Figure 5
Figure 5. In the absence of a platelet thrombus, the anti-β2GP1 autoantibody/β2GP1 complex neither bound the endothelium nor enhanced endothelial calcium mobilization. (A) After blocking platelet accumulation by infusion of eptifibatide (10 μg/g mouse), the median integrated antibody fluorescence (FAntibody) after infusion of anti-β2GP1 F(ab′)2 (24 thrombi, 2 mice) or control F(ab′)2 (26 thrombi, 2 mice) over 150 seconds after vessel wall injury was measured. Anti-β2GP1 F(ab′)2 (green); control F(ab′)2 (black). No significant binding of anti-β2GP1 F(ab′)2 was observed in the absence of a platelet thrombus. (B) After blocking platelet accumulation by infusion of eptifibatide (10 μg/g mouse), the median integrated protein fluorescence (Fβ2GP1 or FHSA) after infusion of Alexa 647 conjugated to β2GP1 (24 thrombi, 2 mice) or Alexa 647 conjugated to HSA (26 thrombi, 2 mice) over 150 seconds after vessel wall injury was measured. β2GP1 (red); control (black). No significant binding of β2GP1 was observed. (C) Fluo-4-AM was delivered into the mouse circulation via the femoral artery, and concurrent platelet aggregation was inhibited by infusion of eptifibatide (10 μg/g mouse) every 15 minutes. After laser-induced vessel wall injury, changes to endothelial Ca2+ levels were observed by excitation at 488 nm and images were recorded over time. Antibody-induced change in endothelial cell activation was examined in the injured vessel performed upstream in 1 arteriole before and 15 minutes after infusion of 10 μg of anti-β2GP1 autoantibodies. Images show calcium elevation in the endothelium in the absence of platelet accumulation obtained at time 0 and at 90 seconds after vessel wall injury before (panel 1) and after (panel 2) injection of 10 μg of anti-β2GP1 antibodies. The fluorescence signal is represented as a pseudocolor intensity map where black represents the least intense and red represents the most intense fluorescence signal. (D) The kinetics of endothelial cell activation at the site of laser-induced injury was determined by calculating median fluorescence values at 488 nm as a function of time. Calcium mobilization is represented by the median fluorescence intensity of Fluo-4-AM–loaded endothelial cells over 3 minutes before (black) and after (gray) infusion of 10 μg of anti-β2GP1 IgG for 15 to 20 thrombi in 2 mice.

In the absence of a platelet thrombus, the anti-β2GP1 autoantibody/β2GP1 complex neither bound the endothelium nor enhanced endothelial calcium mobilization. (A) After blocking platelet accumulation by infusion of eptifibatide (10 μg/g mouse), the median integrated antibody fluorescence (FAntibody) after infusion of anti-β2GP1 F(ab′)2 (24 thrombi, 2 mice) or control F(ab′)2 (26 thrombi, 2 mice) over 150 seconds after vessel wall injury was measured. Anti-β2GP1 F(ab′)2 (green); control F(ab′)2 (black). No significant binding of anti-β2GP1 F(ab′)2 was observed in the absence of a platelet thrombus. (B) After blocking platelet accumulation by infusion of eptifibatide (10 μg/g mouse), the median integrated protein fluorescence (Fβ2GP1 or FHSA) after infusion of Alexa 647 conjugated to β2GP1 (24 thrombi, 2 mice) or Alexa 647 conjugated to HSA (26 thrombi, 2 mice) over 150 seconds after vessel wall injury was measured. β2GP1 (red); control (black). No significant binding of β2GP1 was observed. (C) Fluo-4-AM was delivered into the mouse circulation via the femoral artery, and concurrent platelet aggregation was inhibited by infusion of eptifibatide (10 μg/g mouse) every 15 minutes. After laser-induced vessel wall injury, changes to endothelial Ca2+ levels were observed by excitation at 488 nm and images were recorded over time. Antibody-induced change in endothelial cell activation was examined in the injured vessel performed upstream in 1 arteriole before and 15 minutes after infusion of 10 μg of anti-β2GP1 autoantibodies. Images show calcium elevation in the endothelium in the absence of platelet accumulation obtained at time 0 and at 90 seconds after vessel wall injury before (panel 1) and after (panel 2) injection of 10 μg of anti-β2GP1 antibodies. The fluorescence signal is represented as a pseudocolor intensity map where black represents the least intense and red represents the most intense fluorescence signal. (D) The kinetics of endothelial cell activation at the site of laser-induced injury was determined by calculating median fluorescence values at 488 nm as a function of time. Calcium mobilization is represented by the median fluorescence intensity of Fluo-4-AM–loaded endothelial cells over 3 minutes before (black) and after (gray) infusion of 10 μg of anti-β2GP1 IgG for 15 to 20 thrombi in 2 mice.

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