Contribution of GpIb-V-IX and VWF to fibrin formation in whole blood. Platelets were superfused with recalcified whole blood at a shear rate of 250 s⁻¹ for 5 minutes, after which the flow was stepwise reduced every 2.5 minutes to reach 125 s⁻¹ at 10 minutes. Blood from healthy control subjects (Ctrl) or type 1 VWD patients was supplemented with labeled fibrinogen (150 μg/mL), AF647–annexin A5 (0.5 μg/mL) and/or 12G1 mAb (10 μg/mL). Bright-field and confocal fluorescence images were captured at 10 minutes (t = 0) with a Biorad/Zeiss laser-scanning microscope system (see “Methods”). (A) Representative images taken after 60 seconds, showing clusters of PS-exposing platelets and a growing fibrin network. Arrow indicates thrombus (bars, 10 μm). (B), Time plots showing fluorescence accumulation of AF546-fibrin(ogen) (blue) and AF647–annexin A5 (red). (C), High-resolution confocal images of OG488-fibrin(ogen) and AF647–annexin A5 fluorescence. Data are representative of 3 or more experiments.