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Blood, Vol. 107, Issue 9, 3537-3545, May 1, 2006
Mechanism of platelet adhesion to von Willebrand factor and microparticle formation under high shear stress
Blood Reininger et al.
107: 3537
Supplemental materials for: Reininger et al
These movies represent experiments performed with reflection interference contrast microscopy (RICM), which allows the visualization of unstained cellular structures juxtaposed to an optically transparent surface. Only platelets that adhere to the substrate immobilized on the glass coverslip, but not freely moving cells, are visible at the flow rates utilized in these experiments. Blood flow is from right to left. Note that the brightness and contrast controls on the display used may need adjustment for best visibility of details. Manually advancing the frames of the movie may help visualize events that occur rapidly.
Files in this Data Supplement:
- Video 1. Whole blood containing PPACK perfused over immobilized VWF multimers at γw= 6, 000 s-1 (MOV, 2.36 MB)
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Part 1. The interface between reactive surface and platelets is seen from the onset of perfusion, in real time or at 50% velocity when indicated. Platelets translocate with different speed for variable distance. Some have short tethers and are essentially stationary; others move while membrane tethers connected to stationary upstream DAPs extend for lengths exceeding 30 µm. The shape heterogeneity of platelets reflects changes in the orientation of their discoid body during movement depending on the position of the limited membrane areas that interact with the immobilized ligand. Part 2. The same surface is seen in real time starting 61 s after the onset of perfusion; spread platelets are firmly adherent, and aggregates begin to form.
- Video 2. A detail of the experiment presented in Video 1 seen at 20% velocity (MOV, 5.14 MB)
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A first DAP forms at the origin of a tether (highlight 1). Additional DAPs then form downstream as the tether length increases, and their presence is evidenced by the ability to maintain bends opposing the pull from the platelet body (highlight 2). Successive straightening of a bend indicates the loss of adhesive force in these DAPs (highlight 3).
- Video 3. Washed blood cell suspension with normal platelet count (390, 000/µl) perfused over dVWFA1 at γ= 6, 000 s-1 (MOV, 3.09 MB)
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A detail of the surface shows an interacting platelet that appears in the field of view with a formed tether, which then recoils (highlight) indicating a loss of adhesion in the upstream DAP; repeats of this event are shown, the latter at 50% velocity. A new DAP forms and translocates more slowly than the platelet body causing a lengthening of the tether, which then recoils again (highlight); repeats of this event are also shown, the latter at 50% velocity. Note that, at times, the platelet forms a second tether.
- Video 4. Washed blood cell suspension, with reduced platelet count (11, 000/µl) and 10 µm PG E1 to prevent thrombus formation, perfused over type I collagen at γ= 4, 000 s-1 (MOV, 0.98 MB)
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Larger fibrils are distinctly visible, but other collagen structures that are highly reactive with VWF and platelets cannot be seen by light microscopy [Savage, B., M. H. Ginsberg and Z. M. Ruggeri. 1999. Blood 94: 2704-2715.]. Part 1. In the absence of VWF there is no demonstrable platelet adhesion (time indicated from the onset of flow). Part 2. After addition to the cell suspension of purified VWF (20 µg/ml), whose A3 domain binds to collagen during perfusion, platelets become tethered to the surface with a mechanism similar to that observed when purified VWF is immobilized directly on glass. Two repeats of an initial adhesion event are shown (highlight), the last at 50% velocity. Note that no interaction is observed when a blood cell suspension with purified VWF is perfused on glass not previously coated with collagen (not shown here).
- Video 5. Washed blood cell suspension with normal platelet count (230, 000/µl) perfused over dVWFA1 at γ= 4, 000 s-1 (MOV, 973 KB)
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Part 1. The sequence, in real time or at 50% velocity, highlights the translocation of a platelet through the field of view, beginning with the initial interaction. Several flip and rotational movements are seen, mediated by DAPs that form dynamically and in variable number within the platelet body without tether formation. Flips occur along the longitudinal axis of the discoid platelets whereas rotation indicates movement around an axis perpendicular to the former. The time shown is from the onset of perfusion. Part 2. A rotational movement of the translocating platelet is shown repeatedly at reduced (50%) velocity to highlight the pivoting around a single DAP that supports attachment to the surface.
- Video 6. Washed blood cell suspension with normal platelet count (270, 000/µl) perfused over dVWFA1 at γ = 1, 000 s-1 (MOV, 831 KB)
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The sequence highlights a DAP at the origin of a tether that supports attachment to the surface of an entire platelet, whose body moves freely in the flow field as shown by oscillations and positional changes.
- Video 7. Washed blood cell suspension with reduced platelet count (11, 000/µl), perfused over dVWFA1 at γ = 40, 000 s-1 (MOV, 782 KB)
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The sequence, in real time with highlighted sections at 33% velocity, begins 16 seconds after the onset of flow and demonstrates the presence on the surface of numerous platelet-derived particles. The first highlighted section demonstrates the generation of two microparticles with the size of DAPs from a single translocating platelet. The second highlighted section demonstrates the detachment of a longer tether from another platelet. Isolated DAPs and tether continue to translocate on the immobilized VWFA1 at lower speed than whole platelets. The third highlighted section demonstrates the concurrent formation of multiple tethers in a platelet and their tendency to fuse with one another during translocation.
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