Salt-induced fibrillogenesis of fibrinogen (SAL-FIB): In vitro experiments and simulations

纤维状纤维蛋白原支架对于组织工程应用特别有吸引力，因为它们非常模仿天然血凝块的结构和生化成分。已知在体外条件下诱导纤维蛋白原原纤维形成的不同技术，包括静电纺丝、表面和缓冲液驱动的纤维形成以及盐诱导的自组装。然而，迄今为止，尚不清楚特定的表面相互作用或缓冲条件是否有助于纤维蛋白原的体外纤维形成。此外，目前对纤维蛋白原分子水平的研究缺乏对潜在原子过程的清晰了解。因此，我们的项目将重点研究以下问题：在体外条件下哪种机制驱动纤维蛋白原的纤维形成？为了回答这个问题，我们的项目将使用紧密相连的模拟和实验方法的多尺度组合。我们将首次建立完整的纤维蛋白原分子模型，其中还包括翻译后修饰。基于实验动态光散射研究，我们将分析不同缓冲系统中纤维蛋白原的聚集，该纤维蛋白原模型将用于分子动力学 (MD) 研究，以分析分子周围的局部离子分布。随后，我们将利用 MD 结果来研究盐离子是否在干燥时融入纤维蛋白原分子或纤维蛋白原纤维中。相关的实验研究将涉及自组装纤维蛋白原纳米纤维的形态和元素组成的比浊测量和分析。为了了解纤维组装是否伴随或由纤维蛋白原构象的变化驱动，我们将筛选自组装纤维是否可能发生淀粉样蛋白转变并进行光谱分析。由此产生的圆二色性 (CD) 光谱将与通过引导 MD 模拟获得的具有诱导结构变化的参考单分子 CD 光谱进行比较。该项目的主要挑战将是根据从纤维蛋白原结构/构象集合获得的实验结果来讨论和解释来自 MD 模拟的单分子信息。在这种情况下，流体动力学半径和CD谱将是两个可观察到的量，它们将直接连接我们的模拟和实验研究。只有通过这种组合方法，我们才能提出一种明显依赖于环境参数的纤维蛋白原体外纤维形成的详细机制。因此，拟议的项目将为纤维蛋白原的体外纤维形成提供基本见解，这对于开发具有明确结构功能关系的新型纤维蛋白原纳米纤维是必要的。

Fibrous fibrinogen scaffolds are particularly attractive for tissue engineering applications since they closely mimic the architecture and biochemical composition of native blood clots. Different techniques are known to induce fibrillogenesis of fibrinogen under in vitro conditions including electrospinning, surface- and buffer-driven fiber formation as well as salt-induced self-assembly. However, to date it is not yet understood whether specific surface interactions or buffer conditions contribute to the in vitro fibrillogenesis of fibrinogen. Moreover, current studies on the molecular level of fibrinogen lack a clear understanding of the underlying atomistic processes. Therefore, our project will focus on the following question: Which mechanism drives fibrillogenesis of fibrinogen under in vitro conditions? To answer this question our project will use a multi-scale combination of closely connected simulative and experimental methods. For the first time, we will establish a complete molecular model of fibrinogen, that also includes posttranslational modifications. Based on experimental dynamic light scattering studies, where we will analyze the aggregation of fibrinogen in different buffer systems, this fibrinogen model will be used in molecular dynamics (MD) studies to analyze local ion distributions around the molecule. Subsequently, we will use our MD results to study whether salt ions are incorporated into fibrinogen molecules or into fibrinogen fibers upon drying. The associated experimental studies will involve turbidimetric measurements and analysis of the morphology and elemental composition of self-assembled fibrinogen nanofibers. To understand whether fiber assembly is accompanied or driven by changes in the fibrinogen conformation, we will screen self-assembled fibers for possible amyloid transitions and perform spectroscopic analyses. The resulting circular dichroism (CD) spectra will be compared to reference single molecule CD spectra with induced structural changes obtained by steered MD simulations.A major challenge in this project will be to discuss and interpret the single molecule information from MD simulations with respect to experimental results obtained from an ensemble of fibrinogen structures/conformations. In this context, the hydrodynamic radius and CD spectra will be two observables that will directly link our simulative and experimental studies. Only with this combined approach we will be able to propose a detailed mechanism for the in vitro fibrillogenesis of fibrinogen with a clear dependence on environmental parameters. The proposed project will therefore provide fundamental insights into in vitro fibrillogenesis of fibrinogen, which are necessary to develop a new class of fibrinogen nanofibers with defined structure-function-relationships.