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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:
- Video 6. Visualization of activation-independent platelet adhesion and aggregation by reflection interference contrast microscopy (RICM) (MOV, 3.26 MB)
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Blood containing PPACK and PG E1 was perfused over immobilized VWF at the indicated wall shear rates.
(1). A single platelet (highlighted) establishes contact with the surface and is then stretched by hydrodynamic forces while attached through an upstream point of firm adhesion.
(2). Platelets 1 and 2 (highlighted) adhere to the surface and are stretched by hydrodynamic forces in successive and concurrent steps, indicating that they are linked. The sliding motion during stretching and the oscillatory movements in the flow field indicate that the point of attachment to the surface is upstream of platelet 1 and most of the structure, at this point approximately 40 μm long, is not directly in contact with immobilized VWF. A horizontal arrow that appears in front of platelet 2 indicates a retraction in the direction opposite to flow caused by the adhesion of a rolling aggregate (highlighted by a vertical arrow) that attaches near the body of platelet 1. Shown 3 times faster than real time.
(3). The stage of the microscope is moved in the direction opposite to flow to show the overall length of an activation-independent platelet aggregate (approximately 200 μm). The focal plane changes from the surface to above the surface, as indicated, to show discoid platelets attached to or rolling onto segments of stretched platelets. Shown 2 times faster than real time.
(4). Washed blood cells suspended in buffer (platelet count: 250,000/μl) were perfused over immobilized VWF. Activation-independent aggregates form after addition of soluble VWF (20 μg/ml; lower half of the screen), while in its absence (upper half of the screen) adhesion is limited to single rolling platelets. These form transient, thin membrane tethers while translocating on the surface, but adhesion never becomes stable. Note the fast translocation velocity of single platelets rolling onto immobilized VWF as compared to the prolonged stationary adhesion of platelets within the activation-independent aggregate. Shown 2 times faster than real time. Images obtained with a Plan-Neofluar Ph3 Antiflex 63x/1.25 NA oil immersion objective (Zeiss).
- Video 7. Platelet adhesion and aggregation onto immobilized VWF without inhibition of activation (MOV, 370 KB)
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Blood containing PPACK but no PG E1 was perfused over immobilized VWF. Mepacrine was added for visualization by epifluorescence microscopy (F, upper half of the screen; wall shear rate: 16,000 s-1), or surface events were visualized directly by RICM without mepacrine addition (R, lower half of the screen; wall shear rate: 24,000 s-1). In the absence of activation inhibitors, platelets that adhere firmly to the surface exhibit a darker grey color by RICM because they are spread and have extended areas of close membrane contact with the surface. In contrast, stretched platelets at the tip of a stationary aggregate and those in rolling aggregates exhibit a lighter grey color, indicating that their surface contact area is limited and most of the platelet body is not close to the surface. These platelets are discoid and not activated. Fluorescence images obtained with a Plan-Neofluar 40×/0.75 NA objective (Zeiss); RICM images obtained with a Plan-Neofluar Ph3 Antiflex 63x/1.25 NA oil immersion objective (Zeiss).
- Video 8. Formation of activation-independent platelet aggregates onto extracellular matrix (MOV, 1.78 MB)
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Blood containing PPACK and PG E1 was perfused over extracellular matrix deposited by mouse fibroblasts (see Supplemental Methods) at the indicated wall shear rates. Activation-independent platelet aggregates form above a threshold shear rate, as seen upon blood perfusion over immobilized VWF. Images obtained with a Plan-Neofluar 40×/0.75 NA objective (Zeiss).
- Video 9. Formation of activation-independent platelet aggregates over the surface of activated platelets (MOV, 3.07 MB)
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(1).Whole blood containing PPACK and mepacrine to render platelets fluorescent was initially perfused over type I collagen fibrils at 1,500 s-1 wall shear rate (seen through the green fluorescence channel with a black-and-white videocamera). Platelet thrombi rapidly form onto the surface (shown 10 times faster than real time).
(2). After 150 s, perfusion was continued without interruption replacing whole blood with a washed blood cell suspension containing platelets (250,000/μl) treated with 10 μM PG E1 and labeled with red-orange calcein AM (seen through the red fluorescence channel; note that the mepacrine-labeled thrombi are not visible). The suspension was supplemented with 15 μg/ml purified multimeric VWF. Shown 5 times faster than real time. At the wall shear rate of 1,500 s-1 the activation-inhibited platelets roll onto the formed thrombi without attaching firmly. Note that the values shown are wall shear rates calculated from the flow velocity assuming a perfusion chamber without thrombi.
(3). Overlay of the PG E1-treated platelets (red) rolling over the thrombi formed at the end of whole blood perfusion (green). The image from the green channel is a stationary single frame used to show relative positions and taken immediately before visualizing the red channel; the images from the red channel are taken during perfusion in real time.
(4). PG E1-treated platelets (seen through the red fluorescence channel) form activation-independent rolling aggregates when the shear rate increases, as indicated. Shown 5 times faster than real time.
(5). Overlay of the PG E1-treated platelets (red) forming rolling aggregates over adherent and activated platelets (green; stationary single frame taken immediately before visualizing the red fluorescence channel and used to show relative positions). Shown 5 times faster than real time. Images shown to this point obtained with a Plan-Neofluar 40×/0.75 NA objective (Zeiss).
(6). At the calculated wall shear rate of 25,000 s-1, PG E1-treated platelets (seen through the red fluorescence channel) form rolling as well as stationary activation-independent aggregates over previously adherent platelets. Shown 5 times faster than real time. Images obtained with a Fluar 20×/0.75 NA objective (Zeiss). Note that the size of the initial stable thrombi decreases with time since perfusion of PG E1-treated platelets cannot sustain the stability of aggregates.
- Video 10. Activation-independent platelet aggregation mediated by multimeric VWF or dimeric VWF A1 domain (MOV, 3.68 MB)
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Washed blood cells suspended in buffer (platelet count: 250,000/μl) were perfused over immobilized VWF in a variable shear rate flow chamber (see legend to Fig. 3). The suspension contained either 20 μg/ml soluble VWF multimers (+VWF; top panel in the screen) or dimeric VWF A1 domain at the concentration of 5 μg/ml (+dA1 (a); middle panel) or 50 μg/ml (+dA1 (b); bottom panel). Four positions in the chamber are shown from the inlet, where flow develops from a stagnation point, to the outlet, and the corresponding shear rates are indicated. Rolling aggregates form at lower shear rates in the presence of soluble dimeric A1 domain (aggregates are larger when the concentration of the isolated domain is greater) but not in the presence of multimeric VWF. In contrast, at higher shear rates (23,000 s-1) rolling aggregates form in the presence of multimeric VWF but not A1 domain, even when the latter is added at high concentration (12-fold molar excess over native VWF based on subunit mass). Images obtained with a Plan-Neofluar 40×/0.75 NA objective (Zeiss).
- Video 11. Intravital microscopy evaluation of activation-independent platelet aggregate formation in injured mouse mesenteric vessels (MOV, 2.53 MB)
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(1). Several minutes after induction of a vessel wall injury by exposure to ferric chloride (see Supplemental Methods), mouse platelets labeled with green calcein AM are incorporated into thrombi formed in both an arteriole and a venule (seen through the green fluorescence channel with a black-and-white videocamera). The direction of flow is indicated by arrows.
(2). PG E1-treated mouse platelets labeled with red-orange calcein AM also become localized at the site of growing thrombi (seen through the red fluorescence channel).
(3). Overlay of the images obtained in the green and red fluorescence channels with corresponding color assignment. The image from the green channel is a stationary single frame used to show relative positions and taken immediately before visualizing the red channel; the images from the red channel are taken during perfusion in real time.
(4). The PG E1-treated platelets, which cannot become activated, are not incorporated into thrombi; rather, they appear linked into aggregates that form on the surface of the stable thrombi but adhere only transiently. Note the stretching of activation-independent aggregates that precedes detaching under the influence of shear stress (seen through the red fluorescence channel).
(5). The thrombi formed by platelets not treated with PG E1 remain largely stationary on the vessel wall during the observation period (seen through the green fluorescence channel). Images obtained with an Achroplan 40×/0.80 NA water immersion objective (Zeiss).
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