Figure 5
Figure 5. Retraction of platelet-fibrin clots is attenuated by deletion of Bcl-3 in murine platelets. (A) Platelets were isolated from Bcl-3−/− and WT mice as described in “Materials and methods.” The cells were left quiescent or were activated and were then probed for Bcl-3 by Western analysis. (B) Platelets from Bcl-3–deficient and WT mice were left quiescent or activated with thrombin (0.5 U/mL) for 5 minutes and then examined for surface P-selectin or integrin αIIbβ3 by flow cytometry as described in “Materials and methods.” Blue and red areas indicate staining by anti-αIIbβ3 or anti–P-selectin and as control immunoglobulin, respectively. In the right panel, thrombin (0.1 U/mL) was added to the PRP from WT or Bcl-3−/− animals, and aggregation was monitored as described in “Materials and methods.” Similar results were observed when ADP was used as an agonist (data not shown). (C) PRP was collected from Bcl-3−/− and WT mice and normalized for platelet counts. The PRP was then left quiescent or stimulated with thrombin. After 2 hours, samples of the platelet-fibrin clots were prepared for transmission electron microscopy (“Materials and methods”). (Left) Dense fibrin complexes formed around irregularly shaped, spiculated platelets in the PRP from control animals. The red arrows point to platelets surrounded by electron-dense fibrin complexes, with other platelets enmeshed in dense fibrin nearby. (Right) Dense fibrin polymers were infrequent in suspensions of Bcl-3–deficient platelets. The blue arrowhead points to a small area of dense fibrin mesh adjacent to a Bcl-3−/− platelet, with other platelets and a few scattered fibrin complexes nearby. The Bcl-3−/− platelets (3 are indicated by red arrows) were less spiculated and tended to retain discoid shape. Scale bar, 2 μm. (D) WT or Bcl-3−/− murine platelets were isolated, resuspended in media containing fluorescently labeled fibrinogen, and stimulated with thrombin. Platelet aggregation and the organization of labeled fibrin complexes were examined at the designated time points. WT platelets bound labeled fibrin on their surfaces, giving them a white/orange globular appearance, and retracted fluorescently labeled fibrin into dense, compact masses around the platelet aggregates. A few individual fibrin strands were visible at early time points (blue arrows) but had been retracted into the tight platelet-fibrin mesh by 60 minutes. Incubations with platelets from Bcl-3–deficient animals resulted in loose complexes of labeled fibers. Individual fibrin strands were easily visible at 15 minutes and 60 minutes (blue arrows, bottom panels), in contrast to their paucity the incubations with WT platelets (top panels). Data in this figure are representative of 3 independent experiments. Scale bar, 5 μm.

Retraction of platelet-fibrin clots is attenuated by deletion of Bcl-3 in murine platelets. (A) Platelets were isolated from Bcl-3−/− and WT mice as described in “Materials and methods.” The cells were left quiescent or were activated and were then probed for Bcl-3 by Western analysis. (B) Platelets from Bcl-3–deficient and WT mice were left quiescent or activated with thrombin (0.5 U/mL) for 5 minutes and then examined for surface P-selectin or integrin αIIbβ3 by flow cytometry as described in “Materials and methods.” Blue and red areas indicate staining by anti-αIIbβ3 or anti–P-selectin and as control immunoglobulin, respectively. In the right panel, thrombin (0.1 U/mL) was added to the PRP from WT or Bcl-3−/− animals, and aggregation was monitored as described in “Materials and methods.” Similar results were observed when ADP was used as an agonist (data not shown). (C) PRP was collected from Bcl-3−/− and WT mice and normalized for platelet counts. The PRP was then left quiescent or stimulated with thrombin. After 2 hours, samples of the platelet-fibrin clots were prepared for transmission electron microscopy (“Materials and methods”). (Left) Dense fibrin complexes formed around irregularly shaped, spiculated platelets in the PRP from control animals. The red arrows point to platelets surrounded by electron-dense fibrin complexes, with other platelets enmeshed in dense fibrin nearby. (Right) Dense fibrin polymers were infrequent in suspensions of Bcl-3–deficient platelets. The blue arrowhead points to a small area of dense fibrin mesh adjacent to a Bcl-3−/− platelet, with other platelets and a few scattered fibrin complexes nearby. The Bcl-3−/− platelets (3 are indicated by red arrows) were less spiculated and tended to retain discoid shape. Scale bar, 2 μm. (D) WT or Bcl-3−/− murine platelets were isolated, resuspended in media containing fluorescently labeled fibrinogen, and stimulated with thrombin. Platelet aggregation and the organization of labeled fibrin complexes were examined at the designated time points. WT platelets bound labeled fibrin on their surfaces, giving them a white/orange globular appearance, and retracted fluorescently labeled fibrin into dense, compact masses around the platelet aggregates. A few individual fibrin strands were visible at early time points (blue arrows) but had been retracted into the tight platelet-fibrin mesh by 60 minutes. Incubations with platelets from Bcl-3–deficient animals resulted in loose complexes of labeled fibers. Individual fibrin strands were easily visible at 15 minutes and 60 minutes (blue arrows, bottom panels), in contrast to their paucity the incubations with WT platelets (top panels). Data in this figure are representative of 3 independent experiments. Scale bar, 5 μm.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal