Microfluidic Thrombosis under Multiple Shear Rates and Antiplatelet Therapy Doses

PLOS ONE, Dec 2019

The mainstay of treatment for thrombosis, the formation of occlusive platelet aggregates that often lead to heart attack and stroke, is antiplatelet therapy. Antiplatelet therapy dosing and resistance are poorly understood, leading to potential incorrect and ineffective dosing. Shear rate is also suspected to play a major role in thrombosis, but instrumentation to measure its influence has been limited by flow conditions, agonist use, and non-systematic and/or non-quantitative studies. In this work we measured occlusion times and thrombus detachment for a range of initial shear rates (500, 1500, 4000, and 10000 s−1) and therapy concentrations (0–2.4 µM for eptifibatide, 0–2 mM for acetyl-salicylic acid (ASA), 3.5–40 Units/L for heparin) using a microfluidic device. We also measured complete blood counts (CBC) and platelet activity using whole blood impedance aggregometry. Effects of shear rate and dose were analyzed using general linear models, logistic regressions, and Cox proportional hazards models. Shear rates have significant effects on thrombosis/dose-response curves for all tested therapies. ASA has little effect on high shear occlusion times, even at very high doses (up to 20 times the recommended dose). Under ASA therapy, thrombi formed at high shear rates were 4 times more prone to detachment compared to those formed under control conditions. Eptifibatide reduced occlusion when controlling for shear rate and its efficacy increased with dose concentration. In contrast, the hazard of occlusion from ASA was several orders of magnitude higher than that of eptifibatide. Our results show similar dose efficacy to our low shear measurements using whole blood aggregometry. This quantitative and statistically validated study of the effects of a wide range of shear rate and antiplatelet therapy doses on occlusive thrombosis contributes to more accurate understanding of thrombosis and to models for optimizing patient treatment.

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Microfluidic Thrombosis under Multiple Shear Rates and Antiplatelet Therapy Doses

Citation: Li M, Hotaling NA, Ku DN, Forest CR ( Microfluidic Thrombosis under Multiple Shear Rates and Antiplatelet Therapy Doses Melissa Li 0 Nathan A. Hotaling 0 David N. Ku 0 Craig R. Forest 0 James P. Brody, University of California Irvine, United States of America 0 1 Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology , Atlanta , Georgia , United States of America, 2 George W. Woodruff Department of Mechanical Engineering, Georgia Institute of Technology , Atlanta, Georgia , United States of America The mainstay of treatment for thrombosis, the formation of occlusive platelet aggregates that often lead to heart attack and stroke, is antiplatelet therapy. Antiplatelet therapy dosing and resistance are poorly understood, leading to potential incorrect and ineffective dosing. Shear rate is also suspected to play a major role in thrombosis, but instrumentation to measure its influence has been limited by flow conditions, agonist use, and non-systematic and/or non-quantitative studies. In this work we measured occlusion times and thrombus detachment for a range of initial shear rates (500, 1500, 4000, and 10000 s21) and therapy concentrations (0-2.4 mM for eptifibatide, 0-2 mM for acetyl-salicylic acid (ASA), 3.5-40 Units/L for heparin) using a microfluidic device. We also measured complete blood counts (CBC) and platelet activity using whole blood impedance aggregometry. Effects of shear rate and dose were analyzed using general linear models, logistic regressions, and Cox proportional hazards models. Shear rates have significant effects on thrombosis/dose-response curves for all tested therapies. ASA has little effect on high shear occlusion times, even at very high doses (up to 20 times the recommended dose). Under ASA therapy, thrombi formed at high shear rates were 4 times more prone to detachment compared to those formed under control conditions. Eptifibatide reduced occlusion when controlling for shear rate and its efficacy increased with dose concentration. In contrast, the hazard of occlusion from ASA was several orders of magnitude higher than that of eptifibatide. Our results show similar dose efficacy to our low shear measurements using whole blood aggregometry. This quantitative and statistically validated study of the effects of a wide range of shear rate and antiplatelet therapy doses on occlusive thrombosis contributes to more accurate understanding of thrombosis and to models for optimizing patient treatment. - Funding: This study was made possible by the American Heart Association (10GRNT4430029), the Wallace H. Coulter Foundation Translational Grant, and by a fellowship from the Technological Innovation Generating Economic Results (TI:GER) program at the Georgia Institute of Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Thrombosis, the formation of occlusive platelet aggregates in blood, is the primary cause for the pathology of stroke and heart attack. Thrombosis can be treated with antiplatelet therapies, but these are not effective for many patients, with an estimated 545% still undergoing adverse cardiovascular events after treatment [1,2] depending on the therapy used. Incorrect doses of antiplatelet therapies can have side effects including severe bleeding, gastrointestinal discomfort, and death in some cases. The majority of these cases are due to idiopathic aspirin resistance [36], while the use of GPIIb/IIIa inhibitors, such as eptifibatide/ IntegrilinH, have also contributed. The mechanisms for antiplatelet therapy resistance and guidelines for appropriate doses are poorly understood [7,8]. Thus, instrumentation for evaluating thrombosis before and after application of antiplatelet therapy would provide valuable feedback in clinical studies and personalized patient treatment for optimizing therapies and their respective doses. The International Society on Thrombosis and Hemostasis (ISTH) has recommended criteria for the design of such instrumentation [9]. First, flow conditions in antiplatelet therapy instrumentation should properly reproduce conditions in the vasculature, including multiple shear rates spanning physiological to pathological levels. Further, the flow environment should feature a pathologically relevant eccentric constriction, or stenosis. Within this flow environment, instrumentation should enable continuous monitoring to examine not only fully occlusive thrombosis, but also the possibility of thrombus detachment, which can lead to clinical correlates of stroke or embolism. Instrumentation should be capable of measuring the effects of multiple therapies in combination, as they are commonly prescribed. Finally, although antibody or fluorescence labeling and microscopy are very effective for identi (...truncated)


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Melissa Li, Nathan A. Hotaling, David N. Ku, Craig R. Forest. Microfluidic Thrombosis under Multiple Shear Rates and Antiplatelet Therapy Doses, PLOS ONE, 2014, 1, DOI: 10.1371/journal.pone.0082493