Characterization of the Binding Interaction between ADAMTS13 and von Willebrand Factor (VWF).

VWF multimers are cleaved into smaller less thrombogenic fragments in plasma by ADAMTS13. Deficiency of ADAMTS13 activity leads to thombotic thrombocytopenic purpura (TTP), a disease characterized by thrombocytopenia, microangiopathic hemolytic anemia, fever, neurological decline, and renal insufficiency. ADAMTS13 is a member of the A Disintegrin and Metalloprotease with ThromboSpondin repeats family that has characteristic motifs including a signal sequence, a propeptide, a catalytic metalloprotease domain, a disintegrin domain, a thrombospondin-1 (TSP1) repeat, a cysteine-rich region, and a spacer domain. These domains are followed in ADAMTS13 by seven additional TSP1 repeats and two C-terminal CUB domains. Previous work has shown that ADAMTS13 truncated after the spacer domain retains proteolytic activity towards VWF, but little is known about the potential role of the additional C-terminal TSP1 and CUB domains in VWF binding or cleavage. We therefore developed an enzyme-linked immunosorbent assay (ELISA)-based system to study ADAMTS13-VWF binding. VWF was immobilized in microtiter wells and incubated with plasma or recombinant ADAMTS13 variants. Bound ADAMTS13 was detected directly by solubilization and Western blotting, or indirectly by ELISA. ADAMTS13 proteolytic activity towards VWF is enhanced by denaturation of VWF with urea or guanidine, but ADAMTS13 bound specifically to VWF without prior denaturation. EDTA increased the binding of ADAMTS13 to VWF and prevented proteolysis of the immobilized VWF. Binding was saturable and time-dependent with maximal binding in two hours. Binding was reversible with a half-time for dissociation of four hours. ADAMTS13 in normal human plasma but not in plasma from a patient with TTP bound immobilized VWF. The stoichiometry of VWF monomers to ADAMTS13 at saturation was approximately 2 (range 1–4) and the Kd of recombinant ADAMTS13 binding to VWF was 12 nM (range 5–26 nM). This Kd for binding is similar to the Km for VWF cleavage of 16 nM determined independently, and both are comparable to the estimated plasma ADAMTS13 concentration of 8 nM. The properties of C-terminally truncated ADAMTS13 constructs suggest a regulatory function for certain domains. Truncation after the 8th TSP1 domain decreased the Kd 2-fold. Further truncation after either the 6th or 7th TSP1 domain increased the Kd 2-fold relative to full-length ADAMTS13. Truncation after the spacer domain gave binding properties indistinguishable from full-length ADAMTS13. Truncation after the metalloprotease domain gave no detectable binding to VWF. Therefore, binding of ADAMTS13 to VWF requires sequences in the cysteine-rich or spacer domains, and is modulated by sequences in at least the 8th TSP1 repeat and the CUB domains.