Figure 6. Effect of blocking thiols in rADAMTS-13 on VWF cleavage.

(A) HUVECs were cultured to confluence and then stimulated with 25 mM histamine. Washed platelets with rADAMTS-13 (10 or 20 nM) before and after being treated with 10 mM of IAA (free IAA was removed by dialysis) were perfused over the activated endothelial cells at a 2.5-dyn/cm² shear stress for 3 min. The number of ULVWF strings over 10 continuous review fields was then counted and ADAMTS-13 activity was calculated as a percentage of ULVWF strings cleaved during perfusion (Student's t-test, n = 6). (B) Stimulated HUVECs were incubated with lyophilized human platelets in the presence of buffer control, IAA-treated buffer control, rADAMTS-13, or IAA-treated rADAMTS-13. Platelet-decorated ULVWF strings are visible under a light microscope (200 ×, top two panels), but cleaved and undetectable in the presence of IAA-treated and untreated rADAMTS-13 (bottom two panels, bar = 25 μm). (C) Purified plasma VWF multimers were first partially denatured with urea and then incubated with barium-activated rADAMTS-13 with and without IAA treatment. Aliquots were taken at different incubation intervals to detect cleaved VWF fragments by immunoblotting. The figure represents 4 separate experiments.