Publications by authors named "Marc Jacquemin"

47 Publications

Factor VIII-Fc Activates Natural Killer Cells Fc-Mediated Interactions With CD16.

Front Immunol 2021 28;12:692157. Epub 2021 Jun 28.

Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, United States.

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 <1%) hemophilia A (HA) patients and are a serious hindrance to effective management of HA. Consequently, strategies that can either prevent anti-FVIII inhibitors from developing or "tolerize" individuals who develop such antibodies represent a clinically important unmet need. One intervention for patients with high-titer inhibitors is immune tolerance induction (ITI) therapy. Although ITI therapy is the only clinically proven strategy to eradicate anti-FVIII inhibitors, mechanisms of inhibitor reduction remain unknown. Factor VIII Fc-fusion (rFVIIIFc) is an enhanced half-life antihemophilic factor used in replacement therapy for HA. Fc-fusion is a successful protein bio-engineering platform technology. In addition to enhancement of plasma half-life 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 CD16. We demonstrated rFVIIIFc signaling 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 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 findings suggest a potential use of Fc-fusion proteins acting NK cells to target antigen-specific B-cells, in the management of unwanted immune responses directed against immunogenic self-antigens or therapeutic protein products.
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http://dx.doi.org/10.3389/fimmu.2021.692157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8273617PMC
June 2021

Venous Thromboembolism in Patients Discharged after COVID-19 Hospitalization.

Semin Thromb Hemost 2021 Jun 23;47(4):362-371. Epub 2021 Apr 23.

Department of Cardiovascular Diseases, University Hospitals Leuven, Leuven, Belgium.

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.
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http://dx.doi.org/10.1055/s-0041-1727284DOI Listing
June 2021

Coagulation Factors Accumulate During Normothermic Liver Machine Perfusion Regardless of Donor Type and Severity of Ischemic Injury.

Transplantation 2021 Mar 22. Epub 2021 Mar 22.

Lab of Abdominal Transplantation, Transplantation Research Group, Department of Microbiology Immunology and Transplantation, KU Leuven, Belgium Abdominal Transplantation Surgery and Coordination, University Hospitals Leuven, Leuven, Belgium Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium Clinical Department of Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium Department of HPB and Liver Transplant Surgery, Royal Free Hospital, London, UK Clinique Universitaires Saint-Luc, UCL, Department of Pathology, Brussels, Belgium KU Leuven, Lab of Hepatology, Leuven, Belgium Vascular Medicine and Hemostasis, University Hospitals Leuven, Belgium Biomedical Research Center and Oxford Transplant Center, Nuffield Department of Surgical Sciences, University of Oxford, Oxford, UK.

Background: Coagulation factors may inform on liver function during normothermic machine perfusion (NMP). We investigated whether graft ischemic injury impairs the accumulation of (anti)coagulation factors during NMP of porcine and human livers.

Methods: Dynamics of FV, FVII, FVIII, FIX, 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 minutes 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 4 groups depending on donor type and post-transplant 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 2-6 fold lower in severely injured grafts (all p<0.05). All factors negatively correlated with AST (coefficient range -0.50 to -0.93; all p<0.05) and lactate (range -0.51 to -0.67; all p<0.05). In human livers, no difference in factor accumulation rates and no correlation with other markers was observed. One graft with primary nonfunction had low rate of factor accumulation.

Conclusion: (Anti)coagulation 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.
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http://dx.doi.org/10.1097/TP.0000000000003763DOI Listing
March 2021

Apixaban in patients on haemodialysis: a single-dose pharmacokinetics study.

Nephrol Dial Transplant 2021 04;36(5):884-889

Division of Nephrology, UZ Leuven, Leuven, Belgium.

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.
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http://dx.doi.org/10.1093/ndt/gfaa351DOI Listing
April 2021

Optimization of the detection of inhibitory autoantibodies against the VWF-cleaving protease ADAMTS13 with an automated chemiluminescent ADAMTS13 activity immunoassay.

Int J Lab Hematol 2021 Apr 10;43(2):290-297. Epub 2020 Oct 10.

Department of Cardiovascular Sciences, Centre for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.

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 HemosIL 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 Ca to preserve the activity of the metalloprotease. Using Bethesda assays based on the 3 incubation procedures and the HemosIL AcuStar ADAMTS13 Activity assay, the inhibitor titers were determined in patients' samples with ADAMTS13 antibodies and compared with those determined using the Technozym 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 Ca can be used in a Bethesda assay for quantifying the inhibitory activity of antibodies interfering with ADAMTS13 in the chemiluminescent HemosIL AcuStar ADAMTS13 Activity assay.
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http://dx.doi.org/10.1111/ijlh.13359DOI Listing
April 2021

Von Willebrand factor and ADAMTS13 impact on the outcome of Staphylococcus aureus sepsis.

J Thromb Haemost 2020 03 22;18(3):722-731. Epub 2019 Dec 22.

Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.

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.
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http://dx.doi.org/10.1111/jth.14686DOI Listing
March 2020

Accurate measurement of extended half-life and unmodified factor VIII low levels with one-stage FVIII assays is dependent on the matrix of calibration curves.

Haemophilia 2019 Jan 27;25(1):e19-e26. Epub 2018 Dec 27.

Clinical Department of Laboratory Medicine, University Hospitals of Leuven, Leuven, Belgium.

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.
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http://dx.doi.org/10.1111/hae.13656DOI Listing
January 2019

Pure ultra-fine carbon particles do not exert pro-coagulation and inflammatory effects on microvascular endothelial cells.

Environ Sci Pollut Res Int 2019 Jan 19;26(1):991-999. Epub 2018 Nov 19.

Department of Genetics and Molecular Medicine, School of Medicine, Zanjan University of Medical Sciences (ZUMS), Zanjan, Iran.

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.
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http://dx.doi.org/10.1007/s11356-018-3783-3DOI Listing
January 2019

Does haemophilia slow down the development of liver fibrosis?

Haemophilia 2019 01 14;25(1):e32-e35. Epub 2018 Nov 14.

Cliniques Universitaires Saint-Luc, Brussels, Belgium.

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http://dx.doi.org/10.1111/hae.13630DOI Listing
January 2019

Targeting Coagulase Activity in Staphylococcus aureus Bacteraemia: A Randomized Controlled Single-Centre Trial of Staphylothrombin Inhibition.

Thromb Haemost 2018 05 3;118(5):818-829. Epub 2018 Apr 3.

Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, University of Leuven, Leuven, Belgium.

Background: () bacteraemia is frequent and carries a high morbidity and mortality. Coagulases secreted by 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 infection.

Objective: A single-centre, randomized, controlled feasibility and safety trial of staphylothrombin inhibition with DTIs in patients with bacteraemia.

Patients And Methods: Consecutive eligible adult patients with 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 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;  = 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 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.
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http://dx.doi.org/10.1055/s-0038-1639586DOI Listing
May 2018

Biodistribution of Liver-Derived Mesenchymal Stem Cells After Peripheral Injection in a Hemophilia A Patient.

Transplantation 2017 08;101(8):1845-1851

1 Université Catholique de Louvain, Cliniques Universitaires St Luc, Service de Gastroentérologie & Hépatologie Pédiatrique, Brussels, Belgium.2 Université Catholique de Louvain, Institut de Recherche Expérimentale et Clinique, Brussels, Belgium.3 Centre de Thérapie Cellulaire et Tissulaire, Cliniques Universitaires St Luc, Brussels, Belgium.4 Service de Médecine Nucléaire, Cliniques Universitaires St Luc, Brussels, Belgium.5 Division of Medicine, Institute for Liver and Digestive Health, Royal Free Hospital, University College of London, London, United Kingdom.6 Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, KU Leuven, Belgium.7 Service d'Hématologie, Cliniques Universitaires St Luc, Brussels, Belgium.

Background: 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.

Methods: 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).

Results: 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.

Conclusion: These results suggest the potential use of ADHLSCs in the treatment of hemophilia A.
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http://dx.doi.org/10.1097/TP.0000000000001773DOI Listing
August 2017

Frequency and epitope specificity of anti-factor VIII C1 domain antibodies in acquired and congenital hemophilia A.

Blood 2017 08 15;130(6):808-816. Epub 2017 May 15.

Department of Pediatrics, Clinical and Molecular Hemostasis, Frankfurt University Hospital, Frankfurt am Main, Germany.

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.
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http://dx.doi.org/10.1182/blood-2016-11-751347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553573PMC
August 2017

Idarucizumab for dabigatran overdose.

Clin Toxicol (Phila) 2016 Sep 25;54(8):644-6. Epub 2016 May 25.

a Department of Cardiovascular Sciences , University of Leuven , Leuven , Belgium ;

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 lack of an antidote is a concern in patients who overdose on dabigatran, even though the drug can be eliminated with hemodialysis. Idarucizumab is an antibody fragment that binds dabigatran with high affinity. It reverses the anticoagulant effect of dabigatran within minutes and is approved for the reversal of dabigatran during emergency situations.

Case Details: We describe the use of idarucizumab in the management of a 68-year-old woman who was taking dabigatran 150 mg twice daily and ingested 125 capsules. Despite gastric lavage and administration of activated charcoal within two hours of drug intake, the activated partial thromboplastin time (aPTT) and prothrombin time (PT) remained prolonged. The administration of 5 g of intravenous idarucizumab promptly and completely reversed the anticoagulant activity of dabigatran as assessed by routine and specific coagulation assays (aPTT from to 75 to 26 s, PT from 26 to 11 s and diluted thrombin time from 92 to 27 s). The initially planned emergency hemodialysis was canceled.

Discussion: This case highlights the potential use of idarucizumab for the management of massive dabigatran overdoses.
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http://dx.doi.org/10.1080/15563650.2016.1187737DOI Listing
September 2016

Free hemoglobin: a boost to platelet thrombi.

Blood 2015 Nov;126(20):2262-3

UNIVERSITY OF LEUVEN.

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http://dx.doi.org/10.1182/blood-2015-09-667642DOI Listing
November 2015

T cell response to FVIII.

Cell Immunol 2016 Mar 26;301:8-11. Epub 2015 Sep 26.

University of Leuven, Center for Molecular and Vascular Biology, Herestraat 49 Bus 913, B3000 Leuven, Belgium; ImCyse s.a.-n.v., Bioincubator II, Gaston Geenslaan 1, B-3001 Leuven, Belgium. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cellimm.2015.09.007DOI Listing
March 2016

Factor VIII inhibitor: affinity is the clue.

Blood 2015 Feb;125(7):1056-8

UNIVERSITY OF LEUVEN.

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http://dx.doi.org/10.1182/blood-2014-12-618991DOI Listing
February 2015

Response: Extended analysis of microarray data does not contradict preplasmablast phenotype of human CD20+CD27+CD43+ cells.

Blood 2013 Nov;122(22):3693-4

PharmAbs, The Katholieke Universiteit Leuven Antibody Centre, and Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, Katholieke Universiteit Leuven, Leuven, Belgium.

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http://dx.doi.org/10.1182/blood-2013-09-528091DOI Listing
November 2013

Calibration of the X-ray photoelectron spectroscopy binding energy scale for the characterization of heterogeneous catalysts: is everything really under control?

Chemphyschem 2013 Oct 5;14(15):3618-26. Epub 2013 Sep 5.

Institute of Condensed Matter and Nanosciences (IMCN), Molecules, Solids and Reactivity (MOST), Université catholique de Louvain, Croix du Sud 2/L7.05.17, 1348 Louvain-la-Neuve (Belgium).

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-Al2O3 supports and Pd/SiO2-Al2O3 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.
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http://dx.doi.org/10.1002/cphc.201300411DOI Listing
October 2013

Characterization of proposed human B-1 cells reveals pre-plasmablast phenotype.

Blood 2013 Jun 23;121(26):5176-83. Epub 2013 Apr 23.

PharmAbs, The Katholieke Universiteit Leuven Antibody Centre, Katholieke Universiteit Leuven, Leuven, Belgium.

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.
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http://dx.doi.org/10.1182/blood-2012-12-471953DOI Listing
June 2013

Time course of acquired von Willebrand disease associated with two types of continuous-flow left ventricular assist devices: HeartMate II and CircuLite Synergy Pocket Micro-pump.

J Heart Lung Transplant 2013 May;32(5):539-45

Department of Cardiac Surgery, University Hospital Leuven, Belgium.

Background: Bleeding complications are frequent adverse events in patients supported with axial continuous-flow pumps. Previous retrospective studies demonstrated that bleeding events in patients with the HeartMate II (Thoratec Corp, Pleasanton, CA) were attributed to acquired von Willebrand syndrome. We sought to analyze the von Willebrand factor (VWF) profile in patients receiving a HeartMate II or a CircuLite (Saddle Brook, NJ,) device (Synergy Pocket Micro-pump) prospectively.

Methods: Prospectively analyzed were 34 patients supported with left ventricular assist device (LVAD; 26 with HeartMate II and 8 with CircuLite). The control group comprised 20 patients who underwent heart transplantation (HTx). Blood samples were taken pre-operatively and at 14 days and 3, 6, 9, and 12 months post-operatively.

Results: Patients with LVADs had a high incidence of bleeding complications. From the immediate post-operative phase throughout the entire observation, the VWF ristocetin cofactor activity (Rco)/antigen (Ag) ratio of patients with HeartMate II and CircuLite devices was consistently lower compared with HTx patients. No correlation was found between the individual VWF:Rco/Ag ratio and bleeding events or transfusion requirements. The VWF:Rco/Ag ratio normalized immediately in patients who received HTx.

Conclusions: Acquired von Willebrand syndrome was confirmed to occur immediately after the implantation of both types of LVAD and persisted up to 12 months. A lower VWF:Rco/Ag ratio was associated with larger transfusion requirements. Acquired von Willebrand syndrome resolves after LVAD explantation.
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http://dx.doi.org/10.1016/j.healun.2013.02.006DOI Listing
May 2013

Anticoagulation with fondaparinux for hemodiafiltration in patients with heparin-induced thrombocytopenia: dose-finding study and safety evaluation.

Artif Organs 2013 May 5;37(5):482-7. Epub 2013 Mar 5.

Department of Nephrology, University Hospitals Leuven, Leuven, Belgium.

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.
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http://dx.doi.org/10.1111/aor.12002DOI Listing
May 2013

Increased synapse formation obtained by T cell epitopes containing a CxxC motif in flanking residues convert CD4+ T cells into cytolytic effectors.

PLoS One 2012 9;7(10):e45366. Epub 2012 Oct 9.

Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.

The nature of MHC class II-binding epitopes not only determines the specificity of T cell responses, but may also alter effector cell functions. Cytolytic CD4+ T cells have been observed primarily in anti-viral responses, but very little is known about the conditions under which they can be elicited. Their potential as regulators of immune responses, however, deserves investigations. We describe here that inclusion of a thiol-disulfide oxidoreductase motif within flanking residues of class II-restricted epitopes results, both in vitro and in vivo, in elicitation of antigen-specific cytolytic CD4+ T cells through increased synapse formation. We show that both naïve and polarized CD4+ T cells, including Th17 cells, can be converted by cognate recognition of such modified epitopes. Cytolytic CD4+ T cells induce apoptosis on APCs by Fas-FasL interaction. These findings potentially open the way towards a novel form of antigen-specific immunosuppression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045366PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3467281PMC
April 2013

A randomized clinical trial comparing point-of-care platelet function assays and bleeding time in healthy subjects treated with aspirin or clopidogrel.

Platelets 2012 15;23(4):249-58. Epub 2011 Sep 15.

Merck Research Laboratories, Rahway, NJ 07065-0900, USA.

Traditional assays of the coagulation status of patients, bleeding time assessment (BT) and light transmission aggregometry (LTA), are useful in clinical drug development. However, these assays are both labor intensive and expensive. BT results can be operator dependent and by its nature can inhibit subject enrollment in a clinical trial. The preparation of platelet-rich plasma necessary for LTA requires specialized training and laboratory support. Alternatives to these methods are desirable. The goal of this study was identification of a quantitative, easy-to-use, point-of-care device with minimal technical variables that could facilitate assessment of platelet aggregation in clinical drug development. This was a double-blind, placebo-controlled, randomized, three-period cross-over study in healthy volunteers designed to compare the abilities of BT, LTA, and three point-of-care devices, Multiplate®, Platelet Function Analyzer-100®, and VerifyNow® to quantitate the effects on platelet function of 3 days of treatment with aspirin, clopidogrel, or placebo. The effect size (difference in treatment means divided by the pooled standard deviations [SD]) of the three point-of-care devices was greater than or similar to BT and LTA for all treatment comparisons examined. VerifyNow® had the highest effect size comparing ASA to placebo. Multiplate® had the highest effect size comparing clopidogrel to placebo. From this study, we conclude that any one of the three simple-to-use point-of-care devices can reliably assess the treatment effect of ASA and CLP on platelet function in comparison with BT or LTA at the study population level
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http://dx.doi.org/10.3109/09537104.2011.604806DOI Listing
November 2012

Retroviral vectors induce epigenetic chromatin modifications and IL-10 production in transduced B cells via activation of toll-like receptor 2.

Mol Ther 2011 Apr 14;19(4):711-22. Epub 2010 Dec 14.

Department of Molecular and Cellular Medicine, Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.

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 κ light polypeptide gene enhancer in B-cells inhibitor, α (NFKBIA), thereby preventing the translocation of the nuclear factor-κB (NF-κB) 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.
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http://dx.doi.org/10.1038/mt.2010.275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3070090PMC
April 2011

A membrane-interactive surface on the factor VIII C1 domain cooperates with the C2 domain for cofactor function.

Blood 2011 Mar 14;117(11):3181-9. Epub 2010 Dec 14.

Department of Medicine, Veterans Administration Boston Healthcare System, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

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 V(max) for all mutants but only the apparent membrane affinity for wild-type factor VIII. Monoclonal antibody BO2C11 decreased the V(max) 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.
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http://dx.doi.org/10.1182/blood-2010-08-301663DOI Listing
March 2011

Tolerability and pharmacokinetics of TB-402 in healthy male volunteers.

Clin Ther 2010 Jun;32(6):1205-20

Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.

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 microg/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 microg/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 treatment-emergent 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 approximately 48 hours in the d37.5-microg/kg dose groups. TB-402 was associated with a prolonged APTT at doses >or=2.5 microg/kg approximately 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.
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http://dx.doi.org/10.1016/j.clinthera.2010.06.012DOI Listing
June 2010

Activation of human endothelial cells from specific vascular beds induces the release of a FVIII storage pool.

Blood 2010 Jun 29;115(23):4902-9. Epub 2010 Mar 29.

Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.

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.
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http://dx.doi.org/10.1182/blood-2009-07-232546DOI Listing
June 2010
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