Using radial distribution functions to calculate cellular cross-absorbed dose forßemitters: comparison with reference methods and application for18F-FDG cell labeling.

To further improve the understanding ofin vitrobiological effects of incorporated radionuclides, it is essential to accurately determine cellular absorbed doses. In the case ofßemitters, the cross-dose is a major contribution, and can involve up to millions of cells. Realistic and efficient computational models are needed for that purpose. Conventionally, distances between each cell are calculated and the related dose contributions are cumulated to get the total cross-dose (standard method). In this work, we developed a novel approach for the calculation of the cross-absorbed dose, based on the use of the radial distribution function (rdf)) that describes the spatial properties of the cellular model considered. The dynamic molecular tool LAMMPS was used to create 3D cellular models and computerdfsfor various conditions of cell density, volume size, and configuration type (lattice and randomized geometry). The novel method is suitable for any radionuclide of nuclear medicine. Here, the model was applied for the labeling of cells with18F-FDG used for PET imaging, and first validated by comparison with other reference methods. MeanScrossvalues calculated with the novel approach versus the standard method agreed very well (relative differences less that 0.1%). Implementation of therdf-based approach with LAMMPS allowed to achieved results considerably faster than with the standard method, the computing time decreasing from hours to seconds for 106cells. Therdf-based approach was also faster and easier to accommodate more complex cellular models than the standard and other published methods. Finally, a comparative study of the meanScrossfor different types of configuration was carried out, as a function of the cell density and the volume size, allowing to better understand the impact of the configuration on the cross-absorbed dose.

New challenges and best practices for the laboratory monitoring of factor VIII and factor IX replacement.

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.

The adsorption of dabigatran is as efficient as addition of idarucizumab to neutralize the drug in routine coagulation assays.

INTRODUCTION: The direct thrombin inhibitor dabigatran interferes with thrombophilia screening and with the diagnosis of hemostasis disorders that develop during treatment with the anticoagulant. In vitro addition of idarucizumab, a humanized antibody fragment that binds dabigatran, to plasma samples containing dabigatran fully neutralizes the drug. This study was carried out to determine whether binding of dabigatran on selected insoluble commercial adsorbent material, DOAC-STOPR , was as efficient as idarucizumab to neutralize the anticoagulant activity of the drug in vitro. METHODS: Coagulation assays sensitive to dabigatran were carried out with patient and control plasma samples spiked with dabigatran and supplemented with idarucizumab or incubated with adsorbent material. RESULTS: In samples containing upto 10 000 ng/mL dabigatran, the adsorption procedure was at least as efficient as the addition of idarucizumab to neutralize the activity of the anticoagulant drug. Neither the adsorption procedure nor the addition of idarucizumab did impair routine coagulation assays carried out with plasma devoid of dabigatran, such as the activated partial thromboplastin time, prothrombin time, fibrinogen Clauss, and the thrombophilia screening assays used to detect antiphospholipid antibodies or activated protein C resistance. In addition, the adsorption procedure did not interfere with the detection of lupus anticoagulant samples. CONCLUSIONS: Adsorption of dabigatran in plasma samples containing the drug neutralizes its activity as efficiently as the addition of idarucizumab. This method allows the evaluation of thrombophilia markers without interruption of anticoagulation therapy or the detection of hemostasis disorders in patients treated with the drug.

Measurement of B-domain-deleted ReFacto AF activity with a product-specific standard is affected by choice of reagent and patient-specific factors.

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.

Influence of apixaban on commonly used coagulation assays: results from the Belgian national External Quality Assessment Scheme.

INTRODUCTION: The Belgian national External Quality Assessment Scheme performed a survey to assess the effect of the direct oral anticoagulant apixaban on the coagulation assays prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and antithrombin as performed with a large number of reagent/instrument combinations. METHODS: Four lyophilized plasma samples spiked with apixaban (0, 41, 94 and 225 ng/mL) were sent to the 195 Belgian and Luxembourg clinical laboratories performing coagulation testing. RESULTS: PT and aPTT were barely influenced at the concentrations tested. At 225 ng/mL apixaban, PT and aPTT clotting times were only 1.15 times longer than at 0 ng/mL. Among PT reagents, RecombiPlasTin 2G® showed a slightly higher sensitivity with 225 ng/mL apixaban prolonging the PT clotting time 1.3-fold. Among aPTT reagents, there was no appreciable difference in sensitivity. Fibrinogen results were unaffected by the presence of apixaban, but antithrombin activity was considerably overestimated when measured with a FXa-based assay. At 225 ng/mL apixaban, the median percentage increase in antithrombin level was 31% when measured with the Liquid Antithrombin® reagent and 44% with the Innovance Antithrombin® reagent. CONCLUSION: Our data provide clinical laboratories with useful information on the impact of apixaban on their routine coagulation assays.

The amplitude of coagulation curves from thrombin time tests allows dysfibrinogenemia caused by the common mutation FGG-Arg301 to be distinguished from hypofibrinogenemia.

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.

The addition of idarucizumab to plasma samples containing dabigatran allows the use of routine coagulation assays for the diagnosis of hemostasis disorders.

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.

Human liver sinusoidal endothelial cells but not hepatocytes contain factor VIII.

BACKGROUND: Although the liver is the major site of coagulation factor VIII (FVIII) synthesis, the type of cells producing FVIII within the liver is still unclear. OBJECTIVES: To measure FVIII in extracts of primary liver sinusoidal endothelial cells (LSECs) and hepatocytes, thereby preventing potential bias resulting from the modifications of the cell phenotype that can take place during in vitro culture. METHODS: LSECs were purified by flow cytometry cell sorting on the basis of their coexpression of Tie2 and CD32b. The purity of the cells was controlled by RNA sequencing. FVIII activity (FVIII:C) in extracts of purified cells was measured with a sensitive FVIII chromogenic assay, in which the specificity of the reaction is controlled by neutralization of FVIII activity with specific inhibitor antibodies. RESULTS: The FVIII:C concentration in purified LSECs ranged from 0.3 to 2.8 nU per cell. In contrast, FVIII:C was undetectable in hepatocytes. The intracellular FVIII:C concentrations are therefore at least 10-100-fold higher in LSECs than in hepatocytes. CONCLUSIONS: Our data demonstrate that LSECs, but not hepatocytes, contain measurable amounts of FVIII:C, and suggest that the former are the main cells producing FVIII in the human liver.

Comparing normal saline versus diluted heparin to lock non-valved totally implantable venous access devices in cancer patients: a randomised, non-inferiority, open trial.

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 <1.4. RESULTS: Three hundred and eighty-two patients from the NS group and 383 from the heparin lock group were included in the analysis. The incidence rate of our primary outcome (easy injection, impossible aspiration) was 3.70% (95% CI 2.91%-4.69%) and 3.92% (95% CI 3.09%-4.96%) of accesses in the NS and heparin groups, respectively. The relative risk was 0.94% (95% CI 0.67%-1.32%). Catheter-related bloodstream infection was 0.03 per 1000 catheter days in the NS group and 0.10 per 1000 catheter days in the heparin group. CONCLUSION: NS is a safe and effective locking solution in implantable ports if combined with a strict protocol for device insertion and maintenance.

Practical and cost-effective measurement of B-domain deleted and full-length recombinant FVIII in the routine haemostasis laboratory.

The assessment of recombinant FVIII (rFVIII) activity (FVIII:C) in plasma of patients is dependent on the assay. Notably, a calibration with a product-specific laboratory standard is recommended when measuring Refacto-AF(R) activity in plasma with a one-stage assay. The objective of this study was to facilitate the measurement of rFVIII, taking into account the recent demonstration that a calibration curve does not have to be included in each run. FVIII:C was measured in patients' samples after infusion of different types of rFVIII with a one-stage and a chromogenic assay calibrated either with pooled normal plasma or a product-specific laboratory standard. Results obtained with the one-stage coagulation assay were compared with these provided by a chromogenic assay. We confirmed that a calibration curve can be used for a prolonged period of time without loss of precision and accuracy. In such conditions, a stable relation between the calibration curves generated with a product-specific laboratory standard and plasma can be established. In patients' plasma, Refacto-AF levels measured with a one-stage FVIII assay calibrated with plasma or a product-specific laboratory standard diverged from -58% to -17% and from -25% to +18%, respectively, from the activity determined with a chromogenic substrate assay. By comparison, FVIII:C levels of full-length rFVIII measured with the one-stage assay calibrated with plasma were 6-49% lower than with the chromogenic assay. In a monocentric setting, the long-term stability of the calibration curves allows the implementation of a practical and cost-effective approach to determine rFVIII:C levels.

Antidote strategies to reverse anticoagulation with TB-402, a long-acting partial inhibitor of factor VIII.

BACKGROUND: TB-402 is a partially inhibiting antibody of factor VIII that is under development as a long-acting anticoagulant. PATIENTS AND METHODS: The reversibility of FVIII inhibition by TB-402 was evaluated in vitro after spiking with recombinant human FVIII (rhFVIII), human plasma-derived FVIII (hpdFVIII), recombinant activated human FVII (rhFVIIa), FVIII inhibitor bypassing activity (FEIBA), and prothrombin complex concentrate (PCC). Twelve subjects were randomized to placebo or 35 or 70 IU kg(-1) rhFVIII 48 h after a single dose of 620 µg kg(-1) TB-402. TB-402 concentrations, FVIII activity (FVIII:C), activated partial thromboplastin time (APTT) and thrombin generation were measured over a period of 8 weeks. RESULTS: In spiked samples, TB-402 inhibited FVIII:C by 30%, prolonged APTT by 4.5 s, and reduced the peak height in the thrombin generation assay to 56% ± 13% of the control value. In the presence of 10 µg mL(-1) TB-402, rhFVIII restored FVIII:C and APTT to the values obtained in the absence of TB-402. The inhibitory effect of TB-402 on thrombin generation was entirely reversed by rhFVIII, hpdFVIII, rhFVIIa, FEIBA, and PCC. In men, the mean half-life (t(1/2) ) of TB-402 was 14.2 days. TB-402 lowered the endogenous thrombin potential by 23% for ~ 35 days. Infusion of 35 IU kg(-1) rhFVIII had a marginal effect, whereas 70 IU kg(-1) rhFVIII restored FVIII:C, reduced APTT back to baseline for 9 h, and restored thrombin generation for ~ 3 h. CONCLUSIONS: TB-402 resulted in a stable long-term anticoagulant effect. rhFVIII and other procoagulants counteracted the effect of TB-402 temporarily, and may be effective antidotes for future clinical practice.

Partial versus complete factor VIII inhibition in a mouse model of venous thrombosis.

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 0.1mg/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 1mg/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 (1mg/kg) inhibited thrombus formation to the same extent as BO2C11 (2mg/kg) and enoxaparin (5mg/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.

Successful immune tolerance induction by FVIII in hemophilia A patients with inhibitor may occur without deletion of FVIII-specific T cells.

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 30years 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.

Single intravenous administration of TB-402 for the prophylaxis of venous thromboembolism after total knee replacement: a dose-escalating, randomized, controlled trial.

BACKGROUND: TB-402 is a novel anticoagulant monoclonal antibody with a prolonged antithrombotic effect resulting from its partial factor (F)VIII inhibition and long half-life. We evaluated the efficacy and safety of a single administration of TB-402 for the prevention of venous thromboembolism (VTE) after total knee replacement (TKR). PATIENTS AND METHODS: This was a phase II, dose-escalating, randomized, enoxaparin-controlled, open-label study. Patients were post-operatively assigned to a single dose of TB-402 (0.3, 0.6 or 1.2 mg kg(-1)) or enoxaparin 40 mg for at least 10 days (n = 75 per group; 3:1 TB-402 to enoxaparin). The primary efficacy outcome was total VTE defined as asymptomatic deep vein thrombosis (DVT) detected by bilateral venography and symptomatic VTE by day 7 to 11. The principal safety outcome was the incidence of major bleeding and clinically relevant non-major bleeding. RESULTS: Total VTE was lower in all TB-402 groups compared with enoxaparin: 16.7%(95% CI 9.8-26.9), 23.9%(95% CI 15.3-35.3), 24.1%(95% CI 16.0-34.5) and 39.0%(95% CI 28.8-50.1) for TB-402 0.3, 0.6, 1.2 mg kg(-1) and enoxaparin, respectively (P = 0.003 for TB-402 0.3 mg kg(-1) vs. enoxaparin). The incidence of total VTE in the pooled TB-402 groups was 21.6% (95%CI 16.6-27.5), an absolute risk reduction vs. enoxaparin of 17.4% (95% CI 5.2-29.6). Major or clinically relevant non-major bleeding was observed in 3/75(4.0%), 4/74(5.4%), 7/87(8.0%) and 3/79(3.8%) patients for TB-402 0.3, 0.6, 1.2 mg kg(-1) and enoxaparin, respectively. CONCLUSIONS: TB-402, as a single post-operative administration, was associated with a lower rate of VTE in all doses tested, compared with enoxaparin. The incidence of major and clinically relevant non-major bleeding was similar to enoxaparin 40 mg for TB-402 0.3 and 0.6 mg kg(-1).

Mild haemophilia: a disease with many faces and many unexpected pitfalls.

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.

Variable region heavy chain glycosylation determines the anticoagulant activity of a factor VIII antibody.

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.

A human FVIII inhibitor modulates FVIII surface electrostatics at a VWF-binding site distant from its epitope.

SUMMARY BACKGROUND: BO2C11 is a human monoclonal factor (F) VIII inhibitor. When bound to the C2 domain of FVIII, the Fab fragment of BO2C11 (Fab(BO2C11)) buries a surface of C2 that contains residues participating in a binding site for von Willebrand factor (VWF). BO2C11 has thus been proposed to neutralize FVIII by steric hindrance. OBJECTIVES: The BO2C11 epitope on C2 overlaps with residues located at the periphery of the putative VWF binding site; hence, most of the residues that constitute the VWF binding site on C2 and a3 remain accessible for VWF interaction following BO2C11/FVIII complex formation. We thus investigated the contribution of alternative molecular mechanisms to FVIII inactivation by BO2C11. METHODS: Continuum electrostatic calculations were applied to the crystal structure of C2, free or Fab(BO2C11)-complexed. In silico predictions were confirmed by site-directed mutagenesis and VWF-binding assays of the mutated FVIII. RESULTS: Binding of Fab(BO2C11) to C2 induced perturbations in the electrostatic potential of C2 and in the local electrostatic parameters of 18 charged residues in C2, which are distant from the BO2C11 epitope. Nine of the predicted electrostatic hotspots clustered on the VWF-binding site of C2. Mutation of some of the predicted electrostatic hotspots has been associated with hemophilia A and reduced VWF binding in vitro. CONCLUSIONS: Inhibitors may neutralize FVIII by alteration of protein surface electrostatics at a long distance from their epitope. Perturbation of the electrostatic environment of C2, either upon binding by anti-FVIII antibodies or consecutive to missense mutations in the F8 gene, may lead to hampered VWF binding and reduced FVIII residence time in circulation.

A human monoclonal antibody inhibiting partially factor VIII activity reduces thrombus growth in baboons.

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.

Mild/moderate haemophilia A: new insights into molecular mechanisms and inhibitor development.

In mild/moderate haemophilia A (MHA) patients, many factor VIII (FVIII) gene defects, mainly missense mutations, have been identified and greatly improved the understanding of the structure and function of FVIII molecule. Characterization of the molecular mechanisms involved in MHA has helped to identify regions critical for proper FVIII biosynthesis, thrombin activation, intramolecular stability as well as binding regions for important intermolecular interactions with von Willebrand factor, factor IXa and the phospholipid surface. Some missense mutations were also recognized as contributing factors to inhibitor development in MHA, in parallel to acquired factors such as inflammatory state or intensity of treatment. Treatment of MHA with inhibitor patients raises questions on how best to stop or prevent bleeding episodes and eradicate the inhibitor. Longitudinal data collection is currently being conducted in France and Belgium to enhance our knowledge in this field and to further help make treatment decision. The description of mutations in MHA finally contributed to the identification of epitopes involved in the immune response to FVIII. In some patients, the epitope specificity of inhibitor antibodies recognizing normal exogenous FVIII alone and not patient ('self') FVIII was described. This distinguished epitope specificity could also be demonstrated at the T-cell clonal level. One might expect that these molecular studies will have a major impact on development of new FVIII products in the future.

The use of factor VIII/von Willebrand factor concentrate for immune tolerance induction in haemophilia A patients with high-titre inhibitors: association of clinical outcome with inhibitor epitope profile.

A retrospective chart review of 11 subjects with severe haemophilia A and high-titre inhibitors who received a von Willebrand factor-containing FVIII concentrate (VWF/FVIII) for immune tolerance induction (ITI) was accompanied by B cell inhibitor epitope mapping during 10/11 treatment courses. ITI was successful or partially successful in all seven subjects who received VWF/FVIII for initial ITI, and failed in all four subjects whose ITI with this product was initiated following treatment failure using recombinant factor VIII. Variables including age at inhibitor development and age at ITI initiation, interval between inhibitor detection and ITI initiation, titre at start of ITI, and peak historical titres prior to and during ITI were not statistically significant outcome predictors in this cohort. However, the B cell epitope specificity in all four successful and in one of two partially successful ITI subjects for whom information was available included the C2 and excluded the A2 domains. Conversely, FVIII B cell epitopes in one partially successful ITI and in all three failed ITI subjects for whom data were available mapped to both the C2 and the A2 domains. The FVIII B cell epitope profile was associated with ITI outcome in this VWF/FVIII-treated cohort. Its role in predicting ITI outcome and guiding choice of FVIII product for ITI requires further study.