Von Willebrand factor (VWF) and factor VIII (FVIII) circulate in a tight noncovalent complex. At present, the cells that contribute to the removal of FVIII and VWF are of unknown identity. Here, we analyzed spleen and liver tissue sections of VWFdeficient mice infused with recombinant VWF or recombinant FVIII. This analysis revealed that both proteins were targeted to cells of macrophage origin. When applied as a complex, both proteins were codirected to the same macrophages.Chemical inactivation of macrophages using gadolinium chloride resulted in doubling of endogenous FVIII levels in VWFnull mice, and of VWF levels in wild-type mice. Moreover, the survival of infused VWF was prolonged almost 2-fold in VWFdeficient mice after gadolinium chloride treatment. VWF and FVIII also bound to primary human macrophages in in vitro tests. In addition, radiolabeled VWF bound to human THP1 macrophages in a dosedependent, specific, and saturable manner (half-maximal binding at 0.014 mg/mL). Binding to macrophages was followed by a rapid uptake and subsequent degradation of the internalized protein. This process was also visualized using a VWFgreen fluorescent protein fusion protein.In conclusion, our data strongly indicate that macrophages play a prominent role in the clearance of the VWF/FVIII complex. (Blood. 2008;112:1704-1712)

BackgroundVon Willebrand factor (VWF) is critical for the in vivo survival of factor VIII (FVIII). Since FVIII half-life correlates with VWF-antigen pre-infusion levels, we hypothesized that VWF levels are useful to predict FVIII half-life.MethodologyStandardized half-life studies and analysis of pre-infusion VWF and VWF-propeptide levels were performed in a cohort of 38 patients with severe haemophilia A (FVIII <1 IU/ml), aged 15–44 years. Nineteen patients had blood-group O. Using multivariate linear regression-analysis (MVLR-analysis), the association of VWF-antigen, VWF-propeptide, age and body-weight with FVIII half-life was evaluated.Principal FindingsFVIII half-life was shorter in blood-group O-patients compared to non-O-patients (11.5±2.6 h versus 14.3±3.0 h; p = 0.004). VWF-antigen levels correlated with FVIII half-life considerably better in patients with blood-group non-O than O (Pearson-rank = 0.70 and 0.47, respectively). Separate prediction models evolved from MVLR-analysis for blood-group O and non-O patients, based on VWF-antigen and VWF/propeptide ratio. Predicted half-lives deviated less than 3 h of observed half-life in 34/38 patients (89%) or less than 20% in 31/38 patients (82%).ConclusionOur approach may identify patients with shorter FVIII half-lives, and adapt treatment protocols when half-life studies are unavailable. In addition, our data indicate that survival of FVIII is determined by survival of endogenous VWF rather than VWF levels per se.

Protein A (Spa) is a surface‐associated protein of Staphylococcus aureus best known for its ability to bind to the Fc region of IgG. Spa also binds strongly to the Fab region of the immunoglobulins bearing VH3 heavy chains and to von Willebrand factor (vWF). Previous studies have suggested that the protein A–vWF interaction is important in S. aureus adherence to platelets under conditions of shear stress. We demonstrate that Spa expression is sufficient for adherence of bacteria to immobilized vWF under low fluid shear. The full length recombinant Ig‐binding region of protein A, Spa‐EDABC, fused to glutathione‐S‐transferase (GST), bound recombinant vWF in a dose‐dependent and saturable fashion with half maximal binding of about 30 nm in immunosorbent assays. Full length‐Spa did not bind recombinant vWF A3 domain but displayed binding to recombinant vWF domains A1 and D′‐D3 (half maximal binding at 100 nm and 250 nm, respectively). Each recombinant protein A Ig‐binding domain bound to the A1 domain in a similar manner to the full length‐Spa molecule (half maximal binding 100 nm). Amino acid substitutions were introduced in the GST‐SpaD protein at sites known to be involved in IgG Fc or in VH3 Fab binding. Mutants altered in residues that recognized IgG Fc but not those that recognized VH3 Fab had reduced binding to vWF A1 and D′‐D3. This indicated that both vWF regions recognized a region on helices I and II that overlapped the IgG Fc binding site.