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Blood, Vol. 108, Issue 6, 1903-1910, September 15, 2006
Activation-independent platelet adhesion and aggregation under elevated shear stress
Blood Ruggeri et al.
108: 1903
Supplemental materials for: Ruggeri et al
Note: At the high shear rates used in these experiments, leukocytes do not interact with the surface and only platelets are visualized. To appreciate rapid events and structural details, select “Double Size” from the “Movie” pull-down menu and play sequences frame-by-frame. All fluorescence images (except Video 4, part 3) were acquired with a silicon-intensified (SIT) camera (Hamamatsu Photonics) and RICM images with a CCD camera (DXC-390; Sony).
Files in this Data Supplement:
- Supplemental Methods (PDF, 82.5 KB) -
This document is legal sized, not paper sized.
- Video 1. Distinct modalities of platelet thrombus formation as a function of shear rate (MOV, 587 KB)
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Blood containing PPACK was perfused over type I collagen fibrils. See the legend to Fig. 1 for details. The movie shows concurrently perfusion at the higher (hi) shear rate of 24,000 s-1 (upper half of the screen) and at the lower (lo) shear rate of 3,000 s-1 (lower half). In the upper half, a highlighted area shows a group of linked platelets stretched by hydrodynamic forces to the point of detachment from the forming thrombus. Images obtained with a Plan-Neofluar 40×/0.75 NA objective (Zeiss).
- Video 2. Formation of activation-independent platelet aggregates under elevated shear rate (MOV, 1.21 MB)
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(1). Blood containing PPACK, PG E1 and EDTA was perfused over immobilized VWF. See the legend to Fig. 2 for details. Single translocating platelets are present on the surface when the shear rate is 3,000 s-1, but aggregates form promptly when the shear rate is 20,000 s-1. The progressive growth of one such aggregate is highlighted. Note the transition from an ellipsoidal shape during translocation to an elongated chain-like shape during arrest. An uncoated area of glass at the right hand side of the screen shows no platelet attachment. Images obtained with a Plan-Neofluar 10×/0.30 NA objective (Zeiss).
(2). Washed blood cells suspended in buffer (platelet count: 250,000/μl) were perfused over immobilized VWF with (upper half of the screen) or without (lower half of the screen) the addition of 20 μg/ml soluble VWF. See the legend to Fig. 2 for details. In either experimental condition, single platelets adhere in similar number when the surface is exposed to a shear rate of 3,000 s-1. When the shear rate is 24,000 s-1, fewer single platelets adhere in the absence of soluble VWF, but surface coverage increases and aggregates form in the presence of soluble VWF. Images obtained with a Plan-Neofluar 40×/0.75 NA objective (Zeiss).
- Video 3. Activation-independent platelet aggregates form in pulsatile blood flow (MOV, 625 KB)
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This experiment was conducted as described for Video 2 (1), except that flow was maintained with a syringe pump controlled by a computer. The flow velocity changed linearly with a period of 1 s and ranged between 0 and three selected top values, resulting in the peak shear rates shown in the three panels of the movie. Images obtained with a Fluar 20×/0.75 NA objective (Zeiss).
- Video 4. Formation and stability of activation-independent platelet aggregates as a function of shear rate gradients (MOV, 2.6 MB)
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(1). Blood containing PPACK and PG E1 was perfused over immobilized VWF in a flow chamber in which the shear rate varied continuously from the inlet to the outlet (Usami S, Chen HH, Zhao Y, Chien S, and Skalak R. Design and construction of a linear shear stress flow chamber. Ann. Biomed. Eng. 1993; 21:77-83). Two conditions are shown, in which the shear rate either decreases (higher shear rates upstream; upper half of the screen) or increases (lower shear rates upstream; lower half of the screen) from the inlet (right) to the outlet (left). Minimum and maximum shear rates are 0 and 30,000 s-1, respectively. Four different positions are shown in each case, corresponding to shear rates of 5,000, 12,000, 20,000 and 26,000 s-1, respectively, at the surface. The presence of single rolling platelets, rolling (ellipsoid-like) aggregates or firmly adherent (string-like) aggregates depends mostly on the local shear rate, but is influenced to some extent by upstream shear rate values (see Results). Images obtained with a Plan-Neofluar 40×/0.75 NA objective (Zeiss).
(2). Perfusion of the same blood in a rectangular chamber with constant shear rate. Rolling platelet aggregates are present on the surface exposed to a shear rate of 24,000 s-1. The microscope stage is moved in the direction of flow to keep a group of rolling aggregates in the filed of view. When the shear rate drops to 3,000 s-1, rolling aggregates (one such aggregate is highlighted in yellow) progressively disperse into single platelets. Images obtained with a Fluar 20×/0.75 NA objective (Zeiss).
(3). A firmly adherent platelet aggregate retracts when the shear rate drops from 26,000 to 2,000 s-1, but most platelets remain within the stationary aggregate. When the shear rate returns to 26,000 s-1, the aggregate stretches in the direction of flow. Single platelets roll around and onto the aggregated platelets, and groups of linked platelets are seen detaching from the larger aggregate under the effect of high shear stress. Shown 4 times faster than real time. Images obtained with a Plan-Neofluar 40×/0.75 NA objective (Zeiss) through a color CCD camera (DXC-390; Sony).
- Video 5. Dynamic growth of activation-independent platelet aggregates (MOV, 0.99 MB)
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Blood containing PPACK and PG E1 was perfused over immobilized VWF at the indicated wall shear rates.
(1). Three platelets arrest their motion concurrently (evidence that they are connected) when the link between two of them (not visible by epifluorescence microscopy) wraps around other stationary platelets (colored in yellow). Interplatelet links are stretched by tensile stress, platelets rearrange their position in the chain-like structure, and eventually all six platelets (now linked together) detach forming a rolling aggregate.
(2). A rolling aggregate (highlighted in yellow) wraps around a stationary platelet. While reorienting in the direction of flow, the moving end of the aggregate incorporates three platelets and wraps around a second stationary platelet, thus forming an arch between two points of firm adhesion. An interplatelet link breaks at the upstream end of the arch, and the released extremity reorients in the direction of flow. The interplatelet link wrapped around the second stationary platelet is stretched and breaks while the downstream tip of the aggregate is wrapping around a third stationary platelet. These findings show that stationary platelets on the surface serve as anchoring points for interplatelet links within rolling aggregates, thus providing a mechanism for transient aggregate arrest and growth.
(3). An activation-independent platelet aggregate is seen while stretching under flow until it detaches leaving one platelet behind. A small rolling aggregate attaches to this platelet and is in turn stretched until it detaches. The two-color bar measures changes in the length of aggregates.
(4). Interplatelet links rupture under the effect of tensile stress, and the platelets remaining on the surface recoil in the direction opposite to flow (highlighted by the vertical bar at the rupture point). Stretching and recoiling of interplatelet links under tensile stress demonstrate their elastic properties. Images obtained with a Plan-Neofluar 40×/0.75 NA objective (Zeiss).
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