Microfluidic assays for hyper-reactive platelets in diabetes

 描述(由申请人提供)：糖尿病、肥胖、高血压和其他心血管疾病是血小板高反应性的主要危险因素。尽管这一点很重要，但血小板高反应性的分子机制仍不清楚。我们的长期目标是整合生物力学和生化方法，以了解糖尿病、肥胖症和心血管疾病患者的疾病机制，并利用这些知识设计工具，促进医生对这些疾病的治疗决策-诊断工具，跟踪疾病进展的工具，以及跟踪治疗过程的工具。使用独特的单血小板生物膜力探针(BFP)分析，我们收集了初步证据，表明存在血小板的中间状态(盘状，但表达低水平的激活标志)，这种状态的主要特征是整合素分子采用一种能够产生中等亲和力的构象。我们推测，这种中间状态在糖尿病患者的血小板过度活动中起着重要作用。虽然这种方法对于探测单个血小板上的分子相互作用是敏感和强大的，但它非常劳动密集型和低通量。该项目的目标是设计一种简单易用、高通量和高信息量的微流控方法，以了解导致血小板激活的分子事件序列。我们将使用这种方法获得正常和疾病血小板的中间状态、其稳定性和状态变化的动力学的详细特征。新检测方法的验证和多动假说的证明将使这一检测方法在未来进一步发展，用于临床诊断或患者动脉粥样硬化血栓的治疗。我们已经为这个项目组建了一支工程师和临床医生团队。这项工作具有创新性，因为目前还没有这种高通量的分析方法可以产生机械性的见解(并且只使用一滴血)，而且了解血小板中间状态的作用，特别是在糖尿病中的作用，将导致对血小板多动障碍的诊断和治疗的显著改进。

 DESCRIPTION (provided by applicant): Diabetes, obesity, and hypertension among other cardiovascular diseases are large risk factors for platelet hyperreactivity. Despite the importance, the molecular mechanisms of the platelet hyperreactivity are still unknown. Our long-term goal is to integrate biomechanical and biochemical approaches to understand the disease mechanisms in patients with diabetes, obesity and cardiovascular diseases, and to use this knowledge to design tools that facilitate physicians' decisions on treatment of these diseases - tools to diagnose, tools to follow disease progression, and tools to follow treatment courses. Using a unique single-platelet Biomembrane Force Probe (BFP) assay, we have gathered preliminary evidence that there exists an intermediate state of platelets (discoid in shape but express low-level markers of activation) and this state is primarily characterized by having integrin molecules adopting a conformation that gives rise to intermediate affinity. We hypothesize that this intermediate state plays an important role in platelet hyperactivity in diabetics. While this assay is sensitive and powerful for probing molecular interactions on single platelets, it is very labor-intensive and low throughput. The goal of this project is to design a simple-to-use and yet high-throughput and highly informative microfluidic approach to understand sequences of molecular events that lead to platelet activation. We will obtain detailed characterization of the intermediate state, its stability, and the kinetics of state changs of the normal and diseased platelets using this approach. Validation of the new assay and proof of the hyperactivity hypothesis will allow this assay to be further developed in the future for clinical diagnosis or to follow treatment of atherothrombosis in patients. We have assembled a team of engineers and clinicians for this project. The work is innovative because no such high-throughput assay that yields mechanistic insights (and only using a drop of blood) is currently available, and that understanding the role of the intermediate state of platelets, particularly in diabetes, will lead to a significant improvement in diagnostic and treatment for platelet hyperactivity disorders.