Leveraging platelet signal transduction for personalized antiplatelet therapy

项目概要/摘要 血小板介导的血栓形成导致冠状动脉疾病（CAD）和中风，分别是第一和第五大 美国常见的死亡原因。抗血小板药物，通常是阿司匹林和/或氯吡格雷， 改善CAD或卒中患者的生存率，并且仍然是两种以上治疗的支柱 几十年然而，尽管阿司匹林和/或氯吡格雷抑制血小板可以改善预后， 复发仍然很常见，人们越来越意识到，不适当的抗血小板药物剂量 会导致不良结果。一方面，抗血小板药物剂量不足可导致 冠心病和中风复发的风险另一方面，过量给药可导致出血风险增加，包括致命的 颅内出血这个问题的范围是巨大的。超过5000万美国公民 处方阿司匹林和/或氯吡格雷治疗心血管疾病。对任何一种药物的反应不当 发生在大约25%的患者中。然而，尽管改进监测战略的需要没有得到满足， 血小板功能检测的金标准仍然是血小板聚集测定法，基本上没有改变 自50年前发展以来。较新的床旁（POC）设备已经上市，但需要 昂贵的设备和消耗品和/或具有复杂的读数，并且不适合于 高容量医院和医生办公室的工作流程。因此，迫切需要一种血小板功能测定方法 可以由医生简单地订购，并在中心实验室廉价地批量进行， 类似于CBC或电解质的测试。在评估血小板信号通路对 在抗血小板药物中，我们发现血小板蛋白Drp 1受双重磷酸化的控制， 系统一个磷酸化位点（Drp 1-Ser 616）在血小板激动剂作用下磷酸化， 抗血小板药物第二个位点（Drp 1-Ser 637）在血小板拮抗剂的作用下被磷酸化。这 双重磷酸化系统对抗血小板剂的抑制极其敏感， 格式化以监测抗血小板治疗。我们的测定显示出与两种聚集测定法的强相关性。 正在进行的临床研究;然而，这些研究也强调了重要的机会 改进.我们将追求3个目标来评估Drp 1的磷酸化可以被 用于开发一种新的策略，用于快速，廉价的抗血小板治疗监测。在目标1中，我们目前 将评价检测方法转换为基于荧光的检测方法，以测量S616 pDrp 1 以及同一孔中Drp 1的总水平。在目标2中，我们将建立简单的预分析步骤， 在中心实验室和优化激动剂条件，以精确定义我们的测试面板。试验特性 如pDrp 1检测试剂的灵敏度、特异性、精密度和准确度，将在目标3中确定，以确保 实验室之间的质量控制和标准化，并使大规模的第二阶段研究。

Project Summary/Abstract Platelet-mediated thrombosis causes both coronary arterial disease (CAD) and stroke, the first and fifth most common causes of mortality in the US. Antiplatelet agents, typically aspirin and/or clopidogrel, significantly improve survival in patients with CAD or stroke and have remained a mainstay of treatment for more than two decades. Yet despite the consensus that platelet inhibition with aspirin and/or clopidogrel improves outcomes, recurrence remains common and there is a growing awareness that inappropriate dosing of antiplatelets contributes to poor outcomes. On one hand, under-dosing of antiplatelet agents can result in increased likelihood of CAD and stroke recurrence. On the other hand, over-dosing can lead to increased bleeding risk including fatal intracranial hemorrhage. The scope of this problem is enormous. More than 50 million US citizens have been prescribed aspirin and/or clopidogrel for cardiovascular disease. Inappropriate responsiveness to either drug occurs in approximately 25% of patients. Yet despite the unmet need for improved monitoring strategies, the gold standard for platelet function testing remains platelet aggregometry, which is fundamentally unchanged since its development 50 years ago. Newer point-of-care (POC) devices have been marketed but require expensive equipment and consumables and/or have complex readouts and do not lend themselves to the workflow of high-volume hospitals and doctors’ offices. Thus, there is a vital need for a platelet function assay that can be simply ordered by physicians and batched to be performed inexpensively in a central laboratory, similarly to how a CBC or electrolytes are tested. In evaluating the response of platelet signaling pathways to antiplatelet agents, we have found that the platelet protein Drp1 is under the control of a dual phosphorylation system. One phosphorylation site (Drp1-Ser616) is phosphorylated in response to platelet agonists and inhibited by antiplatelet agents. A second site (Drp1-Ser637) is phosphorylated in response to platelet antagonists. This dual phosphorylation system is extremely sensitive to inhibition by antiplatelet agents and is well-suited for formatting to monitor antiplatelet therapy. Our assay has shown strong correlation with aggregometry in two ongoing clinical studies; however, these studies have also underscored the opportunity for important improvements. We will pursue 3 aims to evaluate the hypothesis that the phosphorylation of Drp1 can be leveraged to develop a new strategy for rapid, inexpensive monitoring of antiplatelet therapy. In Aim 1, our current assay will be evaluated for conversion to a fluorescence-based assay to measure both the level of S616 pDrp1 as well as the total level of Drp1 in the same well. In Aim 2, we will establish simple pre-analytical steps for use in a central laboratory and optimize agonist conditions to precisely define our testing panel. Assay characteristics such as sensitivity, specificity, precision, and accuracy of the pDrp1 assay will be determined in Aim 3 to ensure quality control and standardization between laboratories and enable large-scale phase II studies.