Interaction between VWF and β2-integrin isotypes. (A) Various concentrations of wt-VWF (0-5 μg/well) were immobilized in microtiter wells (2 hours at 37°C) and were then blocked with 3% BSA/0.1% Tween-20/TBS (pH 7.4) for 1 hour at 37°C. Subsequently, immobilized VWF was incubated with GST/αM I-domain (1 μM) in 2 mM Mn2+/TBS for 2 hours at 37°C. Bound GST/αM I-domain was detected using a monoclonal anti-αM antibody. Absorbance versus concentration of immobilized VWF was plotted (inset). Binding of various concentrations of GST/αM I-domain (0-2 μM) to immobilized VWF (1 μg/well) was determined, as described. (B) Binding of GST/αM I-domain (1 μM) to immobilized VWF (1 μg/mL) in the absence or presence of NIF (10 μg/mL) was assessed, as described for panel A. Relative binding was plotted with binding in the absence of NIF as a reference (100%). (C) Transfected fibroblastic L cells expressing αXβ2 or αLβ2 and their nontransfected counterparts (1 × 105 cells/well) were incubated with immobilized wt-VWF, VWF/D′-D3, or VWF/A1-A2-A3 for 60 minutes at 37°C. Wells were gently washed, and adhesion was quantified, as described in the Figure 1 legend. Relative adhesion was plotted with adhesion of αXβ2-expressing L cells to wt-VWF as a reference (100%). Data represent mean ± SEM of 3 to 6 experiments performed in duplicate.
