Leveraging platelet signal transduction for personalized antiplatelet therapy

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.Project Narrative Blood platelets contribute to arterial thrombosis, which causes heart attacks and strokes and is responsible for more deaths in the United States than any other single disease process. Drugs that block the activity of platelets and inhibit arterial thrombosis are available, but they can affect people differently and therefore personalized monitoring of antiplatelet agents is highly desirable. This project will generate an inexpensive beta test kit that will position this novel approach to personalized dosing of life-saving antiplatelet medications for future commercialization and FDA-approval.