Comparison of platelets attachment and translocation velocities on immobilized VWF-D′A3 under shear stress. Fluorescently labeled platelets were perfused over immobilized wtD′A3 or its variants at shear rates from 400 to 2000 s⁻¹ for 4 minutes. (A) After 4 minutes, the number of platelets that remained stationery for the duration of 3 frames (0.15 seconds) on the indicated substrates as a function of shear rate determined using ImageJ. (B) Translocation velocities (μm/s⁻¹) were measured by tracking individual platelets frame by frame at an interval of 0.05 second, in the direction of flow (n = 30 platelets for each 3 fields of view). Values shown are mean ± SEM of at least 4 independent experiments performed in duplicate. (C-D) To ensure equal D′A3 VWF coating, wtD′A3 OLG variants were immobilized onto flow slides. Next, after perfusion of PBS through the slides at 1000 s⁻¹, the bound protein was recovered from the channels using 2× SDS-PAGE reducing buffer and heating for 30 minutes, and then analyzed by SDS-PAGE followed by Western blotting with polyclonal anti–VWF-HRP–conjugated antibodies (C) or anti–c-myc antibodies (D). (E) Intensity of the bands (corresponding to VWF, ∼ 260 kDa) was determined using MacBiophotonics ImageJ software and expressed as a proportion of wtD′A3 (P = .6).