Utility of 96-well plate aggregometry and measurement of thrombi adhesion to determine aspirin and clopidogrel effectiveness

Paul C. J. Armstrong; Al-Rehan Dhanji; Nicola J. Truss; Zetty N. M. Zain; Arthur T. Tucker; Jane A. Mitchell; Timothy D. Warner

doi:10.1160/TH09-04-0215

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2009; 102(04): 772-778
DOI: 10.1160/TH09-04-0215

New Technologies, Diagnostic Tools and Drugs

Schattauer GmbH

Utility of 96-well plate aggregometry and measurement of thrombi adhesion to determine aspirin and clopidogrel effectiveness

Paul C. J. Armstrong

¹William Harvey Research Institute, Barts & the London School of Medicine & Dentistry, Queen Mary University of London, London, UK

,

Al-Rehan Dhanji

¹William Harvey Research Institute, Barts & the London School of Medicine & Dentistry, Queen Mary University of London, London, UK

,

Nicola J. Truss

¹William Harvey Research Institute, Barts & the London School of Medicine & Dentistry, Queen Mary University of London, London, UK

,

Zetty N. M. Zain

¹William Harvey Research Institute, Barts & the London School of Medicine & Dentistry, Queen Mary University of London, London, UK

,

Arthur T. Tucker

¹William Harvey Research Institute, Barts & the London School of Medicine & Dentistry, Queen Mary University of London, London, UK

²Ernest Cooke Clinical Microvascular Unit, St Bartholomew’s Hospital, Barts & The London NHS Trust, London, UK

,

Jane A. Mitchell

³Cardiothoracic Pharmacology, Unit of Critical Care Medicine, National Heart and Lung Institute, Imperial College, London, UK

,

Timothy D. Warner

¹William Harvey Research Institute, Barts & the London School of Medicine & Dentistry, Queen Mary University of London, London, UK

› Institutsangaben

Abstract

Summary

Aspirin and clopidogrel are key anti-thrombotic therapies. Results from platelet reactivity testing during therapy, have been shown to correlate with future events and would allow for the optimisation of therapy. However, there is little agreement among current tests and there remains a clear clinical need for a universal standardised test. It was the objective of this study to explore the potential of 96-well plate aggregometry as a definitive clinical test of platelet reactivity with respect to aspirin and clopidogrel.A small non-blinded trial of 16 healthy male volunteers assigned to seven days of aspirin (75mg/day) or clopidogrel (75mg/day) therapy. Blood was collected before and on day 7 of treatment. Platelet aggregation was measured using a 96-well plate based aggregation method, and thrombi adhesion measured by colourimetric assay. Platelet agonists used were ADP (0.1–30µM), arachidonic acid (0.03–1.3mM), collagen (0.1–30µg/ml), adrenaline (0.001–100µM), ristocetin (0.2–3mg/ ml),TRAP6 amide (0.130µM) and U46619 (0.130µM). Concentration response curves were constructed to each agonist under the various conditions and used to extract data such as log EC₅₀, Hill slope, and area under the curve. These demonstrated low intraand inter-assay variability and strong discrimination of drug effects.This study demonstrates the ability of the 96-well plate based aggregation and adhesion method to detect and differentiate between stable aspirin and clopidogrel treatment in healthy volunteers.Moreover,this assay marries the ability to test subjects or patients using a range of platelet agonists with more rapidity and ease than the current gold standard platelet assay, traditional light transmission aggregometry, making it a serious alternative assay for use in clinical settings.

Keywords

Aggregation - aspirin - clopidogrel - platelets

Volltext

Referenzen

References
1 Cattaneo M. Laboratory detection of ‘aspirin resistance’: what test should we use (if any)?. Eur Heart J 2007; 28: 1673-1675.
2 Lordkipanidze M, Pharand C, Schampaert E. et al. A comparison of six major platelet function tests to determine the prevalence of aspirin resistance in patients with stable coronary artery disease. Eur Heart J 2007; 28: 1702-1708.
3 Serebruany VL, Goto S. The challenge of monitoring platelet response after clopidogrel. Eur Heart J 2008; 29: 2833-2834.
4 Smith Jr. SC, Feldman TE, Hirshfeld Jr. JW. et al. ACC/AHA/SCAI 2005 Guideline Update for Percutaneous Coronary Intervention--Summary Article: A Report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (ACC/AHA/SCAI Writing Committee to Update the 2001 Guidelines for Percutaneous Coronary Intervention). Circulation 2006; 113: 156-175.
5 Gurbel PA, Becker RC, Mann KG. et al. Platelet Function Monitoring in Patients With Coronary Artery Disease. Journal of the American College of Cardiology 2007; 50: 1822-1834.
6 Cairns JA, Eikelboom J. The Pursuit of Clinically Relevant Measures of Platelet Function After Antiplatelet Drug Therapy. J Am Coll Cardiol 2008; 52: 1978-1980.
7 Wang ZJ, Zhou YJ, Liu YY. et al. Impact of clopidogrel resistance on thrombotic events after percutaneous coronary intervention with drug-eluting stent. Thrombosis Research 2009; 124: 46-51.
8 Fratantoni JC, Poindexter BJ. Measuring platelet aggregation with microplate reader. A new technical approach to platelet aggregation studies. Am J Clin Pathol 1990; 94: 613-617.
9 Bednar B, Condra C, Gould RJ. et al. Platelet aggregation monitored in a 96 well microplate reader is useful for evaluation of platelet agonists and antagonists. Thrombosis Research 1995; 77: 453-463.
10 Antithrombotic Trialists’ Collaboration. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ 2002; 324: 71-86.
11 Mehta SR, Yusuf S, Peters RJG. et al. Effects of pretreatment with clopidogrel and aspirin followed by long-term therapy in patients undergoing percutaneous coronary intervention: the PCI-CURE study. The Lancet 2001; 358: 527-533.
12 Saw J, Madsen EH, Chan S. et al. The ELAPSE (Evaluation of Long-Term Clopidogrel Antiplatelet and Systemic Anti-Inflammatory Effects) Study. J Am Coll Cardiol 2008; 52: 1826-1833.
13 Sacco RL, Diener H-C, Yusuf S. et al. Aspirin and Extended-Release Dipyridamole versus Clopidogrel for Recurrent Stroke. N Engl J Med 2008; 359: 1238-1251.
14 Gori AM, Marcucci R, Migliorini A. et al. Incidence and Clinical Impact of Dual Nonresponsiveness to Aspirin and Clopidogrel in Patients With Drug-Eluting Stents. Journal of the American College of Cardiology 2008; 52: 734-739.
15 Bowry ADK, Brookhart MA, Choudhry NK. MetaAnalysis of the Efficacy and Safety of Clopidogrel Plus Aspirin as Compared to Antiplatelet Monotherapy for the Prevention of Vascular Events. The American Journal of Cardiology 2008; 101: 960-966.
16 De Miguel A, Ibanez B, Badimon JJ. Clinical implications of clopidogrel resistance. Thromb Haemost 2008; 100: 196-203.
17 Hankey GJ, Eikelboom JW. Aspirin resistance. The Lancet 2006; 367: 606-617.
18 Hanjis C, Frishman WH, Lerner RG. Aspirin resistance: mechanisms and clinical implications. Cardiol Rev 2006; 14: 18-25.
19 Lev EI, Patel RT, Maresh KJ. et al. Aspirin and Clopidogrel Drug Response in Patients Undergoing Percutaneous Coronary Intervention: The Role of Dual Drug Resistance. Journal of the American College of Cardiology 2006; 47: 27-33.
20 Rocca B, Patrono C. Determinants of the interindividual variability in response to antiplatelet drugs. Journal of Thrombosis and Haemostasis 2005; 3: 1597-1602.
21 Zimmermann N, Hohlfeld T. Clinical implications of aspirin resistance. Thromb Haemost 2008; 100: 379-390.
22 Price MJ, Endemann S, Gollapudi RR. et al. Prognostic significance of post-clopidogrel platelet reactivity assessed by a point-of-care assay on thrombotic events after drug-eluting stent implantation. Eur Heart J 2008; 29: 992-1000.
23 Bonello L, Camoin-Jau L, Arques S. et al. Adjusted Clopidogrel Loading Doses According to VasodilatorStimulated Phosphoprotein Phosphorylation Index Decrease Rate of Major Adverse Cardiovascular Events in Patients With Clopidogrel Resistance: A Multicenter Randomized Prospective Study. Journal of the American College of Cardiology 2008; 51: 1404-1411.
24 Michelson AD. Platelet Function Testing in Cardiovascular Diseases. Circulation 2004; 110: e489-493.
25 Jennings I, Woods TAL, Kitchen S. et al. Platelet function testing: practice among UK National External Quality Assessment Scheme for Blood Coagulation participants, 2006. J Clin Pathol 2008; 61: 950-954.
26 Cattaneo M, Hayward CP, Moffat KA. et al. Results of a worldwide survey on the assessment of platelet function by light transmission aggregometry: a report from the platelet physiology subcommittee of the SSC of the ISTH. J Thromb Haemost 2009; 7: 1029.
27 Harrison P, Frelinger 3rd AL, Furman MI. et al. Measuring antiplatelet drug effects in the laboratory. Thromb Res 2007; 120: 323-336.
28 Rand ML, Leung R, Packham MA. Platelet function assays. Transfus Apheresis Sci 2003; 28: 307-317.
29 Gurbel PA, Bliden KP, DiChiara J. et al. Evaluation of dose-related effects of aspirin on platelet function: results from the Aspirin-Induced Platelet Effect (ASPECT) study. Circulation 2007; 115: 3156-3164.
30 Macchi L, Christiaens L, Brabant S. et al. Resistance to aspirin in vitro is associated with increased platelet sensitivity to adenosine diphosphate. Thrombosis Research 2002; 107: 45-49.
31 Gremmel T, Steiner S, Seidinger D. et al. Comparison of methods to evaluate clopidogrel-mediated platelet inhibition after percutaneous intervention with stent implantation. Thromb Haemost 2009; 101: 333-339.
32 Lordkipanidze M, Pharand C, Nguyen TA. et al. Comparison of four tests to assess inhibition of platelet function by clopidogrel in stable coronary artery disease patients. Eur Heart J 2008; 29: 2877-2885.
33 Angiolillo DJ, Alfonso F. Platelet function testing and cardiovascular outcomes: steps forward in identifying the best predictive measure. Thromb Haemost 2007; 98: 707-709.
34 Armstrong PC, Truss NJ, Ali FY. et al. Aspirin and the in vitro linear relationship between thromboxane A(2) mediated platelet aggregation and platelet production of thromboxane A(2). J Thromb Haemost 2008; 6: 1933-1943.
35 Bellavite P, Andrioli G, Guzzo P. et al. A colorimetric method for the measurement of platelet adhesion in microtiter plates. AnalBiochem 1994; 216: 444-450.
36 Gasparyan AY, Watson T, Lip GYH. The Role of Aspirin in Cardiovascular Prevention: Implications of Aspirin Resistance. Journal of the American College of Cardiology 2008; 51: 1829-1843.
37 Shantsila E, Lip GY. ‘Aspirin resistance’ or treatment non-compliance: which is to blame for cardiovascular complications?. J Transl Med 2008; 6: 47.
38 Vane JR, Botting RM. The mechanism of action of aspirin. Thromb Res 2003; 110: 255-258.
39 Angiolillo DJ, Guzman LA, Bass TA. Current antiplatelet therapies: Benefits and limitations. American Heart Journal 2008; 156: 3S-9S.
40 Kattlove HE, Gomez MH. Studies on the mechanism of ristocetin-induced platelet aggregation. Blood 1975; 45: 91-96.
41 Ali FY, Warner TD, Mitchell JA. Comparison of responses in IP and PPARb deleted murine platelets. Heart Online 2008; 94: e22.
42 Ali FY, Armstrong PC, Dhanji AR. et al. Antiplatelet actions of statins and fibrates are mediated by PPARs. Arterioscler Thromb Vasc Biol 2009; 29: 706-711.