Figure 2.
Interaction of CHO-rVWF with macrophage and hepatocyte clearance receptors. (A) Macrophage and hepatocyte receptors implicated in regulating VWF clearance in vivo. (B) To determine whether macrophages play a role in regulating the clearance of CHO-rVWF, in vivo clearance studies were repeated in VWF–/– mice 24 hours after clodronate-induced macrophage depletion. Data are graphed as percentage residual CHO-rVWF relative to the amount injected. P value is the outcome of extra-sum-of-squares F test. (C) Binding of CHO-rVWF (red) and pdVWF (blue) to murine BMDMs was assessed by flow cytometry. Representative histograms of binding relative to no VWF (gray) are shown. The y-axis represents the binding capacity normalized to the number of cells, and the x-axis represents the fluorescence intensity absorbance at the wavelength of 488 nm, in which higher values indicate more binding. (D) MGL binding was assessed for CHO-rVWF compared to pdVWF using plate-binding assay. Points represent mean ± SEM. P value is the outcome of extra-sum-of-squares F test. (E) BMDMs were isolated from WT and MGL1–/– mice. Binding of pdVWF (blue bars) and CHO-rVWF (red bars) to BMDMs with or without MGL was then assessed by flow cytometry. Data are presented as MFI normalized to controls without VWF. (F) In vivo clearance of pdVWF (blue lines) and CHO-rVWF (red lines) was studied in WT (solid lines) and MGL1–/– mice (dashed lines). Mice were sampled after injection (time = 0) and subsequently sampled at indicated time points. Data are graphed as percentage residual VWF relative to the VWF:Ag at time of 0. (G) Binding of CHO-rVWF (red) and pdVWF (blue) to human hepatocyte HepG2 cells was assessed by flow cytometry. Representative histograms of binding relative to no VWF (gray) are shown. Data were assessed for normality using the Shapiro-Wilk test and compared by the Student t test. The y-axis represents the binding capacity normalized to the number of cells, and the x-axis represents the fluorescence intensity absorbance at the wavelength of 488 nm, in which higher values indicate more binding. (H) ASGPR1 binding was assessed for CHO-rVWF compared to pdVWF using plate-binding assay. Points represent mean ± SEM. P value is the outcome of extra-sum-of-squares F test. (I) In vivo clearance of pdVWF (blue lines) and CHO-rVWF (red lines) was studied in WT (solid lines) and Asgr1–/– mice (dashed lines). Mice were sampled after injection (time = 0) and subsequently sampled at indicated time points. Data are graphed as percentage residual VWF relative to the VWF:Ag at time of 0. (J) LRP1 cluster IV binding was assessed for CHO-rVWF (red) compared to pdVWF (blue) using plate-binding assay. Points represent mean ± SEM. P value is the outcome of extra-sum-of-squares F test. (K) Binding of CHO-rVWF (red) and pdVWF (blue) VWF to HEK293 cells stably transfected with LRP1 (HEK-LRP1) was assessed by flow cytometry. Points represent mean ± SEM. P value is the outcome of extra-sum-of-squares F test. Abs, absorbance; MFI, mean fluorescence intensities; WT, wild-type.

Interaction of CHO-rVWF with macrophage and hepatocyte clearance receptors. (A) Macrophage and hepatocyte receptors implicated in regulating VWF clearance in vivo. (B) To determine whether macrophages play a role in regulating the clearance of CHO-rVWF, in vivo clearance studies were repeated in VWF–/– mice 24 hours after clodronate-induced macrophage depletion. Data are graphed as percentage residual CHO-rVWF relative to the amount injected. P value is the outcome of extra-sum-of-squares F test. (C) Binding of CHO-rVWF (red) and pdVWF (blue) to murine BMDMs was assessed by flow cytometry. Representative histograms of binding relative to no VWF (gray) are shown. The y-axis represents the binding capacity normalized to the number of cells, and the x-axis represents the fluorescence intensity absorbance at the wavelength of 488 nm, in which higher values indicate more binding. (D) MGL binding was assessed for CHO-rVWF compared to pdVWF using plate-binding assay. Points represent mean ± SEM. P value is the outcome of extra-sum-of-squares F test. (E) BMDMs were isolated from WT and MGL1–/– mice. Binding of pdVWF (blue bars) and CHO-rVWF (red bars) to BMDMs with or without MGL was then assessed by flow cytometry. Data are presented as MFI normalized to controls without VWF. (F) In vivo clearance of pdVWF (blue lines) and CHO-rVWF (red lines) was studied in WT (solid lines) and MGL1–/– mice (dashed lines). Mice were sampled after injection (time = 0) and subsequently sampled at indicated time points. Data are graphed as percentage residual VWF relative to the VWF:Ag at time of 0. (G) Binding of CHO-rVWF (red) and pdVWF (blue) to human hepatocyte HepG2 cells was assessed by flow cytometry. Representative histograms of binding relative to no VWF (gray) are shown. Data were assessed for normality using the Shapiro-Wilk test and compared by the Student t test. The y-axis represents the binding capacity normalized to the number of cells, and the x-axis represents the fluorescence intensity absorbance at the wavelength of 488 nm, in which higher values indicate more binding. (H) ASGPR1 binding was assessed for CHO-rVWF compared to pdVWF using plate-binding assay. Points represent mean ± SEM. P value is the outcome of extra-sum-of-squares F test. (I) In vivo clearance of pdVWF (blue lines) and CHO-rVWF (red lines) was studied in WT (solid lines) and Asgr1–/– mice (dashed lines). Mice were sampled after injection (time = 0) and subsequently sampled at indicated time points. Data are graphed as percentage residual VWF relative to the VWF:Ag at time of 0. (J) LRP1 cluster IV binding was assessed for CHO-rVWF (red) compared to pdVWF (blue) using plate-binding assay. Points represent mean ± SEM. P value is the outcome of extra-sum-of-squares F test. (K) Binding of CHO-rVWF (red) and pdVWF (blue) VWF to HEK293 cells stably transfected with LRP1 (HEK-LRP1) was assessed by flow cytometry. Points represent mean ± SEM. P value is the outcome of extra-sum-of-squares F test. Abs, absorbance; MFI, mean fluorescence intensities; WT, wild-type.

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