Figure 3.
Evaluation of the functional properties of 2N VWF and the viability of plasma FVIII activity in VWF2N/2N mice. (A-B) The capacity of 2N VWF in collagen binding. To investigate if VWF in 2N mice can function normally in binding to collagen, we performed collagen III and IV binding assays, in which a 96-well plate was coated with collagen and plasma VWF:Ag bound to collagen was determined using a protocol similar to VWF:Ag ELISA. Plasma VWF:Ag levels were determined by ELISA in parallel. Pooled plasma from wild-type C57BL/6J mice was used as a standard. (A) The ratio of collagen III-bound VWF (CB3) to plasma VWF:Ag. (B) The ratio of collagen IV-bound VWF (CB4) to plasma VWF:Ag. (C) The capacity of 2N VWF in platelet binding. To determine if VWF in 2N mice can bind effectively to platelets, we performed a VWF/platelet-binding assay. VWF was activated by botrocetin in whole blood, and VWF-bound platelets were analyzed by flow cytometry. Samples from VWF−/− and VWF+/+ mice were used as controls in parallel. Data shown are representative histograms and the mean value from 3 mice in each group. (D) The ability of 2N VWF in multimerization. To examine if VWF in 2N mice can fully multimerize, we ran VWF multimers on plasma samples from 2N mice. Plasmas from VWF2N/+ and VWF+/+ mice were run in parallel. (E-F) The viability of plasma FVIII in 2N mice. To investigate if FVIII is viable in VWF2N/2N mice, rhVWF, or mVWF, was infused into VWF2N/2N mice. Blood samples were collected at various time points after infusion, and plasmas were isolated for VWF and FVIII assays. (E) Functional FVIII:C levels in VWF2N/2N mice upon rhVWF infusion. VWF2N/2N mice were infused with 50 U/kg of rhVWF (Vonvendi, Baxalta) via retro-orbital venous plexus injection. Human VWF:Ag levels were determined by VWF:Ag ELISA using anti-human specific antibodies, and pooled human plasma was used as the standard. Plasma FVIII:C levels were determined by a chromogenic assay, and rhF8 was used as the standard. (F) Functional FVIII:C levels in VWF2N/2N mice upon mVWF infusion. VWF2N/2N mice were infused with 200 μL of pooled plasma from FVIII−/− mice. Mouse VWF:Ag levels were determined by ELISA using mouse-specific antibodies, and pooled plasma from wild-type C57BL6 mice was used as the standard. Plasma FVIII:C levels were determined by a chromogenic assay, and rhF8 was used as the standard. These data demonstrate that VWF from 2N mice has normal functional activities in binding to collagen and platelets and in multimerization. The endogenous mouse FVIII in VWF2N/2N mice is bioavailable and can be stabilized in plasma in the presence of normal VWF.

Evaluation of the functional properties of 2N VWF and the viability of plasma FVIII activity in VWF2N/2N mice. (A-B) The capacity of 2N VWF in collagen binding. To investigate if VWF in 2N mice can function normally in binding to collagen, we performed collagen III and IV binding assays, in which a 96-well plate was coated with collagen and plasma VWF:Ag bound to collagen was determined using a protocol similar to VWF:Ag ELISA. Plasma VWF:Ag levels were determined by ELISA in parallel. Pooled plasma from wild-type C57BL/6J mice was used as a standard. (A) The ratio of collagen III-bound VWF (CB3) to plasma VWF:Ag. (B) The ratio of collagen IV-bound VWF (CB4) to plasma VWF:Ag. (C) The capacity of 2N VWF in platelet binding. To determine if VWF in 2N mice can bind effectively to platelets, we performed a VWF/platelet-binding assay. VWF was activated by botrocetin in whole blood, and VWF-bound platelets were analyzed by flow cytometry. Samples from VWF−/− and VWF+/+ mice were used as controls in parallel. Data shown are representative histograms and the mean value from 3 mice in each group. (D) The ability of 2N VWF in multimerization. To examine if VWF in 2N mice can fully multimerize, we ran VWF multimers on plasma samples from 2N mice. Plasmas from VWF2N/+ and VWF+/+ mice were run in parallel. (E-F) The viability of plasma FVIII in 2N mice. To investigate if FVIII is viable in VWF2N/2N mice, rhVWF, or mVWF, was infused into VWF2N/2N mice. Blood samples were collected at various time points after infusion, and plasmas were isolated for VWF and FVIII assays. (E) Functional FVIII:C levels in VWF2N/2N mice upon rhVWF infusion. VWF2N/2N mice were infused with 50 U/kg of rhVWF (Vonvendi, Baxalta) via retro-orbital venous plexus injection. Human VWF:Ag levels were determined by VWF:Ag ELISA using anti-human specific antibodies, and pooled human plasma was used as the standard. Plasma FVIII:C levels were determined by a chromogenic assay, and rhF8 was used as the standard. (F) Functional FVIII:C levels in VWF2N/2N mice upon mVWF infusion. VWF2N/2N mice were infused with 200 μL of pooled plasma from FVIII−/− mice. Mouse VWF:Ag levels were determined by ELISA using mouse-specific antibodies, and pooled plasma from wild-type C57BL6 mice was used as the standard. Plasma FVIII:C levels were determined by a chromogenic assay, and rhF8 was used as the standard. These data demonstrate that VWF from 2N mice has normal functional activities in binding to collagen and platelets and in multimerization. The endogenous mouse FVIII in VWF2N/2N mice is bioavailable and can be stabilized in plasma in the presence of normal VWF.

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