Figure 1
Figure 1. Interaction of VWF and FH. (A) Colocalization of VWF and FH in WPBs of ECs. HUVEC were fixed with PFA (4%), permeabilized with Triton X-100 (0.5%), and stained for intracellular VWF and FH. For FH detection, a pool of 3 biotinylated monoclonal antibodies against FH or a biotinylated mouse IgG1 isotype control, followed by streptavidin-FITC, were used. For VWF, a rabbit polyclonal anti-human VWF IgG or a rabbit IgG control, followed by an Alexa Fluor 555–labeled goat anti-rabbit IgG, were used. Nuclei were stained with Hoechst 33342 dye (blue); a 63× oil-immersion objective was used for photography. (B) Cosecretion of FH and VWF by heme-stimulated HUVECs. Confluent HUVECs were incubated alone or in the presence of 100 µM heme in serum-free medium for 30 minutes at 37°C. VWF and FH were detected in culture supernatant by ELISA using commercially available purified proteins as standards. (C-D,F) Interaction between VWF and FH. VWF (10 nM; C) or anti-VWF antibody (D,F) were coated on 96-well microtiter plates. After blocking with Tris-buffered saline, 3% albumin, purified FH or recombinant FH fragments consisting of domains 1 to 4, 6 to 8, and 19 to 20 (C), or normal plasma (D,F) were incubated. In panel C, plasma was incubated in the presence of 300 mM NaCl to prevent formation of the VWF/FH complex during the incubation time (see supplemental Figure 4, available on the Blood Web site). FH and its fragments were detected with a polyclonal goat anti-FH antibody, a horseradish peroxidase–conjugated rabbit polyclonal anti-goat antibody and substrate. The polyclonal anti-FH IgG recognized equally FH and all recombinant FH fragments (supplemental Figure 1). (E) SPR analysis of FH binding to VWF. Association and dissociation of FH (1.5-3200 nM) with immobilized recombinant VWF (5500 RU) were followed in 10mM HEPES, 150 mM NaCl, Tween 0.005%, pH 7.4, for 360 seconds each, with a flow rate of 10 μL per minute. Curves depict RUs as a function of time. Data were fitted by the 1:1 Langmuir binding model with a drifting baseline. (E, inset) The equilibrium binding as the maximum signal measured at the end of the association phase as a function of the concentration of FH. Data were fitted with a 1-site nonlinear fit. Representative of 2 independent experiments. (F) (Inset) Protein G–coated sepharose beads where incubated with a polyclonal anti-VWF antibody, and then incubated with IgG-depleted normal plasma. FH was detected by western blotting under reduced conditions using an anti-FH antibody (lane 1, normal plasma; lane 2, type 3 VWD plasma; lane 3, FH-depleted serum). FITC, fluorescein isothiocyanate; HEPES, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; PFA, paraformaldehyde; RU, resonance unit.

Interaction of VWF and FH. (A) Colocalization of VWF and FH in WPBs of ECs. HUVEC were fixed with PFA (4%), permeabilized with Triton X-100 (0.5%), and stained for intracellular VWF and FH. For FH detection, a pool of 3 biotinylated monoclonal antibodies against FH or a biotinylated mouse IgG1 isotype control, followed by streptavidin-FITC, were used. For VWF, a rabbit polyclonal anti-human VWF IgG or a rabbit IgG control, followed by an Alexa Fluor 555–labeled goat anti-rabbit IgG, were used. Nuclei were stained with Hoechst 33342 dye (blue); a 63× oil-immersion objective was used for photography. (B) Cosecretion of FH and VWF by heme-stimulated HUVECs. Confluent HUVECs were incubated alone or in the presence of 100 µM heme in serum-free medium for 30 minutes at 37°C. VWF and FH were detected in culture supernatant by ELISA using commercially available purified proteins as standards. (C-D,F) Interaction between VWF and FH. VWF (10 nM; C) or anti-VWF antibody (D,F) were coated on 96-well microtiter plates. After blocking with Tris-buffered saline, 3% albumin, purified FH or recombinant FH fragments consisting of domains 1 to 4, 6 to 8, and 19 to 20 (C), or normal plasma (D,F) were incubated. In panel C, plasma was incubated in the presence of 300 mM NaCl to prevent formation of the VWF/FH complex during the incubation time (see supplemental Figure 4, available on the Blood Web site). FH and its fragments were detected with a polyclonal goat anti-FH antibody, a horseradish peroxidase–conjugated rabbit polyclonal anti-goat antibody and substrate. The polyclonal anti-FH IgG recognized equally FH and all recombinant FH fragments (supplemental Figure 1). (E) SPR analysis of FH binding to VWF. Association and dissociation of FH (1.5-3200 nM) with immobilized recombinant VWF (5500 RU) were followed in 10mM HEPES, 150 mM NaCl, Tween 0.005%, pH 7.4, for 360 seconds each, with a flow rate of 10 μL per minute. Curves depict RUs as a function of time. Data were fitted by the 1:1 Langmuir binding model with a drifting baseline. (E, inset) The equilibrium binding as the maximum signal measured at the end of the association phase as a function of the concentration of FH. Data were fitted with a 1-site nonlinear fit. Representative of 2 independent experiments. (F) (Inset) Protein G–coated sepharose beads where incubated with a polyclonal anti-VWF antibody, and then incubated with IgG-depleted normal plasma. FH was detected by western blotting under reduced conditions using an anti-FH antibody (lane 1, normal plasma; lane 2, type 3 VWD plasma; lane 3, FH-depleted serum). FITC, fluorescein isothiocyanate; HEPES, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; PFA, paraformaldehyde; RU, resonance unit.

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