Functional analysis of human–canine and canine–human chimeras of GPIbα.
(A) Human–canine and canine–human chimeras of GPIbα, where residue numbers correspond to amino acid sequences of human12 and canine13 GPIbα. Canine sequence is represented in black. Chimeras H104, H128, H152, and H176 have been described previously.11 (B) Ristocetin-dependent vWF binding to GPIbα chimeras. Specific binding of 125I-labeled vWF (1 μg/mL) in the presence of ristocetin (1 mg/mL) to CHO βIX cells (5 × 106/mL) co-expressing wild-type human GPIbα, canine GPIbα, or canine–human chimeras of GPIbα for 30 minutes at 22°C. Binding is expressed relative to specific binding of125I-labeled vWF to each cell line in the presence of botrocetin (2.5 μg/mL) in parallel assays.11 Specific binding was calculated from total binding by subtracting nonspecific binding measured in the absence of modulator in parallel assays. Error bars, ± SEM (n = 3). (C) Adhesion of GPIbα chimera-expressing CHO cells to vWF under flow. Grades are defined as follows: +, 1% to 10% of cells; ++, 11% to 79% of cells; +++, more than 80% of cells. (Rolling velocities are ± SEM.)
