von Willebrand factor self-association is regulated by the shear-dependent unfolding of the A2 domain.
von Willebrand factor self-association is regulated by the shear-dependent unfolding of the A2 domain.
复制标题
DOI：
10.1182/bloodadvances.2018030122
复制
发表时间：
2019-04
期刊：
影响因子：
7.5
通讯作者：
Changjie Zhang;Anju Kelkar;S. Neelamegham
中科院分区：
文献类型：
--
作者：
Changjie Zhang;Anju Kelkar;S. Neelamegham
血管性血友病因子（vonWillebrand factor，VWF）自结合导致VWF在暴露于流体剪切时的同型结合。这一过程的分子机制尚未建立。在这项研究中，我们证明了剪切依赖性展开的VWF A2结构域的多聚体蛋白是一个主要的调节蛋白质的自我协会。该机制控制血小板糖蛋白Ibα受体、胶原基质和离体血栓生长期间的自结合。为了支持这一点，防止由于N-和C-末端残基的二硫键桥连引起的结构域解折叠的A2-结构域突变（“Lock-VWF”）在各种实验条件下减少自缔合和血小板活化。相比之下，降低测定钙浓度和通过阻止与钙的配位而使VWF-A2构象不稳定的2个突变（D1498 A和R1597 W VWD 2A型突变）增强自缔合。使用一组缺乏A1结构域的重组蛋白（“ΔA1蛋白”）的研究表明，除了纯同型A2相互作用外，VWF-A2还可能参与其他蛋白质结构域以控制自缔合。添加纯化的高密度脂蛋白和载脂蛋白A1部分阻断了VWF的自结合。总的来说，类似的条件促进VWF自缔合和ADAMTS 13介导的蛋白水解，低钙和A2疾病突变增强这两个过程，并锁定A2阻断它们的同时。因此，VWF似乎已经在单个A2功能域中进化出2种平衡分子功能，以动态调节循环中的蛋白质大小：ADAMTS 13介导的蛋白水解和VWF自缔合。通过靶向VWF-A2调节自缔合速率可能提供调节血栓形成和止血速率的新方法。
von Willebrand factor (VWF) self-association results in the homotypic binding of VWF upon exposure to fluid shear. The molecular mechanism of this process is not established. In this study, we demonstrate that the shear-dependent unfolding of the VWF A2 domain in the multimeric protein is a major regulator of protein self-association. This mechanism controls self-association on the platelet glycoprotein Ibα receptor, on collagen substrates, and during thrombus growth ex vivo. In support of this, A2-domain mutations that prevent domain unfolding due to disulfide bridging of N- and C-terminal residues ("Lock-VWF") reduce self-association and platelet activation under various experimental conditions. In contrast, reducing assay calcium concentrations, and 2 mutations that destabilize VWF-A2 conformation by preventing coordination with calcium (D1498A and R1597W VWD type 2A mutation), enhance self-association. Studies using a panel of recombinant proteins that lack the A1 domain ("ΔA1 proteins") suggest that besides pure homotypic A2 interactions, VWF-A2 may also engage other protein domains to control self-association. Addition of purified high-density lipoprotein and apolipoprotein-A1 partially blocked VWF self-association. Overall, similar conditions facilitate VWF self-association and ADAMTS13-mediated proteolysis, with low calcium and A2 disease mutations enhancing both processes, and locking-A2 blocking them simultaneously. Thus, VWF appears to have evolved 2 balancing molecular functions in a single A2 functional domain to dynamically regulate protein size in circulation: ADAMTS13-mediated proteolysis and VWF self-association. Modulating self-association rates by targeting VWF-A2 may provide novel methods to regulate the rates of thrombosis and hemostasis.