Characterization of VWF and FVIII expression in type 2N VWD model mice. Blood samples were collected from 2 lines of VWF^2N model mice by tail bleeds using 3.8% sodium citrate as an anticoagulant, and plasmas were isolated for VWF and FVIII assays. Plasmas from VWF^+/+ littermates were used as controls in parallel. (A) Plasma VWF antigen (VWF:Ag) levels. Mouse VWF antigen (VWF:Ag) levels were determined by ELISA using anti-mVWF monoclonal antibody 344.2 for capture and biotin-conjugated monoclonal antibody 332.2 for detection. Plasma pooled from our wild-type C57BL/6J colony was used as the standard. (B) Plasma functional FVIII activity (FVIII:C) levels. Plasma FVIII:C levels were determined by a chromogenic assay. Recombinant human B-domain deleted FVIII (rhFVIII, Xyntha) was used as the standard. (C) The capacity of VWF binding to human FVIII. Anti–human FVIII monoclonal antibody 103.1 was coated on a 96-well plate, and rhfFVIII (Kogenate) was captured from a 1-U/mL solution. Plasmas from VWF^2N mice were incubated with the antibody captured rhfF8, unbound mVWF was washed off, and the remaining FVIII-bound mVWF was detected using mVWF ELISA detection reagents. The standard curve was constructed by measuring mVWF binding from serially diluted pooled plasma from our wild-type C57BL/6J colony. (D) The percentage of mVWF capable of binding to captured hFVIII. Data from 2N1 and 2N2 colonies were combined for this analysis. The percentage was calculated by dividing the level of VWF binding to antibody-captured hFVIII by the plasma VWF level in the same animal (using data from Figure 2C). ****P < .0001. n.s., no statistically significant difference between 2 groups. These results demonstrate that VWF2N mice have normal levels of plasma VWF but are incapable of binding FVIII, resulting in severely reduced levels of plasma FVIII:C.