Factor VIII binding affects the mechanical unraveling of the A2 domain of von Willebrand factor.
Factor VIII binding affects the mechanical unraveling of the A2 domain of von Willebrand factor.
复制标题
DOI：
10.1111/jth.14962
复制
发表时间：
2020-09
期刊：
影响因子：
--
通讯作者：
Zhang XF
中科院分区：
文献类型：
--
作者：
Cao W;Cao W;Zhang W;Zheng XL;Zhang XF
血管性血友病因子（vonWillebrand factor，VWF）被ADAMTS 13蛋白水解裂解对正常止血至关重要。我们以前的研究表明，凝血因子VIII（或FVIII）与VWF的结合增强了剪切下ADAMTS 13对VWF的蛋白水解裂解。本研究旨在确定FVIII介导的增强ADAMTS 13对VWF蛋白水解作用的机制。单分子力光谱，原子力显微镜，和表面等离子体共振都采用。使用单分子力谱，我们表明，添加FVIII（~5 nM）到D 'D3或D' D3 A1不会显著改变这些片段的力诱导解折叠;然而，将相同浓度的FVIII添加到D ′ D3 A1 A2中消除了其在~40 nm处的长解折叠事件，提示FVIII与D ′ D3和/或A2的结合可能导致A2结构域中力诱导的构象变化。原子力光谱法进一步证明了FVIII和D 'D3（或A2）之间的直接结合，其固有2D解离速率（k 0）为0.02 ± 0.01 s-1（或0.3 ± 0.1 s-1）。通过表面等离子体共振试验进一步证实了FVIII和A2之间的直接结合相互作用，解离常数（KD）约为0.2 μM;在相同条件下未检测到FVIII和A1之间的结合。我们的研究结果表明，FVIII的D 'D3和/或A2的结合可能会改变中央A2结构域的机械性能。这些发现为机械力下ADAMTS 13对VWF蛋白水解的FVIII依赖性调节的分子机制提供了新的见解。
Proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 is crucial for normal hemostasis. Our previous studies demonstrate that binding of coagulation factor VIII (or FVIII) to VWF enhances the proteolytic cleavage of VWF by ADAMTS13 under shear. Present study aims to determine the mechanism underlying FVIII-mediated enhancing effect on VWF proteolysis by ADAMTS13 under force. Single molecular force spectroscopy, atomic force microscopy, and surface plasmon resonance are all employed. Using the single molecule force spectroscopy, we show that an addition of FVIII (~5 nM) to D’D3 or D’D3A1 does not significantly alter force-induced unfolding of these fragments; however, an addition of FVIII at the same concentration to D’D3A1A2 eliminates its long unfolding event at ~40 nm, suggesting that binding of FVIII to D’D3 and/or A2 may result in force-induced conformational changes in A2 domain. Atomic force spectroscopy further demonstrates the direct binding between FVIII and D’D3 (or A2) with an intrinsic 2D off-rate (k0) of 0.02 ± 0.01 s−1 (or 0.3 ± 0.1 s−1). The direct binding interaction between FVIII and A2 is further confirmed with the surface plasmon resonance assay, with a dissociation constant (KD) of ~0.2 μM; no binding is detected between FVIII and A1 under the same conditions. Our results suggest that binding of FVIII to D’D3 and/or A2 may alter the mechanical property in the central A2 domain. The findings provide novel insight into the molecular mechanism underlying FVIII-dependent regulation of VWF proteolysis by ADAMTS13 under mechanical force.
登录
查看更多内容
影响因子：
20.3
作者：
Chiu, Po-Lin;Bou-Assaf, George M.;Walz, Thomas
通讯作者：
Walz, Thomas
影响因子：
4.8
作者：
Ulrichts, H;Udvardy, MS;Deckmyn, H
通讯作者：
Deckmyn, H
影响因子：
3.4
作者：
Evans, E;Ritchie, K
通讯作者：
Ritchie, K
影响因子：
4.8
作者：
Cao, Wenjing;Sabatino, Denise E.;Zheng, X. Long
通讯作者：
Zheng, X. Long
影响因子：
20.3
作者：
Zhang, Ping;Pan, Weilan;Zheng, X. Long
通讯作者：
Zheng, X. Long