Other studies indicate that VWF modulates the immunogenicity of FVIII concentrates and also protects FVIII from endocytosis by dendritic cells and subsequent presentation to immune effectors . Furthermore, VWF protects FVIII from proteolysis by activated factor X (FXa) and other proteases [7–11]. The overall objective of our in
vitro studies was to estimate the coagulant capacity (FVIII:C capacity) of the rFVIII fraction unable to bind VWF. In in vitro studies, incubation of rFVIII with molar excesses of VWF produces rFVIII/VWF complex and free rFVIII that readily separate via gel filtration (Sephacryl S-500) , and the coagulation activity of the two entities can, in theory, be subsequently Selleck Apitolisib measured. However, in practice, after the incubation of Kogenate® with plasma-derived VWF or Advate® with pdVWF, the VWF-binding (i.e. selleck kinase inhibitor rFVIII:Ag/VWF) and non-binding fractions (i.e. the residual free rFVIII:Ag) were easily separated but ≥95% of the FVIII:C activity loaded was lost after gel filtration. Furthermore, no FVIII:C activity was found in either the rFVIII:Ag/VWF or free rFVIII:Ag fractions
after the two fractions were concentrated. In the absence of VWF, both plasma-derived and rFVIII are readily activated with thrombin or FXa. However, FVIII bound to VWF was activated more slowly with FXa than with thrombin, demonstrating that VWF protects FVIII from proteolysis by FXa but not by thrombin . Based on these prior observations, the abilities
of the following rFVIII products to enhance FX activation by FIXa were compared: Kogenate® (with and without VWF added), Advate® (with or without added VWF) and pdFVIII, Fanhdi® (FVIII/VWF). We assessed each of these FVIII products before and after incubation for 1, 5 or 10 min at 37°C with FXa (1 nm) or thrombin (1 nm), to determine the resultant FVIII cofactor activity (FVIII:C). The relative amount of activated FVIII (FVIIIa) generated in each case was determined by quantifying the FXa produced mafosfamide by FIXa [assessed as enhancement of FX activation (100 nm) by FIXa (1 nm)]. With native Kogenate®, or Advate®, i.e. before pretreatment with FXa or thrombin, a rapid rate of FX activation was observed at 1 min (Table 1). Thus, the low concentration of FXa generated endogenously by FIXa easily activated FVIII:C in situ to provide FVIIIa. In contrast to the two rFVIII products, FX activation with native Fanhdi® at 1 min was very slow (approximately 10% of that seen with Advate® or Kogenate®), and remained slower over the entire 10-min incubation period (Table 1).