Machha Venkata R, Tischer Alexander, Moon-Tasson Laurie, Auton Matthew
Division of Hematology, Departments of Internal Medicine and Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, United States.
Division of Hematology, Departments of Internal Medicine and Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, United States.
J Mol Biol. 2017 Jan 6;429(1):32-47. doi: 10.1016/j.jmb.2016.11.014. Epub 2016 Nov 24.
The blood von Willebrand factor (VWF) mediates platelet adhesion to injured vessels by sequestering platelets from blood flow and depositing them to collagen and other exposed subendothelial matrix proteins. This process of capturing platelets to facilitate formation of platelet plugs occurs through transient interactions with platelet glycoprotein Ibα via the VWF A1 domain which also binds collagen. Using a conformationally diverse collection of natively folded and mutation-induced misfolded von Willebrand disease (VWD) variants, we test a recently proposed affinity up-regulation hypothesis which states that collagen binding changes the conformation of the A1 domain to a high-affinity GPIbα binding competent state. With surface plasmon resonance (SPR), we present this diversified collection to collagen and quantify the kinetics of association and dissociation to ascertain the conformational selectivity of collagen. With analytical rheology, we quantify real-time platelet pause times and translocation velocities across a Cu HisTag-chelated and collagen-bound A1 single domain and A1A2A3 tridomain fragment of VWF under shear stress in an ex vivo shear flow microfluidic chamber. In contrast to expected hypothetical outcomes, collagen has limited conformational selectivity for binding A1. A1-collagen binding is independent of gain- or loss-of-function phenotype and under shear stress, platelet translocation pause times on collagen-bound A1A2A3 are either normal or shorter depending on whether A1 is concertedly bound with the A3 domain to collagen. With respect to A1, collagen has an inhibitory role that provides an explanation for the lack of thrombosis in patients with gain-of-function VWD.
血液中的血管性血友病因子（VWF）通过从血流中隔离血小板并将其沉积到胶原蛋白和其他暴露的内皮下基质蛋白上，介导血小板与受损血管的黏附。通过VWF A1结构域与血小板糖蛋白Ibα的短暂相互作用，发生捕获血小板以促进血小板凝块形成的过程，VWF A1结构域也与胶原蛋白结合。我们使用一系列构象多样的天然折叠和突变诱导的错折叠血管性血友病（VWD）变体，测试了最近提出的亲和力上调假说，该假说认为胶原蛋白结合会将A1结构域的构象改变为高亲和力的GPIbα结合活性状态。利用表面等离子体共振（SPR），我们将这个多样化的集合呈现给胶原蛋白，并量化结合和解离的动力学，以确定胶原蛋白的构象选择性。通过分析流变学，我们在体外剪切流微流体腔室中，在剪切应力下量化了血小板在VWF的铜组氨酸标签螯合和胶原蛋白结合的A1单结构域以及A1A2A3三结构域片段上的实时停顿时间和转运速度。与预期的假设结果相反，胶原蛋白对A1结合的构象选择性有限。A1与胶原蛋白的结合与功能获得或丧失表型无关，在剪切应力下，血小板在胶原蛋白结合的A1A2A3上的转运停顿时间正常或更短，这取决于A1是否与A3结构域协同结合到胶原蛋白上。就A1而言，胶原蛋白具有抑制作用，这为功能获得性VWD患者血栓形成缺乏提供了解释。
