Microfluidic thrombosis under multiple shear rates and antiplatelet therapy doses.

Melissa Li,Craig R Forest,Nathan A Hotaling,David N Ku,James P Brody

doi:10.1371/journal.pone.0082493

Melissa Li, Craig R Forest + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0082493

Copy DOI

Abstract

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.

Highlights

Thrombosis, the formation of occlusive platelet aggregates in blood, is the primary cause for the pathology of stroke and heart attack
The use of heparin does not require the addition of non-physiological adenosine adenosine diphosphate (ADP) or calcium to reactivate platelets in order to form platelet thrombus [24,31], both of which have been found by others [32] to interfere with the function of eptifibatide
We have presented the inverse of occlusion time as more graphically intuitive in that no occlusion tends to zero instead of towards infinity for occlusion time, as described in the statistical methods above

Summary

Introduction

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 5–45% 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’’ [3,4,5,6], while the use of GPIIb/IIIa inhibitors, such as eptifibatide/ IntegrilinH, have contributed. 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

Objectives

Methods

Results

Discussion

Conclusion