Baxalta Innovations GmbH, a member of the Takeda group of companies, Vienna, Austria.
Department of Chemistry, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.
J Thromb Haemost. 2019 Jun;17(6):995-1002. doi: 10.1111/jth.14440. Epub 2019 Apr 21.
The catalytic domain of ADAMTS13 possesses one Zn and up to three putative Ca 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.
To address the stability of ADAMTS13 in citrated human plasma.
ADAMTS13 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.
ADAMTS13 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 Ca or Zn prior to but not postincubation prevented the activity decrease of the enzyme. The SEC-ICP-MS analyses showed that ADAMTS13 only bound Zn and that its reduced activity correlated with a gradual loss of bound Zn . Concomitant higher-order structural analyses demonstrated structural changes in ADAMTS13 that are typical of less-ordered protein structures.
Zn 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.
ADAMTS13 的催化结构域含有一个锌原子和多达三个潜在的钙结合位点，可以被螯合剂失活。虽然认为补充适当的金属阳离子可以完全恢复酶的蛋白水解活性，但尚未研究 ADAMTS13 在金属离子耗尽环境中的稳定性。
研究 ADAMTS13 在柠檬酸盐化的人血浆中的稳定性。
使用 FRETS-VWF73 荧光测定法测定 ADAMTS13 的活性。通过尺寸排阻色谱-电感耦合等离子体质谱（SEC-ICP-MS）测定与 ADAMTS13 结合的金属的摩尔比。使用傅里叶变换红外光谱和动态光散射分析高级结构变化。
ADAMTS13 在室温下稳定长达 24 小时，与是否存在柠檬酸盐（0.38%）无关。然而，在 37°C 时，柠檬酸盐导致活性随时间下降。在肝素化血浆中未见 ADAMTS13 活性下降，但在 37°C 时再次加入柠檬酸盐会导致 ADAMTS13 不稳定。在孵育前而非孵育后添加 Ca 或 Zn 清除柠檬酸盐可防止酶活性下降。SEC-ICP-MS 分析表明，ADAMTS13 仅结合锌，其活性降低与结合锌的逐渐丧失相关。同时进行的高级结构分析表明 ADAMTS13 发生了结构变化，这是结构较为无序的蛋白质的典型特征。
锌是稳定生理温度下 ADAMTS13 结构所必需的，从而防止酶活性不可逆丧失。当在临床环境中使用柠檬酸盐化的人血浆作为 ADAMTS13 的来源时，这一发现尤其重要。
