Impact of plasma histones in human sepsis and their contribution to cellular injury and inflammation.

INTRODUCTION: Circulating histones have been identified as mediators of damage in animal models of sepsis and in patients with trauma-associated lung injury. Despite existing controversies on actual histone concentrations, clinical implications and mechanism of action in various disease conditions, histone levels in human sepsis, association with disease progression and mediated effects on endothelial and immune cells remain unreported. This study aimed to determine histone levels and its clinical implication in septic patients and to elucidate histone-mediated effects ex-vivo. METHODS: Histone levels, endogenous activated protein C (APC) levels and clinical data from two independent cohorts of septic patients were obtained. Histone levels were compared with various control groups including healthy individuals, intensive care unit (ICU) patients without sepsis, ICU patients with multiple organ failure and patients with minor or multiple trauma, all without infection. Endothelial and monocytic cells were stimulated with histones. Cellular integrity and sepsis prototypical cytokines were evaluated. The mechanism of action of histones via Toll-like receptor 4 (TLR4) was evaluated using a function blocking antibody. Histone degradation in plasma was studied by immunoblotting. RESULTS: Histone H4 levels were significantly elevated in patients with sepsis (cohort I; n = 15 and cohort II; n = 19) versus ICU controls (n = 12), patients with multiple organ failure (n = 12) or minor trauma (n = 7), associated with need for renal replacement therapy and decrease in platelet count during disease progression, and remarkably were significantly associated with increased mortality rates in septic patients (ICU-, 28 day- and 90 day mortality rates). There was an inverse correlation between plasma histones and endogenous APC levels. Histone stimulation induced the release of sepsis prototypic cytokines and decreased cell integrity indicated by a significant increase of lactate dehydrogenase (LDH) and propidium iodide (PI) staining. Blocking of TLR4 decreased cellular cytotoxicity on endothelial cells. The calculated half-life of histones in spiked plasma was 4.6 minutes. CONCLUSIONS: Histone levels in septic patients are significantly increased and might mediate disease aggravation by cellular injury and inflammation via TLR4 signaling, which potentially results in multiple organ failure and fatal outcome.

Thrombin generation as marker to estimate thrombosis risk in patients with abnormal test results in lupus anticoagulant routine diagnostics.

BACKGROUND: Lupus anticoagulant (LA) is known to inhibit thrombin generation although patients have an increased risk to develop thrombosis. We tried to determine whether thrombin generation is altered in plasma samples of patients with abnormal test results in LA routine diagnostics and whether its measurement may improve the risk assessment of thrombosis. METHODS: Samples from 63 patients (39 with abnormal test results; 24 controls) were included in the study. Measurement of diluted Russel's viper venom time (dRVVT) was part of the initial guideline conform diagnostic procedure for detection of LA. In addition, measurement of anticardiolipin-IgM, -IgG and ß2-glycoprotein-I-IgM, -IgG were performed. Thrombin generation was measured using two different phospholipid concentrations in the starting reagent. RESULTS: Analyzing all samples by logistic regression, thrombin generation after induction with high phospholipid concentrations was the best predictor of thrombosis. After preselection of samples with alterations in dRVVT, specificity of selected thrombin generation derived parameters for the detection of previous thrombosis increased in this subgroup. CONCLUSIONS: In patients with phospholipid-dependent prolongation of dRVVT, thrombin generation is variably inhibited and the degree of inhibition corresponds to the occurrence of previous thrombosis. Measuring thrombin generation in patients with phospholipid-dependent dRVVT prolongation may improve risk assessment of thrombosis.

Influence of hydroxyethyl starch (HES) 130/0.4 on hemostasis as measured by viscoelastic device analysis: a systematic review.

PURPOSE: Hydroxyethyl starch solutions (HES) are plasma volume expanders which affect hemostasis. Newer HES 130/0.4 is said to be safer. Reevaluation of published evidence is necessary after the recent retraction of studies. METHODS: Systematic review of studies assessing HES 130/0.4 effects on hemostasis by thrombelastography (TEG, ROTEM) or Sonoclot (SCR) in comparison with crystalloid or albumin control fluids was performed. Only studies which provided statistical comparisons between study fluids were analyzed. Studies were divided into in vitro or in vivo hemodilution studies. We assessed study quality, HES effects which differed significantly from controls, values outside normal range, degree of hemodilution, and cumulative HES dose. RESULTS: Seventeen in vitro and seven in vivo hemodilution studies were analyzed. Four studies reported quality control measures. Nineteen studies (all 15 ROTEM studies, 3 of 5 in vitro TEG, and 1 of 2 SCR studies) showed a significant hypocoagulatory effect of HES 130/0.4 on clot formation, while clotting time was not uniformly affected. Three in vivo TEG studies with low HES doses or cancer patients found mixed or nonsignificant results. In studies which provided normal ranges (n = 9), more values were outside normal ranges in the HES than in the control groups (87/122 vs. 58/122, p < 0.001). Dose effects were apparent in the in vitro studies, which investigated higher dilutions up to 80%. In vivo studies were fewer and did not investigate doses >40 ml/kg. CONCLUSIONS: HES 130/0.4 administration results in a weaker and smaller clot. Until results from well-designed clinical trials are available, safer fluids should be chosen for patients with impaired coagulation.