Multi-step binding of ADAMTS-13 to von Willebrand factor.
Multi-step binding of ADAMTS-13 to von Willebrand factor.
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ADAMTS-13与von Willebrand因子的多步结合。
DOI：
10.1111/j.1538-7836.2009.03620.x
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发表时间：
2009-12
期刊：
影响因子：
--
通讯作者：
Sadler JE
中科院分区：
文献类型：
--
作者：
Feys HB;Anderson PJ;Vanhoorelbeke K;Majerus EM;Sadler JE
ADAMTS13的蛋白降解活性受其底物von Willebrand因子(VWF)的构象控制，而VWF二级结构的变化是有效切割的关键。底物识别是通过ADAMTS13中远离活性中心的几个非催化结构域来介导的。我们假设并不是所有ADAMTS13在VWF中的结合位点都是隐蔽的，并分析了天然VWF与ADAMTS13的结合。使用抗VWF抗体和磁珠对VWF-ADAMTS13复合体进行免疫沉淀。用Western blotting和免疫吸附实验检测结合情况。免疫共沉淀实验表明，ADAMTS13与天然多聚体VWF结合，Kd为79±11 nM，未见明显的蛋白降解。在剪切诱导的VWF去折叠后，结合增加了3倍，VWF被切割。与天然VWF的结合是饱和的、依赖于时间的、可逆的，并且不随离子强度(I为50到200)而变化。此外，ADAMTS13缺失突变体的结果表明，与天然VWF的结合是通过ADAMTS13间隔区远端的结构域介导的，很可能是凝血酶敏感蛋白-1重复。有趣的是，这种相互作用发生在正常人血浆中，ADAMTS_(13)与vWF的化学计量比为0.0040±0.0004(平均值±扫描电子显微镜，n=10)。ADAMTS13与循环中的VWF结合，因此可能被合并到富含血小板的血栓中，在那里它可以立即裂解因流体剪应力而展开的VWF。
ADAMTS13 proteolytic activity is controlled by the conformation of its substrate, von Willebrand factor (VWF), and changes in the secondary structure of VWF are essential for efficient cleavage. Substrate recognition is mediated through several non-catalytic domains in ADAMTS13 distant from the active site. We hypothesized that not all binding sites for ADAMTS13 in VWF are cryptic and analyzed binding of native VWF to ADAMTS13. Immunoprecipiation of VWF-ADAMTS13 complexes using anti-VWF antibodies and magnetic beads was used. Binding was assessed by western blotting and immunosorbent assays. Co-immunoprecipitation demonstrated that ADAMTS13 binds to native multimeric VWF (Kd of 79 ± 11 nM) with no measurable proteolysis. Upon shear-induced unfolding of VWF, binding increased 3-fold and VWF was cleaved. Binding to native VWF was saturable, time dependent, reversible, and did not vary with ionic strength (I of 50 to 200). Moreover, results with ADAMTS13 deletion mutants indicated that binding to native VWF is mediated through domains distal to the ADAMTS13 spacer, likely thrombospondin-1 repeats. Interestingly, this interaction occurs in normal human plasma with an ADAMTS13 to VWF stoichiometry of 0.0040 ± 0.0004 (mean ± SEM, n = 10). ADAMTS13 binds to circulating VWF and may therefore be incorporated into a platelet-rich thrombus, where it can immediately cleave VWF that is unfolded by fluid shear stress.
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影响因子：
4.8
作者：
Katsumi, A;Tuley, EA;Sadler, JE
通讯作者：
Sadler, JE
影响因子：
4.8
作者：
Majerus, EM;Zheng, XL;Sadler, JE
通讯作者：
Sadler, JE
影响因子：
10.4
作者：
Varadi, K.;Rottensteiner, H.;Schwarz, H. P.
通讯作者：
Schwarz, H. P.
影响因子：
4.8
作者：
Anderson, PJ;Kokame, K;Sadler, JE
通讯作者：
Sadler, JE
影响因子：
20.3
作者：
Banno, Fumiaki;Chauhan, Anil K.;Miyata, Toshiyuki
通讯作者：
Miyata, Toshiyuki