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Blood, Vol. 113, Issue 7, 1589-1597, February 12, 2009
Integrin  vβ3 on human endothelial cells binds von Willebrand factor strings under fluid shear stress
Blood Huang et al.
113: 1589
Supplemental materials for: Huang et al
Files in this Data Supplement:
- Figure S1. Anti-αv antibody LM142 specificity controls (JPG, 68.4 KB)
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CHO-K1 cells or HUVECs were cultured on glass-bottomed dishes, and incubated with anti-αv LM142 and Alexa Fluor 488-labeled anti-mouse IgG antibody (Invitrogen). DIC and fluorescence images were acquired as described under “Materials and Methods.”
- Figure S2. Sequential perfusion of stimulated HUVECs with platelets and anti-VWF (JPG, 70.8 KB)
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Confluent HUVECs were perfused at a shear stress of 2.5 dyn/cm2 with medium 199 containing 100 µM histamine and 108/ml formalin fixed platelets. After 1 min, the perfusate was supplemented with fluorescent VWF polyclonal antibody. Images were acquired at the times indicated. At 1 min, platelets have adhered to a VWF string in the center of the field. By 3 min, anti-VWF signal is visible for this platelet string, which has accumulated more platelets in the presence of anti-VWF, and for several smaller strings that did not bind platelets. Arrowheads indicate the approximate beginning and end of VWF strings. Flow direction is from lower left to upper right. Bars are 10 µm in length.
- Figure S3. Localization of P-selectin and VWF strings by immunofluorescence microscopy (JPG, 48.2 KB)
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Confluent HUVECs on cover glasses were perfused with medium M199 containing 100 µM histamine at 2.5 dyn/cm2 shear stress for 5 min, then prepared for immunofluorescence microscopy as described under “Materials and Methods.” The antibodies used were mouse monoclonal anti-P-selectin S12 and Alexa Fluor 488-labeled anti-mouse IgG antibody (Invitrogen) (green); and rabbit polyclonal anti-VWF (#082, DAKO) and AlexaFluor 594¬labeled goat anti-rabbit IgG (Invitrogen) (red). Images were prepared as described under “Materials and Methods.”
- Figure S4. CHO-P cell binding to immobilized VWF is inhibited by anti-P-selectin antibody or RGDS (JPG, 30.3 KB)
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96-well plates were coated with polyclonal anti-VWF (#082, DAKO) diluted 1:1000 in 50 mM sodium phosphate, pH 8.5, at 4°C overnight. Confluent HUVECs in serum free EGM-2 were treated with 100 mM histamine for 1 hr at 37°C. Filtered medium containing ultralarge VWF (approximately 1 µg/ml) was added to the 96-well plates for 2 h at RT. Control wells were incubated instead with 1% BSA in DPBS. Plates were washed with DPBS and blocked with 1% BSA in DPBS for 1 h at RT. CHO-P cells in Ca2+ and Mg2+ containing HBSS buffer were treated with or without 10 µg/ml anti-P selectin antibody or 40 µg/ml “RGDS” peptide for 15 min at RT and added to coated wells for 1 h at 37°C. Plates were washed three times with DPBS, and adherent cells were fixed with 2% formaldehyde for 15 min, then stained with 0.2% crystal violet in 2% ethanol for 15 min. After three washes with water, adherent cells were solubilized with 1% SDS for 30 min. Optical density at 562 nm was read with a microplate reader. Samples were analyzed in quadruplicate.
- Figure S5. Localization of integrin αvβ3 and VWF strings by immunofluorescence microscopy (JPG, 77.3 KB)
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Confluent HUVECs in a flow chamber were perfused at 2.5 dyn/cm2 shear stress with medium containing 100 µM histamine and incubated with anti-VWF and anti-integrin αvβ3 antibodies as described under “Materials and Methods.” (A) The same images from Fig. 7 (panels D, E, and F) at higher magnification to better resolve segments of VWF strings that show colocalization with integrin αvβ3 (arrows). (B) Another example of colocalization of VWF and integrin αvβ3. (C) An example in which many sites of intense VWF fluorescence (red) do not colocalize with integrin αvβ3 (green), indicating that VWF and integrin αvβ3 signals are clearly resolved.
- Figure S6. Weibel-Palade bodies in HUVECs transduced with shLuc or sh1129 (JPG, 59.6 KB)
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HUVECs were transduced with recombinant lentivirus shLuc or sh1129 and selected with puromycin as described under “Materials and Methods.” Cells were cultured on glass-bottomed dishes and prepared for immunofluorescence microscopy as described under “Materials and Methods,” but with an additional step of permeabilization with 0.1 % Triton X-100 before the addition of anti-VWF antibody and AlexaFluor 594-labeled goat anti-rabbit IgG (Invitrogen). Abundant rod-shaped Weibel-Palade bodies are present in both cell populations.
- Figure S7. Cleavage of VWF strings by recombinant ADAMTS13 occurs both in the presence and absence of platelets (JPG, 31.2 KB)
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Perfusion assays were conducted at 2.5 dyn/cm2, with either 0.5 U/ml or 1 U/ml recombinant ADAMTS13, with (black bars) or without (white bars) 1 × 108/ml formalin fixed platelets. The reduction in the mean number of VWF strings was calculated for each experiment. Values represent the mean ± SEM for three experiments.
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