Abstract
ADAMTS13 cleaves von Willebrand factor (VWF) between Tyr(1605) and Met(1606) residues at the central A2 subunit. The amino-terminus of ADAMTS13 protease appears to be sufficient to bind and cleave VWF under static and denatured condition. However, the role of the carboxyl-terminus of ADAMTS13 in substrate recognition remains controversial. Present study demonstrates that ADAMTS13 cleaves VWF in a rotation speed- and protease concentration-dependent manner on a mini vortexer. Removal of the CUB domains (delCUB) or truncation after the spacer domain (MDTCS) significantly impairs its ability to cleave VWF under the same condition. ADAMTS13 and delCUB (but not MDTCS) bind VWF under flow with dissociation constants (K(D)) of about 50 nM and about 274 nM, respectively. The isolated CUB domains are neither sufficient to bind VWF detectably nor capable of inhibiting proteolytic cleavage of VWF by ADAMTS13 under flow. Addition of the TSP1 5-8 (T5-8CUB) or TSP1 2-8 repeats (T2-8CUB) to the CUB domains restores the binding affinity toward VWF and the inhibitory effect on cleavage of VWF by ADAMTS13 under flow. These data demonstrate directly and quantitatively that the cooperative activity between the middle carboxyl-terminal TSP1 repeats and the distal carboxyl-terminal CUB domains may be crucial for recognition and cleavage of VWF under flow.