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Previous studies identified nonneutralizing FVIII-specific antibodies in the circulation of severe and nonsevere hemophilia A (sHA and nsHA) patients without FVIII inhibitors and also in some healthy individuals. To gain a better understanding of the nature of these nonneutralizing antibody responses, we analyzed and compared anti-FVIII antibody signatures in 3 study cohorts: previously treated sHA as well as nsHA patients without FVIII inhibitors, and healthy donors. FVIII-binding IgM, IgG1-4, and IgA antibodies were differentiated, FVIII-specificity was assessed, and associated apparent affinity constants were determined. Our results indicate that the nonneutralizing FVIII-specific antibody response in all study cohorts is dominated by IgG1 and IgA. Prevalences, titers, and affinities of these nonneutralizing antibodies were higher in the hemophilia A cohorts than in healthy donors. Stratification for the anti-hepatitis C virus (HCV) antibody status demonstrated the presence of FVIII-specific IgA with elevated titers in sHA patients with an active or past HCV infection when compared with HCV antibody-positive nsHA patients or HCV antibody-negative patients and healthy donors. Increased titers and affinities of FVIII-specific IgG1 antibodies were observed in a considerable number of hemophilia A patients as opposed to healthy subjects independently of the patients’ anti-HCV antibody status. Overall, our findings support the hypothesis that the generation of nonneutralizing anti-FVIII antibodies in healthy individuals and in noninhibitor hemophilia A patients might be based on similar immune mechanisms. However, differences in prevalences, titers, and affinities of these antibodies indicate distinct differences in the antibody evolution between healthy individuals and patients.

Introduction Romiplostim has been approved in Europe since 2009 to treat patients with chronic primary immune thrombocytopenia (ITP). Using real-world data from seven European countries, we measured the effectiveness and safety outcomes within 24 weeks following romiplostim initiation by duration of ITP: less than 3 months (“newly diagnosed”), 3–12 months (“persistent”), and more than 12 months (“chronic”). Methods Adults with ITP and ≥ 1 romiplostim administration between 2009 and 2012 were included. Endpoints included durable platelet response, median platelet count, rescue therapy, bleeding and adverse events. We used inverse probability of censoring weighted estimators to estimate cumulative risk of each outcome. There were 64 newly diagnosed, 50 persistent, and 226 chronic ITP patients at romiplostim initiation. Results Durable platelet response at 24 weeks ranged from 32% [confidence interval (CI): 18–46%] in newly diagnosed patients to 53% (CI 37–68%) in persistent patients. Median platelet count during follow-up ranged from 88 (CI 80–96) × 109/L in chronic patients to 131 (CI 102–160) × 109/L in newly diagnosed patients. Conclusion Regardless of ITP duration, over half of patients discontinued concomitant ITP medications. Few adverse events were observed. Although only approved for chronic patients, estimates of the romiplostim treatment effect were similar across patients being managed in European clinical practice, regardless of ITP duration at romiplostim initiation. Supplementary Information The online version contains supplementary material available at 10.1007/s12325-021-01727-5.

Background Data on the effect of ABO blood group (ABO), von Willebrand factor (VWF) levels, and age on factor VIII (FVIII) in non-severe haemophilia A (HA) is scarce. Objective To investigate if ABO, VWF levels, and age have an influence on the variability of FVIII levels and consequently on the assessment of severity in non-severe HA. Patients/methods Eighty-nine patients with non-severe HA and 82 healthy controls were included. Data on ABO was collected and FVIII clotting activity (FVIII:C) with one-stage clotting assay (FVIII:C OSA) and chromogenic substrate assay (FVIII:C CSA), FVIII antigen (FVIII:Ag) and VWF antigen (VWF:Ag) and activity (VWF:Act) were determined. Results In HA, FVIII:C OSA and CSA and FVIII:Ag were not different between non-O (n = 42, median 15.5, interquartile range 10.4-24.0; 10.0, 6.8-26.0 and 15.2, 10.7-24.9) and O (n = 47, 14.1, 9.0-23.0; 10.0, 5.0-23.0 and 15.2, 9.3-35.5), whereas in healthy controls, non-O individuals had significantly higher FVIII levels. Fviii C showed no relevant correlation with VWF levels in HA, but we observed strong correlations in healthy controls. Age had only a minor influence in HA, but had a considerable impact on FVIII:C in healthy controls. In multivariable regression analysis ABO, VWF:Ag and age were not associated with FVIII:C in HA, whereas this model explained 61.3% of the FVIII:C variance in healthy controls. Conclusions We conclude that in non-severe HA ABO and VWF levels do not substantially influence the variability of FVIII levels and age has only minor effects on it, which is important information for diagnostic procedures.

The increased incidence of inflammatory bowel disease (IBD) has become a global phenomenon that could be related to adoption of a Western life-style. Westernization of dietary habits is partly characterized by enrichment with the ω-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA), which entails risk for developing IBD. Glutathione peroxidase 4 (GPX4) protects against lipid peroxidation (LPO) and cell death termed ferroptosis. We report that small intestinal epithelial cells (IECs) in Crohn’s disease (CD) exhibit impaired GPX4 activity and signs of LPO. PUFAs and specifically AA trigger a cytokine response of IECs which is restricted by GPX4. While GPX4 does not control AA metabolism, cytokine production is governed by similar mechanisms as ferroptosis. A PUFA-enriched Western diet triggers focal granuloma-like neutrophilic enteritis in mice that lack one allele of Gpx4 in IECs. Our study identifies dietary PUFAs as a trigger of GPX4-restricted mucosal inflammation phenocopying aspects of human CD., Dietary lipids are linked to the development of inflammatory bowel diseases through unclear mechanisms. Here, the authors report that dietary polyunsaturated fatty acids trigger intestinal inflammation resembling aspects of Crohn’s disease, which is restricted by glutathione peroxidase 4 in the intestinal epithelium.

We report the case of a 63‐year‐old Caucasian woman with multiple relapsed IgM multiple myeloma (MM) and elevated free kappa light chains (fκLC). Due to hyperviscosity syndrome with visual impairment, regular plasma exchanges were performed. As part of her 11th line of therapy, an experimental protocol consisting of pembrolizumab, pomalidomide, and dexamethasone was initiated. To reduce fκLC and immunoglobulin (Ig) M, we performed immunoadsorption (IA) using columns containing recombinant single domain camelid antibody fragments as ligands. We measured pembrolizumab (humanized IgG4 kappa anti‐PD1 antibody) levels before and after each IA session and found a 98.1% reduction from baseline with five sessions of IA. Comparable elimination kinetics were observed for serum IgG, whereas fκLC and IgM were eliminated to a substantially lesser extent. These findings highlight that in hyperviscosity syndrome due to IgM MM, broad spectrum IA columns might be only moderately effective compared to total plasma exchange or double filtration plasmapheresis. Monoclonal antibodies are efficiently reduced by extracorporeal therapies and re‐dosing is necessary to provide sufficient efficacy. In the case of serious adverse events such as immune‐related adverse events, IA might be used to eliminate the monoclonal antibody. Measuring IgG levels might be a reasonable strategy for monitoring drug levels of monoclonal antibodies during IA.

In the field of thrombosis and hemostasis the focus was laid on the prophylaxis and treatment of venous thromboembolism in patients with active cancer during this years’ meeting of the American Society of Hematology (ASH) in San Diego. The Adam-VTE trial completed the series of studies using Xa-antagonists for the treatment of cancer-associated thromboembolism. Apixaban reduced the number of recurrent thromboembolic events compared to treatment with dalteparin without increasing the bleeding risk. Furthermore, the use of a prophylactic dose of rivaroxaban or apixaban showed positive results in the primary prevention of venous thromboembolism in ambulatory cancer patients being at high risk for thromboembolic events. A safe standardized perioperative management for direct oral anticoagulants was also presented during the meeting in noncancer patients with atrial fibrillation. This short review will summarize the most clinically relevant highlights in the field of thrombosis and hemostasis of the ASH meeting 2018.

Venous thrombosis and pulmonary embolism are common complications in patients with active cancer. For many years low molecular weight heparins (LMWH) have been the preferred treatment option for cancer patients, because of their superiority over vitamin K antagonists, which bear high risk of interaction and unsatisfactory anticoagulation. With the introduction of direct oral anticoagulants (DOAC) a second oral treatment option appeared. However, recommendations for the use of DOACs in the treatment of cancer-associated venous thromboembolic events were only given recently. DOAC possess a different side effect profile regarding drug/drug interactions. Recent publications revealed a slightly increased risk for non major bleedings compared to LMWH, and caution is advised when used in patients with gastrointestinal tract malignancies, especially upper gastro-intestinal tract malignancies. This review depicts actual considerations for anticoagulation in cancer patients to provide a rational for clinicians before treatment initiation.

Introduction Inflammation induces a procoagulant phenotype of endothelial cells (EC) with the exposure of tissue factor (TF), a potent initiator of the extrinsic coagulation cascade. Although systemic inflammation affects the whole vascular system, thrombotic lesions occur particularly in microcirculation. This raises the question of whether TF-procoagulant activity (TF-PCA) differs between EC from arterial, venous, and microvascular beds. Materials and methods Functional coagulation tests, including TF-PCA, and inflammatory responses were investigated on arterial, venous and microvascular endothelial cells. Interleukin-6 (IL-6) and TF-levels were determined in cohort of 59 septic patients. Results We found that tumor necrosis factor alpha (TNFα), lipopolysaccharide, and interleukin-1β induce a solid, dose-dependent increase in TF-PCA, which is highest in microvascular EC. A positive correlation of interleukin-6 (IL-6) with TF levels was observed in a cohort of 59 septic patients. In contrast, TF-PCA was independent of IL-6 concentrations in vitro. Re-analysis of publicly available gene expression data revealed that among the top 50 genes annotated to coagulation, TF is one of three regulated genes common to the three investigated EC subtypes. The response to inflammatory stimuli in terms of exposure of leukocyte-endothelial- and platelet-endothelial adhesion molecules (E-selectin and PECAM-1), remodeling of adherens junctions, co-exposure of negatively charged surfaces nor breakdown of the glycocalyx was comparable between the EC subtypes and did not explain the higher TF-PCA on microvascular cells. We found that the ratio of TF and TFPI exposure on the endothelial membrane significantly differs between the EC subtypes. Conclusions These findings indicate that the ratio of TF to its inhibitor TFPI is a determinant of endothelial TF-PCA, which is most pronounced on microvascular endothelial cells and might explain why the microvascular system is particularly susceptible to inflammation-induced thrombosis.

Patients with haemophilia A (HA) undergoing neurosurgical procedures have a high risk of haemorrhage with potential fatal outcome. Here, we present a successful perioperative haemostatic concept applying an extended half-life factor VIII (EHL FVIII), Efmoroctocog alfa, in two patients with HA undergoing neurosurgery for paramedian right-sided disc herniation (case 1) and astrocytoma (case 2). After adequate EHL FVIII treatment the surgical procedures were performed without any bleeding complications despite the high-risk interventions. Laboratory measurements confirmed stable FVIII levels throughout the hospital stay. We suggest close interdisciplinary collaboration between involved clinicians as mandatory prerequisite for an optimized perioperative management in patients with HA. The presented cases indicate, that the increased stability, safety and fewer injections provide a rationale to use EHL FVIII products in HA patients undergoing surgical interventions with a very high bleeding risk.

Morbidity and mortality of Philadelphia chromosome–negative myeloproliferative neoplasms (MPNs) are mainly determined by thromboembolic complications. Thrombus formation is facilitated by a neutrophil-specific form of cell death linked to neutrophil extracellular trap (NET) formation (NETosis). Preclinical and clinical data suggested a potential link between NETosis and thrombosis in MPNs. In this study, we aimed to define the impact of NETosis on clinical end points in a large MPN cohort. NETosis was induced in vitro by ionomycin and quantified by enzyme-linked immunosorbent assay–based nucleosome release assays as well as fluorescent staining of free DNA in samples from 103 MPN patients and 28 healthy donors. NETosis rate was correlated with a broad set of clinical data, such as MPN subtype, mutational status, laboratory variables, history of thrombotic events, and treatment types. Triggered NETosis levels were clearly higher in MPN patients than in healthy donors. Positivity for JAK2 V617F or exon 12 as well as CALR mutations correlate with increased NET formation. However, neither JAK2 allelic burden nor history of thromboembolic complication nor the presence of other risk factors for thrombosis (eg, leukocytosis) were associated with the rate of NETosis. In addition, none of the analyzed laboratory parameters nor the type of treatment significantly impacted the rate of NETosis formation. The biology of MPNs has an impact on NET formation because genetic driver mutations favor induction of NETosis, but this does not seems to translate into important clinical end points such as thromboembolic complications. Therefore, NETosis may play a role in facilitating thrombosis, but it is not a sole causative determinant in MPN-associated thrombophilia.

To prevent bleeding in severe haemophilia A [SHA, defined as factor VIII (FVIII) activity

Summary Introduction As a typical consequence of bleeding into muscles and joints, patients with severe hemophilia suffer from acute and chronic pain. In spite of its high prevalence, pain in this patient group is not always sufficiently considered or treated in an effective manner. Aim The recommendations presented in this paper address possible improvements in pain management in hemophilia patients and particularities that have to be taken into account in this patient group. Method The manifold aspects of pain management in hemophilia patients were discussed within the framework of an expert meeting. Based on the available literature and the experts’ clinical experience, the participants developed a set of recommendations presented in this paper. Results Pain management in patients with hemophilia is often insufficient, a fact that not only influences the patients’ quality of life but also implies the risk of difficult to manage chronic pain. Both the prevalent polypharmacy (due to comorbidities) as well as the underlying disease itself present special challenges to pain therapy in this patient group. The present review and recommendations are intended to support medical professionals in recognising the risks of pain chronicity, applying basic principles of multimodal pain therapy, including the options of psychological intervention and modalities of physical medicine in therapy concepts, and reaching a comprehensive understanding of the range of analgesic options available.

Introduction Poly-(ADP)-ribose polymerase (PARP) inhibitors are successfully used for treatment of BRCA-mutated (mut) breast cancers and are under extensive evaluation for BRCA- and PALB2-mutated pancreatic ductal adenocarcinoma (PDAC). However, the optimal treatment regimen for BRCA/PALB2-mutated PDCA has yet to be established. Moreover, limited data are available on the association of BRCA/PALB2 gene alterations with other comutations and immunological biomarkers. Material and methods Tumour samples of 2818 patients with PDAC were analysed for BRCA1/2 PALB2 mutations and other genes by next-generation sequencing (NGS) (MiSeq on 47 genes, NextSeq on 592 genes). TMB was calculated based on somatic non-synonymous missense mutations. MSI-H/dMMR was evaluated by NGS, and PD-L1 expression was determined using immunohistochemistry. Results In 4.2% (n=124) of all PDAC samples BRCA mutations have been detected. BRCA2 mutations were more commonly observed than BRCA1 mutations (3.1%(n=89) vs 1.1% [n=35], p1.0% (BRCA: 21.8% vs wt 11.2%, p

Summary During the annual meeting of the American Society of Hematology (ASH) in San Diego/California, novel developments in the field of hemostaseology were presented. Alternative treatment strategies besides factor replacement were discussed for patients with hemophilia. One of the highlights of the meeting in this year’s plenary session was the presentation of successful adeno-associated virus mediated gene transfer in patients with hemophilia B leading to sustained elevation of factor IX:C (FIX:c). Other alternative treatment approaches in patients with hemophilia A may include bispecific antibodies mimicking factor VIIIa (FVIIIa) activity or disrupting anticoagulant proteins. Focusing on anticoagulation, data on the use of direct oral anticoagulants (DOACs) in cancer patients with atrial fibrillation as well as treatment of superficial vein thrombosis with rivaroxaban were presented. In this short review, we try to highlight the most important presentations during the ASH meeting 2016.

International audience; Venous thromboembolism (VTE) is the second leading cause of death in patients with cancer. These patients are at a high risk of VTE recurrence and bleeding during anticoagulant therapy. The International Initiative on Thrombosis and Cancer is an independent academic working group aimed at establishing a global consensus for the treatment and prophylaxis of VTE in patients with cancer. The International Initiative on Thrombosis and Cancer last updated its evidence-based clinical practice guidelines in 2016 with a free, web-based mobile phone application, which was subsequently endorsed by the International Society on Thrombosis and Haemostasis. The 2019 International Initiative on Thrombosis and Cancer clinical practice guidelines, which are based on a systematic review of the literature published up to December, 2018, are presented along with a Grading of Recommendations Assessment Development and Evaluation scale methods, with the support of the French National Cancer Institute. These guidelines were reviewed by an expanded international advisory committee and endorsed by the International Society on Thrombosis and Haemostasis. Results from head-to-head clinical trials that compared direct oral anticoagulant with low-molecular-weight heparin are also summarised, along with new evidence for the treatment and prophylaxis of VTE in patients with cancer.

Background The current standard of care for patients with hemophilia A is regular prophylaxis with factor VIII (FVIII) administered intravenously. Interest in subcutaneous (s.c.) administration, to potentially increase convenience, reduce the treatment burden and improve compliance, is increasing. Objectives Evaluate the pharmacokinetics (PK), immunogenicity, safety, and preliminary efficacy of s.c. administration of turoctocog alfa pegol (s.c. N8‐GP) in adult or adolescent previously treated patients (PTPs) with severe hemophilia A (alleviate 1; NCT02994407). Patients/Methods In part A, 24 PTPs received a single dose of s.c. N8‐GP (12.5, 25, 50, or 100 IU/kg) with 6 patients per cohort. PK modelling of data from part A supported a suitable dose for part B. Part B comprised a multiple dose trial in 26 PTPs; patients

During the 57th annual meeting of the American Society of Hematology 2015 in Orlando, Florida, various aspects in the field of hemostaseology were presented. The Choosing Wisely® campaign pointed out the importance of the critical use of diagnostic tools to rule out pulmonary embolism and questioned the relevance of thrombophilia testing in women undergoing routine infertility evaluation. Furthermore, the approval of idarucizumab, a specific antidote for the reversal of the anticoagulant effects of the direct thrombin inhibitor dabigatran, was highlighted. Finally, hemophilia specialists awaited the results of the SIPPET Trial, which were presented for the first time during the plenary session. Replacement therapy of previously untreated hemophilia A patients with plasma-derived factor VIII containing von Willebrand factor resulted in a lower incidence of inhibitors compared with patients treated with recombinant factor VIII.

Introduction Poly-(ADP)-ribose polymerase (PARP) inhibitors are successfully used for treatment of BRCA-mutated (mut) breast cancers and are under extensive evaluation for BRCA- and PALB2-mutated pancreatic ductal adenocarcinoma (PDAC). However, the optimal treatment regimen for BRCA/PALB2-mutated PDCA has yet to be established. Moreover, limited data are available on the association of BRCA/PALB2 gene alterations with other comutations and immunological biomarkers.Material and methods Tumour samples of 2818 patients with PDAC were analysed for BRCA1/2 PALB2 mutations and other genes by next-generation sequencing (NGS) (MiSeq on 47 genes, NextSeq on 592 genes). TMB was calculated based on somatic non-synonymous missense mutations. MSI-H/dMMR was evaluated by NGS, and PD-L1 expression was determined using immunohistochemistry.Results In 4.2% (n=124) of all PDAC samples BRCA mutations have been detected. BRCA2 mutations were more commonly observed than BRCA1 mutations (3.1%(n=89) vs 1.1% [n=35], p1.0% (BRCA: 21.8% vs wt 11.2%, p

Introduction ABO blood group affects levels of von Willebrand Factor (VWF) and Factor (F) VIII, as individuals with blood group non-O have higher levels of VWF and FVIII. To the best of our knowledge there is no data available regarding the association between blood group and FVIII in non-severe haemophilia. Aim We aimed to explore the influence of ABO blood group on VWF and FVIII levels in patients with non-severe haemophilia A (HA). Methods We recruited persons with non-severe HA (FVIII levels of 1-40%). Adult patients (age ≥ 18 years), with platelet count > 105 /µl, without impaired renal and hepatic function, active cancer, surgery within the last 6 weeks, or overt infection within the last 2 weeks, who gave their informed consent were included. A blood sample was collected for laboratory analysis. The diagnosis of haemophilia was confirmed by molecular analysis of the FVIII gene in all patients. None of the patients had inhibitors against FVIII at the time of sample collection. The lowest FVIII level ever measured in each patient's history served as basis for the assessment of severity. Healthy male persons with a median age comparable to the patient group served as controls. We determined the following parameters: ABO blood group, FVIII, VWF activity (VWF:Act), and VWF antigen (VWF:Ag). FVIII was measured with a one-stage assay (Sysmex-CA7000 - Sysmex, Kobe, Japan, in multi-dilution mode with native FVIII-deficient plasma - Technoclone, Vienna, Austria and Aktin FS activator - Siemens, Marburg, Germany), VWF:Act (BC von Willebrand Reagent or Innovance VWF Ac - Siemens Healthcare, Marburg, Germany) and VWF:Ag levels with a latex agglutination assay (STA LIATEST VWF, Diagnostica Stago). Comparison of VWF and FVIII levels between blood group O and non-O was calculated with Mann-Whitney-U test, correlation between FVIII and VWF levels was calculated with Spearman's correlation, association of FVIII and VWF separated for blood group was calculated with univariable linear regression. Association of FVIII with VWF and blood group was calculated using multivariable linear regression. All calculations were performed with SPSS (IBM Version 25.0). Results A total of 89 persons with HA (71 with mild and 18 with moderate haemophilia) and 82 healthy controls were included. Patient characteristics are listed in Table 1. Median levels of VWF were significantly higher both in patients (p=0.002 for both VWF:Ag and VWF:Act) and healthy controls (p In HA there was no correlation between FVIII and VWF:Act (rho=0.180, p=0.095) or between FVIII and VWF:Ag (rho=0.028, p=0.795, Table 3). In univariable linear regression there was no significant association between VWF and FVIII in HA, neither in those with blood group non-O nor in those with blood group O (Figure 1). Also in multivariable linear regression there was no significant association between VWF (p=0.632) and blood group (p=0.929) with FVIII in patients with HA. In healthy controls, there was a strong positive correlation between FVIII and VWF:Act (rho=0.751, p Conclusions Neither the blood group nor the VWF had an influence on FVIII levels in non-severe HA patients. We conclude that the impact of the genetic mutation of the FVIII gene by far outweighs the influence of VWF levels and the blood group. Thus, for the diagnosis of HA and for determination of HA severity, the blood group needs not to be taken into account. Disclosures Pabinger: Roche: Honoraria; Sobi: Research Funding; Novo Nordisk: Research Funding; CSL Behring: Research Funding; Pfizer: Honoraria; Shire: Honoraria; Bayer: Honoraria; Sobi: Honoraria.

Background Microvesicles (MV) are extracellular vesicles known to be associated with cellular activation and inflammation. Hemofiltration is an effective blood purification technique for patients with renal failure and possibly also eliminates inflammatory mediators in the setting of sepsis. On the other hand, proinflammatory stimuli are induced by blood contacting the artificial membrane during extracorporeal blood purification. In chronic dialysis patients a systemic increase in MV has been described. The aim of the study was to investigate whether hemofilter passage of blood in continuous veno-venous hemofiltration (CVVH) alters MV composition and levels in critically ill patients with sepsis. Methods Pre- and postfilter bloods as well as ultrafiltrate samples from intensive care unit patients with severe sepsis were obtained during CVVH with regional citrate anticoagulation. MV subtypes in blood were analyzed by high-sensitivity flow cytometry. Additionally, tissue factor (TF) levels and MV-associated TF activities as well as MV activities were quantified. All parameters were corrected for hemoconcentration applied during CVVH. Results Twelve patients were analyzed. A significant increase in presumably mostly leukocyte-derived CD31+/CD41− MV (1.32 (1.09–1.93)-fold [median (25th–75th quartiles)], p = 0.021) was observed post- to prefilter, whereas platelet-derived MV as well as AnnexinV-binding MV were unaltered. Increments of AnnexinV+, CD42b+ and CD31+/CD41− MV post- to prefilter correlated with filtration fraction (FF) (all p

Attraction of neutrophils to sites of infection or tissue injury is an essential prerequisite for an efficient innate immune response. Herein, we provide novel evidence that the antimicrobial protein, neutrophil gelatinase associated lipocalin (24p3 or lipocalin-2, Lcn2) is a central regulator of this process. Lcn2 is produced by several cell types but high amounts are released by neutrophils. Using human and murine neutrophils, we found that the addition of recombinant Lcn2 significantly stimulated their migration, which was independent of IL-8/keratinocyte chemokine formation. Mechanistically, this could be traced back to Lcn2-mediated changes of Erk1/2 signaling. Accordingly, the i.p. injection of Lcn2 into C57BL/6 mice stimulated the mobilization of neutrophils while we found a significantly reduced neutrophil chemotactic activity of cells obtained from Lcn2 KO mice. This observation transmitted to a reduced accumulation of neutrophils in intra-dermal lesions infected with Salmonella typhimurium in Lcn2 KO mice as compared to WT mice. This was not only due to a reduced chemotaxis but also to an impaired cellular adhesion of neutrophils in the absence of Lcn2. We herein describe a novel role of Lcn2 as an important paracrine chemoattractant and an indispensable factor for neutrophil function in inflammation.

Essential thrombocythemia (ET) patients are at risk of developing thrombotic events. Qualitative platelet (PLT) abnormalities and activation of endothelial cells (ECs) and PLTs are thought to be involved. Microparticles (MPs) can originate from PLTs (PMPs), ECs (EMPs), or red cells (RMPs). Previous studies have indicated that MPs contribute to ET pathophysiology. Endothelial modulators (eg, nitric oxide [NO], adrenomedullin [ADM], and endothelin-1 [ET-1]) are also involved in the pathophysiology of this condition. We hypothesized that treatments for reducing PLT count might also indirectly affect MP generation and endothelial activity by altering endothelial modulator production. The rationale of this study was that hydroxyurea (HU), a cytostatic drug largely used in ET, induces the production of a potent vasoactive agent NO in ECs. An observational retrospective study was designed to investigate the relationship between MPs, NO, ADM, and ET-1 in ET patients on treatment with HU, anagrelide (ANA), aspirin (ASA), and a group of patients before treatment. A total of 63 patients with ET diagnosis: 18 on HU + ASA, 15 on ANA + ASA, 19 on ASA only, and 11 untreated patients, and 18 healthy controls were included in this study. Blood samples were analyzed for MP (absolute total values) and functional markers (percentage values) by flow cytometry. PLT-derived MPs were studied using CD61, CD62P, CD36, and CD63, whereas endothelial-derived MPs were studied using CD105, CD62E, and CD144. Endothelial modulator markers (NO, ADM, and ET-1) were measured by ELISA. Total MP count was higher in the group treated with ANA + ASA (P 0.01). MP markers modified in ET patients returned to levels of healthy controls following treatment, in particular, in patients on ANA treatment. NO and ADM values were higher in the HU group (P 0.001). HU and ANA treatment also affected MP production in a cell origin-specific manner. HU and ANA, although acting via different pathways, have similar final effects. For instance, HU causes vasodilatation by increasing NO and ADM levels, whereas ANA impairs vasoconstriction by reducing ET-1. In conclusion, therapy with HU cytostatic drugs and ANA can reduce PLT count in ET, and also affect endothelial modulatory agents, with HU sustaining vasodilation and prothrombotic MP concentration, whereas ANA decreases vasoconstriction.

PURPOSE Endothelial pathology is considered to play a key role in septic shock. Since endothelial-derived microvesicles (MV) are elevated in various diseases associated with endothelial pathology, they are considered surrogate markers of the endothelial state. By analyzing the signature of circulating MV with high-sensitivity flow cytometry (hsFC), we wanted to test the hypothesis whether endothelial-derived MV are increased in septic shock. METHODS MV in blood from healthy volunteers and patients with septic shock treated in a medical intensive care unit were quantified by hsFC, which has an improved detection limit of approximately 0.3 μm. RESULTS Patients with septic shock (n = 30) showed 3-fold higher levels of CD31+/CD41- MV (58.5 (26.4-101.2) [median (25th-75th percentile)] vs. 19.5 (12.8-25.4) MV/μL; P

SummaryProtease activated receptor-1 (PAR1) mediates barrier protective signalling of activated protein C (APC) in human endothelial cells in vitro and may contribute to APC’s beneficial effects in patients with severe sepsis. Mouse models are of key importance for translational research but species differences may limit conclusions for the human system. We analysed whether mouse APC can cleave, activate and induce signalling through murine PAR1 and tested in newly established mouse models if long-term infusion of APC prevents from vascular leakage. Cell surface immunoassays demonstrated efficient cleavage of endogenous murine endothelial PAR1 by either murine or human APC. Pharmacological concentrations of APC of either species had powerful barrier protective effects on cultured murine endothelial cells that required PAR1 cleavage. Vascular endothelial growth factor-mediated hyperpermeability in the skin was reduced by either endogenously generated as well as directly infused recombinant mouse APC in wild-type mice. However APC did not significantly alter the vascular barrier function in PAR1-deficient mice. In endotoxin-challenged mice, infused APC significantly prevented from pulmonary fluid accumulation in the wild-type mice but not in mice lacking PAR1. Our results directly show that murine APC cleaves and signals through PAR1 in mouse endothelial cells. APC reduces vascular permeability in mouse models and PAR1 plays a major role in mediating these effects. Our data in vitro and in vivo support the paradigm that PAR1 contributes to protective effects of APC on vascular barrier integrity in sepsis.

Akacid medical formulation (AMF) is an oligoguanidine that exerts biocidal activity against airborne and surface microorganisms including bacteria, viruses, fungi, and molds, while showing relatively low toxicity to humans. We have previously shown that AMF exerts antiproliferative effects on a variety of solid tumor cell lines. In this study we raised the question whether AMF could also substantially inhibit cell growth or induce apoptosis in cell lines derived from hematologic malignancies such as leukemia or lymphoma. We found that AMF has antiproliferative effects on various hematologic cell lines derived from human leukemia and lymphoma. Additionally, we show that AMF induces apoptosis in leukemia cell lines not only via the extrinsic and intrinsic pathway, but also in a caspase-independent manner. This effect was found also in G0-arrested cells. Finally, in our animal experiments utilizing male nu/nu Balb/c mice we found a significant growth retardation, which was immunohistochemically associated with a significantly lower number of KI67-positive cells and caspase-3 induction in AMF-treated mice.

Chronic inflammation is characterized by tissue infiltration with monocytes/macrophages, which possess broad proinflammatory, destructive, and remodeling capacities. Elevated levels of osteoprotegerin, an important regulator of differentiation and activation of osteoclasts that also affects different cells of the immune system, were found in the serum of patients with chronic inflammatory diseases. The study of whether osteoprotegerin affects monocyte locomotion in vitro and the possible mechanisms and pathways involved was investigated using Boyden microchemotaxis chambers and Western blot analyses. Osteoprotegerin significantly stimulated monocyte chemotaxis, whereas preincubation of monocytes with osteoprotegerin inhibited monocyte migration toward optimal concentrations of regulated upon activation normal T cell expressed and secreted, monocyte chemotactic protein -1, and procalcitonin. The effects of osteoprotegerin were abolished by pretreating cells with heparinase I and chondroitinase or antibodies against the ectodomain of syndecan-1. Osteoprotegerin signaling was shown to involve protein kinase C, phosphatidylinositol 3-kinase/Akt, and tyrosine kinase. Data suggest that osteoprotegerin affects monocyte mi-gration and protein kinase C and phosphatidylinositol 3-kinase/Akt activation via syndecan-1. Osteoprotegerin-induced deactivation of monocyte chemotaxis toward different chemokines is due to interaction of osteoprotegerin with heparan sulfate and chondroitin sulfate.

Background Interaction of fibrinogen with specific leukocyte integrins of monocytes may link coagulation and inflammation, however, the precise mechanism of fibrinogen leading to the pro-inflammatory and pro-coagulatory response on monocytes is yet unknown. Results Fibrinogen and its digestion fragment D induced pro-coagulant activation of monocytes as assessed in a cellular coagulation assay by reductions in clotting times. Pro-coagulant activation was reversed by blocking antibodies against Mac-1 or LFA-1. Pre-exposure of monocytes to the p38 MAPK inhibitor SB 202190 and the MEK1.2 inhibitor U0126 led to significant increasees in coagulation times whereas blocking JNKII with its inhibitor had no such effect. Blocking NFκB with MG-132 also inhibited pro-coagulant activation of monocytes by fibrinogen. A selective inhibitor of matrix metalloproteinase-9 increased times to clot formation whereas other matrix metalloproteinase inhibitors did not significantly interfere with fibrinogen-augmented clot formation in this assay. Treatment of monocytes with fibrinogen increased concentrations of matrix metalloproteinase-9 immunoreactivity in their supernatants. Conclusions Fibrinogen induces monocyte pro-coagulant activation in an integrin-, nuclear factor κB-, p38 MAPK-, and MEK1.2-dependent manner. Activation of monocytes by fibrinogen increases metalloproteinase-9 secretion, metalloproteinase-9 itself enhances monocyte coagulation by an autocrine mechanism. Results provide further evidence that mediators of hemostasis have a profound impact on cells of the immune system and are closely related to inflammatory pathways.

The protein C pathway is an important regulator of the blood coagulation system. Protein C may also play a role in inflammatory and immunomodulatory processes. Whether protein C or activated protein C affects lymphocyte migration and possible mechanisms involved was tested. Lymphocyte migration was studied by micropore filter assays. Lymphocytes that were pretreated with protein C (Ceprotin) or activated protein C (Xigris) significantly reduced their migration toward IL-8, RANTES, MCP-1, and substance P, but not toward sphingosine-1-phosphate. The inhibitory effects of protein C or activated protein C were reversed by Abs against endothelial protein C receptor and epidermal growth factor receptor. Evidence for the synthesis of endothelial protein C receptor by lymphocytes is shown by demonstration of receptor mRNA expression and detection of endothelial protein C receptor immunoreactivity on the cells’ surface. Data suggest that an endothelial protein C receptor is expressed by lymphocytes whose activation with protein C or activated protein C arrests directed migration. Exposure of lymphocytes to protein C or activated protein C stimulates phosphorylation of Tyr845 of epidermal growth factor receptor, which may be relevant for cytoprotective effects of the protein C pathway.

Summary. Coagulation and inflammation are intimately linked and cellular signaling by coagulation proteases through protease-activated receptors (PARs) may affect pro- and anti-inflammatory responses. Permeability of the endothelial cell barrier at the blood–tissue interface plays a key role in inflammatory disorders such as sepsis. We have recently shown that PAR1 signaling by activated protein C or low concentrations of thrombin can enhance endothelial barrier integrity. In the present study, we analyzed effects of coagulation factor Xa (FXa), which is known to activate both endothelial cell PAR1 and PAR2, on monolayer integrity using a transformed human umbilical vein endothelial cell (HUVEC) line in a dual-chamber system. Preincubation with FXa potently reduced high-dose thrombin-mediated hyperpermeability and basal permeability. FXa was protective at concentrations of 5 nm or higher and proteolytic activity was required. Barrier protective FXa signaling was not affected by cleavage-blocking anti-PAR1 antibodies or by a PAR1 antagonist. Similarly, cleavage-blocking anti-PAR2 alone had no effect, but blocking both PAR1 and PAR2 inhibited barrier protection by FXa. Incubation of the cell layer with a PAR2-specific agonist peptide reduced thrombin-mediated hyperpermeability and basal permeability similar to FXa. In conclusion, not only PAR1, but also PAR2 can mediate barrier protection in endothelial cells and FXa can use either receptor to enhance barrier integrity. Although it is currently unknown whether PAR signaling by FXa has a physiological role, the results suggest a potential protective effect of FXa and other agonists of endothelial PAR2, which should be explored in models of local and systemic inflammation in vivo.

Endothelial cells normally form a dynamically regulated barrier at the blood-tissue interface, and breakdown of this barrier is a key pathogenic factor in inflammatory disorders such as sepsis. Pro-inflammatory signaling by the blood coagulation protease thrombin through protease activated receptor-1 (PAR1) can disrupt endothelial barrier integrity, whereas the bioactive lipid sphingosine 1-phosphate (S1P) recently has been demonstrated to have potent barrier protective effects. Activated protein C (APC) inhibits thrombin generation and has potent anti-inflammatory effects. Here, we show that APC enhanced endothelial barrier integrity in a dual-chamber system dependent on binding to endothelial protein C receptor, activation of PAR1, and activity of cellular sphingosine kinase. Small interfering RNA that targets sphingosine kinase-1 or S1P receptor-1 blocked this protective signaling by APC. Incubation of cells with PAR1 agonist peptide or low concentrations of thrombin (∼ 40 pM) had a similar barrier-enhancing effect. These results demonstrate that PAR1 activation on endothelial cells can have opposite biologic effects, reveal a role for cross-communication between the prototypical barrier-protective S1P and barrier-disruptive PAR1 pathway, and suggest that S1P receptor-1 mediates protective effects of APC in systemic inflammation.

Albumin dialysis has been shown to improve the outcome in patients with cholestatic liver failure caused by chronic liver disease. This study reports 7 liver transplant recipients who were treated with albumin dialysis for intractable pruritus of different origin (ductopenic graft rejection, non-anastomotic strictures, and recurrence of hepatitis C). Treatment with histamine (H1) blockers, opioid antagonists, and cholestyramine had not been effective. The Molecular Adsorbent Recirculating System (MARS; Teraklin, Rostock, Germany) was used for albumin dialysis. All patients presented with numerous scratch marks, 6 of whom had a pronounced icterus. Six patients (86%) responded to 3 consecutive treatments with significant reduction of pruritus. The mean pruritus score, which was quantified by a visual analog scale (VAS), decreased from 9.7 +/- 0.5 to 3.7 +/- 0.8 (SD). The mean duration of 1 treatment was 15.6 hours. The procedure was well tolerated by all patients. The mean total serum bilirubin in patients who responded to therapy declined from 19.11 +/- 16.96 mg/dL (SD) before NLARS therapy to 9.24 +/- 3.52 mg/dL after treatment. The mean serum concentration of 3alpha-hydroxy bile acids decreased from 192.67 +/- 58.12 mumol/L (SD) to 42.33 +/- 31.58 mumol/L (SD). Follow-up in 3 cases showed sustained improvement of pruritus lasting for more than 3 months. In 3 patients, however, pruritus relapsed. One patient, who showed severe pruritus, without relevant elevation of serum bile acids before treatment, did not respond to albumin dialysis. Our data indicate that MARS is an effective therapeutic option for patients with intractable cholestatic pruritus.

Vascular endothelial growth factor (VEGF) is highly expressed in the airway of patients with asthma. Whether VEGF affects eosinophil function in vitro and if VEGF receptors are involved was tested. Eosinophils were from venous blood of healthy donors. Cell migration was studied by micropore filter assays. Signaling mechanisms required for VEGF-dependent migration were tested using signaling enzyme blockers. Expression of flt-1 and KDR/flk-1 mRNA in eosinophils was demonstrated in reverse transcriptase-polymerase chain reaction, and receptor expression was investigated by fluorescence-activated cell sorting analysis. Eosinophil cationic protein release was measured in eosinophil supernatants by enzyme-linked immunosorbent assay. VEGF significantly stimulated eosinophil chemotaxis via activation of protein kinase C and phosphatidylinositol 3'-kinase. The effect on migration was reversed by an antibody against VEGF receptor flt-1, but not by an antibody against KDR/flk-1. Expression of VEGF receptor flt-1 mRNA was shown and synthesis of VEGF receptor in eosinophils is suggested by detection of VEGF receptor immunoreactivity on the cell surface. Data suggest that VEGF receptor flt-1 is expressed by eosinophils whose activation with VEGF stimulates directed migration and release of eosinophil cationic protein. Thus, VEGF may play an important role in the modulation of eosinophilic inflammation.

Activation of protein C by thrombin bound to thrombomodulin is enhanced by endothelial protein C receptor. This pathway may inhibit inflammation. We investigated effects of protein C and activated protein C on neutrophils as well as whether an endothelial protein C receptor is involved in mediating protein C effects. Neutrophils were from venous blood of healthy donors. Cell migration, respiratory burst, phagocytic activity, and apoptosis were studied by micropore filter assays and fluorometry. Receptor expression was investigated by reverse transcriptase–polymerase chain reaction (PCR) for mRNA, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of immunoprecipitated receptor protein, and fluorescence-activated cell-sorter scanner (FACS) analysis using the anti–endothelial protein C receptor antibody RCR-252. Neither protein C nor activated protein C induced migration, yet both of them inhibited neutrophil chemotaxis triggered by interleukin-8, formyl-Met-Leu-Phe, antithrombin, or C5a. A protein C activation–blocking antibody against endothelial protein C receptor diminished inhibitory effects of protein C or activated protein C on migration. No effect of either protein C preparation was seen in neutrophil's respiratory burst, bacterial phagocytosis, or apoptosis assays. Endothelial protein C receptor immunoreactivity was confirmed on neutrophils by FACS. De novo synthesis is suggested by endothelial protein C receptor mRNA expression as demonstrated by reverse transcriptase PCR and immunoprecipitation SDS-PAGE analyses. Data suggest that an endothelial protein C receptor is expressed by human neutrophils whose active site ligation with either protein C or activated protein C arrests directed cell migration. Inhibitory effects of these components of the protein C pathway on neutrophil function may play a role in the protein C–based treatment of severe sepsis.

Protein C (PC) is activated by thrombomodulin-bound thrombin on the endothelial cell surface and activated protein C (APC) inhibits blood coagulation in a negative feedback loop. Endothelial PC receptor (EPCR) can bind PC/APC and activation of EPCR-bound PC is enhanced. Exogenous APC has barrier protective effects on endothelial cells that depend on EPCR binding and protease activated receptor-1 (PAR1) cleavage and that may contribute to the anti-inflammatory effects of APC. Plasma APC concentrations in vivo are low compared to the substrate PC and in order to induce protective signaling exogenous APC has to compete with PC for EPCR binding. In this study we investigated whether the endogenous PC activation pathway may be linked to efficient protective responses analyzing endothelial barrier permeability in a dual chamber system. When endothelial EA.hy926 cells were incubated for 3 h in the presence of 80 nM purified PC and different concentrations of thrombin a dose-dependent linear increase of APC activity in the cell medium was observed over time. APC generation was detectable upon incubation with 20 pM thrombin or higher and a significant barrier protective response to 20 pM thrombin was found only in the presence of PC. 40 pM thrombin enhanced barrier integrity in the presence and absence of PC, consistent with our previous results. To exclude direct thrombin effects on endothelial permeability and to compare protective effects of exogenous and endogenously generated APC, we used the anticoagulant double mutant thrombin W215A/E217A (WE). WE was about 10 times less active than wildtype thrombin for PC activation in our system. However, PAR1-dependent induction of MAP kinase phosphorylation required more than 1000-fold higher concentrations of the thrombin mutant. Thus, 1–10 nM WE leads to APC generation without directly inducing PAR1-dependent signaling. When cells were incubated with various concentrations of exogenous APC or WE+80 nM PC, barrier protective effects of 5 nM exogenous APC and 2 nM WE+80 nM PC (1.3 nM APC generated after 3 h) were similar. Because APC is generated at a constant rate during the incubation period, the average concentration of generated APC in the cell medium was only about 0.65 nM, suggesting that signaling by endogenously generated APC was significantly more efficient. To conclusively demonstrate that protective effects in response to WE are mediated by APC generation, we used recombinant zymogen wildtype PC and a PC variant with a substitution of the active site serine with alanine (PC S360A). Cells were incubated with control or 80 nM wildtype PC and PC S360A, in the presence or absence of WE (4 nM) and exogenous APC (3.3 nM). WE induced protective signaling only in the presence of wildtype PC but not PC S360A. Barrier protective effects of exogenous APC were blocked by both wildtype PC and PC S360A, consistent with their expected role as competitive inhibitors for APC binding to EPCR. These data demonstrate that efficient barrier enhancement by APC is indeed mechanistically coupled to the PC activation pathway. Signaling by endogenously generated APC may play an important role in the regulation of inflammation.

Thrombin initiates fibrin formation and platelet activation, and activates protein C, generating activated protein C (APC) that inhibits blood coagulation by a negative feedback loop. Thrombin has also proinflammatory effects through activation of cellular protease activated receptor-1 (PAR1). Endothelial cell protein C receptor (EPCR) can bind both protein C and APC and activation of EPCR-bound protein C is enhanced. Results from animal models and clinical trials indicate that APC has potent protective effects in systemic inflammation that are independent from its well established anticoagulant function and recombinant APC was recently approved to treat patients with severe sepsis. The molecular basis for APC’s anti-inflammatory effects is incompletely understood. We have identified PAR1 and EPCR as part of a novel APC signaling pathway in quiescent endothelial cells, raising the question how the same receptor PAR1 can mediate both pro- and anti-inflammatory effects. In an overexpression system in PAR-deficient fibroblasts, wildtype PAR2 but not a PAR2 variant with an Arg36 to Ala substitution at the P1 position was activated by APC, indicating that APC can activate PAR2 in addition to PAR1 through a canonical cleavage mechanism. Therefore, we tested whether endothelial cell PAR2 can be activated by APC under conditions where endogenous PAR2 expression is upregulated. Even when PAR2 expression was highly upregulated in inflammatory cytokine-stimulated human umbilical endothelial cells (HUVECs), signaling by APC was strictly dependent on PAR1 cleavage and signaling. Consistent with these results in HUVECs, intravenous injection of APC in wildtype, PAR1−/−, and PAR2−/− mice demonstrated that PAR1 is the major murine receptor that mediates induction of the transcript for monocyte chemoattractant protein-1 in the lung in response to APC. This indicates that indeed the same receptor PAR1 mediates signaling by APC and thrombin both in vitro and in vivo. To test the possibility that APC diminishes proinflammatory thrombin-PAR1 signaling by downregulating cellular levels of functional PAR1, we tested whether preincubation with APC can desensitize Erk1/2 phosphorylation by thrombin. Phospho-Erk1/2 was induced by APC dependent upon PAR1 cleavage, but APC-pretreated cells still responded to PAR1-dependent thrombin signaling, suggesting that only a fraction of the cellular PAR1 is subject to cleavage by APC. These results indicate that APC does not block thrombin signaling by desensitation at the receptor level. Large-scale gene expression profiling demonstrated that APC and thrombin had specific effects on gene expression in tumor necrosis factor α (TNFα )-perturbed endothelial cells that were not detected in quiescent cells. Transcripts for several proapoptotic genes including p53 and thrombospondin-1 were downregulated by APC but not by thrombin or PAR1 agonist peptides in TNFα-stimulated HUVECs. Western blotting confirmed that in TNFα-perturbed HUVECs pretreatment with APC significantly reduced the increase in cellular p53 protein levels in response to the cytotoxic doxorubicin. This APC effect was dependent on EPCR binding and PAR1 cleavage. Thrombospondin-1 protein levels were similarly downregulated by APC but upregulated by thrombin. Both down- and upregulation by APC and thrombin, respectively, were PAR1 dependent. These findings demonstrate that the same receptor on the same cell type can mediate opposite biological effects and they suggest that EPCR cosignaling may modify PAR1-dependent APC signaling in endothelial cells.