Division of Hematology, Departments of Internal Medicine and Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, USA.
Protein Sci. 2013 Aug;22(8):1049-59. doi: 10.1002/pro.2294.
Platelet attachment to von Willebrand factor (vWF) requires the interaction between the platelet GP1bα and exposed vWF-A1 domains. Structural insights into the mechanism of the A1-GP1bα interaction have been limited to an N-terminally truncated A1 domain that lacks residues Q1238 - E1260 that make up the linker between the D3 and A1 domains of vWF. We have demonstrated that removal of these residues destabilizes quaternary interactions in the A1A2A3 tridomain and contributes to platelet activation under high shear (Auton et al., J Biol Chem 2012;287:14579-14585). In this study, we demonstrate that removal of these residues from the single A1 domain enhances platelet pause times on immobilized A1 under rheological shear. A rigorous comparison between the truncated A1-1261 and full length A1-1238 domains demonstrates a kinetic stabilization of the A1 domain induced by these N-terminal residues as evident in the enthalpy of the unfolding transition. This stabilization occurs through site and sequence-specific binding of the N-terminal peptide to A1. Binding of free N-terminal peptide to A1-1261 has an affinity KD=46±6μM and this binding although free to dissociate is sufficient to suppress the platelet pause times to levels comparable to A1-1238 under shear stress. Our results support a dual-structure/function role for this linker region involving a conformational equilibria that maintains quaternary A domain associations in the inactive state of vWF at low shear and an intra-A1-domain conformation that regulates the strength of platelet GP1bα-vWF A1 domain associations in the active state of vWF at high shear.
血小板与血管性血友病因子（vWF）的结合需要血小板 GP1bα 与暴露的 vWF-A1 结构域之间的相互作用。对 A1-GP1bα 相互作用机制的结构见解仅限于截短的 A1 结构域，该结构域缺乏构成 vWF D3 和 A1 结构域之间连接物的残基 Q1238 至 E1260。我们已经证明，去除这些残基会破坏 A1A2A3 三结构域的四级相互作用，并有助于在高剪切力下血小板的激活（Auton 等人，J Biol Chem 2012；287:14579-14585）。在这项研究中，我们证明了从单个 A1 结构域中去除这些残基可增强在流变剪切下固定 A1 上血小板的停顿时间。对截短的 A1-1261 和全长 A1-1238 结构域的严格比较表明，这些 N 末端残基诱导 A1 结构域的动力学稳定性，这在展开过渡的焓中显而易见。这种稳定性是通过 N 末端肽与 A1 的位点和序列特异性结合而产生的。游离 N 末端肽与 A1-1261 的结合具有亲和力 KD=46±6μM，尽管这种结合可以自由解离，但足以将血小板的停顿时间抑制到与剪切应力下 A1-1238 相当的水平。我们的结果支持该连接区的双重结构/功能作用，涉及一种构象平衡，该平衡在低剪切力下维持 vWF 无活性状态下的四级 A 结构域缔合，以及在高剪切力下调节血小板 GP1bα-vWF A1 结构域缔合强度的 A1 结构域内构象。
