Visual evaluation of ADAMTS13 activity within thrombi generated under high shear rate conditions using a monoclonal antibody (N10) that specifically detects ADAMTS13-cleaved VWF. Experimental conditions were as described in Figure 1, except that platelets were not labeled. Thrombi generated on a collagen-coated glass surface at 3 minutes' perfusion with or without an anti-ADAMTS13 antibody under 12 000 s−1 shear were fixed, reacted with both N10 antibody and anti–whole VWF antibody, double-stained with Cy3 (red)- and FITC (green)-fluorescence, and viewed by CLSM. (A) Three-dimensional images of thrombi, representative of 5 independent flow experiments, and the corresponding fluorescence intensity (mean ± SD of 5 areas randomly selected in a single perfusion) corrected for background value (negative control IgGs), indicate that VWF cleavage by ADAMTS13 (red color) within thrombi is significantly (*; P < .01) reduced in the presence of anti-ADAMTS13 antibody as compared with control thrombi (original magnification; ×600). (B) Merged 3-dimensional images and (C) the corresponding longitudinal views of thrombi; in the merged images, portions stained with both green and red fluorescence basically show the color of the higher pixel value, whereas a yellowish color is seen when both pixel values are nearly equal. Thus, the predominantly reddish appearance of the surface portions of control thrombi suggests that ADAMTS13 is more active on the surface of thrombi forming under very high shear rate conditions, while this tendency is barely visible in the presence of anti-ADAMTS13 antibody. (D) Longitudinal views of thrombi, generated at 3 minutes' perfusion without an anti-ADAMTS13 antibody under various shear rates and double-stained, are representative of 5 separate experiments. Note the prominent red color, especially at the surface portions of thrombi, indicating higher ADAMTS13 activity under higher shear rates.
