Impact of age on the host response to sepsis in a murine model of fecal-induced peritonitis.

INTRODUCTION: Despite older adults being more vulnerable to sepsis, most preclinical research on sepsis has been conducted using young animals. This results in decreased scientific validity since age is an independent predictor of poor outcome. In this study, we explored the impact of aging on the host response to sepsis using the fecal-induced peritonitis (FIP) model developed by the National Preclinical Sepsis Platform (NPSP). METHODS: C57BL/6 mice (3 or 12 months old) were injected intraperitoneally with rat fecal slurry (0.75 mg/g) or a control vehicle. To investigate the early stage of sepsis, mice were culled at 4 h, 8 h, or 12 h to investigate disease severity, immunothrombosis biomarkers, and organ injury. Mice received buprenorphine at 4 h post-FIP. A separate cohort of FIP mice were studied for 72 h (with buprenorphine given at 4 h, 12 h, and then every 12 h post-FIP and antibiotics/fluids starting at 12 h post-FIP). Organs were harvested, plasma levels of Interleukin (IL)-6, IL-10, monocyte chemoattract protein (MCP-1)/CCL2, thrombin-antithrombin (TAT) complexes, cell-free DNA (CFDNA), and ADAMTS13 activity were quantified, and bacterial loads were measured. RESULTS: In the 12 h time course study, aged FIP mice demonstrated increased inflammation and injury to the lungs compared to young FIP mice. In the 72 h study, aged FIP mice exhibited a higher mortality rate (89%) compared to young FIP mice (42%) (p < 0.001). Aged FIP non-survivors also exhibited a trend towards elevated IL-6, TAT, CFDNA, CCL2, and decreased IL-10, and impaired bacterial clearance compared to young FIP non-survivors. CONCLUSION: To our knowledge, this is the first study to investigate the impact of age on survival using the FIP model of sepsis. Our model includes clinically-relevant supportive therapies and inclusion of both sexes. The higher mortality rate in aged mice may reflect increased inflammation and worsened organ injury in the early stage of sepsis. We also observed trends in impaired bacterial clearance, increase in IL-6, TAT, CFDNA, CCL2, and decreased IL-10 and ADAMTS13 activity in aged septic non-survivors compared to young septic non-survivors. Our aging model may help to increase the scientific validity of preclinical research and may be useful for identifying mechanisms of age-related susceptibility to sepsis as well as age-specific treatment strategies.

Development and characterization of a fecal-induced peritonitis model of murine sepsis: results from a multi-laboratory study and iterative modification of experimental conditions.

BACKGROUND: Preclinical sepsis models have been criticized for their inability to recapitulate human sepsis and suffer from methodological shortcomings that limit external validity and reproducibility. The National Preclinical Sepsis Platform (NPSP) is a consortium of basic science researchers, veterinarians, and stakeholders in Canada undertaking standardized multi-laboratory sepsis research to increase the efficacy and efficiency of bench-to-bedside translation. In this study, we aimed to develop and characterize a 72-h fecal-induced peritonitis (FIP) model of murine sepsis conducted in two independent laboratories. The experimental protocol was optimized by sequentially modifying dose of fecal slurry and timing of antibiotics in an iterative fashion, and then repeating the experimental series at site 1 and site 2. RESULTS: Escalating doses of fecal slurry (0.5-2.5 mg/g) resulted in increased disease severity, as assessed by the modified Murine Sepsis Score (MSS). However, the MSS was poorly associated with progression to death during the experiments, and mice were found dead without elevated MSS scores. Administration of early antibiotics within 4 h of inoculation rescued the animals from sepsis compared with late administration of antibiotics after 12 h, as evidenced by 100% survival and reduced bacterial load in peritoneum and blood in the early antibiotic group. Site 1 and site 2 had statistically significant differences in mortality (60% vs 88%; p < 0.05) for the same dose of fecal slurry (0.75 mg/g) and marked differences in body temperature between groups. CONCLUSIONS: We demonstrate a systematic approach to optimizing a 72-h FIP model of murine sepsis for use in multi-laboratory studies. Alterations to experimental conditions, such as dose of fecal slurry and timing of antibiotics, have clear impact on outcomes. Differences in mortality between sites despite rigorous standardization warrants further investigations to better understand inter-laboratory variation and methodological design in preclinical studies.

INVESTIGATION OF THE PATHOLOGICAL EFFECTS OF HISTONES, DNA, AND NUCLEOSOMES IN A MURINE MODEL OF SEPSIS.

ABSTRACT: Background: In sepsis, neutrophil extracellular traps (NETs) are an important interface between innate immunity and coagulation. The major structural component of neutrophil extracellular traps is nucleosomes (DNA-histone complexes). In vitro, DNA and histones exert procoagulant/cytotoxic effects whereas nucleosomes are not harmful. However, whether DNA, histones, and/or nucleosomes exert harmful effects in vivo remain unclear. Objectives: (1) The aims of the study are to investigate the cytotoxic effects of nucleosomes ± DNase I and heparin in vitro and (2) to investigate whether DNA, histones, and/or nucleosomes are harmful when injected into healthy and septic mice. Methods : The cytotoxic effects of DNA, histones, and nucleosomes (± DNaseI or ±heparin) were assessed in HEK293 cells. Mice underwent cecal ligation and puncture or sham surgery and then received injections of DNA (8 mg/kg), histones (8.5 mg/kg), or nucleosomes at 4 and 6 h. Organs and blood were harvested at 8 h. Cell-free DNA, IL-6, thrombin-anti-thrombin, and protein C were quantified from plasma. Results:In vitro , incubation of HEK293 cells with DNaseI-treated nucleosomes reduced cell survival compared with nucleosome-treated cells, suggesting that DNaseI releases cytotoxic histones from nucleosomes. Addition of heparin to DNaseI-treated nucleosomes rescued cell death. In vivo, administration of histones to septic mice increased markers of inflammation (IL-6) and coagulation (thrombin-anti-thrombin), which was not observed in sham or septic mice administered DNA or nucleosomes. Conclusions: Our studies suggest that DNA masks the harmful effects of histones in vitro and in vivo . Although administration of histones contributed to the pathogenesis of sepsis, administration of nucleosomes or DNA was not harmful in healthy or septic mice.

THE EFFECTS OF DNASE I AND LOW-MOLECULAR-WEIGHT HEPARIN IN A MURINE MODEL OF POLYMICROBIAL ABDOMINAL SEPSIS.

ABSTRACT: Introduction: Cell-free DNA (CFDNA) has emerged as a prognostic biomarker in patients with sepsis. Circulating CFDNA is hypothesized to be associated with histones in the form of nucleosomes. In vitro, DNA activates coagulation and inhibits fibrinolysis, whereas histones activate platelets and are cytotoxic to endothelial cells. Previous studies have targeted CFDNA or histones in animal models of sepsis using DNase I or heparins, respectively, which has reduced inflammatory and thrombosis markers, thereby improving survival. In this study, we explored the possibility that the combination of DNase I and a low-molecular weight heparin (LMWH) may be a better therapeutic approach than monotherapy in a murine model of abdominal sepsis. Methods: C57Bl/6 mice (8-12 weeks old, both sexes) were subjected to either cecal ligation and puncture or sham surgery. Mice were given antibiotics, fluids, and either saline, DNase I (intraperitoneally, 20 mg/kg/8 h), LMWH (dalteparin, subcutaneously 500 IU/kg/12 h), or a combination of both (n = 12-31). Mice were monitored over 72 h for survival. Organs and blood were harvested for analysis. Levels of LMWH, CFDNA, IL-6, citrullinated histone-H3, thrombin-antithrombin complexes, and protein C were measured in plasma. Results: Administration of either DNase I (81.8%) or LMWH (83.3%, prophylactic range of 0.12 ± 0.07 IU/mL achieved) improved the survival of septic mice compared with saline- (38.7%) and combination-treated mice (48.8%, P < 0.05). Combination-treated mice also showed a small but insignificant improvement in survival compared with saline-treated cecal ligation and puncture mice. Monotherapies may be improving survival by reducing blood bacterial loads, citrullinated histone-H3, and thrombin-antithrombin complexes, and improving protein C levels. Conclusions: Compared with saline- and combination-treated mice, administration of monotherapies to septic mice improved survival. These findings suggest that there may be a negative drug-drug interaction between DNase I and LMWH when DNase I is administered intraperitoneally in a murine model of polymicrobial abdominal sepsis.

Microfluidic device for single step measurement of protein C in plasma samples for sepsis prognosis.

Protein C is a vitamin K dependant protein in plasma that plays an essential role in regulating the coagulation cascade and inflammatory response. As a result of its importance in these roles, it has been suggested as a biomarker for prognosis of patients affected by sepsis. Sepsis is a dysregulated host response to an infection that is the leading cause of mortality in U.S. hospitals and results in the highest cost of hospitalization. It was found that protein C concentration in non-surviving sepsis patients is significantly lower (1.8 µg mL-1) than in survivors and healthy patients who have a protein C concentration of 3.9-5.9 µg mL-1. Current methods for diagnosing sepsis rely on expensive immunoassays or functional assays that require multiple steps for isolation and activation of protein C. We demonstrate in this paper a low cost, single step assay for detection of protein C in blood plasma. This was done by combining isoelectric gates with barium-immobilized metal affinity trapping. The electric field was optimized for use with immobilized metal affinity using COMSOL simulation. The integrated device was tested with samples containing buffered protein C, protein C in the presence of high concentration bovine serum albumin and alpha 1-proteinase inhibitor, and in blood plasma with spiked protein C. The stability of the measured values was tested by monitoring the intensity of a mixture of protein C with BSA and A1PI every minute to determine that measurement after 40 minutes was optimal. The results showed that the device could be used to distinguish a reduction in protein C from 4.46 µg mL-1 to 1.96 µg mL-1 with greater than 98% confidence in plasma making it suitable for sepsis prognosis.

Investigations of the effectiveness of heparin variants as inhibitors of histones.

BACKGROUND: Extracellular histones exert cytotoxic and procoagulant effects which contribute to immunothrombosis in vascular diseases such as sepsis. Heparin has been shown to neutralize the pathologic effects of histones in vitro and in animal models. OBJECTIVES: To compare the effectiveness of unfractionated heparin (UFH), low-molecularweight heparin (LMWH), Vasoflux (lacks anticoagulant activity), and fondaparinux in neutralizing the cytotoxic and procoagulant activities of histones METHODS: Binding affinities between heparin variants and histone subunits were determined by Bio-layer Interferometry. The ability of heparin variants to diminish the cytotoxic and procoagulant effects of histones was studied by treating endothelial cells or monocytic THP-1 cells with histones ± heparin variants. RESULTS: Unfractionated heparin, LMWH, and Vasoflux bind histone subunits with high affinities (Kd <1 pM-66.7 nM) whereas fondaparinux exhibited a low affinity (Kd of 3.06 µM-81.1 mM). UFH, LMWH, and Vasoflux neutralize histone-mediated cytotoxicity as well as monocytic procoagulant activity (as assessed by cell surface tissue factor and phosphatidylserine). In contrast, fondaparinux has no effect on these activities. All four heparin variants reverse histone-mediated impairment of APC generation in a dose-dependent manner. CONCLUSIONS: The ability of heparin to neutralize the cytotoxic and procoagulant effects of histones require heparin fragments >1.7 kDa and is independent of the antithrombin-binding pentasaccharide. In contrast, the ability of heparin to neutralize histone-mediated impairment of APC generation is independent of size and anticoagulant activity. These findings suggest that heparin variants may have differential therapeutic potential in vascular diseases associated with elevated levels of histones.

Mechanistic Studies of DNase I Activity: Impact of Heparin Variants and PAD4.

Background: Excessive production of neutrophil extracellular traps (NETs) in sepsis contributes to vascular occlusion by acting as a scaffold and stimulus for thrombus formation. Removal of extracellular DNA, the major structural component of NETs, by DNase I may reduce host injury. Objectives: (1) To determine how heparin variants (unfractionated heparin, enoxaparin, Vasoflux, and fondaparinux) affect DNase I activity, (2) to measure temporal changes in circulating DNA and DNase I in septic patients. Methods: DNA­histone complexes were treated with DNase I ± heparin variants and visualized via agarose gels. We compared the ability of DNase I ± heparin variants to digest NETs released by phorbol 12-myristate 13-acetate-stimulated neutrophils versus DNA­histone complexes released by necrotic HEK293 cells. Plasma DNA and DNase I levels were measured longitudinally in 76 septic patients. Results: Heparin enhances DNase I-mediated digestion of DNA­histone complexes in a size-dependent manner that does not require the antithrombin-binding region. In contrast, DNase I alone was able to degrade the DNA­histone component of NETs presumably due to peptidylarginine deiminase 4 (PAD4)-mediated histone citrullination that weakens DNA­histone interactions. In purified systems, PAD4 treatment of DNA­histone complexes enhanced the ability of DNase I to degrade histone-bound DNA. In septic patients, endogenous DNase I levels remained persistently low over 28 days, and there were no significant correlations between DNA and DNase I levels. Conclusion: Heparin enhances DNA-mediated digestion of DNA­histone complexes in a size-dependent manner that is independent of its anticoagulant properties. Citrullination of histones by PAD4 renders DNA­histone complexes susceptible to DNase I digestion. Endogenous DNase I levels are persistently decreased in septic patients, which supports the potential utility of DNase I as a therapy for sepsis.

Biomarkers of coagulation, endothelial function, and fibrinolysis in critically ill patients with COVID-19: A single-center prospective longitudinal study.

BACKGROUND: Immunothrombosis and coagulopathy in the lung microvasculature may lead to lung injury and disease progression in coronavirus disease 2019 (COVID-19). We aim to identify biomarkers of coagulation, endothelial function, and fibrinolysis that are associated with disease severity and may have prognostic potential. METHODS: We performed a single-center prospective study of 14 adult COVID-19(+) intensive care unit patients who were age- and sex-matched to 14 COVID-19(-) intensive care unit patients, and healthy controls. Daily blood draws, clinical data, and patient characteristics were collected. Baseline values for 10 biomarkers of interest were compared between the three groups, and visualized using Fisher's linear discriminant function. Linear repeated-measures mixed models were used to screen biomarkers for associations with mortality. Selected biomarkers were further explored and entered into an unsupervised longitudinal clustering machine learning algorithm to identify trends and targets that may be used for future predictive modelling efforts. RESULTS: Elevated D-dimer was the strongest contributor in distinguishing COVID-19 status; however, D-dimer was not associated with survival. Variable selection identified clot lysis time, and antigen levels of soluble thrombomodulin (sTM), plasminogen activator inhibitor-1 (PAI-1), and plasminogen as biomarkers associated with death. Longitudinal multivariate k-means clustering on these biomarkers alone identified two clusters of COVID-19(+) patients: low (30%) and high (100%) mortality groups. Biomarker trajectories that characterized the high mortality cluster were higher clot lysis times (inhibited fibrinolysis), higher sTM and PAI-1 levels, and lower plasminogen levels. CONCLUSIONS: Longitudinal trajectories of clot lysis time, sTM, PAI-1, and plasminogen may have predictive ability for mortality in COVID-19.

Diagnostic Potential of Coagulation-Related Biomarkers for Sepsis in the Emergency Department: Protocol for a Pilot Observational Cohort Study.

BACKGROUND: Between 75% and 80% of patients with sepsis arrive in the hospital through the emergency department. Early diagnosis is important to alter patient prognosis, but currently, there is no reliable biomarker. The innate immune response links inflammation and coagulation. Several coagulation -related biomarkers are associated with poor prognosis in the ICU. The role of coagulation biomarkers to aid in early sepsis diagnosis has not previously been investigated. The objective of our study is to determine the individual or combined accuracy of coagulation and inflammation biomarkers with standard biochemical tests to diagnose adult septic patients presenting to the emergency department. METHODS: in the Emergency Department is a prospective, observational cohort study with a target enrolment of 250 suspected septic patients from two Canadian emergency departments. The emergency physicians will enroll patients with suspected sepsis. Blood samples will be collected at two time points (initial presentation and 4 hr following). Patients will be adjudicated into septic, infected, or not infected status in accordance with the Sepsis-3 definitions. Patient demographics, cultures, diagnosis, and biomarkers will be reported using descriptive statistics. Optimal cut off values with sensitivity and specificity for each biomarker will be determined using C-statistics to distinguish between septic and nonseptic patients. Stepwise multiple logistic regression analysis with exclusion of nonsignificant covariates from the final model will be used to establish a panel of biomarkers. CONCLUSIONS: Our protocol describes the processes and methods for a pragmatic observational biomarker study in the emergency department. This study will seek to determine the potential diagnostic importance of early coagulation abnormalities to identify additional tools for sepsis diagnosis.

National Preclinical Sepsis Platform: developing a framework for accelerating innovation in Canadian sepsis research.

Despite decades of preclinical research, no experimentally derived therapies for sepsis have been successfully adopted into routine clinical practice. Factors that contribute to this crisis of translation include poor representation by preclinical models of the complex human condition of sepsis, bias in preclinical studies, as well as limitations of single-laboratory methodology. To overcome some of these shortcomings, multicentre preclinical studies-defined as a research experiment conducted in two or more research laboratories with a common protocol and analysis-are expected to maximize transparency, improve reproducibility, and enhance generalizability. The ultimate objective is to increase the efficiency and efficacy of bench-to-bedside translation for preclinical sepsis research and improve outcomes for patients with life-threatening infection. To this end, we organized the first meeting of the National Preclinical Sepsis Platform (NPSP). This multicentre preclinical  research collaboration of Canadian sepsis researchers and stakeholders was established to study the pathophysiology of sepsis and accelerate movement of promising therapeutics into early phase clinical trials. Integrated knowledge translation and shared decision-making were emphasized to ensure the goals of the platform align with clinical researchers and patient partners. 29 participants from 10 independent labs attended and discussed four main topics: (1) objectives of the platform; (2) animal models of sepsis; (3) multicentre methodology and (4) outcomes for evaluation. A PIRO model (predisposition, insult, response, organ dysfunction) for experimental design was proposed to strengthen linkages with interdisciplinary researchers and key stakeholders. This platform represents an important resource for maximizing translational impact of preclinical sepsis research.

Immunothrombosis Biomarkers for Distinguishing Coronavirus Disease 2019 Patients From Noncoronavirus Disease Septic Patients With Pneumonia and for Predicting ICU Mortality.

IMPORTANCE: Coronavirus disease 2019 patients have an increased risk of thrombotic complications that may reflect immunothrombosis, a process characterized by blood clotting, endothelial dysfunction, and the release of neutrophil extracellular traps. To date, few studies have investigated longitudinal changes in immunothrombosis biomarkers in these patients. Furthermore, how these longitudinal changes differ between coronavirus disease 2019 patients and noncoronavirus disease septic patients with pneumonia are unknown. OBJECTIVES: In this pilot observational study, we investigated the utility of immunothrombosis biomarkers for distinguishing between coronavirus disease 2019 patients and noncoronavirus disease septic patients with pneumonia. We also evaluated the utility of the biomarkers for predicting ICU mortality in these patients. DESIGN SETTING AND PARTICIPANTS: The participants were ICU patients with coronavirus disease 2019 (n = 14), noncoronavirus disease septic patients with pneumonia (n = 19), and healthy age-matched controls (n = 14). MAIN OUTCOMES AND MEASURES: Nine biomarkers were measured from plasma samples (on days 1, 2, 4, 7, 10, and/or 14). Analysis was based on binomial logit models and receiver operating characteristic analyses. RESULTS: Cell-free DNA, d-dimer, soluble endothelial protein C receptor, protein C, soluble thrombomodulin, fibrinogen, citrullinated histones, and thrombin-antithrombin complexes have significant powers for distinguishing coronavirus disease 2019 patients from healthy individuals. In comparison, fibrinogen, soluble endothelial protein C receptor, antithrombin, and cell-free DNA have significant powers for distinguishing coronavirus disease 2019 from pneumonia patients. The predictors of ICU mortality differ between the two patient groups: soluble thrombomodulin and citrullinated histones for coronavirus disease 2019 patients, and protein C and cell-free DNA or fibrinogen for pneumonia patients. In both patient groups, the most recent biomarker values have stronger prognostic value than their ICU day 1 values. CONCLUSIONS AND RELEVANCE: Fibrinogen, soluble endothelial protein C receptor, antithrombin, and cell-free DNA have utility for distinguishing coronavirus disease 2019 patients from noncoronavirus disease septic patients with pneumonia. The most important predictors of ICU mortality are soluble thrombomodulin/citrullinated histones for coronavirus disease 2019 patients, and protein C/cell-free DNA for noncoronavirus disease pneumonia patients. This hypothesis-generating study suggests that the pathophysiology of immunothrombosis differs between the two patient groups.

Identification of hemostatic markers that define the pre-DIC state: A multi-center observational study.

BACKGROUND: A limitation of diagnostic scoring systems for disseminated intravascular coagulation (DIC) is that once DIC is identified, it may be in a state of irreversible deterioration. OBJECTIVES: To identify hemostatic markers that can identify the pre-DIC state. METHODS: This was a multi-center observational study of 357 septic patients. The incidence of DIC was determined using the International Society on Thrombosis and Haemostasis (ISTH) DIC Score. Markers of interest include components of the DIC score: protein C (PC), antithrombin (AT), and citrullinated histones (H3Cit), which is a marker of NETosis. RESULTS: Out of 357 sepsis patients, 236 patients did not develop DIC (without-DIC), 79 patients had DIC on Day 1 (overt-DIC), and 42 patients developed DIC after Day 1 (pre-DIC). Compared to without-DIC patients, pre-DIC patients had decreased platelet count, increased international normalized ratio (INR), decreased PC and AT, and increased H3Cit. In contrast, D-dimer and fibrinogen levels did not differ between pre-DIC and without-DIC patients. Using receiver operating characteristics (ROC) analysis, we found that platelet count and INR in combination with PC and AT could discriminate pre-DIC from without-DIC. The area under the curve in the ROC analysis was 0.83 (95% confidence interval, 0.76 to 0.89). CONCLUSION: Our study suggests that platelets and INR in combination with PC and AT can identify the pre-DIC state in septic patients. In contrast, D-dimer increased and fibrinogen decreased in the late (ie, overt) stages of DIC. Our data also suggest that NETosis contributes to the onset of DIC in sepsis.

Epigenetic Profiling in Severe Sepsis: A Pilot Study of DNA Methylation Profiles in Critical Illness.

OBJECTIVES: Epigenetic alterations are an important regulator of gene expression in health and disease; however, epigenetic data in sepsis are lacking. To demonstrate proof of concept and estimate effect size, we performed the first epigenome-wide methylation analysis of whole blood DNA samples from a cohort of septic and nonseptic critically ill patients. DESIGN: A nested case-control study using genomic DNA isolated from whole blood from septic (n = 66) and nonseptic (n = 68) critically ill patients on "Day 1" of ICU admission. Methylation patterns were identified using Illumina 450K arrays with percent methylation expressed as ß values. After quality control, 134 participants and 414,818 autosomal cytosine-phosphate-guanine sites were used for epigenome-wide methylation analyses. SETTING: Tertiary care hospitals. SUBJECTS: Critically ill septic and nonseptic patients. INTERVENTIONS: Observational study. MEASUREMENTS AND MAIN RESULTS: A total of 668 differentially methylated regions corresponding to 443 genes were identified. Known sepsis-associated genes included complement component 3; angiopoietin 2; myeloperoxidase; lactoperoxidase; major histocompatibility complex, class I, A; major histocompatibility complex, class II, isotype DR ß I; major histocompatibility complex, class I, C; and major histocompatibility complex, class II, isotype DQ ß I. When compared with whole blood gene expression data from seven external datasets containing septic and nonseptic patients, 81% of the differentially methylated region-associated genes were differentially expressed in one or more datasets and 31% in three or more datasets. Functional analysis showed enrichment for antigen processing and presentation, methyltransferase activity, cell adhesion, and cell junctions. Analysis by weighted gene coexpression network analysis revealed DNA comethylation modules that were associated with clinical traits including severity of illness, need for vasopressors, and length of stay. CONCLUSIONS: DNA methylation marks may provide important causal and potentially biomarker information in critically ill patients with sepsis.

Does cell-free DNA promote coagulation and inhibit fibrinolysis in patients with unprovoked venous thromboembolism?

INTRODUCTION: Cell-free DNA (CFDNA) is the major structural component of neutrophil extracellular traps (NETs). CFDNA contributes to the prothrombotic potential of NETs by promoting thrombin generation and inhibiting fibrinolysis. Patients with venous thromboembolism (VTE) have elevated circulating nucleosomes (i.e. DNA-histone complexes). In this study, we investigated if CFDNA contributes to a procoagulant and an antifibrinolytic state in patients with unprovoked VTE. MATERIALS AND METHODS: Plasma samples from patients with a first episode of unprovoked VTE were obtained from the D-Dimer Optimal Duration Study (DODS). We measured CFDNA plasma levels in 263 patients while on warfarin and 1-month after stopping. Thrombin generation assays and clot lysis assays were measured in patients after stopping warfarin. Comparisons were made with healthy controls. RESULTS: CFDNA levels in VTE patients who stopped warfarin (5.53 µg/mL; 95%CI: 5.34-5.72) were higher than during warfarin therapy (3.11 µg/mL; 95%CI: 2.98-3.25; p < .001), and higher than in healthy controls (2.77 µg/mL; 95%CI: 2.42-3.11; p < .001). VTE patients had a procoagulant state as evidenced by a shorter lag time (30.8 min; 95%CI: 29.2-32.4) compared to controls (48.2 min; 95%CI :41.0-55.5; p < .001) and a greater endogenous thrombin potential (2656 nM∗min; 95%CI: 2479-2836) compared to healthy controls (1198 nM ∗ min; 95%CI: 793-1603). There was a higher proportion of clots generated from patient plasma that were resistant to lysis (43.7%) compared to healthy controls (46.3%; p < .05). CFDNA levels were not associated with enhanced thrombin generation or impaired fibrinolysis in VTE patients. CONCLUSION: CFDNA levels are elevated in patients with unprovoked VTE but do not correlate with the procoagulant or anti-fibrinolytic properties of patient plasma. This study suggests that additional factors in addition to CFDNA may contribute to the pathogenesis of VTE.

Comparison of the source and prognostic utility of cfDNA in trauma and sepsis.

BACKGROUND: Circulating cell-free DNA (cfDNA) may contribute to the pathophysiology of post-injury inflammation and coagulation in trauma. However, the source and mechanism of release of cfDNA in trauma is not well understood. One potential source of cfDNA is from Neutrophil Extracellular Traps (NETs), released by activated neutrophils during the process of NETosis. The primary objective of our study was to determine if cfDNA has prognostic utility in trauma. The secondary objective of this study was to determine the source of cfDNA in trauma compared to sepsis. METHODS: We studied trauma patients from two prospective observational cohort studies: the DNA as a Prognostic Marker in ICU Patients (DYNAMICS) study and the Endotoxin in Polytrauma (ENPOLY) study. We also studied septic patients from the DYNAMICS study. Citrated plasma samples were collected longitudinally from the patients (days 1 to 7). The following molecules were measured in the plasma samples: cfDNA, protein C (PC), myeloperoxidase (MPO) (a marker of neutrophil activation), citrullinated Histone H3 (H3Cit, a marker of NETosis), cyclophilin A (a marker of necrosis), and caspase-cleaved K18 (a marker of apoptosis). RESULTS: A total of 77 trauma patients were included (n = 38 from DYNAMICS and n = 39 from ENPOLY). The median age was 49 years; 27.3% were female, and mortality was 16.9% at 28 days. Levels of cfDNA were elevated compared to healthy values but not significantly different between survivors and non-survivors. There was a positive correlation between MPO and cfDNA in septic patients (r = 0.424, p < 0.001). In contrast, there was no correlation between MPO and cfDNA in trauma patients (r = - 0.192, p = 0.115). Levels of H3Cit, a marker of NETosis, were significantly elevated in septic patients compared to trauma patients (p < 0.01) while apoptosis and necrosis markers did not differ between the two groups. CONCLUSION: Our studies suggest that the source and mechanism of release of cfDNA differ between trauma and sepsis patients. In sepsis, cfDNA is likely primarily released by activated neutrophils via the process of NETosis. In contrast, cfDNA in trauma appears to originate mainly from injured or necrotic cells. Although cfDNA is elevated in trauma and sepsis patients compared to healthy controls, cfDNA does not appear to have prognostic utility in trauma patients. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01355042 . Registered May 17, 2011.

Mortality Risk Profiles for Sepsis: A Novel Longitudinal and Multivariable Approach.

To determine if a set of time-varying biological indicators can be used to: 1) predict the sepsis mortality risk over time and 2) generate mortality risk profiles. DESIGN: Prospective observational study. SETTING: Nine Canadian ICUs. SUBJECTS: Three-hundred fifty-six septic patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Clinical data and plasma levels of biomarkers were collected longitudinally. We used a complementary log-log model to account for the daily mortality risk of each patient until death in ICU/hospital, discharge, or 28 days after admission. The model, which is a versatile version of the Cox model for gaining longitudinal insights, created a composite indicator (the daily hazard of dying) from the "day 1" and "change" variables of six time-varying biological indicators (cell-free DNA, protein C, platelet count, creatinine, Glasgow Coma Scale score, and lactate) and a set of contextual variables (age, presence of chronic lung disease or previous brain injury, and duration of stay), achieving a high predictive power (conventional area under the curve, 0.90; 95% CI, 0.86-0.94). Including change variables avoided misleading inferences about the effects of day 1 variables, signifying the importance of the longitudinal approach. We then generated mortality risk profiles that highlight the relative contributions among the time-varying biological indicators to overall mortality risk. The tool was validated in 28 nonseptic patients from the same ICUs who became septic later and was subject to 10-fold cross-validation, achieving similarly high area under the curve. CONCLUSIONS: Using a novel version of the Cox model, we created a prognostic tool for septic patients that yields not only a predicted probability of dying but also a mortality risk profile that reveals how six time-varying biological indicators differentially and longitudinally account for the patient's overall daily mortality risk.

Low-density lipoprotein (LDL)-dependent uptake of Gram-positive lipoteichoic acid and Gram-negative lipopolysaccharide occurs through LDL receptor.

Lipoteichoic acid (LTA) and lipopolysaccharide (LPS) are bacterial lipids that stimulate pro-inflammatory cytokine production, thereby exacerbating sepsis pathophysiology. Proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulates uptake of cholesterol by downregulating hepatic lipoprotein receptors, including low-density lipoprotein (LDL) receptor (LDLR) and possibly LDLR-related protein-1 (LRP1). PCSK9 also negatively regulates Gram-negative LPS uptake by hepatocytes, however this mechanism is not completely characterized and mechanisms of Gram-positive LTA uptake are unknown. Therefore, our objective was to elucidate the mechanisms through which PCSK9 regulates uptake of LTA and LPS by investigating the roles of lipoproteins and lipoprotein receptors. Here we show that plasma PCSK9 concentrations increase transiently over time in septic and non-septic critically ill patients, with highly similar profiles over 14 days. Using flow cytometry, we demonstrate that PCSK9 negatively regulates LDLR-mediated uptake of LTA and LPS by HepG2 hepatocytes through an LDL-dependent mechanism, whereas LRP1 and high-density lipoprotein do not contribute to this uptake pathway. Bacterial lipid uptake by hepatocytes was not associated with cytokine production or hepatocellular injury. In conclusion, our study characterizes an LDL-dependent and LDLR-mediated bacterial lipid uptake pathway regulated by PCSK9, and provides evidence in support of PCSK9 inhibition as a potential therapeutic strategy for sepsis.

Body temperature and mouse scoring systems as surrogate markers of death in cecal ligation and puncture sepsis.

BACKGROUND: Despite increasing ethical standards for conducting animal research, death is still often used as an endpoint in mouse sepsis studies. Recently, the Murine Sepsis Score (MSS), Mouse Clinical Assessment Score for Sepsis (M-CASS), and Mouse Grimace Scale (MGS) were developed as surrogate endpoint scoring systems for assessing pain and disease severity in mice. The objective of our study was to compare the effectiveness of these scoring systems and monitoring of body temperature for predicting disease progression and death in the cecal ligation and puncture (CLP) sepsis model, in order to better inform selection of surrogate endpoints for death in experimental sepsis. METHODS: C57Bl/6J mice were subjected to control sham surgery, or moderate or severe CLP sepsis. All mice were monitored every 4 h for surrogate markers of death using modified versions of the MSS, M-CASS, and MGS scoring systems until 24 h post-operatively, or until endpoint (inability to ambulate) and consequent euthanasia. RESULTS: Thirty percent of mice subjected to moderate severity CLP reached endpoint by 24 h post-CLP, whereas 100% undergoing severe CLP reached endpoint within 20 h. Modified MSS, M-CASS, and MGS scores all increased, while body temperature decreased, in a time-dependent and sepsis severity-dependent manner, although modified M-CASS scores showed substantial variability. Receiver operating characteristic curves demonstrate that the last recorded body temperature (AUC = 0.88; 95% CI 0.77-0.99), change in body temperature (AUC = 0.89; 95% CI 0.78-0.99), modified M-CASS (AUC = 0.93; 95% CI 0.85-1.00), and modified MSS (AUC = 0.95; 95% CI 0.88-1.01) scores are all robust for predicting death in CLP sepsis, whereas modified MGS (AUC = 0.78; 95% CI 0.63-0.92) is less robust. CONCLUSIONS: The modified MSS and body temperature are effective markers for assessing disease severity and predicting death in the CLP model, and should thus be considered as valid surrogate markers to replace death as an endpoint in mouse CLP sepsis studies.

The impact of the endothelial protein C receptor on thrombin generation and clot lysis.

INTRODUCTION: When thrombin is bound to thrombomodulin (TM), it becomes a potent activator of protein C (PC) and thrombin-activable fibrinolysis inhibitor (TAFI). Activation of PC is enhanced when PC is bound to the endothelial protein C receptor (EPCR). Activated protein C (APC) inhibits thrombin generation while activated TAFI (TAFIa) attenuates fibrinolysis. To determine the impact of diminished EPCR function on thrombin generation and fibrinolysis we generated cells that expressed TM and a variant of EPCR (R96C) that does not bind PC. METHODS: To determine the impact of EPCR on the generation of APC and TAFIa and how this affects thrombin generation and fibrinolysis we performed thrombin generation and clot lysis assays in the presence of cells expressing wild-type TM and EPCR (WT cells) or wild-type TM and the R96C variant of EPCR (R96C cells). RESULTS: In the presence of R96C cells, thrombin generation in normal plasma is increased, as a result of impaired PC activation when compared to WT cells. In addition, clot lysis is delayed in normal plasma in the presence of R96C cells, despite no increase in TAFI activation. In PC deficient plasma, clot lysis is delayed in the presence of WT and R96C cells as a result of increased TAFI activation. CONCLUSIONS: We demonstrate that impaired EPCR function can be detected by thrombin generation and clot lysis assays on cells expressing TM and EPCR. We also demonstrated that deficiency in EPCR has procoagulant effects that lead to a delay in clot lysis.

Procoagulant effects of lung cancer chemotherapy: impact on microparticles and cell-free DNA.

Lung cancer is the second leading type of cancer, with venous thromboembolism being the second leading cause of death. Studies have shown increased levels of microparticles and cell-free DNA (CFDNA) in cancer patients, which can activate coagulation through extrinsic and intrinsic pathways, respectively. However, the impact of lung cancer chemotherapy on microparticle and/or CFDNA generation is not completely understood. The aim of the study was to study the effects of platinum-based chemotherapeutic agents on generation of procoagulant microparticles and CFDNA in vitro and in vivo. Microparticles were isolated from chemotherapy-treated monocytes, human umbilical vein endothelial cells, or cancer cells. Tissue factor (TF) and phosphatidylserine levels were characterized and thrombin/factor Xa generation assays were used to determine microparticle procoagulant activity. CFDNA levels were isolated from cell supernatants and plasma. A murine xenograft model of human lung carcinoma was used to study the procoagulant effects of TF microparticles and CFDNA in vivo. In vitro, platinum-based chemotherapy induced TF/phosphatidylserine microparticle shedding from A549 and A427 lung cancers cells, which enhanced thrombin generation in plasma in a FVII-dependent manner. CFDNA levels were increased in supernatants of chemotherapy-treated neutrophils and plasma of chemotherapy-treated mice. TF microparticles were elevated in plasma of chemotherapy-treated tumour-bearing mice. Plasma CFDNA levels are increased in chemotherapy-treated tumour-free mice and correlate with increased thrombin generation. In tumour-bearing mice, chemotherapy increases plasma levels of CFDNA and TF/phosphatidylserine microparticles. Platinum-based chemotherapy induces the shedding of TF/phosphatidylserine microparticles from tumour cells and the release of CFDNA from host neutrophils.