Figure 1
Figure 1. Characterization of hemostasis and in vivo thrombosis in SERCA3−/− mice. (A) Western blot of SERCAs in mouse platelets. Control (WT) and SERCA3−/− mouse platelets were collected and solubilized and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, prior to transfer to nitrocellulose and detection by antibodies specific for SERCA3 or SERCA2b.28 After addition of a secondary antibody coupled to horse radish peroxidase, bands were revealed by chemiluminescence. The 14-3-3ζ adaptor was used as an internal standard for normalization. Note the absence of SERCA3 in SERCA3−/− platelets and the same levels of SERCA2b in both control and SERCA3−/− platelets. (B) Tail bleeding time. Tail bleeding was performed as indicated in “Materials and methods,” and bleeding time assessed both on control (WT) and SERCA3−/− mice. Results are presented as mean ± SEM, using the Student t test; ***P < .001. (C) Rebleeding was assessed for 1 minute following initial bleeding arrest. A total of 18 control and 21 SERCA3−/− mice were used. (D) Transmission electron microscopy of resting control and SERCA3−/− platelets. Platelets were subjected to standard transmission electron microscopy. Upper panel, control (WT); lower panel, SERCA3−/− platelets. The scale bar (0.5 µm) is shown in the lower left corner of the WT panel. (E) Kinetics of in vivo ferric chloride-induced thrombosis of mesenteric vessels. Venules (v) or arterioles (a) are shown by fluorescence microscopy (limits outlined with white dashed lines), thrombi being visualized by rhodamine 6G–labeled platelets. Images at 0, 30, and 60 minutes are shown. (F) Quantification of thrombus formation. Time to occlusion was noted for 18 control (WT, closed circles) and 21 SERCA3−/− (open circles) mice up to 60 minutes, the maximal time assessed. Results were analyzed using 1-way ANOVA followed by Tukey’s multiple comparison test; ***P < .001. (G) Quantification of emboli. The number of emboli shedding from thrombi was assessed for 60 minutes, both in venules and arterioles of control and SERCA3−/− mice.

Characterization of hemostasis and in vivo thrombosis in SERCA3−/− mice. (A) Western blot of SERCAs in mouse platelets. Control (WT) and SERCA3−/− mouse platelets were collected and solubilized and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, prior to transfer to nitrocellulose and detection by antibodies specific for SERCA3 or SERCA2b.28  After addition of a secondary antibody coupled to horse radish peroxidase, bands were revealed by chemiluminescence. The 14-3-3ζ adaptor was used as an internal standard for normalization. Note the absence of SERCA3 in SERCA3−/− platelets and the same levels of SERCA2b in both control and SERCA3−/− platelets. (B) Tail bleeding time. Tail bleeding was performed as indicated in “Materials and methods,” and bleeding time assessed both on control (WT) and SERCA3−/− mice. Results are presented as mean ± SEM, using the Student t test; ***P < .001. (C) Rebleeding was assessed for 1 minute following initial bleeding arrest. A total of 18 control and 21 SERCA3−/− mice were used. (D) Transmission electron microscopy of resting control and SERCA3−/− platelets. Platelets were subjected to standard transmission electron microscopy. Upper panel, control (WT); lower panel, SERCA3−/− platelets. The scale bar (0.5 µm) is shown in the lower left corner of the WT panel. (E) Kinetics of in vivo ferric chloride-induced thrombosis of mesenteric vessels. Venules (v) or arterioles (a) are shown by fluorescence microscopy (limits outlined with white dashed lines), thrombi being visualized by rhodamine 6G–labeled platelets. Images at 0, 30, and 60 minutes are shown. (F) Quantification of thrombus formation. Time to occlusion was noted for 18 control (WT, closed circles) and 21 SERCA3−/− (open circles) mice up to 60 minutes, the maximal time assessed. Results were analyzed using 1-way ANOVA followed by Tukey’s multiple comparison test; ***P < .001. (G) Quantification of emboli. The number of emboli shedding from thrombi was assessed for 60 minutes, both in venules and arterioles of control and SERCA3−/− mice.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal