Binding of fluorescently labeled human anti-β2GP1 F(ab′)2, fluorescently labeled β2GP1, or both to the developing thrombus. (A) Binding of fluorescently labeled human anti-β2GP1 F(ab′)2 to the developing thrombus. Anti-β2GP1 F(ab′)2 (2.5 μg/mouse) or control F(ab′)2 (2.5 μg/mouse), each labeled with Alexa 488, and anti-CD42 antibody labeled with Dylight 649 were infused into the mouse 15 minutes before laser-induced arteriolar wall injury. The results shown are for binding of anti-β2GP1 F(ab′)2 derived from sera from APS patient 1. Similar results were obtained with anti-β2GP1 F(ab′)2 derived from sera from APS patient 2. (Top) Representative images of the fluorescence signal associated with anti-β2GP1 F(ab′)2 (green; lane 1) or control F(ab′)2 (green; lane 2) and platelets (red) over 150 seconds after vessel injury are shown within the context of the brightfield histology. Merge (yellow). (Bottom) The median integrated antibody fluorescence (FANTIBODY) associated with thrombus formation after infusion of anti-β2GP1 F(ab′)2 (51 thrombi, 5 mice) or control F(ab′)2 (51 thrombi, 4 mice) over 150 seconds after vessel wall injury. Anti-β2GP1 F(ab′)2 (green); control F(ab′)2 (black). (B) Binding of fluorescently labeled β2GP1 in the presence of human anti-β2GP1 autoantibodies to the developing thrombus. Mice were infused with β2GP1 (25 μg/mouse) or HSA (25 μg/mouse), each labeled with Alexa 647, anti-β2GP1 autoantibodies (10 μg/mouse), and anti-CD42 antibody labeled with Dylight 488 before laser-induced arteriolar wall injury. (Top) Representative images of the fluorescence signal associated with β2GP1 (lane 1, red) or HSA (lane 2, red) and anti-CD42 antibody labeled with Dylight 488 for platelet detection (green) over 150 seconds after vessel injury are shown within the context of the brightfield histology. Merge (yellow). (Bottom) Median integrated protein fluorescence (Fβ2GP1 or FHSA) associated with thrombus formation in 3 wild-type mice after infusion of β2GP1 conjugated to Alexa 647 (27 thrombi, 3 mice) or HSA control conjugated to Alexa 647 (24 thrombi, 3 mice) over 150 seconds after vessel wall injury. β2GP1 (red); HSA (black). (C) Simultaneous binding of fluorescently labeled anti-β2GP1 autoantibodies and fluorescently labeled β2GP1 during thrombus formation after laser-induced injury. Mice were infused with labeled anti-β2GP1 F(ab′)2 (2.5 μg) derived from patient 2 plus labeled β2GP1 (25 μg) or labeled control F(ab′)2 (2.5 μg) plus labeled HSA (25 μg) 15 minutes before laser-induced arteriolar wall injury. (Top) Representative images of the fluorescence signal associated with Alexa 488-labeled anti-β2GP1 F(ab′)2 (lane 1, green) or Alexa 488–labeled control F(ab′)2 (lane 2, green) and Alexa 647–labeled β2GP1 (lane 1, red) or Alexa 647–labeled HSA (lane 2, red) over 150 seconds after vessel injury are shown within the context of the brightfield histology. Merge (yellow). (Bottom) The median integrated fluorescence, (FANTIBODY) and (FPROTEIN), of antibody and protein, respectively, associated with thrombus formation after infusion of Alexa 488–labeled anti-β2GP1 F(ab′)2 and Alexa 647–labeled β2GP1 (25 thrombi, 3 mice) or Alexa 488–labeled control F(ab′)2 and Alexa 647–labeled HSA (26 thrombi, 2 mice) over 150 seconds after vessel wall injury. Anti-β2GP1 F(ab′)2 (green); control F(ab′)2 (blue); β2GP1 (red); HSA (black). (D) High-resolution confocal intravital imaging of binding of the anti-β2GP1 autoantibody/β2GP1 complex during thrombus formation after laser-induced injury. Confocal image of thrombus formation 60 seconds after vessel wall injury. Alexa 488–labeled anti-β2GP1 F(ab′)2 (green); β2GP1 (red); merge (yellow). A single confocal slice through the center of the thrombus is shown.
