Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA 30332.
Shanghai Institute of Applied Mathematics and Mechanics, Shanghai Key Laboratory of Mechanics in Energy Engineering, Shanghai University, Shanghai 200072, People's Republic of China.
Mol Biol Cell. 2019 Jul 22;30(16):1920-1929. doi: 10.1091/mbc.E19-01-0021. Epub 2019 May 8.
Hemodynamic forces activate the Von Willebrand factor (VWF) and facilitate its cleavage by a disintegrin and metalloprotease with thrombospondin motifs-13 (ADAMTS13), reducing the adhesive activity of VWF. Biochemical assays have mapped the binding sites on both molecules. However, these assays require incubation of two molecules for a period beyond the time allowed in flowing blood. We used a single-molecule technique to examine these rapid, transient, and mechanically modulated molecular interactions in short times under forces to mimic what happens in circulation. Wild-type ADAMTS13 and two truncation variants that either lacked the C-terminal thrombospondin motif-7 to the CUB domain (MP-TSP6) or contained only the two CUB domains (CUB) were characterized for interactions with coiled VWF, flow-elongated VWF, and a VWF A1A2A3 tridomain. These interactions exhibited distinctive patterns of calcium dependency, binding affinity, and force-regulated lifetime. The results suggest that 1) ADAMTS13 binds coiled VWF primarily through CUB in a calcium-dependent manner via a site(s) outside A1A2A3, 2) ADAMTS13 binds flow-extended VWF predominantly through MP-TSP6 via a site(s) different from the one(s) at A1A2A3; and 3) ADAMTS13 binds A1A2A3 through MP-TSP6 in a Ca-dependent manner to autoinhibit another Ca-independent binding site on CUB. These data reveal that multiple sites on both molecules are involved in mechanically modulated VWF-ADAMTS13 interaction.
血流动力激活血管性血友病因子 (VWF)，并通过含有血小板反应蛋白基序的解整合素金属蛋白酶 13 (ADAMTS13) 促进其裂解，从而降低 VWF 的黏附活性。生化分析已确定两种分子上的结合位点。然而，这些分析需要将两种分子孵育一段时间，超出了在血流中允许的时间。我们使用单分子技术在力的作用下在短时间内检查这些快速、短暂和受机械调节的分子相互作用，以模拟在循环中发生的情况。野生型 ADAMTS13 和两种截断变体，一种缺乏 C 端血小板反应蛋白基序 7 到 CUB 结构域（MP-TSP6），另一种仅含有两个 CUB 结构域（CUB），用于与卷曲 VWF、流动延伸的 VWF 和 VWF A1A2A3 三结构域相互作用。这些相互作用表现出独特的钙依赖性、结合亲和力和力调节寿命模式。结果表明：1）ADAMTS13 通过 CUB 以钙依赖性方式主要通过 A1A2A3 之外的位点结合卷曲的 VWF，2）ADAMTS13 通过与 A1A2A3 不同的位点主要通过 MP-TSP6 结合延伸的 VWF；3）ADAMTS13 通过 MP-TSP6 以 Ca 依赖性方式结合 A1A2A3，从而自动抑制 CUB 上另一个 Ca 非依赖性结合位点。这些数据表明两种分子上的多个位点都参与了机械调节的 VWF-ADAMTS13 相互作用。
