Maik Sossdorf

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

IntroductionCirculating 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.MethodsHistone 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.ResultsHistone 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.ConclusionsHistone 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.

Variations in the ratio between von Willebrand factor and its cleaving protease during systemic inflammation and association with severity and prognosis of organ failure.

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.

The Balance between von-Willebrand Factor and its Cleaving Protease ADAMTS13: Biomarker in Systemic Inflammation and Development of Organ Failure?

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. ULVWF-multimers 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.

Cell-derived microparticles promote coagulation after moderate exercise.

UNLABELLED Cell-derived procoagulant microparticles (MP) might be able to contribute to exercise-induced changes in blood hemostasis. PURPOSES This study aimed to examine (i) the concentration and procoagulant activity of cell-derived MP after a moderate endurance exercise and (ii) the differences in the release, clearance, and activity of MP before and after exercise between trained and untrained individuals. METHODS All subjects performed a single bout of physical exercise on a bicycle ergometer for 90 min at 80% of their individual anaerobic threshold. MP were identified and quantified by flow cytometry measurements. Procoagulant activity of MP was measured by a prothrombinase activity assay as well as tissue factor-induced fibrin formation in MP-containing plasma. RESULTS At baseline, no differences were observed for the absolute number and procoagulant activities of MP between trained and untrained subjects. However, trained individuals had a lower number of tissue factor-positive monocyte-derived MP compared with untrained individuals. In trained subjects, exercise induced a significant increase in the number of MP derived from platelets, monocytes, and endothelial cells, with maximum values at 45 min after exercise and returned to basal levels at 2 h after exercise. Untrained subjects revealed a similar increase in platelet-derived MP, but their level was still increased at 2 h after exercise, indicating a reduced clearance compared with trained individuals. Procoagulant activities of MP were increased immediately after exercise and remained elevated up to 2 h after exercise. CONCLUSIONS We conclude that increased levels of MP were found in healthy individuals after an acute bout of exercise, that the amount of circulating MP contributes to an exercise-induced increase of hemostatic potential, and that there were differences in kinetic and dynamic characteristics between trained and untrained individuals.

Effects of low-dose acetylsalicylic acid and atherosclerotic vascular diseases on the outcome in patients with severe sepsis or septic shock

Sepsis and its sequelae of multiple organ failure is one of the leading causes of death in the industrial countries. Several studies have shown that patients who are treated with low-dose acetyl salicylic acid (ASA) for secondary prevention of atherothrombosis may have a lower risk to develop organ failure in the case of critical illness. The benefit of ASA is probably due to an inhibition of platelet activation as well as an increase in the formation of anti-inflammatory lipoxin A4. On the other hand, the effect of ASA could be – at least partially – an indirect one, caused by atherosclerotic vascular diseases as the cause of ASA treatment. Atherosclerosis is considered as a moderate systemic inflammation and we hypothesise that this chronic condition could have an impact on the outcome in sepsis. To get more information on the benefit of ASA in critically ill patients and on possible interference with atherosclerotic vascular diseases, we analysed the medical records of 886 septic patients who were admitted to the surgical intensive care unit (ICU) of a university hospital. Logistic regression analysis indicated that patients who were treated during the ICU stay with ASA (100 mg/d) had a significantly lower mortality. Odds ratios (ORs; with 95% confidential intervals) of 0.56 (0.37–0.84) and 0.57 (0.39–0.83) were calculated for ICU and hospital mortality, respectively. In contrast, statin treatment did not have significant effect on mortality. Diagnosis of atherosclerotic vascular diseases according to ICD classification did not influence ICU mortality but lowered hospital mortality (OR = 0.71 (0.52–0.99)). Subgroup analysis provided preliminary evidence that clopidogrel when given as only anti-platelet drug may have a similar benefit as ASA, but the combination of ASA and clopidogrel failed to improve the outcome. The time course of plasma fibrinogen and procalcitonin levels indicate that ASA seems to reduce the activation of haemostasis and increase the resolution of inflammation. It is concluded that prospective interventional studies should be done to test the use of ASA as novel therapeutic approach in critically ill patients.

HES 130/0.4 impairs haemostasis and stimulates pro-inflammatory blood platelet function

IntroductionHydroxyethyl starch (HES) solutions are widely used for volume replacement therapy but are also known to compromise coagulation, impair renal function and increase long-term mortality. To test the hypotheses that HES 130/0.4 has fewer adverse effects than HES 200/0.5 and exerts anti-inflammatory properties, we compared the effects of HES 130/0.4, HES 200/0.5 and saline on in vitro haemostasis and pro-inflammatory platelet function.MethodsWhole blood samples from healthy volunteers were mixed with 6% HES 130/0.4, 10% HES 200/0.5, or normal saline to achieve a final haemodilution rate of 10% or 40%. Haemostatic capacity was characterised by thromboelastography (ROTEM) and measurement for FXIIIa activity. Platelet activation and pro-inflammatory platelet functions were characterised by flow cytometry measuring the platelet activation marker CD62P and binding of fibrinogen to platelets as well as the formation of heterotypic platelet-leukocyte conjugates.ResultsCompared with saline, HES 130/0.4 dose-dependently impaired formation and firmness of the fibrin clot but did not affect the fibrin crosslinking activity of FXIIIa. At 40% but not at 10% haemodilution rate, HES 200/0.5 also increased platelet fibrinogen binding and both HES solutions increased expression of CD62P, the main receptor for platelet-leukocyte adhesion. HES 130/0.4 but not HES 200/0.5 increased formation of platelet-neutrophil conjugates and, to a lesser degree, platelet-monocyte conjugates.ConclusionsOur data demonstrate that HES 130/0.4 has similar adverse effects as HES 200/0.5. In particular, both types of HES impair coagulation capacity and stimulate, rather than attenuate, pro-inflammatory platelet function.

Benefit of low-dose aspirin and non-steroidal anti-inflammatory drugs in septic patients.

Analyzing medical records of 979 patients with severe sepsis or septic shock provided some evidence that the use of low-dose aspirin or non-steroidal anti-inflammatory drugs (NSAIDs) was associated with decreased hospital mortality. However, the benefit was abolished when aspirin and NSAIDs were given together.

Impact of sepsis-associated cytokine storm on plasma NGAL during acute kidney injury in a model of polymicrobial sepsis

The impact of cytokine storm on plasma neutrophil gelatinase-associated lipocalin (NGAL) levels during polymicrobial sepsis is unknown. We demonstrate the advantage of plasma NGAL as marker for acute kidney injury (AKI) during septic cytokine storm and an early correlation of tumor necrosis factor alpha (TNFa) with NGAL during septic AKI.

Release of pro-coagulant microparticles after moderate endurance exercise

To the editor, Elevated plasma concentrations of cell-membranederived microparticles (MP) are found in many diseases such as sepsis, cancer, and diabetes mellitus [1, 2]. Various cell types are known as sources of MP, which play a crucial role in angiogenesis, endothelial cell function as well as coagulation and inflammation. A uniform characteristic of the various cell-specific MP is the surface exposure of negatively charged phospholipids. Due to these phospholipids and also due to the exposure of tissue factor (TF) and/or TF pathway inhibitor, MP are believed to be involved in the regulation of coagulation [3, 4]. Furthermore, MP bear cell-specific antigens enabling them to interact with a variety of target cells. Thus, MP can be considered as intercellular signal transferring entities [5, 6]. Low levels of circulating MP, mainly derived from platelets, are also present in healthy individuals but their functional relevance in haemostasis and inflammation is not well understood [7]. Increased levels of circulating MP are known to be associated with an increased risk of cardiovascular disease and atherothrombotic events [1, 8]. Since moderate physical exercise is known to lower the risk of cardiovascular diseases [9, 10], we were interested to study the effect of moderate physical exercise on both concentration and pro-coagulant activity of circulating MP. After approval by the local Ethics Committee and obtaining written informed consent, 16 healthy male volunteers (age 25 3 years; mean SD) underwent a singular 90 min exercise test on a bicycle ergometer with a constant power of 80% of individual anaerobic threshold (IAT). IAT was determined one week prior to the test according to Stegmann et al. [11]. Blood samples were taken before (rest), immediately post (post) and 45 min (45 min post) as well as 2 hours (2 h post) after exercise. MP were identified by flow cytometry according to their size and the binding of FITC-labelled annexin V as well as by the binding of PE-labelled antibodies against cell-specific antigens: platelet-derived MP (PMP) by anti-CD42a, monocyte-derived MP (MMP) by anti-CD14 and endothelial cell-derived MP (EMP) by anti-CD62E. To estimate procoagulant activities of MP two different tests were performed: The ACTICHROME test (American Diagnostica), which measures the pro-thrombinase activity of FXa/Va of MP bound to an annexin V-coated surface in absence of any other plasma constituents and a one-stage clotting test, in which the formation of a fibrin clot was monitored by measuring the optical density in MP-containing plasma samples after re-calcification and addition of a small amount of TF. The 90 min exercise at 80% IAT (mean power 173 73 W) increased heart rate to 156 16 beats/ min, the systolic blood pressure to 156 25 mm Hg and plasma lactate concentration to 2.17 0.81 mmoles/l. As shown in Table I, the exercise resulted in a significant increase in the numbers of white blood cells and platelets. It has been shown elsewhere that the increase in platelet and leukocyte counts is not only due to a reduction in plasma volume but also due to a release of platelets and leukocytes from various storage sites (spleen, lung, bone marrow etc.) [9]. With respect to MP, we also

Plasma Neutrophil Gelatinase-Associated Lipocalin Is Primarily Related to Inflammation during Sepsis: A Translational Approach

Acute kidney injury (AKI) during sepsis is common and underestimated. Plasma neutrophil gelatinase-associated lipocalin (plasma-NGAL) is discussed as new biomarker for AKI diagnosis, but during inflammation its function and diagnostic impact remain unclear. The association between plasma-NGAL and inflammatory markers in septic patients, but also in healthy controls and patients with chronic inflammation before and after either maximum exercise test or treatment with an anti-TNF therapy were investigated. In-vitro blood stimulations with IL-6, lipopolysaccharide, NGAL or its combinations were performed to investigate cause-effect-relationship. Plasma-NGAL levels were stronger associated with inflammation markers including IL-6 (Sepsis: r=0.785 P<0.001; chronic inflammation after anti-TNF: r=0.558 P<0.001), IL-8 (Sepsis: r=0.714 P<0.004; healthy controls after exercise r=0.786 P<0.028; chronic inflammation before anti-TNF: r=0.429 P<0.041) and IL-10 (healthy controls before exercise: r=0.791 P<0.028) than with kidney injury or function. Correlation to kidney injury or function was found only in septic patients (for creatinine: r= 0.906 P<0.001; for eGFR: r= -0.686 P=0.005) and in patients with rheumatic disease after anti-TNF therapy (for creatinine: r= 0.466 P<0.025). In stimulation assays with IL-6 and lipopolysaccharide plasma-NGAL was increased. Co-stimulation of lipopolysaccharide with plasma-NGAL decreased cellular injury (P<0.05) and in trend IL-10 levels (P=0.057). Septic mice demonstrated a significantly improved survival rate after NGAL treatment (P<0.01). Plasma-NGAL seams to be strongly involved in inflammation. For clinical relevance, it might not only be useful for AKI detection during severe inflammation - indeed it has to be interpreted carefully within this setting - but additionally might offer therapeutic potential.