Shear-dependent interaction of platelets with immobilized wt-A1 and mutants.

Reconstituted blood containing fluorescently labeled platelets (10 000/μL) was perfused over wt-A1 or mutant fusion proteins, immobilized onto glass coverslips coated at 300 μg/mL in a flow chamber (see “Materials and methods”). (A) Number of platelets translocating over the A1 domain mutants per field (205 × 330 μm), after 3 minutes of perfusion, at 200 seconds⁻¹ (■), 1000 seconds⁻¹ (▨), and 1500 seconds⁻¹ (▪). Data are expressed as the means ± SDs of at least 3 separate assays (*P < .05). (B) Median velocities of platelet translocation on immobilized wt-A1 and mutants were determined from real-time movies by measuring the distance traveled by single platelets during 1 second of perfusion at 1500 seconds⁻¹. For each mutant, the velocity of 50 individual platelets was measured in one typical experiment. Ratios of the platelet count to the median velocity of platelets interacting with the mutants are represented as black bars. Median platelet velocity (μm/sec) is indicated in parentheses. ♦ indicates a contact time that was too short to allow velocity measurement. For space considerations, single-letter codes were used for amino acids. (C) Platelets in reconstituted blood were perfused at 1500 seconds⁻¹ over wt-A1 (○) and over Ala587Thr-A1 (●) in the presence of increasing concentrations of AJvW-2. After 3 minutes of perfusion, interacting platelets were counted. Results are expressed as the percentages of platelet numbers in the absence of AJvW-2.