ADAMTS-13 metalloprotease interacts with the endothelial cell-derived ultra-large von Willebrand factor
ADAMTS-13 metalloprotease interacts with the endothelial cell-derived ultra-large von Willebrand factor
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DOI：
10.1074/jbc.m301385200
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发表时间：
2003-08-08
影响因子：
4.8
通讯作者：
Cruz, MA
中科院分区：
文献类型：
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作者：
Dong, JF;Moake, JL;Cruz, MA
血栓性血小板减少性紫癜是由先天性或后天性 ADAMTS-13 缺陷引起的，ADAMTS-13 是一种金属蛋白酶，可裂解内皮源性血管性血友病因子 (ULVWF) 的超大多聚体。蛋白水解作用将高反应性和血栓形成性 ULVWF 转化为更小、粘附性更小的血浆形式。 ADAMTS-13 的活性通常在需要蛋白质变性和长时间孵育的非生理条件下的静态系统中测量。我们之前已经证明，ULVWF 多聚体从内皮细胞释放后，会形成血小板修饰的串状结构，并被 ADAMTS-13 快速裂解。在这里，我们报告了 ADAMTS-13 和 VWF 在静态和流动条件下的直接相互作用。 ADAMTS-13 包被的珠子粘附在由受刺激的内皮细胞呈现的固定 VWF 和 ULVWF 线上。这些珠子在静脉（2.5 达因/厘米（2））和动脉（30 达因/厘米（2））剪切应力下粘附到VWF上。然后我们证明 ADAMTS-13 珠粘附到固定化重组 VWF-A1 和 -A3 结构域，但可溶性金属蛋白酶优先结合到 A3 结构域，表明 VWF A3 结构域可能是金属蛋白酶的主要对接位点。我们建议流体剪切拉伸内皮所施加的拉伸应力结合 ULVWF 多聚体，以暴露 A 结构域内循环 ADAMTS-13 的结合位点。然后，结合的酶在非常接近的 A2 结构域内裂解，并将较小的 VWF 多聚体释放到血浆中。一旦释放，这些切割的 VWF 片段就无法被金属蛋白酶接近，以防止进一步切割。
Thrombotic thrombocytopenic purpura is caused by congenital or acquired deficiency of ADAMTS-13, a metalloprotease that cleaves the endothelium-derived ultra-large multimers of von Willebrand factor (ULVWF). The proteolysis converts hyper-reactive and thrombogenic ULVWF into smaller and less adhesive plasma forms. Activity of ADAMTS-13 is usually measured in a static system under non-physiological conditions that require protein denaturation and prolonged incubation. We have demonstrated previously that ULVWF multimers, upon release from endothelial cells, form platelet-decorated string-like structures that are rapidly cleaved by ADAMTS-13. Here we report the direct interaction between ADAMTS-13 and VWF under both static and flowing conditions. ADAMTS-13-coated beads adhered to both immobilized VWF and ULVWF strings presented by stimulated endothelial cells. These beads adhered to VWF under both venous (2.5 dynes/cm(2)) and arterial (30 dynes/cm(2)) shear stresses. We then demonstrated that ADAMTS-13 beads adhered to immobilized recombinant VWF-A1 and -A3 domains, but soluble metalloprotease bound preferentially to the A3 domain, suggesting that the VWF A3 domain may be the primary docking site for the metalloprotease. We suggest that tensile stresses imposed by fluid shear stretch endothelial bound ULVWF multimers to expose binding sites within the A domains for circulating ADAMTS-13. The bound enzyme then cleaves within the A2 domain that lies in close proximity and releases smaller VWF multimers into the plasma. Once released, these cleaved VWF fragments become inaccessible for the metalloprotease to prevent further cleavage.