CAREER: Understanding the Contraction Biomechanics of Platelets at the Single-Cell Level

[150235]这个项目的目标是了解单个血小板的收缩力学和动力学，血小板是负责开始凝块形成的血细胞，以改善医学和生物工程领域。在血栓形成过程中，活化的血小板与生长的纤维蛋白聚合物网络相互作用，并与纤维蛋白支架发生收缩。虽然已知这种血小板驱动的凝块收缩是肌球蛋白介导的，但由于技术限制，对血小板收缩的潜在生物力学方面知之甚少。存在大量的凝块收缩试验，但不能直接测量单细胞水平的血小板收缩力，这对于获得收缩过程的机制理解是必要的。此外，由于纤维蛋白最近被证明具有极其复杂的材料和机械特性，在检查凝块形成的细胞生物力学时，单血小板研究将使纤维蛋白效应与血小板解耦。因此，存在一个明确的需要，稳健，单细胞测定血小板收缩。我们最近发表了第一项实现这一目标的技术，使用改进的原子力显微镜系统直接定量测量单个血小板的收缩力学和动力学。尽管原子力显微镜技术是高度敏感的，但考虑到单个血小板之间已知的广泛的生理异质性和可变性，必须提高通量，以全面了解血小板收缩的潜在细胞生物力学机制。为此，威尔伯·林博士和他的实验室将应用微加工技术开发一种高通量的“生物力学流式细胞仪”，在一次实验中同时测量多个血小板的收缩性。然后，林博士和他的同事将定量研究血小板收缩的生物力学和生物学方面的机制关系，以获得对这一过程的全面的细胞生物力学理解。该提案的教育目标是：为住院儿童创建一项K-12科学推广方案，将其自身的特定疾病作为学习科学的动力和跳板；使用这些儿童习惯的医院用品和设备，作为这种外联活动的一部分，进行动手科学丰富；使本科生和研究生能够实施这一外展计划；并将细胞生物力学概念融入本课程，强调医学是跨学科的，涉及生物、物理、化学和数学。智力优势：血栓形成发生在三个阶段：1)损伤部位的血小板聚集，2)在该部位嵌入血小板的纤维蛋白聚合物的形成，以及3)血小板驱动的血栓收缩。虽然前两个阶段已经很好地描述了，但对最后一个阶段知之甚少。由于血块在血流动力学环境中受到各种外力的影响，并且在空间上不均匀，导致血小板可能遇到的机械微环境具有异质性，因此将细胞生物力学的概念应用于血小板收缩将极大地提高对血块形成的总体基本理解。事实上，我们之前的数据表明血小板收缩依赖于底层基质的机械性能。我们将测试机械微环境与介导血小板收缩的已知信号通路相互作用的假设。为此，我们将以林博士的原子力显微镜技术为基础，使定量研究血小板收缩机制所需的更高通量实验成为可能。更广泛的影响：这将是首次报道的定量研究血小板收缩的细胞生物力学的实验，其结果将显著提高对血小板生理学和凝块形成的整体理解。此外，这些研究将具有广泛的意义，因为血小板不仅参与凝血，还参与许多其他生物过程（例如，感染、炎症、心血管疾病、中风和癌症）和植入生物材料的生物相容性。目前评估血小板功能的诊断仅基于血小板聚集，因此，提出的微系统将形成血小板功能测试新类别的基础。此外，该微系统将潜在地作为血小板收缩功能障碍相关疾病的药物发现平台，如心血管疾病和中风。结合面向住院儿童科学教育的创新教育计划，该跨学科计划将对细胞生物力学，基础血液学，生物医学工程和生物材料产生持久的影响。

1150235 lamThe goal of this project is to understand the contraction mechanics and dynamics of individual platelets, the blood cells responsible for the initiation of clot formation, in order to improve the fields of medicine and bioengineering. During the formation of blood clots, activated platelets interact with growing networks of fibrin polymers and contract against this fibrin scaffold. Although this platelet-driven clot retraction is known to be acto-myosin mediated, extremely little is known about the underlying biomechanical aspects of platelet contraction, due in part to technological limitations. Bulk assays of clot retraction exist but cannot directly measure platelet contractility at the single cell level, which is necessary to obtain a mechanistic understanding of the contraction process. In addition, as fibrin has recently been shown to have extremely complex material and mechanical properties, single platelet studies would enable the decoupling of the fibrin effects from platelets when examining the cellular biomechanics of clot formation. Therefore, a clear need exists for robust, single cell assays of platelet contraction. We recently published the first technique to achieve this goal using a modified atomic force microscopy system to directly and quantitatively measure the contraction mechanics and dynamics of single platelets. Although the atomic force micrscopy technique is highly sensitive, given the known wide physiologic heterogeneity and variability among individual platelets, the throughput must be improved to obtain a comprehensive understanding of the underlying cellular biomechanical mechanisms of platelet contraction. To those ends, Dr. Wilbur Lam and his laboratory will apply microfabrication techniques to develop a high-throughput "biomechanical flow cytometer" that simultaneously measures the contractility of multiple platelets in a single experiment. Then, Dr. Lam and his colleagues will quantitatively investigate the mechanistic relationship between the biomechanical and biological aspects of platelet contraction to obtain a comprehensive, cellular biomechanical understanding of that process.The education objectives of this proposal are to: create a K-12 science outreach program for hospitalized children in which their own specific diseases are used as motivation and springboards for learning about science; use hospital-based supplies and equipment these children are accustomed to for hands-on science enrichment as part of this outreach; enable undergraduate and graduate students to implement this outreach program; and integrate cellular biomechanics concepts into this program, emphasizing that medicine is interdisciplinary and involves biology, physics, chemistry, and math.Intellectual Merit: Clot formation occurs in three phases: 1) platelet aggregation at the site of injury, 2) formation of a fibrin polymer embedding platelets at that site, and 3) platelet-driven clot retraction/contraction. While the first two phases have been well characterized, extremely little is known about the last phase. As clots are exposed to a wide range of external forces in a hemodynamic environment and are spatially non-uniform, leading to a heterogeneity of mechanical microenvironments platelets might encounter, applying the concepts of cellular biomechanics to platelet contraction will vastly improve the overall basic understanding of clot formation. Indeed, our previous data suggest that platelet contraction is dependent on the mechanical properties of the underlying substrate. We will test the hypothesis that the mechanical microenvironment interacts with the known signaling pathways that mediate platelet contraction. To that end, we will build upon Dr. Lam's atomic force microscopy technique to enable the higher throughput experiments needed to quantitatively investigate the mechanics of platelet contraction.Broader Impact: These will be the first reported experiments that quantitatively investigate the cellular biomechanics of platelet contraction and the results will significantly improve the overall understanding of platelet physiology and clot formation. In addition, these studies will have broad reaching implications as platelets are not only involved in clotting but also in numerous other biological processes (e.g., infections, inflammation, cardiovascular disease, stroke, and cancer) and the biocompatibility of implanted biomaterials. Diagnostics assessing platelet function are currently based only on platelet aggregation and as such, the proposed microsystem will form the basis for a new category of platelet function testing. Furthermore, the microsystem will potentially serve as a drug discovery platform for diseases associated with dysfunction in platelet contractility, such as cardiovascular disease and stroke. Combined with the innovative education program geared towards science education of hospitalized children, this interdisciplinary program will have a lasting impact on cellular biomechanics, basic hematology, and biomedical engineering, and biomaterials.