Fig 1.
Circulating VWF levels decrease the clearance of rFVIII and rFVIIIFc.
Plasma activity of rFVIII and rFVIIIFc versus time profile is shown for (A) FVIII-KO and (B) FVIII/VWF-DKO mice (note the time scale difference). Mice are dosed with 250 IU/kg rFVIII or rFVIIIFc and the FVIII activity remaining in plasma determined by the chromogenic activity assay (3–4 samples per time point, mean ± SD). Significance between plasma levels for individual time points on the PK curves is determined by an unpaired 2-tailed student t-test. A significant difference is indicated between PK curves with one or more significant time point differences (p<0.05).
Fig 2.
Biodistribution studies demonstrate the liver is the major clearance organ for both rFVIII and rFVIIIFc in mice.
(A-C) Quantitation of the radioactivity remaining in perfused organs for 125I-rFVIII and 125I-rFVIIIFc. (A) rFVIII in FVIII-KO mice (15 and 60 minute) and (B) rFVIIIFc in FVIII-KO mice (5, 15 and 120 minute) show similar biodistributions. (C) rFVIIIFc in FVIII/VWF-DKO mice at early time points (5 and 15 minute) chosen to match the rapid clearance of rFVIIIFc in these mice. (D, E) Biodistribution of 125I-rFVIIIFc by QWBA. (D) FVIII-KO mice (5, 30 minute, 3, 16, 32 hour) and (E) FVIII/VWF-DKO (5, 15 minute, 1, 2, 6 hour). Liver is the major clearance organ for both rFVIII and rFVIIIFc and in the absence of endogenous VWF there is a 3-fold increased signal in the liver of rFVIIIFc (C and E). FVIII proteins are labeled using iodination conditions optimized to preserve 80% cofactor activity (Panels A-C; n = 2).
Table 1.
Expression of FcRn and candidate clearance receptors for FVIII and VWF in mouse liver.
Fig 3.
The half-life of rFVIIIFc is predominantly extended by FcRn expressed in somatic cells, but not in hematopoietic cells.
The clearance of the following proteins is determined in bone marrow FcRn chimeric mice. (A) rFVIII (250 IU/kg), which clearance is not affected by FcRn (B) rFVIIIFc (250 IU/kg) which is predominantly protected from clearance by FcRn expressed in somatic cells (KO→WT). (C) rFIXFc (250 IU/kg) is mostly protected from clearance by FcRn expressed in somatic cells (KO→WT) (D) human IgG1 (5 mg/kg) is protected from clearance by FcRn expressed in hematopoietic cells (WT→KO). Plasma levels measured at 5 minute are set to 100% and the calculated mean residence times (MRT) are show in the graph insert, n = 4 for each data-point. Significance between plasma levels for individual time points on the PK curves is determined by an unpaired 2-tailed student t-test. A significant difference is indicated between PK curves with one or more significant time point differences (p<0.05).
Fig 4.
In FVIII-KO mice, both rFVIII and rFVIIIFc predominantly co-localize with VWF in liver Kupffer cells.
In liver sections from FVIII-KO mice staining of both rFVIII (A and C, green) and rFVIIIFc (B and D, green) is intensely localized to Kupffer cells. In addition, rFVIII, but not rFVIIIFc, shows a distinct punctate staining associated with hepatocytes. Staining for LSEC and endothelium (A’ and B’, CD31, red) reveals the central vein (CV) and the diffuse sinusoidal network and confirms that the punctate staining of rFVIII (A’, green) is not associated with LSEC, while some rFVIIIFc staining (B’, green) localizes with the liver sinusoid. Nuclei are stained blue with DAPI (A’ and B”). Additional stainings confirm co-localization of rFVIII (C’) and rFVIIIFc (D’) in Kupffer cells (CD68, red). VWF (C” and D”, magenta) is also associated with FVIII staining in Kupffer cells (arrows), however VWF in Weibel-Palade bodies in the endothelial lining of the central vein is not associated with FVIII. For orientation in liver lobules: CV, central vein; S, sinusoid; KC, Kupffer cell and HC, plate of hepatocytes (scale bar, 20 μm).
Fig 5.
In the absence of VWF, rFVIII localizes to hepatocytes and rFVIIIFc is found in the liver sinusoid.
Sections from FVIII/VWF-DKO mice lacking both VWF and FVIII, show strong punctate vesicular staining of rFVIII (A, A’, green) associated with hepatocytes, but not LSEC. In contrast, rFVIIIFc (B, B’, green) stains the liver sinusoid. Staining for LSEC and endothelium (A’, B’, CD31, red) confirms that the punctate staining of rFVIII is localized to the hepatocyte plate, while rFVIIIFc co-stains with the diffuse sinusoidal network of LSEC. (CV, central vein; S, sinusoid and HC, plate of hepatocytes; scale bar, 20 μm).
Fig 6.
Distinct localization patterns for FcRn and FcRγ binding mutants of rFVIIIFc in the absence of VWF.
Sections from FVIII/VWF-DKO mice lacking both VWF and FVIII, stained for Kupffer cells (A’, B’, C’, D’, CD68, red) show strong punctate vesicular staining of both rFVIII (A, A’, green) and the rFVIIIFc-IHH mutant (C, C’, green) that is incompetent to bind FcRn. This punctate staining is associated with hepatocytes and not LSEC. In contrast, both rFVIIIFc (B, B’, green) and the Fcγ-receptor binding mutant, rFVIIIFc-N297A (D, D’ green) localize to the liver sinusoid. In the absence of VWF, rFVIII, rFVIIIFc and the rFVIIIFc mutants are not associated with Kupffer cells (CD68, red). (CV, central vein; S, sinusoid; KC, Kupffer cell and HC, plate of hepatocytes; scale bar, 20 μm).
Fig 7.
Scheme for two parallel uptake pathways in liver.
Fig 8.
Model of two parallel liver clearance pathways for VWF-bound rFVIIIFc and free rFVIIIFc.
In the presence of the dominant FVIII clearance determinant, VWF (A, B), the majority of either rFVIII (A) or rFVIIIFc (B) is in a dynamic complex with VWF (double black arrow). This FVIII/VWF complex is mainly cleared by hematopoietic derived Kupffer cells (KC, open blue arrow), where rFVIIIFc is not recycled by FcRn to prevent degradation. In contrast, VWF-free FVIII (C) or FVIIIFc (D), found either in the absence of VWF or transiently dissociated from the VWF complex, can enter hepatocytes (HC) after diffusion through fenestrae (light blue with open double arrow). While rFVIII is degraded in hepatocytes, free rFVIIIFc entering hepatocytes is cycled by FcRn (red arrows) into the Space of Disse lined by LSEC. This leads to the liver sinusoidal staining pattern observed for rFVIIIFc in FVIII/VWF-DKO mice and allows it to reenter circulation, thereby improving the half-life of FVIIIFc.