Temperature-Dependent Irreversible Conformational Change of ADAMTS13 upon Metal Ion Chelation

Thrombotic thrombocytopenic purpura (TTP) is characterized by a functional deficiency in the plasma metalloprotease ADAMTS13, caused by mutations in the ADAMTS13 gene or by autoantibody inhibition. ADAMTS13 is the key regulator of the hemostatic activity of von Willebrand factor (VWF), accomplished by cleavage of a single site within the A2 domain of VWF. The catalytic domain of ADAMTS13 possesses various binding sites for metallic cations including one Zn²⁺ binding site composed of three histidine residues within the sequence HEXXHXXGXXHD and up to three putative calcium ion-binding sites. The dependence of ADAMTS13 activity on zinc and calcium ions is reflected in its inactivation by chelating agents such as EDTA and doxycycline. Although replenishment with an appropriate metallic cation is thought to fully restore the proteolytic activity of the enzyme, the stability of ADAMTS13 in a Ca²⁺-depleting environment has not yet been explored. This aspect, however, is clinically relevant, as citrated human plasma serves as the standard source for testing ADAMTS13-specific parameters, where the chelator citrate not only prevents activation of the coagulation cascade, but also renders ADAMTS13 inactive.

Here, we addressed the stability of plasma ADAMTS13 in the presence or absence of citrate (0.38%) at various temperatures. While ADAMTS13 proved stable at 4-8°C and at room temperature for up to 24 h irrespective of the presence of citrate, a time-dependent decrease in activity was observed at 37°C in the presence but not in the absence of citrate. No decrease in activity was seen when heparinized plasma was used as source of ADAMTS13, but the addition of 0.38% citrate again caused ADAMTS13 instability at 37°C. Similar results were observed when using the purified recombinant protein as source for ADAMTS13. Higher order structural analyses using Fourier-transformed infrared spectroscopy and dynamic light scattering demonstrated citrate-dependent structural changes in ADAMTS13 at 37°C that are typical for less-ordered protein structures. Addition of 5 mM Ca²⁺, Zn²⁺, or both metal ions prior to incubation completely restored ADAMTS13 stability, but these compounds failed to restore activity when added post incubation even after prolonged periods, suggesting that the loss in activity was irreversible.

We conclude that Zn²⁺ and/or Ca²⁺ ions are required to stabilize the structure of ADAMTS13 at physiological temperature. This finding needs to be considered when using citrated plasma as source of ADAMTS13 in clinical settings such as for plasma exchange to treat acute episodes of TTP.