Leveraging platelet signal transduction for personalized antiplatelet therapy

利用血小板信号转导进行个性化抗血小板治疗

• 批准号: 10480329
• 负责人: David Barrios
• 金额: $ 25.96万
• 依托单位: PLATELETDIAGNOSTICS, LLC
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-06 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10480329
• 关键词: Affect; Agitation; Agonist; Antibodies; Antiplatelet Drugs; Aspirin; Awareness; Biological Assay; Blood; Blood Platelet Antagonists; Blood Platelet Disorders; Blood Platelets; Cardiovascular Diseases; Cardiovascular system; Caring; Cessation of life; Characteristics; Clinical Data; Clinical Research; Clinical Trials; Clinics and Hospitals; Complex; Consensus; Consumption; Coronary Arteriosclerosis; Data; Detection; Development; Devices; Diagnostic; Diagnostic Reagent Kits; Disease; Dose; Drug Monitoring; Effectiveness; Electrolytes; Ensure; Enzyme-Linked Immunosorbent Assay; Enzymes; Equipment; Fluorescence; Future; Goals; Gold; Health Personnel; Hemorrhage; Hospitals; Human; Immunoassay; Immunoglobulin G; Intracranial Hemorrhages; Ketorolac; Knowledge; Laboratories; Lead; Life; Measures; Mechanics; Mediating; Monitor; Myocardial Infarction; Obesity; Oryctolagus cuniculus; Outcome; Pathologist; Pathway interactions; Patients; Performance; Persons; Pharmaceutical Preparations; Phosphorylation; Phosphorylation Site; Physicians; Platelet Activation; Platelet Function Tests; Platelet aggregation; Positioning Attribute; Preparation; Process; Proteins; Protocols documentation; Quality Control; Reagent; Recurrence; Reproducibility; Resistance; Risk; Role; Running; Savings; Sensitivity and Specificity; Signal Pathway; Signal Transduction; Site; Specificity; Standardization; Stroke; System; Testing; Thrombosis; Time; Training; United States; assay development; base; clopidogrel; commercialization; cost; detection limit; fluorophore; improved; improved outcome; instrumentation; laboratory equipment; mortality; non-compliance; novel strategies; patient responsibilities; phase 2 study; platelet function; point of care; point of care testing; response

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.

项目摘要/摘要 血小板介导的血栓形成导致冠状动脉疾病(CAD)和中风，分别位居第一和第五位 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，显著 提高冠心病或中风患者的存活率，两年多来一直是治疗的主要手段 几十年。然而，尽管人们一致认为阿司匹林和/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剂量的抗血小板药物 会导致糟糕的结果。一方面，剂量不足的抗血小板药物可能导致更大的可能性。 冠心病和中风复发的风险。另一方面，过量服用会导致出血风险增加，包括致命的 颅内出血。这个问题的范围是巨大的。已有5000多万美国公民 开出阿司匹林和/或氯吡格雷治疗心血管疾病。对任何一种药物的不适当反应 发生在大约25%的患者中。然而，尽管对改进监测战略的需求尚未得到满足，但 血小板功能检测的金标准仍然是血小板聚集率，这一点从根本上没有变化 从50年前的发展到现在。较新的医疗点(POC)设备已经上市，但需要 昂贵的设备和消耗品和/或有复杂的读数，并不适合 大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。 这可以由医生简单地订购并在中心实验室廉价地进行批量处理， 类似于CBC或电解质的测试方法。在评估血小板信号通路对 抗血小板药物，我们发现血小板蛋白drp1处于双重磷酸化的控制之下。 系统。一个磷酸化位点(Drp1-Ser616)被血小板激动剂磷酸化并被抑制 用抗血小板药物治疗。第二个位点(Drp1-Ser637)是对血小板拮抗剂的反应而被磷酸化。这 双重磷酸化系统对抗血小板药物的抑制极其敏感，非常适合于 格式化以监测抗血小板治疗。我们的化验结果显示，在两种情况下，与聚集度测定法有很强的相关性 正在进行的临床研究；然而，这些研究也强调了重要的 改进。我们将追求3个目标来评估Drp1的磷酸化可以是 开发一种新的战略，快速、廉价地监测抗血小板治疗。在目标1中，我们目前的情况 将评估方法转换为基于荧光的方法以测量S616 pDrp1的水平 以及同一井中DRP1的总水平。在目标2中，我们将建立简单的分析前步骤以供使用 并优化激动剂条件，以精确定义我们的测试小组。化验特征 例如pDrp1检测的敏感性、特异性、精密度和准确性将在目标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成为可能。