Quantification of ADAMTS13 cleavage sites 1605-6 in VWF A2 domains in VWF strings secreted by stimulated and unstimulated HUVECs. HUVECs grown in 24-well plates were incubated with 200 μL of serum-free media or stimulated with 100μM histamine in the same media at 37°C. Culture media was collected after 60 minutes or 48 hours into 10mM EDTA and analyzed for total VWF antigen and VWF 176-kD fragments by immunoassay. (A) VWF 176-kD fragment levels (pg/mL) were quantified using Mab LJ176 against amino acids M¹⁶⁰⁶-P¹⁶²⁰ of VWF monomers, and total VWF antigen levels (ng/mL) were measured using a polyclonal antibody against purified VWF. (B) Ratio of VWF 176-kD fragments per total VWF antigen after 60 minutes and 48 hours from histamine-stimulated and unstimulated HUVECs. Results are means ± SD (n = 12) and demonstrate that there is more ADAMTS-13–mediated cleavage of the few VWF strings secreted at a slow rate from unstimulated HUVECs than the many VWF strings secreted rapidly in response to cell stimulation at 60 minutes. (C) VWF multimer immunoblot representative of soluble VWF collected 1, 24, and 48 hours after HUVEC histamine stimulation in serum-free media. (D) Cartoon illustrating the relationship between rate of VWF string secretion and VWF cleavage multimer length. WPBs, represented by a spool of thread, release longer VWF strings before ADAMTS-13 cleavage under stimulation (rapid release rates) than under unstimulated conditions (basal rate). HUVEC-released ADAMTS-13, represented by the pair of scissors, is slow and continuous and remains constant regardless of the rate of VWF string secretion.