Mutation of His86 to either Ala or Glu leads to gain of VWF binding function. Mutants His86Ala and His86Glu were tested for their ability to bind VWF in the presence of ristocetin (A) or botrocetin (B) at different modulator concentrations and a constant VWF concentration (4.0 μg/mL). The protocol for measuring the modulator-induced VWF binding is similar to that depicted in the legend to Figure 4. (C-D) In the assay of cell adhesion under flow, the cells expressing wild-type or mutant GP Ibα were incubated on immobilized VWF (20 μg/mL) for 1 minute in a parallel-plate flow chamber, after which the chamber was perfused with PBS at flow rates that generated wall shear stresses of 2.5, 10, or 20 dyne/cm2. Rolling velocities of the mutant-expressing cells were measured and compared with those of cells expressing wild-type GP Ibα. (C) Effects of VWF coating density on cell rolling at 20 dyne/cm2. (D) Effects of shear stress on the rolling velocity of mutant-expressing cells at a constant VWF-coating density (20 μg/mL VWF). Error bars indicate SEM.
