formed from γ′-fibrinogen (γ′-Fg), a variant with an elongated γ-chain, are resistant to lysis when compared with clots formed from the predominant γA-Fg, a finding previously attributed to differences in clot structure due to delayed thrombin-mediated fibrinopeptide (FP) B release or impaired cross-linking by factor XIIIa. We investigated whether slower lysis of γ′-Fn reflects delayed plasminogen (Pg) binding and/or activation by tissue plasminogen activator (tPA), reduced plasmin-mediated proteolysis of γ′-Fn, and/or altered cross-linking. Clots formed from γ′-Fg lysed more slowly than those formed from γA-Fg when lysis was initiated with tPA/Pg when FPA and FPB were both released, but not when lysis was initiated with plasmin, or when only FPA was released. Pg bound to γ′-Fn with an association rate constant 22% lower than that to γA-Fn, and the lag time for initiation of Pg activation by tPA was longer with γ′-Fn than with γA-Fn. Once initiated, however, Pg activation kinetics were similar. Factor XIIIa had similar effects on clots formed from both Fg isoforms. Therefore, slower lysis of γ′-Fn clots reflects delayed FPB release, which results in delayed binding and activation of Pg. When clots were formed from Fg mixtures containing more than 20% γ′-Fg, the upper limit of the normal level, the delay in lysis was magnified. These data suggest that circulating levels of γ′-Fg modulate the susceptibility of clots to lysis by slowing Pg activation by tPA and provide another example of the intimate connections between coagulation and fibrinolysis.

Can thrombophilia predict recurrent catheter-related deep vein thrombosis in children?

The role of thrombophilia testing in predicting catheter-related deep vein thrombosis (DVT) after an incident (ie, first) catheter-related DVT in children remains unclear. The present study investigated the association between thrombophilia and recurrent catheter-related DVT. Children with thrombophilia testing, performed according to the clinicians judgment and the familys preference, and a history of objectively confirmed catheter-related DVT were included in the study. Recurrent catheter-related DVT after placement of a new catheter was the main outcome. Thrombophilia was classified as minor, major, or none. Analysis was conducted using mixed effect logistic regression. A total of 245 patients had 1,365 catheters inserted; 941 of these catheters were placed after the incident catheter-related DVT. Anticoagulants as treatment or prophylaxis were administered in 78.1% of inserted catheters for at least 50% of the time they were in place. Minor thrombophilia was found in 12.7% of patients, whereas major thrombophilia was seen in 8.2% of children. The incidence rate of recurrent events was 0.23/100 catheter-days (95% confidence interval, 0.19-0.28 catheter-days); 34.3% (95% confidence interval, 28.6%-40.0%) of patients requiring a new catheter after their incident thrombotic event had at least 1 recurrent event. The incidence proportion of bleeding complications was 4.6/100 patients receiving anticoagulation. Young age of the patient at the time of catheter insertion and lack of administration of treatment or prophylactic doses of anticoagulant were predictive of recurrent events. In contrast, thrombophilia was not predictive of recurrent catheter-related DVT during subsequent catheter insertions among tested patients. Our findings suggest that thrombophilia testing to predict recurrence in these patients may be unnecessary.