BackgroundThe catalytic domain of ADAMTS13 possesses one Zn2+ and up to three putative Ca2+ binding sites and can be inactivated by chelating agents. Although replenishment with an appropriate metallic cation is thought to restore the enzyme's proteolytic activity fully, ADAMTS13 stability in a metal ion‐depleting environment has not been explored.ObjectivesTo address the stability of ADAMTS13 in citrated human plasma.MethodsADAMTS13 activity was measured using the FRETS‐VWF73 fluorogenic assay. The molar ratio of metals bound to ADAMTS13 was determined by size exclusion chromatography inductively coupled plasma mass spectrometry (SEC‐ICP‐MS). Higher‐order structural changes were analyzed using Fourier‐transformed infrared spectroscopy and dynamic light scattering.ResultsADAMTS13 was stable at room temperature for up to 24 hours irrespective of the presence of citrate (0.38%). However, at 37°C, citrate caused a time‐dependent activity decrease. No ADAMTS13 activity decrease was seen in heparinized plasma, but the addition of citrate again caused ADAMTS13 instability at 37°C. Scavenging of citrate by the addition of Ca2+ or Zn2+ prior to but not postincubation prevented the activity decrease of the enzyme. The SEC‐ICP‐MS analyses showed that ADAMTS13 only bound Zn2+ and that its reduced activity correlated with a gradual loss of bound Zn2+. Concomitant higher‐order structural analyses demonstrated structural changes in ADAMTS13 that are typical of less‐ordered protein structures.ConclusionsZn2+ is required to stabilize ADAMTS13 structure at physiologic temperature, thereby preventing irreversible loss of enzyme activity. This finding is particularly important to consider when using citrated human plasma as a source of ADAMTS13 in clinical settings.
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. Disclosures Grillberger: Baxalta Innovations GmbH: Employment. Kaufmann:Baxalta Innovations GmbH: Employment. Kink:Baxalta Innovations GmbH: Employment. Matthiessen:Baxalta Innovations GmbH: Employment. Plaimauer:Baxalta Innovations GmbH: Employment. Scheiflinger:Baxalta Innovations GmbH: Employment. Rottensteiner:Baxalta Innovations GmbH: Employment.
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