5 min
In vivo FVIII recovery in a model of Hemophilia A mice with inhibitors
treated with different FVIII concentrates. Impact of VWF María Isabel Bravo, Bibiana Da Rocha-Souto, Salvador Grancha, Juan Ignacio Jorquera Research & Development, Instituto Grifols S.A., Parets del Vallès, Barcelona, Spain
BACKGROUND/AIMS
The role of von Willebrand factor (VWF) in the treatment of hemophilia A with inhibitors is being extensively studied. Several authors described the presence of VWF in coagulation factor VIII (FVIII) concentrates to be beneficial due to its protective effect against inhibitors. This study was aimed to evaluate the differences of FVIII in vivo recovery between FVIII concentrates –containing or not VWF– in the presence of human inhibitors using a mouse model of hemophilia A.
MATERIAL AND METHODS
In vivo FVIII recovery was evaluated in a severe hemophilia A mice model (FVIIInull E16 KO). Different FVIII therapeutic concentrates (plasma-derived FVIII/VWF complex [pdFVIII/VWF], recombinant [rFVIII] and isolated pdFVIII) were used as FVIII source. In addition, mice were treated with the same FVIII concentrates previously mixed with therapeutic pdVWF concentrate (rFVIII+pdVWF and pdFVIII+pdVWF) in 1IU:1IU ratio. Inhibitor IgG was purified from a pool of hemophilic plasmas with inhibitors (Technoclone) using protein G Sepharose chromatography (GE Healthcare). This pool contains antibodies reacting against both Heavy and Light Chain of FVIII. In order to reproduce inhibitor presence in severe hemophilia A mice, FVIIInull E16 KO animals were infused with inhibitor IgG at 100 BU/kg. Afterwards, KO mice were administered with FVIII concentrates (100 IU/kg). Plasma samples were obtained 5 min post-FVIII administration and FVIII activity (FVIII:C) was measured by chromogenic assay (Chromogenix) (See Figure 1). The basal levels of both inhibitor and FVIII activity were determined. For this, severe hemophilia A mice were infused with inhibitor IgG (100 BU/kg) and plasma samples were obtained 5 min post-inhibitor administration to measure the inhibitor titer by Bethesda assay (See figure 2A). Secondly, mice previously infused with vehicle were administered with FVIII concentrates (100 IU/kg) and plasma samples were obtained to measure FVIII:C (see Figure 2B). FVIII:C recovery was estimated according on the empirical finding that 1IU FVIII/kg body weight raises the plasma FVIII activity by 2.1±0.4% of normal activity. th 8
RESULTS
After 5 min post-inhibitor infusion the titer obtained was 3.2±0.4 BU/ml (n=6). Also, in the absence of inhibitor, basal levels of FVIII:C recovery in FVIIInull mice was similar for all FVIII concentrates, with values ranging from 107% to 124% (n=6) (see Figure 3). In contrast, in the presence of inhibitors the FVIII:C recovery in the same model was higher for VWF containing FVIII concentrates (71.1±17.4% for pdFVIII/VWF; n=6) when compared to concentrates composed of isolated FVIII (31.4±9.9% for rFVIII n=5 and 25.0±2.7% for pdFVIII n=5). When the isolated FVIII products were premixed with pdVWF prior to infusion, the FVIII recovery was only partially restored (67.1±15.1% for rFVIII+pdVWF n=6 and 45.2±8.8% for pdFVIII+pdVWF n=6) (See Figure 4). The FVIII:C recovery data were analyzed using a Student’s ttest. Statistical significance was set at p
treated with different FVIII concentrates. Impact of VWF María Isabel Bravo, Bibiana Da Rocha-Souto, Salvador Grancha, Juan Ignacio Jorquera Research & Development, Instituto Grifols S.A., Parets del Vallès, Barcelona, Spain
BACKGROUND/AIMS
The role of von Willebrand factor (VWF) in the treatment of hemophilia A with inhibitors is being extensively studied. Several authors described the presence of VWF in coagulation factor VIII (FVIII) concentrates to be beneficial due to its protective effect against inhibitors. This study was aimed to evaluate the differences of FVIII in vivo recovery between FVIII concentrates –containing or not VWF– in the presence of human inhibitors using a mouse model of hemophilia A.
MATERIAL AND METHODS
In vivo FVIII recovery was evaluated in a severe hemophilia A mice model (FVIIInull E16 KO). Different FVIII therapeutic concentrates (plasma-derived FVIII/VWF complex [pdFVIII/VWF], recombinant [rFVIII] and isolated pdFVIII) were used as FVIII source. In addition, mice were treated with the same FVIII concentrates previously mixed with therapeutic pdVWF concentrate (rFVIII+pdVWF and pdFVIII+pdVWF) in 1IU:1IU ratio. Inhibitor IgG was purified from a pool of hemophilic plasmas with inhibitors (Technoclone) using protein G Sepharose chromatography (GE Healthcare). This pool contains antibodies reacting against both Heavy and Light Chain of FVIII. In order to reproduce inhibitor presence in severe hemophilia A mice, FVIIInull E16 KO animals were infused with inhibitor IgG at 100 BU/kg. Afterwards, KO mice were administered with FVIII concentrates (100 IU/kg). Plasma samples were obtained 5 min post-FVIII administration and FVIII activity (FVIII:C) was measured by chromogenic assay (Chromogenix) (See Figure 1). The basal levels of both inhibitor and FVIII activity were determined. For this, severe hemophilia A mice were infused with inhibitor IgG (100 BU/kg) and plasma samples were obtained 5 min post-inhibitor administration to measure the inhibitor titer by Bethesda assay (See figure 2A). Secondly, mice previously infused with vehicle were administered with FVIII concentrates (100 IU/kg) and plasma samples were obtained to measure FVIII:C (see Figure 2B). FVIII:C recovery was estimated according on the empirical finding that 1IU FVIII/kg body weight raises the plasma FVIII activity by 2.1±0.4% of normal activity. th 8
RESULTS
After 5 min post-inhibitor infusion the titer obtained was 3.2±0.4 BU/ml (n=6). Also, in the absence of inhibitor, basal levels of FVIII:C recovery in FVIIInull mice was similar for all FVIII concentrates, with values ranging from 107% to 124% (n=6) (see Figure 3). In contrast, in the presence of inhibitors the FVIII:C recovery in the same model was higher for VWF containing FVIII concentrates (71.1±17.4% for pdFVIII/VWF; n=6) when compared to concentrates composed of isolated FVIII (31.4±9.9% for rFVIII n=5 and 25.0±2.7% for pdFVIII n=5). When the isolated FVIII products were premixed with pdVWF prior to infusion, the FVIII recovery was only partially restored (67.1±15.1% for rFVIII+pdVWF n=6 and 45.2±8.8% for pdFVIII+pdVWF n=6) (See Figure 4). The FVIII:C recovery data were analyzed using a Student’s ttest. Statistical significance was set at p
