Femke Stephan

Circulating Nucleosomes and Neutrophil Activation as Risk Factors for Deep Vein Thrombosis

Objective—The formation of neutrophil extracellular traps and the exposure of nucleosomes on these neutrophil extracellular traps contribute to coagulation activation and the propagation of deep vein thrombosis (DVT) in animal models. However, no data are available on the role of neutrophil extracellular traps or nucleosomes in patients with thrombosis. Methods and Results—We conducted a case–control study, in which levels of circulating nucleosomes and neutrophil elastase–&agr;1-antitrypsin complexes were assessed in plasma from 150 patients with objectified symptomatic DVT (cases) and compared with 195 patients with a clinical suspicion of DVT but in whom DVT was excluded (controls). We explored the association between both nucleosomes and elastase–&agr;1-antitrypsin complexes, and the presence of DVT by calculating the odds ratio with corresponding 95% CIs. Elevated levels of both circulating nucleosomes and elastase–&agr;1-antitrypsin complexes were associated with a 3-fold risk of DVT, and the associations remained similar after adjustment for potential confounders (malignancy, smoking, recent immobilization, recent hospitalization). The risk increased with higher nucleosome and elastase–&agr;1-antitrypsin complex levels, suggesting a dose-dependent relationship among circulating nucleosomes, activated neutrophils, and DVT. Conclusion—Our study suggests an association among circulating nucleosomes, activated neutrophils, and presence of DVT in humans, which might have implications for treatment and prevention.

Factor VII-Activating Protease Is Activated in Multiple Trauma Patients and Generates Anaphylatoxin C5a

Severe tissue injury results in early activation of serine protease systems including the coagulation and complement cascade. In this context, little is known about factor VII-activating protease (FSAP), which is activated by substances released from damaged cells such as histones and nucleosomes. Therefore, we have measured FSAP activation in trauma patients and have identified novel FSAP substrates in human plasma. Mass spectrometry-based methods were used to identify FSAP binding proteins in plasma. Anaphylatoxin generation was measured by ELISA, Western blotting, protein sequencing, and chemotaxis assays. Plasma samples from trauma patients were analyzed for FSAP Ag and activity, nucleosomes, C5a, and C3a. Among others, we found complement components C3 and C5 in FSAP coimmunoprecipitates. C3 and C5 were cleaved by FSAP in a dose- and time-dependent manner generating functional C3a and C5a anaphylatoxins. Activation of endogenous FSAP in plasma led to increased C5a generation, but this was not the case in plasma of a homozygous carrier of Marburg I single nucleotide polymorphism with lower FSAP activity. In multiple trauma patients there was a large increase in circulating FSAP activity and nucleosomes immediately after the injury. A high correlation between FSAP activity and C5a was found. These data suggest that activation of FSAP by tissue injury triggers anaphylatoxin generation and thereby modulates the posttraumatic inflammatory response in vivo. A strong link between C5a, nucleosomes, and FSAP activity indicates that this new principle might be important in the regulation of inflammation.

Activation of factor VII-activating protease in human inflammation: a sensor for cell death

IntroductionCell death is a central event in the pathogenesis of sepsis and is reflected by circulating nucleosomes. Circulating nucleosomes were suggested to play an important role in inflammation and were demonstrated to correlate with severity and outcome in sepsis patients. We recently showed that plasma can release nucleosomes from late apoptotic cells. Factor VII-activating protease (FSAP) was identified to be the plasma serine protease responsible for nucleosome release. The aim of this study was to investigate FSAP activation in patients suffering from various inflammatory diseases of increasing severity.MethodsWe developed ELISAs to measure FSAP-C1-inhibitor and FSAP-α2-antiplasmin complexes in plasma. FSAP-inhibitor complexes were measured in the plasma of 20 adult patients undergoing transhiatal esophagectomy, 32 adult patients suffering from severe sepsis and 8 from septic shock and 38 children suffering from meningococcal sepsis.ResultsWe demonstrate plasma FSAP to be activated upon contact with apoptotic and necrotic cells by an assay detecting complexes between FSAP and its target serpins α2-antiplasmin and C1-inhibitor, respectively. By means of that assay we demonstrate FSAP activation in post-surgery patients, patients suffering from severe sepsis, septic shock and meningococcal sepsis. Levels of FSAP-inhibitor complexes correlate with nucleosome levels and correlate with severity and mortality in these patients.ConclusionsThese results suggest FSAP activation to be a sensor for cell death in the circulation and that FSAP activation in sepsis might be involved in nucleosome release, thereby contributing to lethality.

Nucleosome-releasing factor: a new role for factor VII-activating protease (FSAP)

Plasma proteins such as early comple ment components and IgM are involved in the removal of late apoptotic or secondary necrotic (sn) cells. We have recently described how a plasma protease that could be inhibited by the protease inhibitor aprotinin was essential to remove nucleosomes from sn cells. An obvious candidate, plasmin, did indeed have nucleo some‐releasing factor (NRF) activity. However, recalci fied plasma (r‐plasma) retained its NRF activity after plasminogen depletion, which suggests the existence of another protease responsible for NRF activity in plasma. In this study we have used size‐exclusion and anion‐exchange chromatography to purify the protease responsible for NRF activity in plasma. SDS‐PAGE analysis of chromatography fractions containing NRF activity revealed a protein band corresponding with NRF activity. Sequence analysis showed this band to be factor VII‐activating protease (FSAP). We developed monoclonal antibodies to FSAP and were able to com pletely inhibit NRF activity in plasma with monoclonal antibodies to FSAP. Using affinity chromatography we were able to purify single‐chain (sc) FSAP from r‐ plasma. Purified scFSAP efficiently removes nucleo‐ somes from sn cells. We report that factor VII‐activat‐ ing protease may function in cellular homeostasis by catalyzing the release of nucleosomes from secondary necrotic cells.— Zeerleder, S., Zwart, B., te Velthuis, H., Stephan, F., Manoe, R., Rensink, I., Aarden, L. A. Nucleosome‐releasing factor: a new role for factor VII‐activating protease (FSAP). FASEB J. 22, 4077–4084 (2008)

Circulating nucleosomes and severity of illness in children suffering from meningococcal sepsis treated with protein C

Objective:Cell death leading to circulating nucleosomes and histones is a critical step in the pathogenesis of sepsis and contributes to lethality. Activated protein C was demonstrated to attenuate the harmful effects of histones. The objective of this retrospective study was to evaluate whether nucleosomes correlate with the severity of the inflammatory response and mortality in children suffering from severe meningococcal sepsis. Furthermore, we wanted to study the effects of infusion of protein C on nucleosome levels in children with septic purpura. Design:Retrospective analysis of nucleosome levels in children suffering from meningococcal sepsis treated with either placebo or protein C. Setting:Pediatric intensive care unit of a tertiary care university center. Patients:In a randomized, placebo-controlled study, either protein C or placebo was administered to 38 children suffering from meningococcal sepsis. Nucleosome levels have been measured retrospectively in these 38 children suffering from meningococcal sepsis. Measurements and Main Results:Twenty-eight children were treated with protein C and 10 received placebo. Nucleosome levels were significantly higher in nonsurvivors (n = 9) at any time point measured as compared to survivors (n = 29). Nucleosome levels significantly correlated with organ dysfunction scores, cytokines, and parameters for coagulation. Patients treated with protein C had significantly higher activated protein C levels than children receiving placebo. We could not find a clear effect of activated protein C on nucleosome levels in these patients. Conclusion:Circulating nucleosomes correlated with the severity of the inflammatory response and were associated with mortality in children suffering from meningococcal sepsis. We show that protein C administration does not decrease nucleosome levels in these patients.

C1-esterase inhibitor concentrate rescues erythrocytes from complement-mediated destruction in autoimmune hemolytic anemia

To the editor: In autoimmune hemolytic anemia (AIHA), autoantibody-mediated complement activation results in C3 deposition on RBCs and possibly the formation of the membrane attack complex cumulating in intravascular hemolysis.[1][1][⇓][2][⇓][3]–[4][4] In case of acute signs of tissue hypoxia

Cooperation of factor VII-activating protease and serum DNase I in the release of nucleosomes from necrotic cells.

Removal of dead cells is essential in the maintenance of tissue homeostasis, and efficient removal prevents exposure of intracellular content to the immune system, which could lead to autoimmunity. The plasma protease factor VII–activating protease (FSAP) can release nucleosomes from late apoptotic cells. FSAP circulates as an inactive single‐chain protein, which is activated upon contact with either apoptotic cells or necrotic cells. The purpose of this study was to investigate the role of FSAP in the release of nucleosomes from necrotic cells.

Tissue factor pathway inhibitor is an inhibitor of factor VII-activating protease

Background: Factor VII‐activating protease (FSAP) is a serine protease that circulates in plasma in its inactive single‐chain form and can be activated upon contact with dead cells. When activated by apoptotic cells, FSAP leads to the release of nucleosomes. The serpins C1‐inhibitor and α2‐antiplasmin are reported to be the major inhibitors of FSAP. However, regulation of FSAP activity by Kunitz‐type inhibitors is not well studied.

Neutrophil extracellular traps in the host defense against sepsis induced by Burkholderia pseudomallei (melioidosis)

BackgroundNeutrophil extracellular traps (NETs) are a central player in the host response to bacteria: neutrophils release extracellular DNA (nucleosomes) and neutrophil elastase to entrap and kill bacteria. We studied the role of NETs in Burkholderia pseudomallei infection (melioidosis), an important cause of Gram-negative sepsis in Southeast Asia.MethodsIn a prospective observational study, circulating nucleosomes and neutrophil elastase were assayed in 44 patients with Gram-negative sepsis caused by B. pseudomallei (melioidosis) and 82 controls. Functional assays included human neutrophil stimulation and killing assays and a murine model of B. pseudomallei infection in which NET function was compromised using DNase. Specified pathogen-free 8- to 12-week-old C57BL/6 mice were sacrificed post-infection to assess bacterial loads, inflammation, and pathology.ResultsNucleosome and neutrophil elastase levels were markedly elevated in patients compared to controls. NETs killed B. pseudomallei effectively, and neutrophils stimulated with B. pseudomallei showed increased elastase and DNA release in a time- and dose-dependent matter. In mice, NET disruption with intravenous DNase administration resulted in decreased nucleosome levels. Although DNase treatment of mice resulted in diminished liver inflammation, no differences were observed in bacterial dissemination or systemic inflammation.ConclusionB. pseudomallei is a potent inducer of NETosis which was reflected by greatly increased levels of NET-related components in melioidosis patients. Although NETs exhibited antibacterial activity against B. pseudomallei, NET formation did not protect against bacterial dissemination and inflammation during B. pseudomallei-induced sepsis.

Increased Nucleosomes and Neutrophil Activation Link to Disease Progression in Patients with Scrub Typhus but Not Murine Typhus in Laos.

Cell-mediated immunity is essential in protection against rickettsial illnesses, but the role of neutrophils in these intracellular vasculotropic infections remains unclear. This study analyzed the plasma levels of nucleosomes, FSAP-activation (nucleosome-releasing factor), and neutrophil activation, as evidenced by neutrophil-elastase (ELA) complexes, in sympatric Lao patients with scrub typhus and murine typhus. In acute scrub typhus elevated nucleosome levels correlated with lower GCS scores, raised respiratory rate, jaundice and impaired liver function, whereas neutrophil activation correlated with fibrinolysis and high IL-8 plasma levels, a recently identified predictor of severe disease and mortality. Nucleosome and ELA complex levels were associated with a 4.8-fold and 4-fold increased risk of developing severe scrub typhus, beyond cut off values of 1,040 U/ml for nucleosomes and 275 U/ml for ELA complexes respectively. In murine typhus, nucleosome levels associated with pro-inflammatory cytokines and the duration of illness, while ELA complexes correlated strongly with inflammation markers, jaundice and increased respiratory rates. This study found strong correlations between circulating nucleosomes and neutrophil activation in patients with scrub typhus, but not murine typhus, providing indirect evidence that nucleosomes could originate from neutrophil extracellular trap (NET) degradation. High circulating plasma nucleosomes and ELA complexes represent independent risk factors for developing severe complications in scrub typhus. As nucleosomes and histones exposed on NETs are highly cytotoxic to endothelial cells and are strongly pro-coagulant, neutrophil-derived nucleosomes could contribute to vascular damage, the pro-coagulant state and exacerbation of disease in scrub typhus, thus indicating a detrimental role of neutrophil activation. The data suggest that increased neutrophil activation relates to disease progression and severe complications, and increased plasma levels of nucleosomes and ELA complexes represent independent risk factors for developing severe scrub typhus.