Figure 3.
Figure 3. VWF fiber formation on collagen in stenosed channel. (A) Schematic design of flow chamber used for fiber formation assay. (B) Citrated human plasma, supplemented with either 1.5 M CaCl2 or 10 mM EDTA, was perfused over type I collagen substrates for 10 minutes at 100 000/s. A FITC-coupled anti-VWF Ab was added postperfusion to visualize VWF fibers. Representative images in different parts of the flow chamber are show. VWF fiber formation was promoted upon calcium chelation. (C) Fluorescence intensity of fibers formed was measured in different parts of the flow device: inlet transition from 100 µm to 50 µm width, 50 μm middle section, and outlet from 50 μm to 100 μm as shown in panel B. (D-F) VWF variants (WT and Lock) were created with a Cerulean (CFP)-insert prior to the A1 domain in WT and Lock-VWF (D). Protein multimer distribution following expression in HEK293T-furin cells (E), and susceptibility to proteolysis by 1 U/mL ADAMTS13 overnight at 37°C in the presence of 1.6 M urea (F). (G-H) Human plasma was mixed with 5 μg/mL WT-Cer or Lock-Cer VWF variants in the presence of 10 mM EDTA, and perfused over collagen at 100 000/s. Fiber formation was measured in different regions of the flow device. FITC-conjugated anti-VWF Ab measured total VWF deposits, and cerulean fluorescence assayed only recombinant-protein accumulation. Representative images (G) and quantitative analysis (H) are presented. (I) Approximately10 µg/mL WT- or Lock-VWF were repeatedly perfused in the collagen coated stenosed flow chamber at a maximum wall shear rate of 100 000/s for 10 minutes. This allowed 20 passages of VWF through the flow constriction. Solution concentration of VWF was measured before and after shear application. Greater amounts of WT-VWF was lost compared with Lock-VWF. All microfluidics assays were performed 3 times, each with 2 to 4 repeats. *P < .05 with respect to all other treatments. VWF self-association was diminished in the presence of calcium, and upon use of Lock-variants. (B,G) Scale bars, 50 µm; VWF labeled with FITC (green) or Ceruelan (cyan).

VWF fiber formation on collagen in stenosed channel. (A) Schematic design of flow chamber used for fiber formation assay. (B) Citrated human plasma, supplemented with either 1.5 M CaCl2 or 10 mM EDTA, was perfused over type I collagen substrates for 10 minutes at 100 000/s. A FITC-coupled anti-VWF Ab was added postperfusion to visualize VWF fibers. Representative images in different parts of the flow chamber are show. VWF fiber formation was promoted upon calcium chelation. (C) Fluorescence intensity of fibers formed was measured in different parts of the flow device: inlet transition from 100 µm to 50 µm width, 50 μm middle section, and outlet from 50 μm to 100 μm as shown in panel B. (D-F) VWF variants (WT and Lock) were created with a Cerulean (CFP)-insert prior to the A1 domain in WT and Lock-VWF (D). Protein multimer distribution following expression in HEK293T-furin cells (E), and susceptibility to proteolysis by 1 U/mL ADAMTS13 overnight at 37°C in the presence of 1.6 M urea (F). (G-H) Human plasma was mixed with 5 μg/mL WT-Cer or Lock-Cer VWF variants in the presence of 10 mM EDTA, and perfused over collagen at 100 000/s. Fiber formation was measured in different regions of the flow device. FITC-conjugated anti-VWF Ab measured total VWF deposits, and cerulean fluorescence assayed only recombinant-protein accumulation. Representative images (G) and quantitative analysis (H) are presented. (I) Approximately10 µg/mL WT- or Lock-VWF were repeatedly perfused in the collagen coated stenosed flow chamber at a maximum wall shear rate of 100 000/s for 10 minutes. This allowed 20 passages of VWF through the flow constriction. Solution concentration of VWF was measured before and after shear application. Greater amounts of WT-VWF was lost compared with Lock-VWF. All microfluidics assays were performed 3 times, each with 2 to 4 repeats. *P < .05 with respect to all other treatments. VWF self-association was diminished in the presence of calcium, and upon use of Lock-variants. (B,G) Scale bars, 50 µm; VWF labeled with FITC (green) or Ceruelan (cyan).

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