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Blood, Vol. 109, Issue 12, 5260-5269, June 15, 2007
Ligand density dramatically affects integrin  IIbß3-mediated platelet signaling and spreading
Blood Jirousková et al.
109: 5260
Supplemental materials for: Jiroušková et al, Vol 109, Issue 12, 5260-5269
Files in this Data Supplement:
- Figure S1. Platelet adhesion to low-density fibrinogen is faster and results in greater platelet spreading and recruitment of additional layers of platelets (JPG, 56 KB)
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Surfaces were coated with fibrinogen at concentrations from 1 to 100 µg/mL and blocked with 0.35% albumin; gel-filtered platelets in HBMT containing 2 mM Mg2+ were then added and incubated for 1 h. (A) Platelet adhesion to fibrinogen immobilized at different densities. Adhesion was measured by assessing the endogenous acid phosphatase activity. Data are expressed as mean ± SD, n = 4. Insert: Fibrinogen adsorption to polystyrene. 125I-labelled fibrinogen was allowed to adsorb to microtiter wells for 1 h and the radioactivity was counted with a gamma counter after washing (mean ± SD, n = 3). (B) Images from time lapse phase-contrast video microscopy taken at 10, 30, and 60 min after initiation of adhesion. Platelet adhesion to high-density fibrinogen was completed by 40 minutes, with very little platelet-platelet interaction observed, whereas adhesion to low-density fibrinogen was a slower and more dynamic process where spreading of platelets and recruitment of additional platelets on top of already adherent platelets continued for up to 2 hours (Video 1).
- Figure S2. PAC-1 binding to platelets adherent to high-density fibrinogen can be induced by LIBS antibody D3 (JPG, 50.7 KB)
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Platelets were allowed to adhere to high-density fibrinogen for 1 hour and, after washing, treated with activating mAb D3 (1 µg/mL) for an additional 30 minutes. Platelets were then stained with PAC-1 and images were acquired with fixed fluorescence exposition to compare the intensity of staining. D3 increased PAC-1 staining in platelets on high-density fibrinogen compared to control (buffer-treated) platelets, suggesting that the low level of staining of platelets adherent to high-density fibrinogen is due to  IIb 3 adopting an inactive conformation on the platelet surface rather than redistribution of receptors due to ligand engagement.
- Figure S3. 7H2 stains β3 receptors on the basal side of platelets in a punctate pattern on both low- and high-density fibrinogen (JPG, 47 KB)
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Platelets were allowed to adhere to fibrinogen-coated wells in the presence of a very low concentration of Alexa 488-7H2-Fab (20 ng/mL) for 1h, fixed after washing and imaged by TIR-FM. Bar, 10 µm.
- Figure S4. Platelets adherent to both low- and high-density fibrinogen contain granules, which release after addition of thrombin receptor activating peptide (TRAP) (JPG, 102 KB)
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Platelets labeled with the dense granule indicator mepacrine (10 µM) were allowed to adhere for 60 minutes and then imaged while 20 µM TRAP was added. Addition of TRAP led to almost constant degranulation (fewer green granules in the panels on right) and to further platelet spreading on high-density fibrinogen only.
- Video 1. Time lapse video microscopy of platelet adhesion to low- and high-density fibrinogen over the course of 2 hours (MOV, 4.73 MB)
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Platelets were allowed to adhere to low- or high-density fibrinogen and the adhesion process was recorded using phase-contrast time lapse video microscopy.23 Images were obtained automatically and concurrently from 2 channels for 2 hours at 2.4-minute intervals. The timer shows elapsed time during the adhesion (0 to 2 hours). On high-density fibrinogen, platelet adhesion was faster than on low-density fibrinogen, morphological changes were completed more rapidly and adherent platelets did not recruit additional platelet layers.
- Video 2. TIRF time-lapse microscopy of platelets adhering to low- and high-density fibrinogen revealed biphasic mode of adhesion to low-density fibrinogen (MOV, 3.85 MB)
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Platelets were allowed to adhere in the presence of Alexa488-7H2 (50 ng/mL) and the adhesion process was recorded every 10 seconds for 30 minutes using TIR-FM. The time stamp shows real time during the acquisition. On low-density fibrinogen, filopodia and lamellipodia formation were two sequential processes. On high-density fibrinogen fillopodia and lamellipodia formed almost simultaneously.
- Video 3. AP5 binding to the basal side of platelets during adhesion to low- and high-density fibrinogen studied by TIRF time-lapse microscopy (MOV, 3.67 MB)
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Platelets were allowed to adhere to fibrinogen in the presence of Alexa594-7H2 for 1 h and then Alexa488-AP5 was added and TIR-FM images of both channels were acquired simultaneously every 5 s for additional 5 min. Time stamp shows real time during the acquisition. Examination of ligand-bound (AP5 labeled – right panels) and all (7H2 labeled – left panels) IIb3 receptors by TIRF time-lapse microscopy showed a very thin, almost stationary ring of engaged IIb3 receptors on low-density fibrinogen (top panels), whereas the labeling on high-density fibrinogen was more dispersed and dynamic (lower panels).
- Video 4. Calcium flux in platelets adherent to low- and high-density fibrinogen (AVI, 3.82 MB)
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Platelets loaded with the calcium indicator dyes were added to the wells coated with low- or high-density fibrinogen. Calcium oscillations were monitored using confocal microscopy by acquiring images simultaneously in both fluorescence channels over a period of 140 seconds at 2-second intervals. Time stamp shows real time during the acquisition. Sustained calcium oscillations were triggered on low-density fibrinogen in platelets from the very beginning of contact with the surface; in contrast, platelet interaction with high-density fibrinogen resulted mostly in only transient increases in intracellular calcium concentration. Increase in intracellular calcium is reflected by increase in green and decrease in red fluorescence.
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