ADAMTS13 is proteolyzed following thrombin generation in human plasma. Recombinant ADAMTS13 (final concentration 80 nM) was added to defibrinated normal pooled citrated human plasma. (A) Thrombin generation was initiated by the addition of 8 pM TF, Ca²⁺, and phospholipids (PLs; PS/PC/PE, 20:60:20). The amount of thrombin generated in defibrinated ADAMTS13-spiked plasma was monitored in triplicate in real-time by a Fluoroscan plate reader using a fluorogenic thrombin substrate. Readings were quantified with a standard curve generated in parallel. (B) Parallel plasma samples were also analyzed for ADAMTS13 fragmentation. At 0, 10, 20, and 30 minutes, ADAMTS13 degradation was assessed by Western blotting under reducing conditions using an anti-ADAMTS13 protease domain antibody. (C) Citrated whole human plasma (containing 5 mg/mL GPRP-amide) was incubated with either TF/PLs/Ca²⁺ (lane 1), 125 nM ADAMTS13 (lane 2), TF/PLs/Ca²⁺ and 125 nM ADAMTS13 (lane 3), or TF/PLs/Ca²⁺, 125 nM ADAMTS13, and 100 U/mL hirudin (lane 4) for 30 minutes at 37°C. Samples were analyzed by Western blotting using an anti-ADAMTS13 protease domain antibody.