Identification of ADAMTS13 fragments generated by thrombin. Recombinant ADAMTS13 (250 nM) was incubated with 9 nM human thrombin at 37°C for 30 minutes in 20 mM Tris-HCl (pH 7.8) containing 150 mM NaCl and 5 mM Ca2+, causing partial proteolysis of ADAMTS13. Samples were analyzed by Western blotting. (A) ADAMTS13 was detected using the anti-ADAMTS13 protease domain antibody. ADAMTS13 (lane 1) was incubated with thrombin (lane 2). Increased quantities of ADAMTS13 (400 nM) were incubated with thrombin and extended development of blots (lane 3). (B) Probing of samples from panel A using the anti-myc antibody. ADAMTS13 (lane 1); ADAMTS13 treated with thrombin (lanes 2, 3, and 4). Lanes 3 and 4 represent the same sample; lane 3 contains 25% of the volume loaded in lane 4. (C) The predicted identities of the cleavage fragments in panel A (Frags 1-6) and panel B (Frags A-D) are represented. The locations of the predicted cleavage sites are approximate. Fragment boundaries and sizes were estimated from the molecular weights calculated from panel A and panel B. The fragments generated by cleavage at the favored cleavage sites are shown in bold.
