Replacement therapy in hemophilia A with exogenous coagulation factor VIII (FVIII) often results in the development of FVIII-neutralizing antibodies, referred to as inhibitors. Despite of large ...number of studies on the functional properties of FVIII inhibitors, detailed physicochemical characterization of their interactions is not available. Here we studied the biophysical mechanism of the interaction between a human pathogenic antibody—BO2C11 and its target antigen—FVIII. Kinetic and thermodynamic analyses implied that this interaction is not accompanied by significant conformational changes in the proteins. The data also suggested that association of BO2C11 to FVIII is driven mainly by a hydrophobic effect. The protein electrostatics however played a decisive role in this association. Thus, a gradual increase in ionic strength resulted in a considerable increase in the association rate of binding of BO2C11 to FVIII. Such an ionic strength-dependency is uncommon for other antibody–antigen interactions. Our data suggest that electrostatic effects observed for BO2C11–FVIII association may arise from high-energy penalty of desolvation of the charged residues at the binding interfaces. We hypothesize that untypical ionic strength dependence of association of BO2C11 to FVIII reflects the nature of the recognized epitope, namely a molecular surface involved in the binding of FVIII to phospholipids. The presented data provide mechanistic information about FVIII neutralization by an inhibitory antibody and also contribute to the understanding of the general mechanisms of antibody–antigen interactions.
Interactions between platelet glycoprotein (Gp) IIb/IIIa and plasma proteins mediate platelet cross-linking in arterial thrombi. However, GpIIb/IIIa inhibitors fail to disperse platelet aggregates ...after myocardial infarction or ischemic stroke. These results suggest that stability of occlusive thrombi involves additional and as-yet-unidentified mechanisms. In the present study, we investigated the mechanisms driving platelet cross-linking during occlusive thrombus formation. Using computational fluid dynamic simulations and in vivo thrombosis models, we demonstrated that the inner structure of occlusive thrombi is heterogeneous and primarily determined by the rheological conditions that prevailed during thrombus growth. Unlike the first steps of thrombus formation, which are GpIIb/IIIa-dependent, our findings reveal that closure of the arterial lumen is mediated by GpIbα-von Willebrand Factor (VWF) interactions. Accordingly, disruption of platelet cross-linking using GpIbα-VWF inhibitors restored vessel patency and improved outcome in a mouse model of ischemic stroke, although the thrombi were resistant to fibrinolysis or traditional antithrombotic agents. Overall, our study demonstrates that disruption of GpIbα-VWF interactions restores vessel patency after occlusive thrombosis by specifically disaggregating the external layer of occlusive thrombi, which is constituted of platelet aggregates formed under very high shear rates.
•Following endothelial damage, platelet cross-linking during closure of the vessel lumen is mediated by GpIbα-VWF interactions.•Disruption of GpIbα-VWF interactions restores vessel patency by specifically disaggregating the external layer of occlusive thrombi.
To evaluate class III effects of clinically relevant concentrations of dofetilide (5 and 10 nmol/l) and the effects of extracellular potassium K
+
o modulation of arrhythmias onset at the level of ...the “border zone,” we used a previously reported in vitro model whereby normoxic and ischemic/reperfused zones were studied.
Guinea-pig right ventricular strips (driven at 1 Hz at 36.5
±
0.5 °C) were superfused with Tyrode's solution in oxygenated (HCO
3
− 25 mmol/l, K
+ 4 mmol/l, pH 7.35
±
0.05, glucose 5.5 mmol/l: normal zone) and ischemia-simulating conditions (HCO
3
− 9 mmol/l, pH 6.90
±
0.05, no oxygen and no glucose: altered zone) having either K
+
o 4 (
n
=
20), 8 (
n
=
20) or 12 (
n
=
20) mmol/l. Action potentials in normal and altered zones were recorded simultaneously during 30 min of simulated-ischemia and after 30 min of reperfusion with oxygenated Tyrode's solution. Each preparation served as control for successive phases of dofetilide studies (at 5 and 10 nmol/l) and action potential values were normalized to those present at the beginning of the experiment.
During simulated-ischemia, the higher the K
+
o the worse were action potential changes, although full recovery was seen upon 30 min of reperfusion in all K
+
o groups. A high incidence of ischemia/reperfusion arrhythmias was observed in 4 and 12 mmol/l K
+
o groups as opposed to a low incidence of arrhythmias in 8 mmol/l K
+
o group. Dofetilide at 5 and 10 nmol/l with all K
+
o explored: (i) exhibited class III effects, (ii) was effective (or neutral) against ventricular arrhythmias during both simulated-ischemia and reperfusion, and (iii) did not globally increase the dispersion of action potential durations between normal and altered zones.
Different arrhythmogenic mechanisms are involved in this model at different K
+
o with 8 mmol/l providing relative protection. Class III effects of dofetilide are evident in the normal zone when in the ischemic-like zone K
+
o ranges from 4 to 12 mmol/l. Thus dofetilide did not increase dispersion of repolarization and had either an antiarrhythmic or a neutral effect during ischemia/reperfusion.
Background: The use of extending half-life (EHL) FVIII or FIX products is today a current strategy in Hemophilia A (HA) patients for improving prophylaxis and reducing the number of IV injections. Fc ...fusion technology is based on the use of the neonatal Fc receptor and endogenous Fc recycling pathway, thereby prolonging the half-life (T1/2) of rFVIII-Fc. A single dose phase 1/2 pharmacokinetic (PK) study performed in 16 severe HA patients demonstrated a prolonged T ½ of rFVIII-Fc equal to 18.8 hours (mean) compared to 12.2 hours with one conventional rFVIII (Malhangu et al. Blood 2014).
The aim of the present study was to analyze PK data collected with Elocta® in “real life” i.e. in a large cohort of patients treated in 13 different French hemophilia care centers, and results were compared to those obtained with conventional FVIII, when available. Importantly, this study was performed without any involvement of Sobi, the pharmaceutical company that provides Elocta® in France.
Patients and methods: 113 severe Hemophilia A (HA) patients with the following characteristics were included: mean age 30 years (range 3 - 70); weight 65 Kg (17-125); total FVIII-Fc dose injected 2650 IU (500-5750); FVIII-Fc IU/Kg: 41 (25 - 59); VWF Ag 98% (41-279). The FVIII recovery (R) was calculated as follows: (body weight (Kg) x observed increased in FVIII (%))/administered dose (IU/Kg). The T1/2 was calculated with the following formula: Ln2/((Ln FVIII% T1 - Ln FVIII%T2)/T2 - T1)), with T1 ≥ 4 hours and T2 ≥ 24 hours.
Results were compared to those performed with conventional FVIII (non EHL-FVIII) in 48 patients (Advate® n = 14, Refacto® n = 2, Helixate®/Kogenate®/Kovaltry® n = 29, Factane® n = 3)
Results: rFVIII-Fc activity measured by one stage clotting assay (OSA) was 20% lower than those obtained with chromogenic assay (CSA) in samples with FVIII levels higher than 20%, but this difference was lower than 10% when FVIII levels < 20%. Therefore, rFVIII-Fc recovery (R) always appeared lower when measured with OSA (Mean 2.38, range 1.33 - 5.7) than with CSA (mean 2.82, ranges 1.35 - 5.5) (p < 0.0001). No correlation was found between this recovery and age, weight, injected doses or VWF Ag levels. Mean T1/2 measured with rFVIII-Fc equaled 15 hours whatever the measurement method used (OSA or CSA), and was strongly correlated with vWFAg levels (R2 = 0.57). Using OSA, significantly lower recovery (1.86 vs. 2.49, p = 0.0002) and T1/2 values (11.75 vs. 15.13 hours, p = 0.0004) were measured in children (< 10 years, n = 19) compared to adults. Similar differences were evidenced with data obtained by CSA (recovery : 2.26 vs. 2.93, p = 0.0009 and T1/2 : 11.4 vs. 15.6 h, p = 0.004, n = 14 children < 10 years).
PK parameters of FVIII-Fc were compared to those obtained with non EHL-FVIII (rFVIII or pdFVIII) in 47 patients (mean T1/2 equal to 10.0 hours; range 5.3 - 21.2), and half-lives of these two categories of products were well correlated (r2 = 0.57). However, the apparent benefit provided by FVIII-Fc was variable from one patient to another, with a mean T1/2 rFVIII-Fc / T1/2 FVIII ratio ranging from 0.6 to 2.4 (mean 1.4). Interestingly, the increase in T1/2 with FVIII-Fc was lower than 20% only in patients previously treated with BHK-derived rFVIII i.e. Helixate®/Kogenate®/ Kowaltry® (n=10). Whatever the FVIII injected (FVIII-Fc or other non EHL-FVIII), the T1/2 measured was also strongly correlated to vWF levels, which were significantly lower in patients for whom the mean T1/2 rFVIII Fc / T1/2 FVIII ratio was > 1.3 (mean 79% vs 116% in the others, p=0.017).
Conclusion: This study is the first to report PK data obtained with rFVIII-Fc (Elocta®) in a large group of HA patients. Our results confirm the benefit of rFVIII-Fc in most HA patients, adults or children, but also emphasize the impact of vWF on half-life of rFVIII-Fc or conventional non EHL-FVIII. Indeed, the benefit of rFVIII-Fc clearly appears higher in patients with lower vWF levels, with a more significant prolongation of T1/2.
No relevant conflicts of interest to declare.
The use of enhanced half‐life (EHL) FVIII has improved the quality of prophylaxis in haemophilia A, but with a benefit that may vary from one patient to another. We analysed the pharmacokinetic data ...obtained with efmoroctocog alfa (rFVIII‐Fc) in 114 patients and, in 47 cases, compared them to those previously measured with non‐EHL FVIII. The in vivo recovery (IVR) of rFVIII‐Fc measured with one stage clotting assay (OSA) and chromogenic assay (CSA) was 2.2 and 2.8 IU/mL per IU/kg, respectively. The median half‐life (T1/2) of rFVIII‐Fc was 14.5 hours whatever the FVIII:C assay used, but variable and correlated with preinfusion VWF:Ag levels (r = .76). Both IVR and T1/2 were lower in patients under 12 years old (2.4 IU/mL per IU/kg and 11.1 hours, respectively; CSA). PK study of rFVIII‐Fc vs non‐EHL FVIII showed a T1/2 ratio of 1.4 in favour of rFVIII‐Fc, regardless of the patient's age. However the relative increase in T1/2 with rFVIII‐Fc was lower than 30% in one‐third of patients evaluated, particularly when the previous FVIII administered was a BHK‐derived product. This study therefore suggests that analysis of individual PK profile in response to a specific FVIII concentrate is potentially useful before a switch in haemophilia A patients.
Replacement therapy in severe hemophilia A leads to factor VIII (FVIII) inhibitors in 30% of patients. Factor VIII gene (F8) mutation type, a family history of inhibitors, ethnicity and intensity of ...treatment are established risk factors, and were included in two published prediction tools based on regression models. Recently investigated immune regulatory genes could also play a part in immunogenicity. Our objective is to identify bio-clinical and genetic markers for FVIII inhibitor development, taking into account potential genetic high order interactions. The study population consisted of 593 and 79 patients with hemophilia A from centers in Bonn and Frankfurt respectively. Data was collected in the European ABIRISK tranSMART database. A subset of 125 severely affected patients from Bonn with reliable information on first treatment was selected as eligible for risk stratification using a hybrid tree-based regression model (GPLTR). In the eligible subset, 58 (46%) patients developed FVIII inhibitors. Among them, 49 (84%) were "high risk" F8 mutation type. 19 (33%) had a family history of inhibitors. The GPLTR model, taking into account F8 mutation risk, family history of inhibitors and product type, distinguishes two groups of patients: a high-risk group for immunogenicity, including patients with positive HLA-DRB1*15 and genotype G/A and A/A for IL-10 rs1800896, and a low-risk group of patients with negative HLA-DRB1*15 / HLA-DQB1*02 and T/T or G/T for CD86 rs2681401. We show associations between genetic factors and the occurrence of FVIII inhibitor development in severe hemophilia A patients taking into account for high-order interactions using a generalized partially linear tree-based approach.
Despite a high prevalence of angiodysplasia, no specific guidelines are available for the modalities of endoscopic exploration of gastrointestinal (GI) bleeding in von Willebrand disease (VWD). ...Whether VWD patients could benefit from video capsule endoscopy (VCE) looking for angiodysplasia eligible to endoscopic treatment or at high risk of bleeding is unknown.
To assess the diagnostic efficacy for angiodysplasia and the prognostic value of VCE on top of conventional endoscopy in VWD patients with GI bleeding.
A survey was sent to the 30 centers of the French-network on inherited bleeding disorders to identify VWD patients referred for endoscopic exploration of GI bleeding from January 2015 to December 2017. Data obtained included patient characteristics, VWD phenotype/genotype, GI bleeding pattern, results of endoscopic investigations, and medical management applied including endoscopic therapy. We assessed by Kaplan-Meier analysis the recurrence-free survival after the first GI bleeding event according to endoscopic categorization and, in patients with angiodysplasia, to the presence of small-bowel localizations on VCE exploration.
GI bleeding source localization was significantly improved when including VCE exploration (P < .01), even in patients without history of angiodysplasia (P < .05). Patients with angiodysplasia had more GI bleeding recurrences (P < .01). A lower recurrence-free survival was observed in patients with angiodysplasia (log-rank test, P = .02), and especially when lesions were located in the small bowel (log-rank test, P < .01), even after endoscopic treatment with argon plasma coagulation (log-rank test, P < .01).
VCE should be more systematically used in VWD patients with unexplained or recurrent GI bleeding looking for angiodysplasia eligible to endoscopic treatment or at high risk of relapse.
Cancer patients display cognitive impairment due, at least partly, to the treatments. Additionally, chemotherapeutic treatments can lead to organ injury, limiting their use, and are likely to have ...negative impacts on patients' quality of life. The aim of this study was to investigate the toxicity of 3-Deazaneplanocin A (DZNep) on several tissues and organs, as well as on cognitive functions. DZNep is an inhibitor of S-adenosylmethionine-dependent methyltransferase (in particular of the histone methyltransferase EZH2) which showed antitumoral functions in preclinical trials but whose effects on behavior and on organs (side effects) are not known. Chronic injections of DZNep were performed intraperitoneally in male NMRI mice (2 mg/kg; i.p.; three times per week) during 8 weeks. A follow-up of body weight was assessed during all experiments. Histological analysis were performed on several organs. EZH2 expression and H3K27me3 were assayed by western-blot. Several behavioral tests were performed during treatment and 2 weeks after. A particular focus was made on spontaneous locomotor activity, cognitive functions (spontaneous alternation and recognition memory), and anxiety- and depression-related behavior. Hematological modifications were also assessed. Chronic DZNep treatment transiently reduced animal growth. It had no effect on most organs but provoked a reversible splenomegaly, and persistent testis reduction and erythropoiesis. DZNep administration did not alter animal behavior. In conclusion, this study is encouraging for the use of DZNep for cancer treatment. Indeed, it has no effect on animal behavior, conferring an advantageous safety, and induces irreversible side effects limited on testis which are unfortunately found in most chemotherapy treatments.
Summary
Acquired factor V inhibitor (AFVI) is an extremely rare disorder that may cause severe bleeding. To identify factors associated with bleeding risk in AFVI patients, a national, multicentre, ...retrospective study was made including all AFVI patients followed in 21 centres in France between 1988 and 2015. All patients had an isolated factor V (FV) deficiency <50% associated with inhibitor activity. Patients with constitutional FV deficiency and other causes of acquired coagulation FV deficiencies were excluded. The primary outcome was incident bleeding and factors associated with the primary outcome were identified. Thirty‐eight (74 36–100 years, 42·1% females) patients with AFVI were analysed. Bleeding was reported in 18 (47·4%) patients at diagnosis and in three (7·9%) during follow‐up (7 0·2–48.7 months). At diagnosis, FV was <10% in 31 (81·6%) patients. Bleeding at diagnosis was associated with a prolonged prothrombin time that strongly correlated with the AFVI level measured in plasma {r = 0·63, 95% confidence interval (CI) 0·36–0·80, P < 0·05}. Bleeding onset during follow‐up was associated with a slow AFVI clearance (P < 0·001). The corresponding receiver operating characteristics curve showed that AFVI clearance was predictive of bleeding onset with an AFVI clearance of seven months with a sensitivity of 100% (95% CI: 29–100) and a specificity of 86% (95% CI: 57–98, P = 0·02). Kaplan–Meier analysis showed that AFVI clearance >7 months increased the risk of bleeding by 8 (95% CI: 0·67–97, P = 0·075). Prothrombin time at diagnosis and time for clearance of FV inhibitor during follow‐up are both associated with bleeding in patients with AFVI.
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