Mechanosensitive Von Willebrand Factor Protein-Protein Interactions Regulate Hemostasis
Mechanosensitive Von Willebrand Factor Protein-Protein Interactions Regulate Hemostasis
复制标题
机械敏感冯维勒布兰德因子蛋白质-蛋白质相互作用调节止血
DOI：
10.1016/j.bpj.2014.11.2764
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发表时间：
2015
影响因子：
3.4
通讯作者：
Gräter F
中科院分区：
文献类型：
--
作者：
Aponte-Santamaría C;Huck V;Posch S;Bronowska AK;Grässle S;Brehm AM;Obser T;Schneppenheim R;Hinterdorfer P;Schneider SW;Baldauf C;Gräter F
机械敏感蛋白的一个反复出现的主题是一个神秘的结合位点，被外力发现。在这里，我们测试的作用，这种机制在血小板的粘附在血管损伤的网站，一个关键的过程中介导的血管性血友病因子（VWF）。我们从原子模拟，原子力显微镜（AFM），和微流控实验的数据表明，VWF A2域结合到VWF A1域，使它埋葬血小板结合位点位于A1。这意味着通过VWF A1和A2结构域之间的直接蛋白质-蛋白质相互作用使VWF与血小板结合失活。在力探针模拟和AFM实验中，拉伸力通过解离A1-A2复合物来揭示血小板结合位点。该过程伴随着A2结构域的仅部分解折叠，导致其切割位点的轻微暴露。因此，我们的数据表明，血小板结合和裂解降解的激活是通过A1与A2的相互作用偶联的，力保证VWF在裂解前准备好激活。A2缺失VWF突变体的微流控实验证实了A2结构域在体外的关键失活作用。总的来说，失活的VWF的力依赖性域间A1-A2的相互作用回答了血小板如何被阻止在平衡条件下结合到VWF的问题。一个隐藏的蛋白结合位点的概念，发现力不仅提供了长期观察的剪切依赖性血小板结合在血液凝固过程中的分子基础，但它也可能有助于解释剪切诱导的VWF自组装。
A recurring theme of mechanosensitive proteins is a cryptic binding site that gets uncovered by an external force. Here, we test the role of such mechanism in the adhesion of platelets at sites of vascular injury, a key process mediated by the von Willebrand factor (VWF). Our data from atomistic simulations, atomic force microscopy (AFM), and microfluidic experiments demonstrate that the VWF A2 domain binds to the VWF A1 domain, such that it buries the platelet binding site located at A1. This implies inactivation of VWF for the binding of platelets by a direct protein-protein interaction between the VWF A1 and A2 domains. During force-probe simulations and AFM experiments, a stretching force uncovered the platelet binding site, by dissociating the A1-A2 complex. This process was accompanied with only a partial unfolding of the A2 domain causing minor exposure of its cleavage site. Our data thus suggest that activation for platelet binding and degradation by cleavage are coupled through the interaction of A1 with A2, and force guarantees that VWF gets ready for activation before cleavage. Microfluidic experiments with an A2-deletion VWF mutant corroborate the critical inactivation role of the A2 domain in vitro. Overall, inactivation of VWF by force-dependent inter-domain A1-A2 interactions answers the question of how platelets are prevented to bind to VWF under equilibrium conditions. The notion of a cryptic protein binding site uncovered by force does not only provide the molecular basis for the long-standing observation of shear-dependent platelet binding during blood clotting, but it might also help to explain shear-induced VWF self-assembly.