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Blood, Vol. 109, Issue 2, 610-612, January 15, 2007
VWF protects FVIII from endocytosis by dendritic cells and subsequent presentation to immune effectors
Blood Dasgupta et al.
109: 610
Supplemental materials for: Dasgupta et al, Vol 109, Issue 2, 610-612
Files in this Data Supplement:
- Figure S1. FVIII and FVIII-FITC interact with VWF (JPG, 49.4 KB)
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FVIII-FITC was prepared by dialyzing recombinant human FVIII (1000 IU, Helixate NexGen; ZLB-Behring, Bern, Switzerland) against bicarbonate buffer (pH 9.2) containing 5 mM CaCl2 at 4°C for 2 hours, followed by coupling with fluorescein isothiocyanate (FITC) for 7 to 8 hours at 4°C (unconjugated FITC was removed by dialyzing against RPMI 1640 media). Polystyrene microtitration plates were coated with VWF at 2 µg/mL in PBS for 1 hour at 37°C. After saturation with PBS–1% BSA, FVIII and FVIII-FITC (0-17.9 nM) were incubated for 1 hour at 37°C. The binding of FVIII/FVIII-FITC to coated VWF was detected by the addition of a monoclonal anti-FVIII heavy-chain antibody mAb2 (3 µg/mL) followed by incubation with a rabbit anti–murine IgG antibody coupled to streptavidin peroxidase. The reaction was revealed using OPD as substrate (absorbance 492 nm).
- Figure S2. Dissociation of the FVIII/VWF complex restores endocytosis of FVIII by DCs (JPG, 48.8 KB)
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FVIII-FITC (107 nM), preincubated in medium alone or in the presence of a 25-fold molar excess of VWF for 1 hour at 37°C, was incubated with DCs for 2 hours at 37°C or 4°C. When indicated, FVIII-FITC was pretreated with the F(ab′)2 fragments of a monoclonal anti-FVIII IgG (BO2C11, 176 nM), and VWF with the F(ab′)2 fragments of a monoclonal anti-VWF IgG (Ac418, 516 nM; a kind gift from Dr J. P. Girma, INSERM U770, Bicêtre, France), for 1 hour at 37°C. Control experiments validated the inhibitory effects of BO2C11 and Ac418 on the interaction between FVIII and VWF (Figure S3). FVIII-FITC+ cells were analyzed as described. Average of 3 individual experiments. The statistical significances were assessed using the Mann-Whitney test.
- Figure S3. F(ab′)2 fragments of BO2C11 and Ac418 inhibit the interaction between FVIII and VWF (JPG, 52 KB)
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ELISA plates were coated with VWF at 2 µg/mL/well. FVIII (1 µg/mL) was preincubated with F(ab′)2 fragment of BO2C11 (0.04-20 µg/mL) and loaded on the wells. The bound FVIII was revealed as described. In parallel wells, Ac418 (0.03-20 µg/mL) was added to VWF-coated wells and then incubated with biotin labeled FVIII (2 µg/mL). Biotin was revealed by the addition of horseradish peroxidase and a specific substrate (R&D Systems, Lille, France). Values are expressed as percentage of the absorbance (arbitrary units) obtained when FVIII was incubated alone with VWF.
- Figure S4. Soluble VWF inhibits the binding to VWF of both unconjugated and FITC-conjugated FVIII (JPG, 58.9 KB)
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FVIII (0.55 nM; ■) and FVIII-FITC (0.55 nM; ○) were preincubated alone or in the presence of increasing concentrations of soluble VWF or HSA (ie, molar ratios of 1:1 to 1:130) followed by incubation on ELISA plates coated with 2 µg/mL VWF. The bound FVIII was detected by the addition of a monoclonal antibody mAb2 (an anti-FVIII heavy chain) followed by incubation with a rabbit anti–murine IgG antibody coupled to streptavidin peroxidase. The reaction was revealed using OPD as substrate (absorbance at 492 nm). Results depict percentage of FVIII/FVIII-FITC binding to coated VWF considering 100% when preincubation was done with FVIII/FVIII-FITC alone.
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