Assaying the efficacy of dual-antiplatelet therapy: use of a controlled-shear-rate microfluidic device with a well-defined collagen surface to track dynamic platelet adhesion

Lucitt, Margaret B.; O’Brien, Sarah; Cowman, Jonathan; Meade, Gerardene; Basabe‐Desmonts, Lourdes; Somers, Martin; Kent, Nigel; Ricco, Antonio J.; Kenny, Dermot

doi:10.1007/s00216-013-6897-y

Cited by 12 publications

(15 citation statements)

References 32 publications

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“…We report findings with some similarities and differences to the prior flow studies of Lucitt et al [17] and Menolicchio et al [18]. Lucitt et al found no effect on the rate of platelet coverage of the collagen surface with in vitro ASA addition at 1500 s −1 .…”

Section: Discussionsupporting

confidence: 82%

Detection of platelet sensitivity to inhibitors of COX-1, P2Y1, and P2Y12 using a whole blood microfluidic flow assay

Diamond

2014

Thrombosis Research

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BACKGROUND Microfluidic devices recreate the hemodynamic conditions of thrombosis. METHODS Whole blood inhibited with PPACK was treated ex vivo with inhibitors and perfused over collagen for 300 s (wall shear rate = 200 s−1) using a microfluidic flow assay. Platelet accumulation was measured in the presence of COX-1 inhibitor (aspirin, ASA), P2Y1 inhibitor (MRS 2179), P2Y12 inhibitor (2MeSAMP) or combined P2Y1 and P2Y12 inhibitors. RESULTS High dose ASA (500 μM), 2MeSAMP (100 μM), MRS 2179 (10 μM),or combined 2MeSAMP and MRS 2179 decreased total platelet accumulation by 27.5%, 75.6%, 77.7%, and 87.9% (p < 0.01), respectively. ASA reduced secondary aggregation rate between 150 and 300 s without effect on primary deposition rate on collagen from 60 to 150 s. In contrast, 2MeSAMP and MRS 2179 acted earlier and reduced primary deposition to collagen between 60 and 105 s and secondary aggregation between 105 and 300 s. RCOX and RP2Y (defined as a ratio of secondary aggregation rate to primary deposition rate) demonstrated 9 of 10 subjects had RCOX < 1 or RP2Y < 1 following ASA or 2MeSAMP addition, while 6 of 10 subjects had RP2Y < 1 following MRS 2179 addition. Combined MRS 2179 and 2MeSAMP inhibited primary platelet deposition rate and platelet secondary aggregation beyond that of each individual inhibitor. Receiver-Operator Characteristic area under the curve (AUC) indicated the robustness of RCOX and RP2Y to detect inhibition of secondary platelet aggregation by ASA, 2MeSAMP, and MRS 2179 (AUC of 0.874 0.966, and 0.889, respectively). CONCLUSIONS Microfluidic devices can detect platelet sensitivity to antiplatelet agents. The R-value can serve as a self-normalized metric of platelet function for a single blood sample.

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Section: Discussionsupporting

confidence: 82%

Detection of platelet sensitivity to inhibitors of COX-1, P2Y1, and P2Y12 using a whole blood microfluidic flow assay

Diamond

2014

Thrombosis Research

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“…A role of the α2β1 receptor in the inhibition by clopidogrel and ticagrelor of platelet adhesion and further activation is less possible since this receptor must be initially activated by inside-out signaling [34], and in our adhesion experiments we did not use any platelet agonist. Finally, platelet inhibition by aspirin may not be involved in the above effects of clopidogrel and ticagrelor, since aspirin has no effect on the initial stage of platelet adhesion [3,37], consistent with our findings. Therefore, we can assume that our findings are only the result of the effect of P2Y 12 antagonists.…”

Section: Discussionsupporting

confidence: 91%

“…Platelet adhesion, under arterial flow conditions, is initiated by platelet interactions with extracellular matrix proteins such as von Willebrand factor (vWf) and collagen exposed during atherosclerotic plaque rupture [2]. The adherent platelets recruit additional platelets into a growing thrombus by secreting autocrine factors, such as adenosine diphosphate (ADP) and thromboxane A 2 (TxA 2 ) that induce further platelet activation and aggregation [3].…”

Section: Introductionmentioning

confidence: 99%

Dynamic platelet adhesion in patients with an acute coronary syndrome: The effect of antiplatelet therapy

et al. 2016

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Platelet adhesion and aggregation are key functions leading to thrombus formation. The effect of aspirin, clopidogrel, and ticagrelor on platelet aggregation has been well established, however, there is limited data on the effect of these drugs on platelet adhesion. We therefore evaluated the effect of these drugs on platelet adhesion in acute coronary syndrome (ACS) patients. Citrated blood was collected from 50 ACS patients loaded with 325 mg of aspirin (baseline) and at 5 days after the administration of aspirin 100 mg/day and clopidogrel (600 mg loading dose, 75 mg/day) (n = 26) or ticagrelor (180 mg loading dose, 90 mg × 2/day) (n = 24). High on-treatment platelet reactivity (HTPR) to clopidogrel was estimated by vasodilator stimulated phosphoprotein (VASP) phosphorylation assay. Platelet adhesion to collagen was studied for 6 min under high shear stress and was evaluated using the time to platelet recruitment (TPR), the perimeter and average area of each adherent object, number of adherent objects, and the total percent of surface coverage (SC%). Six ACS patients exhibited HTPR to clopidogrel and excluded from the platelet adhesion assays. TPR and SC% values were similar among patient groups at baseline and controls. However, all other adhesion parameters were different in ACS patients, indicating the formation of more aggregates in regard to controls. At 5 days post-treatment with either clopidogrel or ticagrelor, the TPR values were increased and the SC% values were reduced to a similar extent compared with baseline. However, significant differences were observed in the ticagrelor group in the perimeter, number of adherent objects, and the average area of each adherent object indicating a more potent inhibition of adherence-induced platelet aggregation than clopidogrel. In conclusion, aspirin does not affect platelet adherence to collagen, whereas clopidogrel and ticagrelor inhibit to a similar extent dynamic platelet adhesion at 5 days post-treatment in ACS patients. However, ticagrelor exhibits a greater inhibitory effect on reducing adhesion-induced platelet aggregation.

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“…Our results are also supported by in-vivo clinical trials which have reported “no antithrombotic benefit…in patients with severe atherosclerosis and stenoses…however anti-thrombotic protection…in patients with less severe lesions is reported” and that high dose ASA therapy was ineffective at preventing re-occlusion in high shear stenoses (>90%) [40], [55]. Previous groups have also used microfluidic flow systems to show similarly low efficacy of ASA at arterial shear rates (1500 s −1 ) in comparison to other GPIIb/IIIa inhibitors such as abciximab [26] or P 2 Y 12 inhibitors such as MeSAMPs [56]. Thus, our results show good agreement with a variety of other in-vivo and ex-vivo studies on the effects of ASA on thrombus detachment and on the relative magnitude of ASA in comparison to alternative therapies including GPIIb/IIIa inhibitors.…”

Section: Discussionmentioning

confidence: 99%

Microfluidic Thrombosis under Multiple Shear Rates and Antiplatelet Therapy Doses

Hotaling²,

Ku³

et al. 2014

PLoS ONE

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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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Assaying the efficacy of dual-antiplatelet therapy: use of a controlled-shear-rate microfluidic device with a well-defined collagen surface to track dynamic platelet adhesion

Cited by 12 publications

References 32 publications

Detection of platelet sensitivity to inhibitors of COX-1, P2Y1, and P2Y12 using a whole blood microfluidic flow assay

Detection of platelet sensitivity to inhibitors of COX-1, P2Y1, and P2Y12 using a whole blood microfluidic flow assay

Dynamic platelet adhesion in patients with an acute coronary syndrome: The effect of antiplatelet therapy

Microfluidic Thrombosis under Multiple Shear Rates and Antiplatelet Therapy Doses

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