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Nonacog alfa, a standard half-life recombinant factor IX (FIX), is used as a prophylactic treatment in severe haemophilia B (SHB) patients. Its half-life determined in clinical studies involving a limited sampling (72 h) was shown to be rather short. In our clinical practice, we suspected that its half-life could have been underestimated.We aimed to evaluate nonacog alfa pharmacokinetics in real world clinical practice based on FIX levels in patients receiving prophylaxis.We retrospectively collected data on patients with SHB receiving prophylaxis from eight centres across France. The terminal half-life (THL), time to reach 5-2 IU/dl and FIX activity at 48, 72 and 96 h were derived by Bayesian estimations using NONMEM analysis.Infusion data (n = 455) were collected from 64 patients with SHB. The median THL measured in 92 pharmacokinetic (PK) studies was 43.4 h. In 26 patients ≤12 years of age, 51 PK studies showed a median time to reach 5 IU/dl of FIX of 70.5 h and a median time to reach 2 IU/dl of 121.5 h. In 38 patients 13-75 years of age, 41 PK studies showed a median time to reach 5 IU/dl of FIX of 92.0 h and a median time to reach 2 IU/dl of 167.5 h. Extending the sampling beyond 72 h makes it possible to observe a plateau, with FIX remaining between 2 and 5 IU/dl for several days and shows that the THL of nonacog alfa might be longer than previously described.Nonacog alfa terminal half-life (THL) in patients receiving regular prophylaxis was evaluated in clinical practice. The median THL was estimated to be 36.9 h for patients aged .8-12 years. The median THL was estimated to be 49.9 h for patients aged 13-75 years. For patients aged ≤12 and 12 years, the median times to reach 5 IU/dl were 70.5 and 92 h, respectively; to reach 3 IU/dl, 95.5 and 131.5 h, respectively; to reach 2 IU/dl, 121.5 and 167.5 h, respectively. We suggest that the half-life of nonacog alfa might be longer than previously described in both younger and older patients.

Introduction Two meta-analyses showed lower bone mineral density (BMD) in patients with haemophilia (haemophilia type and severity were often not specified) compared with healthy controls. This finding could be related to reduced mobility and sedentary lifestyle, and/or hepatitis C or HIV infection. The aim of this study is to determine osteoporosis prevalence in patients with haemophilia classified in function of the disease type (A or B) and severity, and to evaluate the potential role of regular prophylactic factor replacement (early vs delayed initiation) in preserving or restoring BMD. Methods and analysis The haemoPHILia and ostEoporOSis Study is a prospective, controlled, multicentre study that will include patients in France (13 haemophilia treatment centres), Belgium (1 centre) and Romania (1 centre). In total, 240 patients with haemophilia and 240 matched healthy controls will be recruited (1:1). The primary objective is to determine osteoporosis prevalence in patients with severe haemophilia A and B (HA and HB) without prophylaxis, compared with healthy controls. Secondary outcomes include: prevalence of osteoporosis and osteopenia in patients with mild, moderate and severe HA or HB with prophylaxis (grouped in function of their age at prophylaxis initiation), compared with healthy subjects; BMD in patients with HA and HB of comparable severity; correlation between BMD and basal factor VIII/IX levels and thrombin potential; and quantification of plasmatic markers of bone remodelling (formation and resorption) in patients with haemophilia. Ethics and dissemination The protocol was approved by the French Ethics Committee and by the French National Agency for Medicines and Health Products Safety (number: 2019-A03358-49). The results of this study will be actively disseminated through scientific publications and conference presentations. Trial registration number NCT04384341.

Emicizumab is a prophylaxis for patients with severe haemophilia A with and without inhibitor. Despite weekly administration of emicizumab, coagulation states stay below normal value and cannot be assessed by standard haemostasis tests. In our two patients, we used the thrombin-generation assay (endogenous thrombin potential and Peak) to monitor the patient's clotting status. Under emicizumab, it is necessary to add a bypassing agent (BPA) such as rFVIIa (Novoseven) to avoid bleeding before surgery. The BPA dosage was based on a thrombin-generation assay and collegial consultation.

The thrombin generation (TG) assay is a well-established tool to capture the clotting potential of any healthy or haemophiliac subject. It measures ex vivo the kinetics of thrombin activation throughout the coagulation. Clinical studies allowed to create two databases gathering the coagulation factor levels and the thrombin generation profile of 40 healthy and 40 haemophiliac A (HA) subjects. Besides, portions of all HA samples were spiked with increasing levels of a TFPI antibody (considered as a possible therapeutic target) and corresponding TG profiles were determined. The non-linear mixed-effect (NLME) modelling aims at describing and explaining the experimentally observed important variability of the TG curves between subjects and the individual effects of spiking with a TFPI antibody. The models consist of an empirical description of the TG kinetics, accounting for an additive residual error and between-subject variability on its parameters. Factor VIII and TFPI were found to significantly explain and reduce the variability of the TG of haemophilia A samples. Besides, the model is shown to correctly reproduce the variability in the response to the ex vivo spiking with the TFPI antibody, by combining the empirical description of TG to a simple Hill equation that accounts for the binding between TFPI and different doses of its antibody. Such models can be useful for clinical practice, with the analysis and comparison of the distributions of TG profiles in healthy and haemophilia populations; and also for research, with the analysis of the effect of TFPI and its neutralization on individual TG profiles.

The thrombin generation (TG) assay evaluates haemostatic balance, which is influenced by the levels of many coagulation factors and inhibitors. Our objective was to identify the determinant factors of TG in haemophilia A (HA) and haemophilia B (HB) patients and to compare them to those in healthy controls. Coagulation factor and inhibitor levels, and TG, were measured in platelet-poor plasma from 40 patients with HA, 32 patients with HB and 40 healthy subjects. Data were analysed using multiple regression models. In HA patients, factor VIII was a positive determinant of endogenous thrombin potential (ETP) and peak, whereas tissue factor pathway inhibitor (TFPI) and factor V were negative determinants of ETP and peak. In HB patients, FIX was a positive determinant of ETP and peak, FVII being a positive determinant of peak. Antithrombin and protein S (PS) were negative determinants of ETP while FX was a negative determinant of peak. Above all, in HB patients, TFPI was a negative determinant of ETP and peak. In healthy subjects, FVIII was a positive determinant of ETP and peak, whereas FX and protein S were negative determinants of these parameters. TFPI was not a negative determinant of either peak or ETP. In haemophilic patients, the determinant factors of TG are all implicated in FXa generation and inhibition, the crucial determinant factor being TFPI whatever the type of haemophilia, A or B. These findings contribute to the rationale that recently place TFPI as a target for innovative therapies of haemophilia.

Background Multiple myeloma (MM) is associated with a high risk of thrombosis, particularly during the first months of treatment including immunomodulatory drugs (IMiDs). There is no consensus on prevention of thromboembolic risk in patients with de novo MM, and identification of patients requiring anticoagulant thromboprophylaxis remains challenging. Evaluating coagulability by an in vitro thrombin generation (TG) test might be a way of identifying such patients. Objective To determine whether TG assessment could reveal an increase in coagulability during the first three chemotherapy cycles. Methods This prospective and longitudinal observational study included patients newly diagnosed with MM. TG was determined in platelet-rich and platelet-poor plasma using calibrated automated thrombography with a low tissue factor (TF) concentration. Results Seventy-one patients were enrolled, allowing TG analysis during 213 chemotherapy cycles. TG remained unchanged throughout follow-up irrespective of treatment regimen, but values determined before cycles 2 and 3 were significantly higher in patients receiving iMiDs-containing regimens. No association was found between TG and its changes and thrombosis occurrence during follow-up: venous thrombosis in eight patients; no cardiovascular event. A significantly (87%) lower risk of venous thrombosis was observed in patients receiving prophylaxis with a low-molecular-weight heparin (LMWH; OR: 0.13 (95% CI: 0.02-0.76). Neither bortezomib- nor dexamethasone-containing regimens were associated with thrombotic risk. Changes in TG, as studied, were not associated with thrombotic events. Conclusions The only factor associated with a reduction in early thrombotic risk was prophylaxis with LMWH. The issue of how to identify patients requiring prophylactic anticoagulation remains unresolved.

Background and purpose: Biological response to clopidogrel prescribed after a non-cardioembolic ischemic stroke or transient ischemic attack (TIA) has been little studied. The aim of our study (AAPIX) was to assess this response and investigate the agreement between different biological assays in revealing poor responders. Methods: Patients hospitalized following a non-cardioembolic ischemic stroke or transient ischemic attack (TIA) and prescribed clopidogrel were consecutively included from September 2013 to November 2015 in the Stroke Center of Saint-Etienne Hospital. Blood was drawn after 5 to 8 days of standard-dose clopidogrel. Light transmission aggregometry (LTA) and flow cytometric assays, using vasodilator-stimulated phosphoprotein [VASP] and CD62P, were accomplished for all patients. Transmission electron microscopy (TEM) was performed for a poor clopidogrel-responder and for a patient with discordant platelet assay results (platelet reactivity index (PRI) >50% and maximum platelet aggregation

BACKGROUND The thrombin generation (TG) assay can assess individual clotting potential. The thrombin generation potential is correlated with the patient's bleeding phenotype and varies from one patient to the other for the same degree of factor VIII or IX deficiency. OBJECTIVE To define in vitro for individual haemophilic patients the target factor VIII or IX level required to normalize their TG. PATIENTS/METHODS Plasmas from 20 haemophilic patients were spiked with increasing levels of the deficient coagulation factor and TG parameters were measured. The relationships between factor levels and TG parameters were determined by linear regression. The normal range of thrombin generation was defined in 39 healthy male volunteers. RESULTS Despite inter-individual heterogeneity in basal TG and responses to spiking, a linear relationship was found between factor VIII or IX levels and TG parameters for individual patients. Based on the individual responses of patient plasmas to spiking, it is possible to define in vitro the target factor VIII or IX levels needed to normalize the TG parameters. For both haemophilic A and haemophilic B patients, significant correlations were found between basal peak values and their correction slopes. The correction slope was steeper in haemophilic B patients, so the factor IX level needed to normalize the TG parameters was lower than for haemophilic A patients. CONCLUSIONS The TG assay could be used to determine in vitro the patient-specific factor VIII or IX level to be reached to effectively normalize their TG. These in vitro results should be confirmed by ex-vivo studies.

Background and purpose: Low biological response to Clopidogrel prescribed after non cardioembolic ischemic stroke or transient ischemic attack (TIA) is a major clinical problem and could explain the recurrence of vascular events. Platelet α2-adrenoreceptors are involved in the high residual platelet reactivity in stable coronary artery disease patients on dual antiplatelet therapy. In the present study we investigated the impact of platelet α2-adrenoreceptors on ADP-induced platelet aggregation and on ADP-induced platelet membrane CD62P (P-selectin) expression, a marker of platelet activation on blood samples from patients hospitalized at the acute phase of a non cardioembolic ischemic stroke or TIA. Methods: 72 consecutive patients were prospectively recruited over the course of two years in a monocentric study. Patients received a daily 75 mg-dose of Clopidogrel. ADP-induced platelet aggregation was measured alone, with low dose epinephrine or with atipamezole, a selective α blocker of α2-adrenoreceptors, by Light Transmittance Aggregometry (LTA). Platelet membrane expression of P-selectin was measured by flow cytometry with either ADP alone or combined with epinephrine. Results: Epinephrine at low dose stimulated ADP-induced platelet membrane expression of CD62P whereas Atipamezole significantly inhibited 10 µM ADP-induced platelet aggregation. Conclusions: Our study showed the role of platelet α2-adrenoreceptors in biological low response to Clopidogrel for patients hospitalized for a non-cardioembolic ischemic stroke or TIA. Atipamezole could improve the status of biological response to Clopidogrel.

The coagulation cascade comprises numerous chemical reactions between many proteins, that finally lead to the formation of a clot to stop bleeding. Many numerical models have attempted to translate understanding of this cascade into mathematical equations that simulate the chain reactions. However, their predictions have not been validated against clinical data stemming from patients. In this paper, we propose an extensive validation of five available models, by comparing in healthy and haemophilic subjects, thrombin generation measured in vitro to thrombin generation predicted by the models in silico. In order to render the models more predictive, we calibrated the models to have an acceptable agreement between the experimental and estimated data. Optimization processes based on genetic algorithms were developed to search for those calibrated kinetic parameters. Our results show that the thrombin generation kinetics are so complex that they cannot be predicted by a unique set of kinetic parameters for all patients: the calibration of only three parameters in a subject-specific way allows reaching good model estimations for different experimental conditions realized on the same patient.

Delayed-onset heparin-induced thrombocytopenia without thrombosis in a patient receiving postoperative thromboprophylaxis with rivaroxaban

HSP90 targeting is a promising therapeutic approach in cancer. 17-AAG is an HSP90 inhibitor with completed Phase I trials in patients with advanced cancer and recently published Phase II trials. The aim of this work was to study the expression of HSP 90 and apoptotic proteins, the effects in culture of 17-AAG on cell survival and apoptosis and to compare Philadelphia-positive (Ph+) ALL to common B cell ALL, in ALL cell lines and in patients’ cells collected at ALL diagnosis. We analysed 2 ALL cell lines and 63 leukaemic samples from patients treated in our institution (44 common B cell ALL and 19 Ph+ ALL). We performed flow cytometry analysis of bone marrow aspiration and cell lines with a combination of anti-HSP90, Bax, Bcl-2 and Bcl-xl antibodies. Apoptosis after cell culture (in presence or not of 17-AAG) was assessed using Annexin V and activated caspase-3 staining. Ph+ ALL cells appeared to be more sensitive to 17-AAG cytotoxicity with a 100 % mortality rate after exposure to 10 μM for 24 h (vs. 62 % for B-common ALL). A high percentage of HSP90-positive cells (in Ph+ ALL samples) was associated with high sensitivity to 17-AAG. 17-AAG induced apoptosis in a dose-dependent manner and was associated with down-regulation of Bcl-2 and Bcl-Xl expression and up-regulation of Bax expression. Considering that Bcr-Abl constitutes HSP 90 substrates, HSP 90 inhibition could be of particular interest for Ph+ ALL disease, even in patients harbouring resistance to tyrosine kinase inhibitor therapy.