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Background: Chronic thromboembolic pulmonary hypertension (CTEPH) is a life-threatening condition and rare complication of acute pulmonary embolism. Mechanisms underlying impaired clot resolution and in sustained fibrothrombotic obstruction of the pulmonary arterial bed remain poorly understood. Since defective angiogenesis correlated to defective clot resolution based on observations in surgical material from patients with CTEPH, we aimed to validate its crucial pathogenic role by intrathrombus inhibition of angiogenesis in a novel CTEPH rabbit model. Methods: We aimed to compare whether intrathrombus administration of an antifibrinolytic agent, tranexamic acid, or an inhibitor of angiogenesis, SU5416, would contribute to CTEPH progression. Both products were administered on a weekly basis by autologous clot embolization in rabbits. Right ventricular pressure was monitored by telemetry, right ventricular function by transthoracic echocardiography, and a complete pulmonary hemodynamic evaluation was obtained through right heart catheterization. Markers of inflammation, endothelial dysfunction, heart failure, and fibrinolysis were measured in plasma. Pulmonary vessel remodeling was analyzed by immunohistochemistry. Results: Impairing intrathrombus angiogenesis by repeatedly embolizing autologous blood clots containing SU5416 resulted in elevated mean pulmonary arterial pressure (38 mm Hg), increased indexed pulmonary vascular resistance, and enhanced right ventricular hypertrophy (80%, 1.9-fold, 36%, respectively, compared with rabbits embolized with clots containing an antifibrinolytic agent). This was caused by both obstruction of large pulmonary arteries with fibrothrombotic material and muscularization of pulmonary microvessels, and accompanied by inflammatory cell infiltration and increased circulating endothelin-1. Conclusions: The key role of angiogenesis-driven clot resolution was validated in a reliable small-animal model reproducing the major pathophysiological hallmarks of CTEPH.

The splenic endothelial Weibel-palade bodies are one of the most important candidate organelles to release von Willebrand factor upon stimulation with desmopressin. However, the presence of functional desmopressin-specific receptor has not yet been demonstrated on endothelial cells. Experimental evidences are in favour of an indirect pro-haemostatic effect of desmopressin, but the exact mediator and its cellular origin are largely elusive. Here, we report partially hampered desmopressin response in a splenectomised severe haemophilia A/Beta Thalassemia patient without any genetic variant relevant to his incomplete desmopressin response. To further investigate the role of the spleen in this phenomenon, the release of VWF from desmopressin-treated human splenic endothelial cells was assessed in vitro. As a result, desmopressin induced the release of VWF from endothelial cells when the cells were co-cultured with non-classical (CD14

BACKGROUND: Coagulation factors may inform on liver function during normothermic machine perfusion (NMP). We investigated whether graft ischemic injury impairs the accumulation of anticoagulation factors during NMP of porcine and human livers. METHODS: Dynamics of FV, FVII, FVIII, FIX, and FX during NMP and their correlation with graft injury was investigated in porcine livers with minimal (no warm ischemia, n = 5) or severe injury (60 min warm ischemia, n = 5). Next, FV, FVIII, FIX, fibrinogen, and antithrombin were measured in 35 matched human liver NMPs from the COPE trial. Correlation of these factors with outcomes was explored. Livers were categorized in to 4 groups depending on donor type and posttransplant peak aspartate aminotransferase (AST) as surrogate of minimal (peak < 500 IU/L) or moderate injury (peak > 1000 IU/L). RESULTS: Factor concentrations increased significantly during NMP regardless of severity of injury. In porcine livers, factor concentrations were 2- to 6-fold lower in severely injured grafts (all P < 0.05). All factors negatively correlated with AST (coefficient range: from -0.50 to -0.93; all P < 0.05) and lactate (range: from -0.51 to -0.67; all P < 0.05). In human livers, no difference in factor accumulation rates and no correlation with other markers were observed. One graft with primary nonfunction had low rate of factor accumulation. CONCLUSIONS: Anticoagulation factors accumulate during NMP regardless of donor type and severity of injury. In pigs, severe ischemic injury resulted in significantly lower factor concentrations. In human livers with life-sustaining function, they do not correlate with hepatic injury. Whether low concentrations predict nonfunction in high-risk livers with severe injury requires further investigation. ispartof: TRANSPLANTATION vol:106 issue:3 pages:510-518 ispartof: location:United States status: published

The most challenging complication associated with Factor VIII (FVIII) replacement therapy is the development of neutralizing anti-drug antibodies, or inhibitors, which occur in 23-35% of severe (FVIII level via neonatal Fc receptor (FcRn) binding, other Fc-mediated interactions, including engagement with Fc gamma receptors (FcγR), may have immunological consequences. Several case reports and retrospective analyses suggest that rFVIIIFc offers superior outcomes with respect to ITI compared to other FVIII products. Previously we and others demonstrated rFVIIIFc interactions with activating FcγRIIIA/CD16. Here, we investigated if rFVIIIFc activates natural killer (NK) cells via CD16. We demonstrated rFVIIIFc signaling via CD16 independent of Von Willebrand Factor (VWF):FVIII complex formation. We established that rFVIIIFc potently activated NK cells in a CD16-dependent fashion resulting in IFNγ secretion and cytolytic perforin and granzyme B release. We also demonstrated an association between rFVIIIFc-mediated NK cell IFNγ secretion levels and the high-affinity (158V) CD16 genotype. Furthermore, we show that rFVIIIFc-activated CD16+ NK cells were able to lyse a B-cell clone (BO2C11) bearing an anti-FVIII B-cell receptor in an antibody-dependent cellular cytotoxicity (ADCC) assay. These in vitro findings provide an underlying molecular mechanism that may help explain clinical case reports and retrospective studies suggesting rFVIIIFc may be more effective in tolerizing HA patients with anti-FVIII inhibitors compared to FVIII not linked to Fc. Our in vitro findings suggest a potential use of Fc-fusion proteins acting via NK cells to target antigen-specific B-cells, in the management of unwanted immune responses directed against immunogenic self-antigens or therapeutic protein products.

Background Apixaban, a direct oral anticoagulant inhibiting factor Xa, has been proven to reduce the risk of atrial fibrillation-related stroke and thromboembolism in patients with mild to moderate renal insufficiency. Patients on renal replacement therapy, however, were excluded from randomized controlled trials. Therefore, uncertainty remains concerning benefits, dosing and timing of intake in haemodialysis population. Methods We conducted a Phase II pharmacokinetics study in which 24 patients on maintenance haemodialysis were given a single dose (2.5 mg or 5 mg) of apixaban, either 30 min before or immediately after dialysis on the mid-week dialysis day. Results Apixaban 5 mg resulted in higher area under the curve (AUC0–48) in comparison with 2.5 mg, although significance could only be reached for dosing pre-dialysis (2.5 mg versus 5 mg, P = 0.008). In line, peak concentrations (Cmax) after dosing pre-dialysis were significantly higher in the 5 mg than in the 2.5 mg groups (P = 0.02). In addition, dialysis resulted in significant reduction of drug exposure. AUC0–48 pre-dialysis were on average 48% (2.5 mg) and 26% (5 mg) lower than the AUC0–48 post-dialysis, in line with Cmax. As a result, a dose of 2.5 mg post-dialysis and a dose of 5 mg pre-dialysis resulted in similar AUC0–48. In contrast, significant differences were found between the 5 mg group post-dialysis and the 2.5 mg group pre-dialysis (P = 0.02). Conclusions Our data suggest that exposure to apixaban in patients on maintenance haemodialysis is dependent not only on drug dose but also on timing of intake relative to the haemodialysis procedure.

Background Venous thromboembolism (VTE) is a frequent complication of COVID-19, so that the importance of adequate in-hospital thromboprophylaxis in patients hospitalized with COVID-19 is well established. However, the incidence of VTE after discharge and whether postdischarge thromboprophylaxis is beneficial and safe are unclear. In this prospective observational single-center study, we report the incidence of VTE 6 weeks after hospitalization and the use of postdischarge thromboprophylaxis. Methods Patients hospitalized with confirmed COVID-19 were invited to a multidisciplinary follow-up clinic 6 weeks after discharge. D-dimer and C-reactive protein were measured, and all patients were screened for deep vein thrombosis with venous duplex-ultrasound. Additionally, selected high-risk patients received computed tomography pulmonary angiogram or ventilation–perfusion (V/Q) scan to screen for incidental pulmonary embolism. Results Of 485 consecutive patients hospitalized from March through June 2020, 146 patients were analyzed, of which 39% had been admitted to the intensive care unit (ICU). Postdischarge thromboprophylaxis was prescribed in 28% of patients, but was used more frequently after ICU stay (61%) and in patients with higher maximal D-dimer and C-reactive protein levels during hospitalization. Six weeks after discharge, elevated D-dimer values were present in 32% of ward and 42% of ICU patients. Only one asymptomatic deep vein thrombosis (0.7%) and one symptomatic pulmonary embolism (0.7%) were diagnosed with systematic screening. No bleedings were reported. Conclusion In patients who had been hospitalized with COVID-19, systematic screening for VTE 6 weeks after discharge revealed a low incidence of VTE. A strategy of selectively providing postdischarge thromboprophylaxis in high-risk patients seems safe and potentially effective.

Introduction Contact of blood with artificial surfaces such as mechanical support devices, catheters, and mechanical heart valves activates the contact activation (CA) pathway of coagulation. Furthermore, recent animal data and clinical studies suggest a more important contribution of CA in pathological thrombus formation in other cardiovascular diseases. Direct oral anticoagulants (DOACs) are recommended as first-line treatment in most patients who require long-term anticoagulation. However, because DOACs directly inhibit a single downstream coagulation factor (thrombin (fXIIa) or factor Xa (fXa)), it has been suggested that their efficacy could be reduced in the presence of strong activation of the CA pathway as compared to anticoagulants that target multiple, more upstream located coagulation factors. Purpose To compare the efficacy of a DOAC (apixaban) and heparin to suppress thrombin generation in the presence of strong CA pathway activation. Methods Pooled platelet-poor plasma was spiked with either apixaban (dissolved in DMSO and PBS) or unfractionated heparin to achieve therapeutic plasma levels. SynthASil, a commercially available mixture of phospholipids and silica, was used to stimulate the CA pathway in two different dilutions (1–80 and 5–80). Downstream coagulation was accessed by Thrombin Generation Test using Thrombinoscope by Stago and associated Thrombin Calibrator (activity 640 nM). The endogenous thrombin potential (area under the thrombin generation curve; ETP), peak thrombin generation (PTG), time to peak (ttPeak) and time to start (ttStart) were accessed. Results With decreasing concentrations of apixaban, stimulation with the lower dose SynthASil reveals an increasing ETP and PTG. As expected, ttPeak and ttStart decreased. Even supratherapeutic levels of apixaban (i.e. 1120 ng/mL) could not inhibit thrombin from being generated, in striking contrast with UFH where no thrombin was formed. Using a five times higher dose of SynthASil showed comparable ETP for all concentrations of apixaban, allocated around the control value. PTG, however, slightly increased with decreasing concentrations of apixaban. ttPeak and ttStart slightly decreased. Except for the subtherapeutic UFH concentration of 0,114 IU/mL, no thrombin was generated with UFH. Conclusion UFH is more effective in inhibiting downstream thrombin generation compared to apixaban as a response to activation of the CA pathway in vitro. These findings could help explain why direct inhibitors were not able to show non-inferiority in patients with mechanical heart valves and support the development of specific CA pathway inhibitors for patients with conditions that activate the CA pathway. Thrombin generation curves Funding Acknowledgement Type of funding source: None

Background Bleeding is a main cause of morbidity and mortality in critically ill cardiogenic shock patients, supported by short-term percutaneous mechanical circulatory support (pMCS) devices. Bleeding not only occurs because of obligatory heparin and antiplatelet therapy (both required in the prevention of pump and stent thrombosis) but possibly also results from device-related coagulopathy. Similar to long-term ventricular assist devices, mechanical shear-induced acquired von Willebrand syndrome (AVWS) might further increase the bleeding risk. Therefore, we aimed to investigate the effect of left Impella percutaneous continuous flow pumps on the development of AVWS due to shear-induced excessive cleavage of large vWF multimers by the metalloproteinase ADAMTS-13, resulting in loss of high-molecular-weight vWF multimers. Methods Between March 2019 and January 2020, all cardiogenic shock patients supported by a left Impella and referred to a single tertiary ICU were studied. Both vWF Antigen (vWF:Ag) and vWF:GPIbR (ristocetin-induced binding of vWF to a recombinant wildtype Glycoprotein Ib fragment) levels were measured by chemiluminescent immunoassays using an AcuStar (Werfen) assay to determine the vWF:GPIbR /vWF:Ag ratio (normal range ≥1.0). VWF multimer analysis was performed by electrophoresis. On-pump analyses were performed 12h after implantation and off-pump analyses 12h after Impella explantation. Patients who died on-pump were excluded because of lack of paired data after explantation. Results Eight left Impella patients (four Impella CP, four Impella 5.0) were analyzed for AVWS. The vWF:GPIbR /vWF:Ag ratio was Conclusions Our data highlight the rapid onset and reversal of AVWS in all studied cardiogenic shock patients, supported by a left Impella pump. The determination of the GPIbR /vWF:Ag ratio with the AcuStar appears a reliable and faster test to detect AVWS as compared to vWF multimers electrophoresis. Further research into innovative pharmacological interventions (e.g. ADAMTS-13 inhibitors) should target pMCS-induced AVWS in an effort to reduce hemostatic complications in this critically ill ICU population. AVWS in Impella supported patients Funding Acknowledgement Type of funding source: None

Introduction Acquired thrombotic thrombocytopenic purpura is a rare disease associated with the production of autoantibodies against the VWF-cleaving protease ADAMTS13. The detection of these antibodies is made difficult by the instability of ADAMTS13 in citrated plasma and the time-consuming ADAMTS13 assays. The aim of our study was to evaluate the optimal conditions for detecting anti-ADAMTS13 inhibitory antibodies with the novel automated chemiluminescent immunoassay HemosILR AcuStar ADAMTS13 Activity assay. Methods The parallelism between the AcuStar ADAMTS13 calibration curve and ADAMTS13 concentrations in serially diluted citrated plasma was evaluated after 2 hours incubation at 25°C, 37°C, or 37°C after addition of Ca2+ to preserve the activity of the metalloprotease. Using Bethesda assays based on the 3 incubation procedures and the HemosILR AcuStar ADAMTS13 Activity assay, the inhibitor titers were determined in patients' samples with ADAMTS13 antibodies and compared with those determined using the TechnozymR ADAMTS13 activity ELISA. Results The criterion of parallelism was respected for the 3 incubation methods over the range of ADAMTS13 concentrations relevant for the detection of ADAMTS13 inhibitor antibodies in a Bethesda assay. In agreement with this observation, all the incubation methods permitted the accurate detection and quantification of inhibitory anti-ADAMTS13 antibodies in the samples from patients with acquired thrombotic thrombocytopenic purpura. Conclusion Incubation of plasma samples with normal plasma at 25°C, 37°C, or 37°C after addition of Ca2+ can be used in a Bethesda assay for quantifying the inhibitory activity of antibodies interfering with ADAMTS13 in the chemiluminescent HemosILR AcuStar ADAMTS13 Activity assay.

BACKGROUND: Previous clinical evidence correlates levels of von Willebrand factor (VWF) and its cleaving protease ADAMTS13 with outcome in septic patients. No previous studies addressed if VWF and ADAMTS13 affected the outcome of Staphylococcus aureus sepsis. OBJECTIVES: We studied the role of VWF and ADAMTS13 in S. aureus sepsis both in patients and in mice. METHODS: VWF levels and ADAMTS13 activity levels were measured in plasma samples from 89 S. aureus bacteremia patients by chemiluminescent assays and were correlated with clinical sepsis outcome parameters. In wild-type mice and mice deficient in VWF and ADAMTS13, we investigated the outcome of S. aureus sepsis and quantified bacterial clearance and organ microthrombi. RESULTS: In patients with S. aureus bloodstream infections, high VWF levels and low ADAMTS13 activity levels correlated with disease severity and with parameters of inflammation and disseminated intravascular coagulation. In septic mice, VWF deficiency attenuated mortality, whereas ADAMTS13 deficiency increased mortality. Bacterial clearance was enhanced in VWF-deficient mice. The differences in mortality for the studied genotypes were associated with differential loads of organ microthrombi in both liver and kidneys. CONCLUSIONS: In conclusion, this study reports the consistent relation of VWF, ADAMTS13 and their ratio to disease severity in patients and mice with S. aureus sepsis. Targeting VWF multimers and/or the relative ADAMTS13 deficiency that occurs in sepsis should be explored as a potential new therapeutic target in S. aureus endovascular infections. ispartof: JOURNAL OF THROMBOSIS AND HAEMOSTASIS vol:18 issue:3 pages:722-731 ispartof: location:England status: published

Several recombinant factor VIII and factor IX concentrates with extended half-life (EHL) have recently been validated by clinical studies. The availability of these novel concentrates is expected to significantly facilitate the treatment of patients with hemophilia A and B. However, the modification applied to these molecules has introduced variations in their activity measurement in routine coagulation assays. Depending on the assays, underestimations of up to 10-fold or overestimations of up to approximately 30-fold in the measurements of the recovery have been reported in some factor concentrates. Such biases in monitoring may lead to major under- or overtreatment, as well as unnecessary searching for inhibitor antibodies. In this review, we discuss the guidelines and recommendations that allow the selection of optimal strategies to monitor patients treated with these novel factor concentrates. Based on the specificities of the assays and on local regulations, different chromogenic substrate assays in addition to one-stage clotting assays may be validated to allow the accurate measurement of all novel products. An efficient communication between the clinical laboratory and the clinicians is essential to ensure that the appropriate assays are carried out in laboratories and that the clinicians correctly evaluate the data. Further laboratory and clinical studies are still required for the optimization of the laboratory assays that can be used in the measurement of novel factor VIII and factor IX concentrates with EHL.

Background Staphylococcus aureus (S. aureus) bacteraemia is frequent and carries a high morbidity and mortality. Coagulases secreted by S. aureus initiate blood coagulation by directly activating prothrombin. This pathogen-activated coagulation is insensitive to most antithrombotic drugs, with the exception of small molecule direct thrombin inhibitors (DTIs). DTIs inhibit the coagulase-prothrombin complex, or staphylothrombin, and improve outcome in preclinical models of S. aureus infection. Objective A single-centre, randomized, controlled feasibility and safety trial of staphylothrombin inhibition with DTIs in patients with S. aureus bacteraemia. Patients and Methods Consecutive eligible adult patients with S. aureus positive blood cultures in the University Hospitals Leuven (Belgium) were randomized 1:1 to DTI (oral dabigatran 110 mg twice daily or intravenous argatroban according to activated partial thromboplastin time [aPTT]) for 7 to 10 days, or subcutaneous enoxaparin 40 mg once daily. Primary outcomes were feasibility and safety of DTI in patients with S. aureus bacteraemia. Secondary outcomes include D-dimer evolution (day 0–4) as marker of coagulation activation; inflammatory and microbiological parameters; and clinical outcomes including metastatic infections. Results Thirty-one percent (94/303) of screened patients were enrolled. Dabigatran plasma levels inhibited staphylothrombin. Clinically relevant bleeding (5/47 vs. 5/47) and thrombotic (7/47 vs. 7/47) complications were similar in both groups. Coagulase inhibition with DTIs was associated with a trend towards faster D-dimer decrease at day 4 (–662 ± 249 ng/mL vs. –40 ± 213 ng/mL for DTI-treated patients vs. control; p = 0.06) and a numerically lower number of persistently positive blood cultures. No differences in inflammatory parameters or other clinical outcomes were observed. Conclusion Targeting staphylothrombin with DTIs is feasible in a subset of S. aureus bacteraemic patients, with comparable safety to standard thromboprophylaxis. In future studies of staphylothrombin inhibition, feasibility can be further improved by rapid diagnostics and by strategies without concomitant anticoagulant effect.

Introduction Thrombin time (TT) tests are useful for diagnosing coagulation disorders involving abnormal fibrinogen but do not allow us to distinguish between qualitative and quantitative defects. However, with the widening availability of optical coagulation automates, more information about the coagulation process is becoming increasingly accessible. Methods In this study, we compared the coagulation curves of TT tests carried out with plasma from healthy donors with those from patients with acquired low Clauss fibrinogen levels or with dysfibrinogenemia caused by a heterozygous point mutation in the fibrinogen γ-chain that results in a p.Arg301(275)Cys substitution. The functional fibrinogen levels of these three groups of samples were also measured with the Clauss method, and their fibrinogen protein levels were determined by ELISA. Results Our data indicate that the amplitude and maximal velocity of coagulation curves from plasma samples from FGG p.Arg301(275)Cys dysfibrinogenemic patients were comparable to those from plasma samples with fibrinogen in the normal range, whereas the amplitude of coagulation curves from patients with acquired low fibrinogen levels was lower. Conclusions Examination of the amplitude of coagulation curves generated during TT tests may provide additional information to enable the differential diagnoses of diseases following a low fibrinogen measurement by the Clauss method.

Dear Editor, Congenital factor X (FX) deficiency is an extremely rare, autosomal recessive inherited condition with an estimated incidence of 1:1 000 000 with an eightfold to 10‐fold increase in frequency in populations with consanguineous marriages.1 It is a heterogeneous bleeding disorder that, dependent on the residual FX level, can be asymptomatic or present with grade I (minor provoked), grade II (minor spontaneous; eg, epistaxis) to grade III (severe spontaneous) bleeding (eg, haematomas, haemarthrosis, central nervous, umbilical cord and gastrointestinal bleeding). [...]

Suzuki coupling is a crucial reaction for the preparation of many high value compounds, especially for the synthesis of pharmaceuticals. Pd-based heterogeneous catalysts are highly regarded for achieving this reaction. However, the knowledge of the Suzuki mechanism with this type of catalysts is relatively limited. In the present paper, the influence of the acidity of heterogeneous catalysts on the Suzuki coupling has been methodically investigated on palladium supported on silica–alumina. A Suzuki coupling model reaction was used to assess if any correlation exists between the performances of the prepared catalysts and their acidity. The results have demonstrated that, the Pd being in an oxidized state, the acid sites at the heterogeneous catalyst surface participate actively to the mechanism of the Suzuki coupling. An optimum of total acidity of the catalysts leading to a peak of performances (conversion and selectivity) was identified. Several assumptions concerning the implication of the catalytic support in the different steps of the Suzuki coupling mechanism are proposed and discussed. Furthermore, the recyclability of the catalyst has been demonstrated, revealing that our catalysts really behave heterogeneously.

Several lines of evidence indicate that the immune response to Factor VIII (FVIII) in patients with hemophilia A is T cell-dependent. This review highlights the link between the epitope specificity of FVIII-specific T cells and their potential roles in different categories of patients. FVIII-specific T cells able to recognize wild-type (i.e. therapeutic) FVIII but not the mutated self FVIII of hemophilia patients have been identified in patients with mild/moderate hemophilia carrying some point mutations. Such T cells likely contribute to the higher frequency of neutralizing anti-FVIII antibodies (inhibitors) development in these patients. In contrast, as yet no T cells have been identified that can differentiate between FVIII molecules with non-hemophilia-causing single amino acid variants encoded by non-synonymous single-nucleotide polymorphisms in the F8 gene. Other mechanisms are therefore still to be identified that will explain the clinically noted differences in the incidence of inhibitor development between patients of different races who are known to have differences at these sites. Beside information about the mechanism of inhibitor development, the analysis of FVIII-specific T cells has provided tools to develop novel diagnostic and therapeutic approaches, such as the generation of FVIII-specific regulatory T cells that may be useful in preventing or suppressing the immune response to FVIII.

INTRODUCTION: The monitoring of factor VIII (FVIII) replacement therapy relies on the accurate measurement of FVIII activity over a large concentration range. However, unexplained overestimation of low FVIII levels has recently been reported with extended half-life recombinant FVIIIs. AIM: The objective of this study was to confirm previous publications indicating that the reagents used to generate the calibration curves determine the accuracy of the measurement of low FVIII levels. METHODS: We generated FVIII calibration curves with FVIII-deficient plasmas or a commercial diluent buffer. We then measured FVIII levels in FVIII-deficient plasma spiked with plasma FVIII, a full-length recombinant FVIII (Advate® , Shire, Brussels, Belgium) and two extended half-life recombinant FVIIIs (Elocta® , Swedish Orphan Biovitrum, Woluwe Saint-Lambert, Belgium and Afstyla® , CSL Behring, Mechelen, Belgium). FVIII levels were also analyzed in spiked samples prediluted two- and fourfold, either in diluent buffer or in FVIII-deficient plasma to evaluate parallelism. RESULTS: Coagulation times of calibration curves generated with diluent buffer were longer than those with FVIII-deficient plasmas. This resulted in an overestimation of FVIII levels lower than 25 IU/dL in spiked samples and in detection of FVIII activity (≥1 IU/dL) in FVIII-deficient plasma. Predilution of samples with diluent buffer rather than with FVIII-deficient plasma also led to discordant results. CONCLUSION: Our data confirm that the generation of calibration curves by dilution in FVIII-deficient plasma is crucial for the accurate measurement of low FVIII levels. When serial dilutions of samples are analyzed, predilution in FVIII-deficient plasma is required to respect the parallelism criteria. These methods should be more generally implemented for coagulation instruments. ispartof: HAEMOPHILIA vol:25 issue:1 pages:E19-E26 ispartof: location:England status: published

Dear Editor, Haemophilia is an X‐linked recessive bleeding disorder, resulting from deficient or dysfunctional coagulation factor VIII (haemophilia A) or IX (haemophilia B). Depending on the severity of their factor deficiency, patients present with a range of bleeding diathesis from traumatic injury or surgically induced bleeding to spontaneous haemorrhage in the soft tissues, joints or muscles. Before the 1960s, the prognosis of severe haemophilia A patients was just 11 years or less,1 then the introduction of fresh‐frozen plasma, cryoprecipitate, and finally blood‐pooled clotting factor concentrates considerably improved the survival and quality of life of patients with haemophilia or with other bleeding disorders.2 However, the epidemic of HCV and human immunodeficiency virus (HIV) then erupted with severe consequences. Until the 1989 discovery of HCV,3 almost all haemophilia patients treated with clotting factor products were infected with HCV.4 The clinical presentation of HCV is characterized by chronic liver disease that can progress to fibrosis with development of (de)compensated cirrhosis and hepatocellular carcinoma. [...]

Pro-thrombotic and inflammatory changes play an important role in cardiovascular morbidity and mortality, resulting from short-term exposure to fine particulate air-pollution. Part of those effects has been attributed to the ultra-fine particles (UFPs) that pass through the lung and directly contact blood-exposed and circulating cells. Despite UFP-induced platelet activation, it is unclear whether the penetrated particles exert any direct effect on endothelial cells. While exposure levels are boosting as a result of world-wide increases in economic development and desertification, which create more air-polluted regions, as well as increase in demands for synthetic UFPs in medicine and various industries, further studies on the health effects of these particles are required. In this study, human pulmonary and cardiac microvascular endothelial cells (MECs) have been exposed to 0.1, 1, 10, and 100 μg/ml suspensions of either a natural (carbon black) or a synthetic (multi-walled carbon nano-tubes) type of UFPs, in vitro. As a result, no changes in the levels of coagulation factor VIII, Von Willebrand factor, Interleukin 8, and P-selectin measured in the cells’ supernatant were observed prior to and 6, 12, and 24 h after exposure. In parallel, the spatio-temporal effect of UFPs on cardiac MECs was evaluated by Transmission Electron Microscopy. Despite phagocytic uptake of pure UFPs observed on cellular sections of the treated cells, Weibel-Palade bodies remained intact in shape and similar in number when compared with the untreated cells. Our work shows that carbon itself is a non-toxic carrier for endothelial cells.

Summary Background The anticoagulant effect of dabigatran can be approximated by its prolongation of routine coagulation assays. Consequently, dabigatran also interferes with thrombophilia screening or with diagnosing hemostasis disorders that have developed after the initiation of anticoagulant treatment, such as vitamin K deficiency or acquired hemophilia A. Objectives This study was carried out to determine whether idarucizumab, a humanized antibody fragment that binds dabigatran, could fully neutralize dabigatran in routine diagnostic coagulation assays conducted in vitro, thereby preventing false-positive or false-negative diagnostic readouts. Methods Preliminary experiments identified coagulation assays that were sensitive to dabigatran, and identified a concentration of idarucizumab that neutralized the effects of dabigatran. These assays were then carried out with patient and control plasma samples spiked with dabigatran, with or without a molar excess of idarucizumab. Results Dabigatran altered the prothrombin time, activated partial thromboplastin time and thrombin time, and the measurement of intrinsic and extrinsic factor levels. Screening and confirmation tests used for lupus anticoagulant detection were prolonged by dabigatran, falsely suggesting the presence of lupus anticoagulant. Conversely, the addition of dabigatran falsely corrected an abnormal activated protein C resistance ratio. Addition of idarucizumab completely normalized these measurements, and allowed the correct identification of normal and abnormal samples with these assays. Conclusions In vitro addition of idarucizumab to plasma samples containing dabigatran fully neutralizes the drug, and facilitates the use of routine coagulation assays to allow the diagnosis of hemostasis disorders that may be concurrently present in patients taking dabigatran.

Context: An overdose of oral anticoagulants represents a challenging scenario for emergency physicians. Dabigatran, an oral direct thrombin inhibitor, is increasingly used in place of warfarin. The...

With the exception of liver transplantation, there is no cure for hemophilia, which is currently managed by preemptive replacement therapy. Liver-derived stem cells are in clinical development for inborn and acquired liver diseases and could represent a curative treatment for hemophilia A. The liver is a major factor VIII (FVIII) synthesis site, and mesenchymal stem cells have been shown to control joint bleeding in animal models of hemophilia. Adult-derived human liver stem cells (ADHLSCs) have mesenchymal characteristics and have been shown able to engraft in and repopulate both animal and human livers. Thus, the objectives were to evaluate the potency of ADHLSCs to control bleeding in a hemophilia A patient and assess the biodistribution of the cells after intravenous injection.A patient suffering from hemophilia A was injected with repeated doses of ADHLSCs via a peripheral vein (35 million In-oxine-labeled cells, followed by 125 million cells the next day, and 3 infusions of 250 million cells every 2 weeks thereafter; total infusion period, 50 days).After cell therapy, we found a temporary (15 weeks) decrease in the patient's FVIII requirements and severe bleeding complications, despite a lack of increase in circulating FVIII. The cells were safely administered to the patient via a peripheral vein. Biodistribution analysis revealed an initial temporary entrapment of the cells in the lungs, followed by homing to the liver and to a joint afflicted with hemarthrosis.These results suggest the potential use of ADHLSCs in the treatment of hemophilia A.

Investigations of X-ray photoelectron spectra from solid samples need corrections for the surface charging effect. For powder samples such as heterogeneous catalysts and their supports, the C(C,H) component of the C 1s peak is often used as an internal standard for the calibration of the binding energy scale. Although this method is widely recognized as suitable for the study of heterogeneous catalysts, we show that a significant calibration bias can be encountered upon comparing samples with different bulk composition. In this paper, a series of SiO2 -Al2 O3 supports and Pd/SiO2 -Al2 O3 catalysts with various Si/Al ratios were studied. The spectra issued from these samples were processed with the classical calibration method on the basis of the carbon peak. Important discrepancies in the relative position of the photoelectron peaks were noticed. After systematically discarding instrument-related issues, a true chemical influence of the bulk matrix on the analyzed surface species was evidenced. The extent of this chemical effect was dependent on the composition of the sample and more precisely on its ionicity. Two possible mechanisms for this chemical effect were proposed and discussed. Finally, an alternative calibration method was offered.

BACKGROUND: Heparin has been used for years as a locking solution in totally implantable venous access devices. Normal saline (NS) might be a safe alternative for heparin. However, evidence of non-inferiority of NS versus heparin is lacking. PATIENTS AND METHODS: We randomly allocated 802 cancer patients with a newly inserted port either to heparin lock (300 U/3 ml) or to NS lock groups in a 1:1 assignment ratio. The primary outcome was the number of functional complications, which was defined as 'easy injection, impossible aspiration' at port access. Secondary outcomes included all functional problems and catheter-related bacteraemia. We hypothesised that NS locks do not cause more functional problems and catheter-related bacteraemia than heparin locks. Non-inferiority is established if the upper limit of the confidence interval (CI) for the relative risk of NS versus heparin is

Controversy has arisen about the nature of circulating human CD20(+)CD27(+)CD43(+)CD70(-)CD69(-) B cells. Although originally described as being the human counterpart of murine B-1 B cells, some studies have raised the possibility that these might instead be plasmablasts. In this article, we have further characterized the putative B-1 cells and compared them directly with memory B cells and plasmablasts for several functional characteristics. Spontaneous antibody production of different isotypes as well as the induced production of antigen-specific antibodies after vaccination with a T-cell-dependent antigen did not reveal differences between the putative B-1 cells and genuine CD20(-) plasmablasts. Gene expression profiling of different B-cell subsets positioned the phenotype of putative B-1 cells closer to CD20(-) plasmablasts than to memory B cells. Moreover, putative B-1 cells could be differentiated into CD20(-) plasmablasts and plasma cells in vitro, supporting a pre-plasmablast phenotype. In conclusion, characterization of the putative B-1 cells revealed a functional phenotype and a gene expression profile that corresponds to cells that differentiate into CD20(-) plasmablasts. Our data offer perspectives for the investigation of differentiation of B cells into antibody secreting cells.

The optimal anticoagulation regimen for hemodialysis (HD) in patients with heparin-induced thrombocytopenia (HIT) has not been defined. Hemodiafiltration (HDF) adds a large convective component to HD, thereby changing the pharmacokinetics of most anticoagulants. Data on coagulation regimens for HDF are scant. We therefore aimed to study the feasibility, effectiveness, tolerability, and pharmacokinetics of fondaparinux anticoagulation in HDF. This was a prospective observational dose-finding study. Patients were started on fondaparinux at a dose of 0.05 mg/kg postdialysis body weight. Per protocol dose escalation was performed when significant clotting was observed and reduced when the anti-Xa activity postdialysis exceeded 0.4 IU/mL. Dose adjustments were made by steps of 0.01 mg/kg postdialysis weight. Anti-Xa activity was measured using a chromogenic method calibrated with low-molecular-weight heparin and validated against fondaparinux-calibrated anti-Xa activity. Four patients with HIT were followed for 160 sessions in total. At the end of the dose titration study, three patients ended at a maintenance dose of 0.03 mg/kg and one patient at 0.04 mg/kg of fondaparinux. Significant bleeding attributable to fondaparinux did not occur. The occurrence of clotting increased parallel to the reduction of fondaparinux dose, from 0/53 and 0/15 sessions at the higher doses (0.04 and 0.05 mg/kg) to 3/75 (4%) at 0.03 mg/kg and 1/17 (6%) at 0.02 mg/kg. Fondaparinux may be safely used and provides adequate anticoagulation for HDF in patients with HIT. We recommend to adjust dosage of fondaparinux to body weight and to initiate therapy at a dose of 0.03 mg/kg to prevent accumulation. Dose titration can be achieved by targeting postdialysis anti-Xa activity.

Several studies showed that neutralizing anti-factor VIII (anti-fVIII) antibodies (inhibitors) in patients with acquired hemophilia A (AHA) and congenital hemophilia A (HA) are primarily directed to the A2 and C2 domains. In this study, the frequency and epitope specificity of anti-C1 antibodies were analyzed in acquired and congenital hemophilia inhibitor patients (n = 178). The domain specificity of antibodies was studied by homolog-scanning mutagenesis (HSM) with single human domain human/porcine fVIII proteins and antibody binding to human A2, C1, and C2 domains presented as human serum albumin (HSA) fusion proteins. The analysis with HSA-fVIII domain proteins confirmed the results of the HSM approach but resulted in higher detection levels. The higher detection levels with HSA-fVIII domain proteins are a result of antibody cross-reactivity with human and porcine fVIII leading to false-negative HSM results. Overall, A2-, C1-, and C2-specific antibodies were detected in 23%, 78%, and 68% of patients with AHA (n = 115) and in 52%, 57%, and 81% of HA inhibitor patients (n = 63). Competitive binding of the human monoclonal antibody (mAb) LE2E9 revealed overlapping epitopes with murine C1-specific group A mAbs including 2A9. Mutational analyses identified distinct crucial binding residues for LE2E9 (E2066) and 2A9 (F2068) that are also recognized by anti-C1 antibodies present in patients with hemophilia. A strong contribution of LE2E9- and 2A9-like antibodies was particularly observed in patients with AHA. Overall, our study demonstrates that the C1 domain, in addition to the A2 and C2 domains, contributes significantly to the humoral anti-fVIII immune response in acquired and congenital hemophilia inhibitor patients.

Introduction Postinfusion ReFacto AF levels can be difficult to measure accurately due to discrepancies between one-stage and chromogenic FVIII assays. To overcome this, the use of the ReFacto AF laboratory standard (RAFLS) is recommended, but there are discordant reports regarding its usefulness. Aim We investigated whether calibration with RAFLS and measurement of ReFacto AF levels are influenced by the choice of reagents and patient-specific factors in one-stage FVIII assays. Methods Calibration curves were generated with both the RAFLS and a plasma standard using different F8DPs and one-stage FVIII assay reagents. This selection of reagents was then used to determine FVIII levels in the plasma of patients repeatedly treated with ReFacto AF. Results were compared with those obtained with a chromogenic assay. Results F8DP devoid of von Willebrand factor (VWF) falsely increased the values of RAFLS pro-coagulant activity generated using the APTT reagent. The resulting RAFLS calibration curve underestimated ReFacto AF levels to be half of their true concentration. The use of RAFLS with F8DP containing VWF reduced the discrepancy observed between the one-stage and chromogenic FVIII assays. However, the mean difference between the two assays still varied up to 50% depending on the patient. Conclusions The RAFLS is a suitable calibrator for one-stage FVIII assays carried out with F8DP containing VWF. However, calibration with the RAFLS does not avoid the effect of patient-specific variables that contribute to discrepancies between the measurements of ReFacto AF levels with one-stage and chromogenic FVIII assays.

Nowadays, the control of CO2 emissions is still a challenge. A few alternatives exist but nothing concrete seems to be developed. Instead of catching and storing CO2, one possibility would be its transformation into value added molecules as methane. Rhodium catalysts are active in CO2 methanation reaction. But it seems that a competitive adsorption exist between the two reactants: CO2 and hydrogen. In order to surpass this trouble and increase hydrogen adsorption, a known active catalyst in methanation (Rh/γ-Al2O3) was put into contact with a known active catalyst in hydrogen activation (Ni/activated carbon). Catalysts were prepared by the mechanical mixing of the latter two in different proportions. Catalysts were tested in the low temperature methanation reaction using CO2 and H2. Methane is produced in all cases with a 100% of selectivity. A significant synergy appears in the catalytic activity of this mixed catalyst. Production of methane in mixtures is largely higher than the theoretical predicted values considering the individual performances. Catalysts were characterized before and after reaction by ICP-AES, N2 physisorption, XRD, CO2 chemisorption, ToF-SIMS, XPS and TPR. The synergy is due to the increase of H2 adsorption and promoting the carbon hydride formation. Furthermore the suggested hydrogen spill-over maintains Rh particles in a metallic state necessary for the reaction.

Introduction Partial inhibition of Factor VIII (FVIII) may provide antithrombotic efficacy whilst avoiding excessive anticoagulation. Materials and Methods We studied the anticoagulant effects of a partial (TB-402) and a complete (BO2C11) FVIII-inhibiting monoclonal antibody (MAb) on FVIII, aPTT, thrombin generation and fibrin deposition in a flow chamber model. The antithrombotic efficacy of TB-402 and BO2C11 was compared in a mouse model of venous thrombosis. Results Both in vitro and ex vivo , the maximally achievable FVIII inhibition by TB-402 was about 25 to 30%. The degree of inhibition reached a plateau in vitro at 0.316 μg/mL and ex vivo after administering 0.1 mg/kg and higher doses. BO2C11 strongly inhibited FVIII:C, up to 91% at 100 μg/mL in vitro , and by 88% ex vivo 1 hour after administering 1 mg/kg to the mice. Whereas BO2C11 also markedly prolonged the aPTT and completely inhibited thrombin generation in vitro and ex vivo, the effect of TB-402 on the aPTT and on thrombin generation was limited. Similarly, in a dynamic flow chamber model, TB-402 and BO2C11 inhibited tissue factor-induced human fibrin deposition by 40% and 76%, respectively. In a mouse model of FeCl 3 -induced venous thrombosis, TB-402 (1 mg/kg) inhibited thrombus formation to the same extent as BO2C11 (2 mg/kg) and enoxaparin (5 mg/kg), with a mean (± SD) occlusion time of 51 ± 13 minutes for TB-402, compared to 28 ± 6 minutes for the controls, 51 ± 13 minutes for BO2C11 and 55 ± 11 minutes for enoxaparin. Conclusions In this mouse model of venular thrombosis, partial FVIII inhibition yielded similar antithrombotic effects as nearly complete FVIII inhibition. These preclinical data are indicative of a therapeutic potential of partial FVIII inhibition in the management of venous thromboembolism.

The methanation of CO2 was performed on Rh/γ-Al2O3 catalyst at temperatures between 50 and 150 °C at 2 bar of pressure in a pulse reactor. Experiments confirm the formation of methane at low temperature and pressure. The formation of formiate species during the adsorption of CO2 can be excluded. After reaction with CO2, the catalyst is oxidized. Oxidation is not observed in the presence of CO. The CO2 is adsorbed dissociatively, forming CO (ads) and O (ads). Gem-dicarbonyl Rh(CO)2 species are more reactive than the Rh–CO linear species. The type of adsorbed species depends on the Rh oxidation state. The formation of methane by hydrogenation of CO2 and CO is carried out with 100% selectivity. The activation energy for the hydrogenation of CO2 and CO is lower than values presented in the literature which have been obtained at higher temperature. In the presence of CO2 and CO, the reaction of methanation of CO2 seems to be inhibited by CO. When oxygen is added in low amount in the reactant gas feed, a positive effect on methanation is observed. When the amount of oxygen is too high, oxygen has a negative effect. These results are in agreement with thermodynamics equilibrium calculations, except when O2 is present, confirming the importance of kinetic effects in the reaction. These results open new perspectives of application of catalysis, in order to recycle CO2 in the presence of H2.

Summary. Background: The development of an inhibitor is the major complication facing patients with hemophilia A treated by administration of factor (F) VIII concentrates. Restoration of tolerance to FVIII can be achieved by prolonged administration of FVIII (immune tolerance induction, ITI). Although ITI has been used for more than 30 years in patients with hemophilia A and inhibitor, its mechanism of action is still poorly understood. Objectives: As administration of high doses of antigen can induce the apoptosis of the T cells recognizing the antigen, a potential mechanism of action of ITI may be the deletion of FVIII-specific T cells. Patients/Methods: We studied the CD4+ T-cell response to FVIII in five (one mild, one moderate and three severe) patients successfully desensitized by administration of FVIII and in control subjects. Results: Following repeated stimulation with autologous dendritic cells loaded with FVIII, FVIII-specific T oligoclonal cell lines were expanded from the blood of one of the successfully desensitized patients. The FVIII-specific T cells produced IL-5, IL-13 and IL-2. By contrast, FVIII-specific T-cell lines could not be derived from three patients with mild hemophilia A without inhibitor or from four normal control subjects. Conclusions: These data represent the first analysis of the cellular mechanisms regulating the induction of tolerance to FVIII. They demonstrate that successful tolerance induction may occur without deletion of FVIII-specific T cells.

The immune response toward viral vectors used for gene therapy and genetic vaccination appears to be critically important in determining the therapeutic outcome. However, the mechanisms that control the immune response following gene transfer are poorly understood. Unexpectedly, we found that integrating retroviral vector particles induce stable interleukin-10 (IL-10) production in murine (BALB/c H-2 d) transduced B cells. This requires a novel mechanism whereby the interaction of retroviral vector particle with its cognate cellular receptor activates intracellular signaling pathways resulting in stable epigenetic modifications. Murine B cells exposed to retroviral vector particles triggered the colocalization of the retroviral cellular receptor mouse cationic amino acid transporter 1 (mCAT1) and Toll-like receptor 2 (TLR2) into lipid microrafts, which in turn activated TLR2 signaling pathways. TLR2 activation induced STAT3 phosphorylation and increased phosphorylated histone 3 (H3) at the STAT3-binding site of the IL-10 promoter. In addition, TLR2 activation during transduction activates nuclear factor of light polypeptide gene enhancer in B-cells inhibitor, α (NFKBIA), thereby preventing the translocation of the nuclear factor-B (NF-B) complex to the nucleus and the transcription of proinflammatory cytokines. These findings open new perspectives for controlling immune responses following gene therapy and genetic vaccination. © The American Society of Gene & Cell Therapy., SCOPUS: ar.j, info:eu-repo/semantics/published

Factor VIII binds to phosphatidylserine (PS)-containing membranes through its tandem, lectin-homology, C1 and C2 domains. However, the details of C1 domain membrane binding have not been delineated. We prepared 4 factor VIII C1 mutations localized to a hypothesized membrane-interactive surface (Arg2090Ala/Gln2091Ala, Lys2092Ala/Phe2093Ala, Gln2042Ala/Tyr2043Ala, and Arg2159Ala). Membrane binding and cofactor activity were measured using membranes with 15% PS, mimicking platelets stimulated by thrombin plus collagen, and 4% PS, mimicking platelets stimulated by thrombin. All mutants had at least 10-fold reduced affinities for membranes of 4% PS, and 3 mutants also had decreased apparent affinity for factor X. Monoclonal antibodies against the C2 domain produced different relative impairment of mutants compared with wild-type factor VIII. Monoclonal antibody ESH4 decreased the Vmax for all mutants but only the apparent membrane affinity for wild-type factor VIII. Monoclonal antibody BO2C11 decreased the Vmax of wild-type factor VIII by 90% but decreased the activity of 3 mutants more than 98%. These results identify a membrane-binding face of the factor VIII C1 domain, indicate an influence of the C1 domain on factor VIII binding to factor X, and indicate that cooperation between the C1 and C2 domains is necessary for full activity of the factor Xase complex.

Experimental evidences show that it is possible to produce methane from an exclusively inorganic way using CO 2 and H 2 on a Rh/γ-Al 2 O 3 catalyst at low temperature and atmospheric pressure. New insights on the mechanism of methanation are given. The first step of the mechanism in the methanation reaction could be the chemisorption of CO 2 on the catalyst. The second step is the dissociation of CO 2 into CO and O adsorbed on the surface. The third step is the reaction of dissociated species with H 2 . Adsorption, dissociation of CO 2 and the reaction of dissociated species with H 2 are evidenced by in situ DRIFT experiments. The reaction could occur between the adsorbed species. However a reaction between adsorbed species and gaseous hydrogen cannot be excluded. XPS shows that the CO 2 oxidizes the catalyst and this oxidation deactivates the catalyst. The conversion of CO 2 also increases with temperature. Methane is the unique hydrocarbon molecule formed and obtained with almost 100% selectivity. No other hydrocarbonated products have been observed.

Summary. Despite major advances in diagnosis and treatment, the management of patients with mild haemophilia (MH) remains a major challenge. Mild haemophilia is defined by factor levels between 0.05 and 0.40 IU mL−1. The bleeding associated with mild haemophilia is most frequently episodic, occurring during surgery or following trauma. Spontaneous bleeding is rare. Diagnosis is sometimes delayed because of insensitivity of screening clotting assays or discrepancies in factor VIII activity as measured by different assays. The treatment of choice in mild haemophilia A is desmopressin, which typically induces a 2–6-fold increase of factor VIII over baseline. However, desmopressin has its limitations in this setting such as the occurrence of tachyphylaxis and failure to respond in an undetermined proportion of patients. Factors underlying poor biological response or magnitude of response to desmopressin are incompletely understood. Inhibitor development in mild haemophilia is particularly distressing. This complication arises at an older age in this patient group because of infrequent need for factor VIII replacement. Inhibitors in mild haemophilia patients often cross-react with endogenous factor VIII resulting in severe spontaneous bleeding frequently in a postoperative setting. Intensive perioperative use of factor VIII and some specific mutations induce a particularly high risk for inhibitor development, but risk factors are incompletely understood. For reasons of the older age of the patients, treatment of bleeding with bypassing agents may cause major thrombotic complications. Data on therapeutic options for inhibitor eradication in patients with mild haemophilia are particularly scarce. With increased life-expectancy for all haemophilia patients, the group of elderly patients with mild haemophilia requiring major surgery will further increase. Prevention of inhibitors, particularly in this patient group, should be a major topic of interest in both clinic and research.

Although the liver is known to be the main site of factor VIII (FVIII) production, other organs are probably also important for the regulation of FVIII secretion. However, the study of the regulation of extrahepatic FVIII production has been hampered by the lack of definitive identification of human tissues able to secrete FVIII. Recent studies have shown that lung endothelial cells can synthesize FVIII. We therefore studied the production of FVIII by endothelial cells purified from other vascular beds. Because physiologic stress results in a rapid elevation of FVIII, we also investigated whether endothelial cells can store FVIII and secrete it after treatment with agonists. Microvascular endothelial cells from lung, heart, intestine, and skin as well as endothelial cells from pulmonary artery constitutively secreted FVIII and released it after treatment with phorbol-myristate acetate and epinephrine. By contrast, endothelial cells from the aorta, umbilical artery and umbilical vein did not constitutively secrete FVIII or release it after treatment with agonists, probably because of a lack of FVIII synthesis. Extrahepatic endothelial cells from certain vascular beds therefore appear to be an important FVIII production and storage site with the potential to regulate FVIII secretion in chronic and acute conditions.

Background: TB-402, a human monoclonal antibody that partially inhibits Factor VIII activity (FVIII:C), is being developed as a long-acting antithrombotic agent. Objectives: The primary goal of this study was to investigate the tolerability of TB-402 in healthy male volunteers. Secondary objectives were to determine the pharmacokinetics and pharmacodynamics of TB-402. Methods: In this ascending-dose study, healthy subjects aged 18 to 45 years were randomly assigned in a 2:1 ratio to receive TB-402 administered as a single intravenous bolus at 0.015, 0.1, 0.5, 2.5, 12.5, 37.5, 188, 620, or 1860 μg/kg or matching inactive vehicle (placebo). An older group (55–75 years) was also administered the highest dose that was well tolerated in the younger group (1860 μg/kg). Adverse events (AEs) were obtained from spontaneous reporting and from answers to nonleading questions asked by the principal investigator and study staff during follow-up visits on days 4, 7 (±1 day), 14 (±1 day), 21 (±2 days), 28 (±3 days), 42 (±3 days), and 56 (±3 days) after TB-402 administration. AEs were monitored up to the last study visit on day 56 after the administration of TB-402 or placebo, with special attention to bleeding events. The pharma-codynamic assessment of TB-402 included changes in FVIII:C, activated partial thromboplastin time (APTT), and prothrombin time (PT). Results: The study enrolled 56 subjects (mean ages: younger group, 28 years [range, 20–45 years]; older group, 65 years [range, 58–76 years]; weight, 79 kg [range, 60–104 kg] and 81 kg [range, 64–94 kg], re-spectively). Thirty-one of the 38 subjects who received TB-402 (82%) experienced a total of 85 treatmentemergent AEs (TEAEs), and 14 of 18 subjects who received placebo (78%) experienced 35 TEAEs. A total of 34 bleeding events were reported in 13 of 38 subjects (34%) who received TB-402 and 7 of 18 subjects (39%) who received placebo. Most common AEs reported in subjects who received TB-402 were headache (11 [29%]), vessel puncture-site hematoma (7 [18%]), and traumatic hematoma (5 [13%]); with placebo, these AEs were vessel puncture-site hematoma (4 [22%]), headache (3 [17%]), vasovagal reaction (3 [17%]), and hematuria (3 [17%]). No serious AEs considered to be related to TB-402 were reported, and no dose-dependent increases in bleeding events were observed. On pharmacokinetic analysis of TB-402, the t 1/2 values across doses were 22.9 days (age 18–45 years) and 19.5 days (age 55–75 years). TB-402 was associated with a reduction in FVIII:C over a period of ~48 hours in the d37.5-μg/kg dose groups. TB-402 was associated with a prolonged APTT at doses ≥2.5 μg/kg ~1.1–1.2-fold predose APTT). Administration of a higher dose of TB-402 was associated with an extended duration of APTT prolongation. No significant effect on PT was found. Conclusions: In this study in healthy male volunteers, TB-402 was well tolerated in the population studied. Based on the findings from this study, the long t 1/2 of TB-402 may allow a pharmacodynamic effect over a prolonged period after single-dose administration. Further trials are needed to address the tolerability and efficacy of this agent in preventing thromboembolism. Clinicaltrials.gov identifier: NCT00612196.

Regulatory T cells (Tregs) hold much promise for the therapy of allergy and autoimmunity, but their use is hampered by lack of Ag specificity (natural Tregs) and difficulty to expand in vitro or in vivo (adaptive Tregs). We designed a method for in vivo induction of Ag-specific Tregs, in BALB/c H-2d, that share characteristics with type 1 Tregs (Tr1). A retroviral vector was constructed encoding a major T cell epitope of a common allergen, Der p 2, fused to an endosomal targeting sequence (gp75) for efficient MHC class II presentation. B cells transduced with such construct were adoptively transferred to BALB/c mice before or after peptide immunization. Long-lasting Ag-specific immune tolerance was achieved in both cases. Genetically modified B cells constitutively expressed the transgene for at least 3 mo. B cells from IL-10(-/-) mice were unable to induce tolerance. Upon transfer, B cells induced Foxp3(-)CD4(+) T cells showing phenotypic and functional characteristics comparable to Tr1-cells, including production of IL-10 but not of TGF-beta, and high expression of CTLA-4. Adoptive transfer of such T cells conferred unresponsiveness to allergen immunization and prevented the development of Der p 2-induced asthma. Functional Tr1-like cells can therefore be induced in vivo using retrovirally transduced B cells. ispartof: American Journal of Immunology pages:1107-1118 ispartof: Journal of Immunology vol:183 issue:12 pages:8232-8243 ispartof: location:United States status: published

BACKGROUND: The inhibitory activity of an anti-factor VIII (FVIII) antibody can be modulated through glycosylation of the antigen binding site, as has recently been described. This offers the opportunity to develop an optimized anticoagulant agent targeting partial FVIII inhibition. OBJECTIVES: We investigated in non-human primates the antithrombotic activity, pharmacokinetics,and pharmacodynamics of a human monoclonal antibody, Mab-LE2E9Q, inhibiting FVIII activity partially. METHODS: The ability of Mab-LE2E9Q to prevent thrombosis was evaluated in baboons after administration of 1.25 and 5 mg kg(-1) antibody or saline as a single intravenous (i.v.) bolus. Thrombus development was recorded in expansion ('venous') and in Dacron ('arterial') thrombosis chambers incorporated in an extracorporeal arteriovenous shunt implanted between the femoral vessels 1 h, 24 h and 7 days after the administration of Mab-LE2E9Q. RESULTS: Mab-LE2E9Q reduced thrombus growth to a similar extend 1 h, 1 day and 1 week after administration of the antibody. Ex vivo pharmacodynamic analysis indicated that the evaluation of the residual FVIII activity was strongly dependent on the type of FVIII assay and on the phospholipid concentration in the assay. No significant difference in bleedings was observed between animals treated with Mab-LE2E9Q or with saline. CONCLUSIONS: Understanding the role of glycosylation in FVIII inhibition by a human monoclonal antibody allowed selection of an antibody inhibiting only moderately FVIII activity while significantly reducing thrombus development in a baboon extracorporeal model. As that antibody did not increase the bleeding tendency, it may represent a novel type of a long-acting antithrombotic agent with an optimal safety/efficacy profile. ispartof: Journal of Thrombosis and Haemostasis vol:7 issue:3 pages:429-437 ispartof: location:England status: published

The mechanism of action of antibodies inhibiting partially factor VIII (FVIII) activity (type II inhibitor) is still poorly understood. We produced an unusual type II monoclonal antibody, called LE2E9, derived from a patient with mild haemophilia A. The antibody displayed several unexpected structural and functional properties such as glycosylation in the variable region, binding to the FVIII C1 domain, inhibition of maximum 80-90% FVIII activity when in excess over FVIII, and prevention of FVIII binding to von Willebrand factor (VWF). Those unusual characteristics of the antibody prompted multidisciplinary studies to determine its mechanism of action and the role of the FVIII C1 domain. Enzymatic deglycosylation and site-directed mutagenesis indicated that the oligosaccharides do not determine the affinity of the antibody but enhanced its FVIII neutralizing activity. Modification of glycosylation in the variable region of antibodies therefore contributes to the diversity of FVIII type II inhibition and provides a novel strategy with which to modulate the functional activity of antibodies. Investigation of the FVIII C1 domain function led to identification of mutations located in that domain and impairing FVIII binding to VWF as a common cause of mild/moderate haemophilia A. Finally, the cloning of human monoclonal antibodies inhibiting partially FVIII activity opened the way to evaluate such antibodies as a novel type of anticoagulant drug. This concept was validated in animal models of thrombosis. Those studies are expected to have a significant impact on the optimization of treatments for patients with inhibitor as well as for patients with thrombophilia.

This concise review is focused on genetic, molecular and clinical aspects of von Willebrand disease (VWD) type 2N and of mild hemophilia A due to mutations impairing FVIII-von Willebrand factor (VWF) interactions. Missense mutations in the VWF gene impairing the binding to FVIII do not impair the structure of VWF multimers nor the ability of VWF to aggregate platelets but causes an accelerated clearance of FVIII. Missense mutations in the FVIII gene impairing the binding to VWF are a common cause of mild/moderate hemophilia A. The implications of these observations for the treatment of patients with coagulation factor concentrates and desmopressin are discussed.

Patients with mild or moderate haemophilia A usually have a mild bleeding disorder requiring only occasional treatment with factor VIII (FVIII) concentrates. The frequency of inhibitor development in such patients has been the subject of several recent surveys, which significantly modified our appreciation of this complication. Studies of the anti-FVIII antibodies provided an explanation for the different bleeding phenotypes observed in mild/moderate haemophilia A patients with inhibitors. Antibodies distinguishing between the patient's mutant FVIII and the normal wild-type FVIII were characterized, in addition to antibodies inhibiting completely or only partially FVIII activity. T lymphocytes recognizing FVIII and likely involved in the development of the immune response to FVIII were successfully identified. The FVIII peptides recognized by those FVIII-specific cells bind to many major histocompatibility complex (MHC) class II molecules, which may provide an explanation for the lack of strong association between MHC haplotypes and inhibitor development. Although these studies have advanced our understanding of the conditions leading to inhibitor development, further work is required to determine whether the mode of FVIII administration significantly influences inhibitor development. Further studies of the genetic factors are also required to fully understand the mechanisms leading to inhibitor development in patients with mild/moderate haemophilia A.