Proteolytic cleavage of full-length VWF by ADAMTS13 and ADAMTS13-RYY. (A) Processing activity of ADAMTS13 (WT) and ADAMTS13-RYY (RYY) toward denatured, multimeric recombinant VWF was determined essentially as described previously.30 Proteolysis of VWF multimers was determined at different time points (0, 7.5, and 15 minutes) and samples analyzed by Western blot. These experiments were performed in the presence of a control, monoclonal, antipneumococcal antibody (see Figure 2 legend), which does not affect proteolysis. Cleavage of full-length VWF results in a reduction in the largest VWF multimers and the appearance of characteristic triplet bands. (B) Experiments were conducted as in panel A, except the patient derived inhibitory monoclonal antibody II-1 directed to the spacer domain was used instead of the control antibody. (C) Western blot analysis of ADAMTS13 preparations in conditioned media. This demonstrates the fidelity of ELISA quantitation of these 2 proteins used in this figure. (D) Processing activity of ADAMTS13 (WT), ADAMTS13-RYY (RYY), and control medium (−ve Ctrl) under flow on the surface of endothelial cells. The number of UL-VWF strings after the addition of ADAMTS13 is expressed as a percentage of the number of UL-VWF strings that were present at the onset of the experiment. The number of UL-VWF strings was monitored for 5 minutes at 10-second intervals. A decline in the number of UL-VWF strings also was observed in the absence of ADAMTS13, reflecting spontaneous detachment of UL-VWF strings from the surface of endothelial cells (−ve Ctrl). The inset shows the percentage of VWF strings remaining at 2.5 minutes, when repeated (n = 3) experiments were analyzed. *P < .05 determined by Student paired t test. (E) Experiments conducted as in panel D but in the presence of monoclonal antibody II-1.
