Time-course of [Ca²⁺]i changes during a platelet adhesive and cohesive process on a vWF-coated surface under a shear rate of 1,500 s⁻¹. Reconstituted blood containing platelets loaded with Ca²⁺ indicators was perfused over a vWF-coated surface with a shear rate of 1,500 s⁻¹. (Upper panels) Images obtained at 10 seconds, 1 minute, 4 minutes, and 8 minutes of perfusion (original magnification × 1,000). To distinguish translocating platelets from those firmly adhering to the surface, images were reconstructed by superimposition of 10 frames (2,380 μm² each) obtained every 0.1 seconds (total, 1 seconds) at each time point indicated. Thus, individual platelets translocating along the surface are seen as multiple images in a line (arrow 1), linked with a motion trace that appears as an artifact in a digitized video image when platelets are moving in a manner beyond the limit of time resolution of the videotape, whereas noncohered platelets not moving for at least 1 second are seen as single entities (arrows 2 and 3). Translocating platelets are reddish, whereas cohering platelets preferentially seen at late stages of platelet adhesive and cohesive processes (4 and 8 minutes of perfusion) are green (arrow 4). Single platelets firmly adhering to but not yet cohering on the surface are heterogeneous in color, with some appearing reddish (arrow 2) and others greenish (arrow 3). These time-course images were taken from the different locations in the same perfusion to minimize a possible photo-activation of platelets. (Lower panels) Time-course changes of G/R ratio (left) and platelet surface coverage (right). Data represent the mean and SD of results obtained from 5 independent perfusions at a shear rate of 1,500 s⁻¹ (see Fig 1 legend). Note that the G/R ratio and surface coverage of platelets increase as a function of time.