Key Laboratory of Thrombosis and Hemostasis of Ministry of Health, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, China.
Thromb Res. 2010 Oct;126(4):e260-5. doi: 10.1016/j.thromres.2010.07.009. Epub 2010 Aug 11.
ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 repeat motif. 13) is the major metalloprotease for VWF degradation. ADAMTS13 deficiency causes the accumulation of uncleaved VWF and might lead to a lethal thrombotic thrombocytopenic purpura (TTP). Thrombospondin-1 (TSP1) is considered as a reductase of VWF (von Willebrand factor) which can mildly downregulate the size of VWF by targeting on disulfide bond between VWF dimers. It was reported that TSP1 might protected VWF from cleaving by ADAMTS13, yet the underlying mechanism of this VWF protection has remained unknown.
Full-length ADAMTS13 and different domains (A1,A2,A3) of human VWF were constructed and expressed respectively. The binding ability of TSP1 or ADAMTS13 with each VWF domain or full-length VWF was investigated by using enzyme linked immunosorbent assay. The inhibition of ADAMTS13 activities by the different concentrations of TSP1 were observed by western blot and residual-collagen binding assay (R-CBA) under the denaturing condition.
We found that ADAMTS13 interacted with the rVWF A1, A2, A3 domains and full-length VWF, while TSP1 also bound to three A domains, especially to A2 and A3 domains. We observed that TSP1 partially blocked ADAMTS13 binding to A2 domain, A3 domain and full length VWF. The results of our assays showed that TSP1 could restrain ADAMTS13 activity up to 70%.
Our study suggested that TSP1 played competitively inhibitory role in ADAMTS13 binding and cleaving of VWF, and the potential competition might happen within A2 and A3 domains.
ADAMTS13（一种解整合素样金属蛋白酶与血小板反应蛋白 1 型重复基序 13）是 VWF 降解的主要金属蛋白酶。ADAMTS13 缺乏会导致未切割的 VWF 积聚，并可能导致致命性血栓性血小板减少性紫癜（TTP）。血小板反应蛋白 1（TSP1）被认为是 VWF（血管性血友病因子）的还原酶，它可以通过靶向 VWF 二聚体之间的二硫键，轻度下调 VWF 的大小。据报道，TSP1 可能通过 ADAMTS13 保护 VWF 不被切割，但这种 VWF 保护的潜在机制尚不清楚。
构建并表达全长 ADAMTS13 和人 VWF 的不同结构域（A1、A2、A3）。通过酶联免疫吸附试验（ELISA）研究 TSP1 或 ADAMTS13 与每个 VWF 结构域或全长 VWF 的结合能力。在变性条件下，通过 Western blot 和剩余胶原结合分析（R-CBA）观察不同浓度的 TSP1 对 ADAMTS13 活性的抑制作用。
我们发现 ADAMTS13 与 rVWF A1、A2、A3 结构域和全长 VWF 相互作用，而 TSP1 也与三个 A 结构域结合，特别是 A2 和 A3 结构域。我们观察到 TSP1 部分阻断 ADAMTS13 与 A2 结构域、A3 结构域和全长 VWF 的结合。我们的实验结果表明，TSP1 可以抑制 ADAMTS13 的活性达 70%。
我们的研究表明，TSP1 在 ADAMTS13 结合和切割 VWF 中发挥竞争性抑制作用，潜在的竞争可能发生在 A2 和 A3 结构域内。
