Cofactors of thrombin and ADAMTS13 proteolysis. Recombinant ADAMTS13 (250 nM) was treated with 50 nM thrombin that had been preincubated at 37°C with either equimolar or a 10-fold molar excess of either soluble rabbit TM (A) or heparin (B) in 20 mM Tris-HCl (pH 7.8) containing 150 mM NaCl and 5 mM Ca²⁺. ADAMTS13 fragmentation was monitored under reducing conditions using the anti-protease domain antibody. (A) Thrombin was preincubated with either 50 nM or 500 nM soluble rabbit TM for 5 minutes. Thereafter, the ability of each thrombin/TM mix to cleave ADAMTS13 was monitored at 0 minutes, 30 minutes, and 1 hour time points, and was compared with that of thrombin alone. (B) Thrombin was preincubated with either 50 nM or 500 nM heparin and analyzed as in panel A. (C) The specificity of thrombin for ADAMTS13 relative to other ADAMTS-family members was gauged by incubating 250 nM ADAMTS4 in 20 mM Tris-HCl (pH 7.8) containing 150 mM NaCl and 5 mM Ca²⁺ with 9 nM thrombin for 0 to 6 hours. Samples were analyzed by Western blotting using a polyclonal anti-ADAMTS4 protease domain antibody.