polyclonal rabbit anti-human vwf/hrp detecting ab  (Agilent technologies)

Journal: Blood Advances

Article Title: von Willebrand factor self-association is regulated by the shear-dependent unfolding of the A2 domain

doi: 10.1182/bloodadvances.2018030122

Figure Lengend Snippet: Role of VWF-A2 in self-association. (A) Schematic of VWF proteins. A1 domain is deleted in ΔA1-VWF. WT protein (WT-VWF) has vicinal Cys in the A2 domain. The disulfide bond links the N and C terminus of VWF-A2 in Lock-VWF. (B) Protein multimer distribution of VWF variants expressed in HEK293T-furin cells. (C) Time course of VWF cleavage by 1 U/mL ADAMTS13 in the presence of 1.6 M urea at 37°C. Minimal cleavage of Lock-VWF is noted. (D-E) Five × 107/mL washed platelets were shear mixed at 9600/s in a cone-plate viscometer along with 10 μg/mL ΔA1-488, either in the absence of VWF or upon addition of 10 μg/mL WT-VWF or Lock-VWF. Buffer calcium concentration was varied from 0 to 1.5M CaCl2 (EDTA was not added). VWF self-association (D) and percentage platelet activation (E) were measured using flow cytometry for samples withdrawn at 5 minutes. VWF self-association quantifies the percentage of platelets binding more than basal (t = 0) levels of Alexa 488-conjugated ΔA1-VWF (ΔA1-488). Platelet activation measures the percentage of platelets binding PE-conjugated Annexin V. (F-G) VWF self-association (F) and platelet activation (G) triggered by WT-VWF was blocked by 20 µg/mL mAbs AK2 (anti-GpIbα) and AVW-3 (anti-VWF A1 domain). Data in panels D-G are from 3 to 4 independents runs, each containing 3 technical replicates. *P < .05 with respect to all other treatments at that calcium concentration. VWF self-association and platelet activation are higher for runs performed with WT-VWF compared with Lock-VWF.

Article Snippet: 23 A polyclonal rabbit anti-VWF Ab from Dako was used in western blots, enzyme-linked immunosorbent assay, and cytometry-bead assays to measure VWF concentration.

Techniques: Concentration Assay, Activation Assay, Flow Cytometry, Binding Assay

Journal: Blood Advances

Article Title: von Willebrand factor self-association is regulated by the shear-dependent unfolding of the A2 domain

doi: 10.1182/bloodadvances.2018030122

Figure Lengend Snippet: 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).

Article Snippet: 23 A polyclonal rabbit anti-VWF Ab from Dako was used in western blots, enzyme-linked immunosorbent assay, and cytometry-bead assays to measure VWF concentration.

Techniques: Tube Formation Assay, Fluorescence, Expressing, Recombinant, Concentration Assay, Labeling