Sensitivity to FVIII inhibitors of the native plasma-derived (pd) FVIII/VWF complex vs. the complexes formed after exogenous FVIII infusion in the haemophilic patient has not been thoroughly studied. The role of VWF in the interaction of FVIII with inhibitors was studied in vitro using different combinations of VWF and FVIII concentrates. Normal plasma, pdFVIII/VWF and isolated FVIII (recombinant FVIII, B-domain deleted and pdFVIII) were used. Titre (BU) was kinetically determined (up to 2 h) in serial dilutions of inhibitor IgG (purified from a pool of plasmas with inhibitors) mixed with VWF and then incubated with the different FVIII. Inhibitor was also added to previously mixed VWF+FVIII. Residual FVIII:C was determined. TGA assays were performed with FVIII-deficient plasma spiked with the FVIII-VWF mixtures with/without an ESH-8 antibody. Inhibitor titres for plasma and pdFVIII/VWF were comparable at all time points. Titres for all concentrates of isolated FVIII were significantly higher than those for plasma or pdFVIII/VWF (1.4–1.9 fold) even after preincubation with VWF. At t = 0 h, titres for plasma or pdFVIII/VWF were unquantifiable, but were detectable for isolated FVIII (0.6–1.6 BU). In contrast to pdFVIII/VWF, the decrease in thrombin generation parameters by isolated FVIII in the presence of ESH-8 was significant (P < 0.01) even when previously combined with VWF. In conclusion, VWF protection against FVIII inhibitor activity might be higher with native pdFVIII/VWF complex than with the corresponding compound formed from the isolated proteins. Bethesda assay titration using different FVIII concentrates would be advisable to guide the treatment of inhibitor patients.
Background: Emicizumab is an alternative non-factor approach for treating patients with hemophilia A. However, there is a potential risk of thrombotic events when emicizumab is concomitantly administered with pro-hemostatic therapies.Objectives: To assess the hemostatic effect in vitro when a plasma-derived factor VIII concentrate containing von Willebrand factor (pdFVIII/VWF) was added to hemophilia A plasma (HAp) in combination with emicizumab.Methods: HAp and HAp with FVIII inhibitors (HAp-i) samples with different concentrations of emicizumab (50 and 100 μg/mL) were combined with activated prothrombin complex concentrate at 0.5 to 1 U/mL, recombinant activated factor VII (rFVIIa) at 0.5 to 7 μg/mL, and pdFVIII/VWF at 0.1 to 4.5 IU/mL. Thrombin generation (TG; thrombin peak and endogenous thrombin potential) was determined using a Calibrated Automated Thrombogram assay.Results: When activated prothrombin complex concentrate was added to HAp and HAp-i with emicizumab, TG dramatically increased (multiplier effect > 4.5×). Addition of rFVIIa to HAp or HAp-i with emicizumab moderately increased TG in a concentration-related manner compared with rFVIIa alone. Addition of pdFVIII/VWF to HAp or HAp-i with emicizumab induced a TG response equivalent to those samples without emicizumab. In an in vitro model of immune tolerance induction with bleeds (HAp-i 15 Bethesda units), combination of pdFVIII/VWF, emicizumab, and rFVIIa did not trigger a multiplying effect on TG.Conclusions: pdFVIII/VWF showed a non-additive effect on TG when combined in vitro with emicizumab. This finding suggests that emicizumab has limited ability to promote factor X activation in the presence of pdFVIII/VWF, thus reducing the risk of thrombotic events.
About 25% of severe hemophilia A patients undergoing factor VIII (FVIII) replacement therapy develop antibodies against FVIII (FVIII inhibitors). The formation of inhibitor is generally accepted as the most common and challenging complication of hemophilia treatment. In the circulation, FVIII is tightly bound to von Willebrand factor (VWF), forming a complex that plays a significant role in FVIII protection from premature degradation (Delignat S et al, Haemophilia 2012;18:248-54). Several laboratory and clinical studies suggest that the presence of VWF in FVIII concentrates might have some benefits due to the protective effect against antibodies (Shi Q et al, J Thromb Haemost 2012;10:2328-37; Mannucci PM, Haemophilia 2012;18 Suppl 2:2-7). Plasma-derived (pd) FVIII/VWF concentrates show higher residual FVIII activity and more thrombin generation after incubation with inhibitors compared to isolated FVIII concentrates (pdFVIII or recombinant, rFVIII). The protective effect of VWF observed in native pdFVIII/VWF complex is not completely restored for isolated FVIII even after mixing with VWF before analyzing the reaction with inhibitors (Bravo MI et al, Haemophilia 2014; in press). This study analyzes the impact of VWF on FVIII protection against inhibitors in an in vivo model, by evaluating the differences of FVIII in vivo recovery between FVIII concentrates with or without VWF in FVIIInull E16Knockout (KO) mice infused with inhibitors purified from hemophilic patients’ plasma. Inhibitor IgG was purified from a pool of human plasmas from hemophilic patients (Technoclone GmbH, Vienna, Austria) by using Protein G Sepharose chromatography as described elsewhere. IgGs were tail vein infused to achieve 3.2 BU/ml (or buffer as a control) prior to FVIII treatment. Five minutes after IgG infusion, animals (n=5-6 for each treatment group) were infused with FVIII therapeutic concentrates (100 IU/kg) from different sources, including native pdFVIII/VWF complex; isolated pdFVIII and full-length recombinant FVIII (rFVIII). In addition, some animal groups were treated with in vitro preformed complexes with pdVWF (therapeutic concentrate for von Willebrand disease) and FVIII (rFVIII+pdVWF and pdFVIII+pdVWF mixed at 1IU:1IU ratio; 15 min at 25ºC). After 5 min post-FVIII infusion, plasma samples were obtained by abdominal cave vein sampling and residual FVIII:C was measured using chromogenic assay. FVIII recovery was estimated according on the empirical finding that 1IUFVIII/kg body weight raises the plasma FVIII activity by 2.1±0.4% of normal activity. In the absence of inhibitor, in vivo FVIII recovery in FVIIInull E16 KO mice was similar for all FVIII concentrates as expected, with values ranging from 107% to 124% (see table). In contrast, in the presence of inhibitors the FVIII recovery showed marked differences among treatment groups. Recovery was higher for the native VWF-containing FVIII concentrate (pdFVIII/VWF: 71.1±17.4%) when compared to concentrates composed of isolated FVIII (rFVIII: 31.4±9.9%; pdFVIII: 25.0±2.7%). When the products containing isolated FVIII were premixed with VWF prior to infusion, FVIII recovery was only partially restored (rFVIII+VWF: 67.1±15.1%; pdFVIII+VWF: 45.2±8.8%). FVIII:C recovery and ratio FVIII:C, 5 min post-infusion of FVIII products (100 IU/kg), without inhibitor and with inhibitor (3.2±0.4 BU/ml) in severe hemophilic mice. Abstract 2826. TableFVIII productsFVIII:C Recovery (%) [mean±SD (n)]FVIII:C ratio (IU) in absence vs. presence of inhibitorWithout inhibitorWith inhibitorpdFVIII /VWF107.9 ± 6.1 (6)71.1 ± 17.4 (5)1.52rFVIII106.9 ± 8.3 (6)31.4 ± 9.9 (5)3.41pdFVIII120.5 ± 5.1 (6)25.0 ± 2.7 (5)4.77rFVIII+pdVWF124.3 ± 11.2 (6)67.1 ± 15.1 (6)1.84pdFVIII+pdVWF111.8 ± 5.9 (6)45.2 ± 8.8 (6)2.47 Our data indicate that VWF-containing FVIII concentrates are more efficient in the restoration of FVIII circulating levels in the FVIIInull E16 KO mice model with inhibitors. Moreover, results also suggest that this protection would be higher in native pdFVIII/VWF complex that in the complex of FVIII+VWF formed from the isolated proteins. Disclosures Bravo: Instituto Grifols: Employment. Da Rocha-Souto:Instituto Grifols: Employment. Grancha:Instituto Grifols: Employment. Jorquera:Grifols Bioscience: Employment.
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