Figure 3
Figure 3. Endothelial cell activation is enhanced at the site of vascular injury in the presence of anti-β2GP1 autoantibodies in vivo. (A) Representative images of ICAM-1 and platelets in the developing thrombus from time 0 to 120 seconds obtained before (left panel, lane 1) and 15 minutes after (right panel, lane 2) infusion of 10 μg of human anti-β2GP1 autoantibodies. ICAM-1, green; platelets, red; merge, yellow. (B) The median integrated ICAM-1 fluorescence (F ICAM-1) associated with thrombus formation before (19 thrombi, 4 mice; blue) and 15 minutes after (27 thrombi, 4 mice; red) infusion of 10 μg of anti-β2GP1 IgG over 150 seconds after vessel wall injury. An irrelevant IgG in place of the anti-ICAM-1 antibody is shown (21 thrombin, 2 mice; green). (C) Comparison of the kinetics of ICAM-1 expression (green) and platelet accumulation (red) during thrombus formation in the absence of anti-β2GP1 IgG. (D) Confocal imaging of ICAM-1 after vascular injury indicates ICAM-1 is localized on the endothelium and not the platelet thrombus. Confocal images of ICAM-1 and platelets were obtained 60 seconds after laser injury during thrombus formation. (Left) ICAM-1 was visualized using anti-ICAM-1 labeled with Alexa 488 (0.4 μg/g mouse) (green) and platelets were visualized using anti-CD42 antibody labeled with Dylight 649 (0.1 μg/g mouse) (red). Merge, yellow. (Right) In the presence of eptifibatide (10 μg/g mouse) and its elimination of platelets, ICAM-1 was visualized on the endothelial surface. Confocal images were obtained through a central section of the thrombus. These confocal images are obtained at high speed in a live mouse where there is minor vessel movement with during each systole. Furthermore, the green and the red images are obtained near simultaneously but not simultaneously. Therefore, the register of the composite image is not perfect. Finally, there is low background noise that we elected not to subtract. We quantitated the fluorescence corresponding to total ICAM-1 fluorescence in the image and quantitated the fluorescence corresponding to the ICAM-1 fluorescence within the thrombus, as defined by platelet fluorescence. (E) F(ab′)2 fragments of anti-β2GP1 autoantibodies enhance activation of endothelial cells similarly to intact anti-β2GP1 autoantibodies. Endothelial cell activation was monitored using anti-ICAM-1 conjugated to Alexa 488 (0.5 μg/g) and platelets were monitored using anti-CD42 antibody conjugated with Dylight 649 (0.1 μg/g) before and 20 minutes after infusion of 12 μg of F(ab′)2 fragments of anti-β2GP1. ICAM-1 expression in endothelial cells was observed in an arteriole before (blue; 19 thrombi, 4 mice) and 20 minutes after infusion of 12 μg of F(ab′)2 fragments of anti-β2GP1 (black; 14 thrombi, 2 mice) or 10 μg of intact anti-β2GP1 (red; 27 thrombi, 4 mice).

Endothelial cell activation is enhanced at the site of vascular injury in the presence of anti-β2GP1 autoantibodies in vivo. (A) Representative images of ICAM-1 and platelets in the developing thrombus from time 0 to 120 seconds obtained before (left panel, lane 1) and 15 minutes after (right panel, lane 2) infusion of 10 μg of human anti-β2GP1 autoantibodies. ICAM-1, green; platelets, red; merge, yellow. (B) The median integrated ICAM-1 fluorescence (F ICAM-1) associated with thrombus formation before (19 thrombi, 4 mice; blue) and 15 minutes after (27 thrombi, 4 mice; red) infusion of 10 μg of anti-β2GP1 IgG over 150 seconds after vessel wall injury. An irrelevant IgG in place of the anti-ICAM-1 antibody is shown (21 thrombin, 2 mice; green). (C) Comparison of the kinetics of ICAM-1 expression (green) and platelet accumulation (red) during thrombus formation in the absence of anti-β2GP1 IgG. (D) Confocal imaging of ICAM-1 after vascular injury indicates ICAM-1 is localized on the endothelium and not the platelet thrombus. Confocal images of ICAM-1 and platelets were obtained 60 seconds after laser injury during thrombus formation. (Left) ICAM-1 was visualized using anti-ICAM-1 labeled with Alexa 488 (0.4 μg/g mouse) (green) and platelets were visualized using anti-CD42 antibody labeled with Dylight 649 (0.1 μg/g mouse) (red). Merge, yellow. (Right) In the presence of eptifibatide (10 μg/g mouse) and its elimination of platelets, ICAM-1 was visualized on the endothelial surface. Confocal images were obtained through a central section of the thrombus. These confocal images are obtained at high speed in a live mouse where there is minor vessel movement with during each systole. Furthermore, the green and the red images are obtained near simultaneously but not simultaneously. Therefore, the register of the composite image is not perfect. Finally, there is low background noise that we elected not to subtract. We quantitated the fluorescence corresponding to total ICAM-1 fluorescence in the image and quantitated the fluorescence corresponding to the ICAM-1 fluorescence within the thrombus, as defined by platelet fluorescence. (E) F(ab′)2 fragments of anti-β2GP1 autoantibodies enhance activation of endothelial cells similarly to intact anti-β2GP1 autoantibodies. Endothelial cell activation was monitored using anti-ICAM-1 conjugated to Alexa 488 (0.5 μg/g) and platelets were monitored using anti-CD42 antibody conjugated with Dylight 649 (0.1 μg/g) before and 20 minutes after infusion of 12 μg of F(ab′)2 fragments of anti-β2GP1. ICAM-1 expression in endothelial cells was observed in an arteriole before (blue; 19 thrombi, 4 mice) and 20 minutes after infusion of 12 μg of F(ab′)2 fragments of anti-β2GP1 (black; 14 thrombi, 2 mice) or 10 μg of intact anti-β2GP1 (red; 27 thrombi, 4 mice).

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