Mechanical properties of VWF in single molecule and cell adhesion force experiments using AFM

已知VWF是剪切力敏感性分子，并且被设计为根据血流中的实际剪切情况通过构象变化来执行其编码的功能。尽管剪切流和生化方法下的血小板聚集的广泛研究阐明了VWF的这些构象变化对血小板粘附的重要性，但在很大程度上缺少对详细机制及其动力学的直接实验研究。事实上，需要对VWF进行生物物理学研究，用于（i）机械弹性表征（例如，载荷-延伸响应、弛豫时间等），和（ii）关于粘附强度和粘附位点（例如内皮细胞、血小板等）定位的分子识别性质。由于VWF和胶原之间的相互作用是止血的关键初始步骤，因此应进行VWF/胶原结合研究。利用原子力显微镜（AFM）可以直接探测单分子水平上特定VWF结构域/胶原相互作用的力和动力学。作为林茨和慕尼黑的两个专业小组，我们特别专注于我们的AFM专业知识的各种相互作用的VWF与细胞和组织components.In第一个资助期内，我们应用AFM作为一种新的方法来探测血小板粘附VWF，VWF的大小分布，展开的VWF-二聚体，和动态的结合选定的VWF域的其他VWF域和胶原蛋白III和VI。在第二个资助期内，我们将通过与Netz（B4）和Gräter/Baldauf（C1）建立纳米力学和计算机模拟研究的独特组合，详细了解VWF蛋白在单域水平上的生物物理功能。根据主要任务，探索机械敏感性VWF分子的机械特性及其与血液成分相互作用的功能之间的联系，我们将与Schneppenheim（A1），Wilmanns（C3）Schneider（A2），Wixforth（B1），Rädler（B3）密切合作研究VWF-血小板网络的形成和病理性VWF突变的影响。因此，我们将扩大我们的调查，胶原蛋白I型和IV型，整合素介导的粘附VWF血小板，和内皮细胞炎症条件下。此外，我们将通过在炎症条件下拉伸VWF和VWF突变体来直接测量力签名的变化。我们希望我们的研究结果的临床相关性将有助于检测疾病和改善VWF患者的治疗。

VWF is known to be a shear force sensitive molecule and designed to perform his encoded functions by conformational changes according to the actual shear situation in the blood stream. Even though extensive studies on blood platelet aggregation under shear flow and biochemical approaches elucidated the importance of these conformational changes of VWF for platelet adhesion, direct experimental investigations on the detailed mechanism and its dynamics are largely missing. In fact, there is a need of biophysical investigations of VWF for (i) the mechano-elastic characterization (e.g. load-extension response, relaxation times etc.), and (ii) the molecular recognition properties with respect to adhesion strength and localization of adhesion sites (e.g. endothelial cells, platelets etc.). Since interactions between VWF and collagen are a key initial step of hemostasis, VWF/collagen binding studies should be performed. Utilizing atomic force microscopy (AFM) the forces and dynamics of specific VWF-domain/collagen interactions on the single molecular level can directly be probed. As two specialized subgroups in Linz and Munich, we specifically focus our AFM expertise on the various interactions of VWF with cells and tissue components.In the first funding period we applied AFM as a new approach to probe the platelet adhesion to VWF, the size distribution of VWF, the unfolding of VWF-dimers, and the dynamics of binding of selected VWF domains to other VWF domains and to collagen III and VI. For the second funding period we will gain a detailed understanding of the biophysical functionalities of the VWF protein on the single domain level by establishing a unique combination of nano-mechanics and computer simulation studies with Netz (B4), and Gräter/Baldauf (C1). According to the main task, exploring the links between the mechanical properties of the mechano-sensitive VWF molecule and its function in the interplay with blood components, we will study the formation of VWF-platelet networks and the impact of pathologic VWF mutations in close collaboration with groups Schneppenheim (A1), Wilmanns (C3) Schneider (A2), Wixforth (B1), Rädler (B3). We will thus extend our investigations to collagen type I and IV, to integrin mediated adhesion of VWF to platelets, and to endothelial cells under inflammatory conditions. Furthermore, we will directly measure changes in the force signature by stretching VWF and VWF mutants under inflammatory conditions. We expect that the clinical relevance of our findings will aid to detect disease and improve the treatment of VWF patients.