Laboratory for Thrombosis Research, K.U. Leuven Campus Kortrijk, Kortrijk, Belgium.
J Thromb Haemost. 2010 Oct;8(10):2305-12. doi: 10.1111/j.1538-7836.2010.04008.x.
The multidomain metalloprotease ADAMTS13 regulates the size of von Willebrand factor (VWF) multimers upon their release from endothelial cells. How the different domains in ADAMTS13 control VWF proteolysis in vivo remains largely unidentified.
Seven C-terminally truncated murine ADAMTS13 (mADAMTS13) mutants were constructed and characterized in vitro. Their ability to cleave VWF strings in vivo was studied in the ADAMTS13(-/-) mouse.
Murine MDTCS (devoid of T2-8 and CUB domains) retained full enzyme activity in vitro towards FRETS-VWF73 and the C-terminal T6-8 (del(T6-CUB)) and CUB domains (delCUB) are dispensable under these assay conditions. In addition, mADAMTS13 fragments without the spacer domain (MDT and M) had reduced catalytic efficiencies. Our results hence indicate that similar domains in murine and human ADAMTS13 are required for activity in vitro, supporting the use of mouse models to study ADAMTS13 function in vivo. Interestingly, using intravital microscopy we show that removal of the CUB domains abolishes proteolysis of platelet-decorated VWF strings in vivo. In addition, whereas MDTCS is fully active in vivo, partial (del(T6-CUB)) or complete (delCUB) addition of the T2-8 domains gradually attenuates its activity.
Our data demonstrate that the ADAMTS13 CUB and T2-8 domains influence proteolysis of platelet-decorated VWF strings in vivo.
多结构域金属蛋白酶 ADAMTS13 在血管性血友病因子（VWF）从内皮细胞释放后调节其多聚体的大小。ADAMTS13 中的不同结构域如何在体内控制 VWF 蛋白水解在很大程度上仍未被确定。
构建并体外鉴定了七个 C 端截断的鼠 ADAMTS13（mADAMTS13）突变体。在 ADAMTS13(-/-) 小鼠中研究了它们在体内切割 VWF 串的能力。
鼠 MDTCS（缺乏 T2-8 和 CUB 结构域）在体外对 FRETS-VWF73 仍保持完全酶活性，并且在这些测定条件下，C 端 T6-8（del(T6-CUB)）和 CUB 结构域（delCUB）是可有可无的。此外，没有间隔结构域的 mADAMTS13 片段的催化效率降低。因此，我们的结果表明，鼠和人 ADAMTS13 中的相似结构域在体外活性中是必需的，支持使用小鼠模型来研究 ADAMTS13 在体内的功能。有趣的是，使用活体显微镜我们显示 CUB 结构域的缺失可消除体内血小板修饰的 VWF 串的蛋白水解。此外，虽然 MDTCS 在体内完全活跃，但 T2-8 结构域的部分（del(T6-CUB)）或完全（delCUB）添加逐渐减弱其活性。
我们的数据表明 ADAMTS13 的 CUB 和 T2-8 结构域影响体内血小板修饰的 VWF 串的蛋白水解。
