Enhanced platelet activation at the site of vascular injury with infusion of anti-β2GP1 autoantibodies. Mice were infused with platelets loaded with fura-2-AM (250 × 106/mouse). Monitoring fluorescence at 380 nm allowed quantitation of platelet accumulation (unliganded fura-2, green) and monitoring fluorescence at 340 nm allowed quantitation of activated platelets (Ca+2 liganded fura-2, red; yellow = merge). (A) Images of the developing thrombus at time 0 and at 90 seconds obtained without (panel 1), with infusion of 10 μg anti-β2GP1 IgG (panel 2) and with infusion of 12 μg anti-β2GP1 F(ab′)2 (panel 3). Resting platelets, green; activated platelets, yellow. (B) Platelet accumulation after laser-induced injury is represented by the median fluorescence intensity of loaded platelets excited at 380 nm over 3 minutes in 20 thrombi in 4 mice before (black) and in 25 thrombi after (gray) infusion of 10 μg of anti-β2GP1 IgG. (C) Platelet activation at the site of laser-induced injury is represented by the median fluorescence intensity of fura-2-AM–loaded platelets excited at 340 nm over 3 minutes in 20 thrombi in 4 mice before (black) and 25 thrombi after (gray) infusion of 10 μg of anti-β2GP1 IgG. (D) F(ab′)2 of anti-β2GP1 IgG-induced enhancement of platelet accumulation. (E) F(ab′)2 of anti-β2GP1 IgG-induced enhancement of fibrin generation. Platelet accumulation and fibrin generation at the site of laser-induced injury were measured before and 20 minutes after infusion of 12 μg of F(ab′)2 of anti-β2GP1 IgG. Platelet and fibrin labeling were performed using anti-CD42 antibody labeled with Dylight 488 (0.1 μg/g) and anti-fibrin antibody labeled with Alexa 647 (0.5 μg/g). F(ab′)2 of anti-β2GP1 IgG-induced changes in platelet thrombus size and fibrin generation were observed in thrombi performed upstream in a single arteriole before (blue, 16 thrombi, 3 mice) and 20 minutes after infusion of 12 μg of F(ab′)2 of anti-β2GP1 IgG (red, 12 thrombi, 2 mice).
