Figure 6.
Figure 6. PP2A inhibition prevents vascular PP2A activation and thrombus formation induced by aPL in mice. (A) Male C57BL/6 mice (4-6 weeks old) were injected with NHIgG or aPL (100 µg IP) in conjunction with the PP2A inhibitor endothall (10 µmol/kg body weight) or the control compound 1,4-dimethyl-endothall (Dm-Endo), and, 24 hours later, aortas were isolated and PP2A activity was quantified; N = 5. (B-D) Mice were treated as described in panel A, and thrombus formation following ferric chloride initiation was evaluated in the mesenteric microcirculation by intravital microscopy. (B) Representative still images taken 9 minutes after ferric chloride application are shown. The images were captured with a Nikon Eclipse Ti microscope and its camera system (Quantem 512SC, Plan Fluor, 10×, 0.3 aperture) at room temperature with fluorescein isothiocyanate as fluorochrome. NIS Elements software was used to capture and process the images. Scale bars, 50 µm. (C-D) The size of the largest thrombi formed within 9 minutes of initiation (C) and time to total occlusion were evaluated (D). In panels C and D, N = 5-6. Values are mean ± SEM, ****P < .0001. Scale bars, 50 µm. (E) Molecular basis of APS. PP2A subunits A, Bδ, B′α, and C are shown in magenta, and posttranslational modifications are shown in red. The superscript numbers indicate the figure in which data are presented about the molecule or structural feature of apoER2, and its participation in the molecular basis of APS. In response to aPL recognition of cell surface β2GPI, resulting β2GPI dimerization and interaction with apoER2, an apoER2-Dab2-SHC1 complex forms in endothelial cells to assemble and activate the heterotrimeric protein phosphatase PP2A. Dab2 recruitment to the apoER2 NPXY motif enables activating L309 methylation of PP2A-C by recruited LCMT-1. Concurrently, SHC1 recruits the PP2A-A to the proline-rich apoER2 C terminus along with 2 distinct regulatory PP2A subunits (PP2A-B), Bδ and B′α, to cause inhibitory dephosphorylation of Akt and eNOS, respectively. The resulting NO deficiency promotes thrombosis.

PP2A inhibition prevents vascular PP2A activation and thrombus formation induced by aPL in mice. (A) Male C57BL/6 mice (4-6 weeks old) were injected with NHIgG or aPL (100 µg IP) in conjunction with the PP2A inhibitor endothall (10 µmol/kg body weight) or the control compound 1,4-dimethyl-endothall (Dm-Endo), and, 24 hours later, aortas were isolated and PP2A activity was quantified; N = 5. (B-D) Mice were treated as described in panel A, and thrombus formation following ferric chloride initiation was evaluated in the mesenteric microcirculation by intravital microscopy. (B) Representative still images taken 9 minutes after ferric chloride application are shown. The images were captured with a Nikon Eclipse Ti microscope and its camera system (Quantem 512SC, Plan Fluor, 10×, 0.3 aperture) at room temperature with fluorescein isothiocyanate as fluorochrome. NIS Elements software was used to capture and process the images. Scale bars, 50 µm. (C-D) The size of the largest thrombi formed within 9 minutes of initiation (C) and time to total occlusion were evaluated (D). In panels C and D, N = 5-6. Values are mean ± SEM, ****P < .0001. Scale bars, 50 µm. (E) Molecular basis of APS. PP2A subunits A, Bδ, B′α, and C are shown in magenta, and posttranslational modifications are shown in red. The superscript numbers indicate the figure in which data are presented about the molecule or structural feature of apoER2, and its participation in the molecular basis of APS. In response to aPL recognition of cell surface β2GPI, resulting β2GPI dimerization and interaction with apoER2, an apoER2-Dab2-SHC1 complex forms in endothelial cells to assemble and activate the heterotrimeric protein phosphatase PP2A. Dab2 recruitment to the apoER2 NPXY motif enables activating L309 methylation of PP2A-C by recruited LCMT-1. Concurrently, SHC1 recruits the PP2A-A to the proline-rich apoER2 C terminus along with 2 distinct regulatory PP2A subunits (PP2A-B), Bδ and B′α, to cause inhibitory dephosphorylation of Akt and eNOS, respectively. The resulting NO deficiency promotes thrombosis.

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