Expression and PK properties of the VWF D′D3 domain. (A) Plasmids expressing VWF fragments were administered to VWF-knockout mice by hydrodynamic injection (HDI),60 to identify the VWF domains necessary for optimal endogenous FVIII stabilization. Plasma FVIII activity was measured in an FVIII chromogenic activity assay before and 48 hours after DNA injection. Individual data points are shown with the horizontal lines depicting the mean. (B) The rFVIIIFc half-life was compared in FVIII/VWF DKO mice expressing VWF-010 and VWF-013 to determine whether the dimeric (VWF-010) or monomeric (VWF-013: D1D2DʹD3C1099A/C1142A) DʹD3 is comparable in protecting FVIII. rFVIIIFc (200 IU/kg) was administered via the tail vein, and the plasma FVIII activity was measured with the chromogenic assay. FVIII activity in mU/mL was plotted against time. Data are means ± SD; n = 4 mice per time point. (C) The PKs of rFVIIIFc-VWF(DʹD3; 200 IU/kg IV) were evaluated in HemA and DKO mice, using a methodology similar to those described in panel B. Plasma FVIII activity was normalized to the dose of factor administered and was plotted against time. Factor half-life (t1/2) was calculated by noncompartmental modeling using WinNonLin, version 5.2, (Pharsight Corp, Mountain View, CA). Data are means ± SD; n = 4 mice per time point.