The Middle and Distal Carboxyl Terminal Domains of ADAMTS13 Are Critical for Docking ADAMTS13 to von Willebrand Factor.
Authors: Zhang Ping , Weilan Pan , Rux Ann H. , Sachais Bruce S. , Zheng X. Long 
Published: October 2019

ADAMTS13 cleaves von Willebrand factor (VWF) at the Tyr1605-Met1606 bond of the central A2 domain. However, the domains of ADAMTS13 required for recognition and cleavage of VWF remain controversial. On the one hand, we and others have shown that the proximal carboxyl terminal domains (i.e. Cys-rich and spacer domains) of ADAMTS13 appeared to be sufficient for recognition of VWF under the static and denatured conditions (Zheng et al, JBC, 2003; Soejima et al, Blood, 2003, Ai et al, JBC, 2005). On the other hand, the distal carboxyl terminus (i.e. the first CUB domain) of ADAMTS13 has been proposed to dock ADAMTS13 to VWF under flow (Tao et al, Blood, 2005). To determine the interaction between ADAMTS13 and native VWF in real time, we employed Biacore technology based on surface plasmon resonance. To avoid conformational change or activation of VWF during the ligand immobilization, we chose to flow purified VWF or A2 fragment (VWF73) over the carboxymethylated dextran (CM5) surface to which was covalently attached via amine coupling either purified full-length recombinant ADAMTS13 (FL-A13) (~8,200 RU), the variant truncated after the 8th thrombospondin type 1 repeat (delCUB) (~7,200 RU), or that truncated after the spacer domain (MDTCS) (~4,200 RU). The control surface was prepared similarly by activation and deactivation processes without protein being coupled. The VWF at various concentrations in 20 mM HEPES buffer, pH 7.5, 150 mM NaCl, 0.02% Tween 20 and 0.05% BSA was flown at 20 μl/min over the surfaces as prepared above. The sensorgrams were recorded simutaneouly in all four microfluid cells and the kinetic parameters were determined. We showed that VWF bound immobilized FL-A13 in a dose and time-dependent manner with an equilibrium dissociation constant (Kd) of 50.0 ± 21.8 nM (n=6). Removal of the CUB domains (construct delCUB) reduced its affinity for VWF by ~5.5 fold (Kd=274 ± 158 nM (n=6). Further removal of the TSP1 2~8 repeats (construct MDTCS) almost completely abolished its binding to VWF (n=4) (Kd not determined). The affinity between ADAMTS13 (or variants) and VWF was not significantly altered by addition of 10 mM EDTA. Pre-denaturization of VWF with 1.5 M guanidine-HCl at 37 °C for 1 h, however, modestly enhanced VWF binding to immobilized delCUB (Kd=185 ± 96 nM, n=4) and MDTCS (Kd not determined), but reduced VWF binding to FL-A13 by ~2 fold, perhaps due to the mild denaturization of FL-A13 by 0.15 M guanidine-HCl in the flowing buffer. Interestingly, VWF73 bound FL-A13, delCUB and MDTCS with the Kd (s) of 2.6 ± 0.5 nM, 4.9 ± 1.0 nM and 3.0 ± 0.5 nM, respectively, which was at least 20~55 fold higher than native VWF did, suggesting that the protein structure surrounding the cleavage site at the central A2 domain of VWF negatively regulates ADAMTS13-VWF interaction. Moreover, the Michael-Menton constant Km (s) for cleavage of the denatured VWF (or VWF73) by FL-A13, delCUB and MDTCS were 260 ± 117 nM, 276 ± 118 nM and 534 ± 657 nM (or 1.8 ± 1.5 μM, 0.3 ± 0.1 μM and 0.6 ± 0.2 μM), respectively. These data demonstrate that the interaction between ADAMTS13 and native VWF is a high affinity one and that the middle and distal carboxyl terminal domains of ADAMTS13 may be critical for initial docking of ADAMTS13 to the exosites of native VWF in solution.