Figure 2
Figure 2. Patterns of total and activated αIIbβ3 in platelets adherent to low- and high-density fibrinogen. Platelets were added to wells coated with fibrinogen and incubated for 1 hour. Wells were then washed and adherent platelets incubated with fluorescently labeled antibodies as described in “Materials and methods.” (A) By confocal microscopy, 7H2 and AP5 staining produced similar patterns on platelets adherent to both low- and high-density fibrinogen, with AP5 staining being more pronounced in the granulomere region in platelets on low-density fibrinogen. (B) PAC-1, mAb specific for activated αIIbβ3, intensely stained the surface of spread platelets on low-density fibrinogen but, under the same conditions, stained only weakly the surface of platelets spread on high-density fibrinogen. Differential interference contrast images (DIC) are shown on the right side for comparison of the platelet morphology. (C) TIR-FM of platelets double stained with AP5 and 7H2 revealed that AP5 staining on the basal surface of platelets spread on low-density fibrinogen appears in a very thin rim at the edge, whereas AP5 staining of spread platelets on high-density fibrinogen is much thicker and diffuse. Cells marked with arrows are magnified in panel D. Bars represent 10 μm. Images shown are representative of at least 2 independent experiments. (D) TIR-FM images were analyzed for the width of AP5 staining by line scan analysis. The box plot shows the median and the 25th and 75th percentiles for 21 and 16 platelets on low- and high-density fibrinogen, respectively, from 3 independent experiments, *P < .001. (E) 7H2 staining of receptors on a platelet spread on high-density fibrinogen is static, whereas AP5 staining shows radial movement. Alexa 546-7H2–labeled platelets were allowed to adhere for 1 hour and then Alexa 488-AP5 was added and both antibodies were imaged using TIR-FM for 5 minutes. The image of a platelet spread on high-density fibrinogen (Video S3) was taken at the beginning of the acquisition period (red) and then 4 minutes later (green). On both low- and high-density fibrinogen, 7H2 staining did not change between the first and last frame as demonstrated by overlay (yellow). AP5 staining of platelet on high-density fibrinogen showed radial extension as judged by the appearance of strong green ring outside the red/yellow staining on the overlaid image. AP5 staining on low-density fibrinogen was without a change between the first and last frame (yellow overlay).

Patterns of total and activated αIIbβ3 in platelets adherent to low- and high-density fibrinogen. Platelets were added to wells coated with fibrinogen and incubated for 1 hour. Wells were then washed and adherent platelets incubated with fluorescently labeled antibodies as described in “Materials and methods.” (A) By confocal microscopy, 7H2 and AP5 staining produced similar patterns on platelets adherent to both low- and high-density fibrinogen, with AP5 staining being more pronounced in the granulomere region in platelets on low-density fibrinogen. (B) PAC-1, mAb specific for activated αIIbβ3, intensely stained the surface of spread platelets on low-density fibrinogen but, under the same conditions, stained only weakly the surface of platelets spread on high-density fibrinogen. Differential interference contrast images (DIC) are shown on the right side for comparison of the platelet morphology. (C) TIR-FM of platelets double stained with AP5 and 7H2 revealed that AP5 staining on the basal surface of platelets spread on low-density fibrinogen appears in a very thin rim at the edge, whereas AP5 staining of spread platelets on high-density fibrinogen is much thicker and diffuse. Cells marked with arrows are magnified in panel D. Bars represent 10 μm. Images shown are representative of at least 2 independent experiments. (D) TIR-FM images were analyzed for the width of AP5 staining by line scan analysis. The box plot shows the median and the 25th and 75th percentiles for 21 and 16 platelets on low- and high-density fibrinogen, respectively, from 3 independent experiments, *P < .001. (E) 7H2 staining of receptors on a platelet spread on high-density fibrinogen is static, whereas AP5 staining shows radial movement. Alexa 546-7H2–labeled platelets were allowed to adhere for 1 hour and then Alexa 488-AP5 was added and both antibodies were imaged using TIR-FM for 5 minutes. The image of a platelet spread on high-density fibrinogen (Video S3) was taken at the beginning of the acquisition period (red) and then 4 minutes later (green). On both low- and high-density fibrinogen, 7H2 staining did not change between the first and last frame as demonstrated by overlay (yellow). AP5 staining of platelet on high-density fibrinogen showed radial extension as judged by the appearance of strong green ring outside the red/yellow staining on the overlaid image. AP5 staining on low-density fibrinogen was without a change between the first and last frame (yellow overlay).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal