Introduction: Recent models capturing the pathophysiology of sepsis and ex-vivo data from patients are speculating about immunosuppression in the so-called late phase of sepsis. Clinical data regarding survival and microbiological burden are missing. The aim of this study was to determine the clinical significance of the 'late phase' of sepsis with respect to overall survival and occurrence of microbiological findings. Methods: In a retrospective trial, 16,041 patient charts from a university intensive care unit were screened, and 999 patients with severe sepsis or septic shock were identified. Three phases were established according to the mortality peaks which were separated by two distinct nadirs: phase I (days 1 to 5), phase II (days 6 to 15) and phase III (days 16 to 150). Patients were analyzed for outcome, SOFA scores, procalcitonin levels, antimicrobial treatment, dialysis, mechanical ventilation and results of blood cultures during their hospital stay. Results: Out of 999 enrolled patients, 308 died during the course of sepsis presenting a characteristic mortality rate (30.8%) with three distinct mortality peaks (at days 2, 7 and 17). Overall 36.7% of all deaths occurred in the early phase (phase I) and 63.3% during the later phases (phase II + III). In total 2,117 blood cultures were drawn. In phase I, 882 blood cultures were drawn, representing a sampling rate of 88% with a positive rate of 14.9%. In phase II, 461 samples were taken, indicating a sampling rate of 52% and a positive rate of 11.3%. Within phase III, 524 samples were obtained representing a sampling rate of 66% with a positive rate of 15.3%, which was significantly higher compared to the positive rate of phase II and similar to phase I. In particular, the rate of typically opportunistic bacteria increased significantly from 9% in phase I up to 18% in phase III. The same is true for Candida spp. (phase I 13%, phase III 30%). Conclusions: The later phase of sepsis is associated with a significant re-increase of positive blood culture results, especially regarding opportunistic bacteria and fungi. These observations warrant further studies focusing on the underlying mechanisms resulting in this outcome burden in the later phase of sepsis.

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.

In a prospective, longitudinal study, we investigated the association between decreased ADAMTS13 activity and impaired hemostasis, as well as organ dysfunctions in patients with systemic inflammation due to extracorporeal cardiopulmonary circuit or with severe sepsis. Similar to negative acute phase proteins, ADAMTS13 activity declined stepwise according to the extent of inflammatory responses. A marked imbalance between ADAMTS13 activity and VWF antigen level was associated with the appearance of ultra-large VWF multimers in plasma, with organ dysfunction and lethality. Our data support the view that systemic inflammation results in an ADAMTS13 deficiency which activates hemostasis.

Summary: Von Willebrand factor (VWF) and related parameters as well as the protease activity regulating its biological activity were measured in plasma of healthy controls and patients with different cause and severity of systemic inflammation to examine the efficacy of the measures to detect highly prothrombotic states including thrombotic microangiopathy (TMA), one of the sequelae of sepsis. Plasma levels of VWF increased with increasing severity of systemic inflammation, probably due to activation of the endothelium. In parallel, the proteolytic activity of VWF inactivating protease, ADAMTS13, stepwise declined with the severity of inflammation, emphasizing the role of VWF-triggered platelet aggregation on the endothelium subsequently followed by development of TMA. As a consequence, the ratio of VWF antigen level and ADAMTS13 activity was significantly higher in patients with inflammation and sepsis, suggesting that this ratio might be more useful for the diagnosis of highly prothrombotic states including TMA than VWF multimer analysis alone. These findings suggest that ADAMTS13, VWF and related parameters, even in a combined approach, might be useful for the diagnosis and the therapeutic monitoring of patients with sepsis associated thrombotic microangiopathy.

Purpose: This review investigates and highlights the activity of Willebrand factor (VWF) and its cleaving protease as biomarkers of the development of multiple organ dysfunction in infectious and noninfectious systemic inflammatory response syndrome. State of the Art: Ultra-large VWF (ULVWF) multimers activate platelets resulting in a prothrombotic situation. Systemic inflammation is associated with increased ULVWF plasma level and a decreased ADAMTS13 activity. The potential role of ADAMTS13 as a diagnostic and prognostic marker of disseminated intravascular coagulopathy is largely underestimated. Summary: VWF is an acute phase protein and its plasma level increases in systemic inflammation. When released from endothelial cells and platelets, the native multimeric glycoprotein is mostly present in the ultralarge form (ULVWF), which may have a major clinical significance under proinflammatory conditions. ULVWFmultimers may activate endothelial cells and platelets simultaneously. The multimers undergo limited proteolysis by a specific plasma metalloprotease known as ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motif), thus, in healthy individuals only marginal amounts of circulating ULVWF are detectable. Severe hereditary or acquired ADAMTS13 deficiency causes thrombotic thrombocytopenic purpura (TTP), which contributes to prothrombotic coagulation abnormalities preceding organ dysfunction systemic inflammatory response syndrome (SIRS). In proinflammatory conditions, ADAMTS13 activity decreases due to various mechanisms, (i) down regulation on a transcriptional level, (ii) proteolytic degradation, and (iii) consumption due to the high substrate level. Marked dysbalance as found in patients with severe sepsis or septic shock results in substantial amounts of plasma ULVWF. This level of dysbalance is negatively correlated with platelet count and positively correlated with the severity of inflammation and the degree of organ failure.