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

职业：在单细胞水平上了解血小板的收缩生物力学

• 批准号: 1150235
• 负责人: Wilbur Lam
• 金额: $ 45万
• 依托单位: Emory University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2018-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1150235&HistoricalAwards=false
• 关键词: CAREER; Understanding; Contraction; Biomechanics; Platelets

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.

1150235拉姆该项目的目标是了解单个血小板的收缩机制和动力学，血小板是负责启动凝块形成的血细胞，以改善医学和生物工程领域。在血凝块的形成过程中，活化的血小板与纤维蛋白聚合物的生长网络相互作用，并与该纤维蛋白支架收缩。尽管已知这种血小板驱动的凝块收缩是肌动蛋白介导的，但部分由于技术限制，对血小板收缩的潜在生物力学方面知之甚少。存在血块收缩的批量测定，但不能在单细胞水平上直接测量血小板收缩性，这是获得收缩过程的机械理解所必需的。此外，由于纤维蛋白最近已被证明具有极其复杂的材料和机械特性，因此当检查凝块形成的细胞生物力学时，单个血小板研究将使纤维蛋白效应与血小板脱钩。因此，显然需要对血小板收缩进行稳健的单细胞测定。我们最近发表了第一个技术，以实现这一目标，使用修改后的原子力显微镜系统，直接和定量测量的收缩力学和动力学的单个血小板。虽然原子力显微镜技术是高度敏感的，已知广泛的生理异质性和个体血小板之间的变异性，吞吐量必须得到改善，以获得一个全面的了解血小板收缩的基本细胞生物力学机制。为此，Wilbur Lam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”，在一个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之间的机制关系，以获得对该过程的全面的细胞生物力学理解。这项建议的教育目标是：为住院儿童创建K-12科学外展计划，其中他们自己的特定疾病作为学习科学的动力和跳板;使用这些孩子习惯的医院用品和设备，作为这项推广活动的一部分，让本科生和研究生能够实施这项推广计划;并将细胞生物力学概念融入这项计划，强调医学是跨学科的，涉及生物学，物理学，化学和数学。智力优势：凝块形成分为三个阶段：1）损伤部位的血小板聚集，2）在该部位形成包埋血小板的纤维蛋白聚合物，和3）血小板驱动的凝块收缩/收缩。虽然前两个阶段已经得到很好的表征，但对最后一个阶段知之甚少。由于凝块在血液动力学环境中暴露于各种外力，并且在空间上是不均匀的，导致血小板可能遇到的力学微环境的异质性，将细胞生物力学的概念应用于血小板收缩将大大提高对凝块形成的整体基本理解。事实上，我们以前的数据表明，血小板收缩是依赖于底层基板的机械性能。我们将测试的假设，即机械微环境与已知的信号通路，介导血小板收缩相互作用。为了达到这个目的，我们将在原子力显微镜技术的基础上，进行更高通量的实验来定量研究血小板收缩的机制。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，而且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。评估血小板功能的诊断目前仅基于血小板聚集，因此，所提出的微系统将形成一种新的血小板功能测试类别的基础。此外，该微系统将有可能作为药物发现平台，用于与血小板收缩功能障碍相关的疾病，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，这个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。