Role of the platelet cytoskeleton in regulating tether formation.

(A) Washed platelets were perfused through VWF-coated microcapillary tubes at 150 s⁻¹ for 5 seconds, after which the wall shear rate was increased to 600 s⁻¹. Translocating platelets containing either membrane tethers (Ai) or filopodial projections (Aii) were fixed and stained with FITC-conjugated phalloidin (F-actin) and an anti–β-tubulin (MT) antibody. Platelet morphology was visualized by DIC. The percentage of platelets with F-actin or microtubular staining within membrane tethers and filopodia was quantified from 105 platelets (bar graph). (B,C) Washed platelets were pretreated with vehicle alone or cytochalasin D (5 μM) prior to perfusion through VWF-coated microcapillary tubes at 150 s⁻¹ for 5 seconds. The wall shear rate was either maintained at 150 s⁻¹ or increased to 600 s⁻¹, 1800 s⁻¹, 5000 s⁻¹, or 10 000 s⁻¹. (B) Scanning electron micrographs of representative cells forming tethers at 600 s⁻¹ and 5000 s⁻¹ in the absence (-CD) or presence (+CD) of cytochalasin D (scale bar equals 1 μm). (C) 600 s⁻¹: Digitized images (left panels) and corresponding schematics (right panels) demonstrating that cytochalasin D has no effect on tether release or retraction under low shear conditions. Once released, the tether contracts into flat ball-like structures (arrowheads) that gradually recede into the cell body. 5000 s⁻¹: Cytochalasin D treatment of platelets results in the formation of unstable tethers at higher wall shear rates. Initially a fine, long membrane tether is pulled from the platelet surface that becomes progressively thicker as membrane pulls from the cell body into the tether. Further extension of this thicker membrane tether results in the formation of rounded ball-like structures along the length of the tether resulting in a beadlike appearance that becomes increasingly unstable. Ultimately the cell body detaches from the tether and reinitiates its surface translocation.