Differential sensitivity and kinetics of response of different ex vivo tests monitoring functional variability of platelet response to clopidogrel

Claire Bal dit Sollier; Natacha Berge; Bernadette Boval; Michel Dubar; Ludovic Drouet

doi:10.1160/TH09-11-0803

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2010; 104(03): 571-581
DOI: 10.1160/TH09-11-0803

Platelets and Blood Cells

Schattauer GmbH

Differential sensitivity and kinetics of response of different ex vivo tests monitoring functional variability of platelet response to clopidogrel

Claire Bal dit Sollier

¹IVS, Hôpital Lariboisière, Paris, France

,

Natacha Berge

¹IVS, Hôpital Lariboisière, Paris, France

,

Bernadette Boval

²Haematology Laboratory, Hôpital Lariboisière, Paris, France

,

Michel Dubar

³Sanofi-Aventis Recherche, Chilly-Mazarin, France

,

Ludovic Drouet

²Haematology Laboratory, Hôpital Lariboisière, Paris, France

› Author Affiliations

Abstract

Summary

We previously showed that variability of response to clopidogrel is linked to occupancy of the P2Y₁₂ receptor by clopidogrel active-metabolite, and that maximal platelet aggregation intensity (MAI) measured by light transmission aggregometry (LTA) correlates with occupancy. The present study compared a range of ex vivo platelet tests at various levels of P2Y₁₂ occupancy. After screening with clopidogrel 75 mg/day for seven days, subjects were selected to obtain ‘low’, ‘average’ and ‘high’ responders and randomised to clopidogrel (75 mg/day days 1–10; 300 mg day 11), or placebo. Assays were LTA in platelet-rich plasma using 2, 5 and 20 μM ADP, VerifyNow^® P2Y₁₂, flow cytometric analysis of platelet activation markers and vasodilator-stimulated phospho-protein (VASP) index, and a thromboelastographic test that is sensitive to clopidogrel. The reference test was P2Y₁₂ receptor occupancy, measured by binding of ³³P-2MeS-ADP to platelets. MAI showed the best correlation with P2Y₁₂ occupancy. Similar results were seen with different ADP concentrations and when LTA data were expressed as inhibition of platelet aggregation. A plot of free receptors/cell versus VASP index was biphasic, with poor correlation for low-level P2Y₁₂ occupancy. Sensitivity of the VerifyNow P2Y₁₂ assay decreased at higher clopidogrel responses. Thromboelastography and P-selectin expression had poor correlation with receptor occupancy. In conclusion, LTA data correlate best with P2Y₁₂ occupancy, the gold standard for detecting clopidogrel’s effect at the receptor level. Our results highlight a limitation of the VASP index, which appears unable to distinguish low, average and high responders early after clopidogrel initiation when P2Y₁₂ occupancy is still low.

Keywords

Clopidogrel - flow cytometry - platelet

Full Text

References

References
1 Bassand J-P. et al. Guidelines for the diagnosis and treatment of non-ST-segment elevation acute coronary syndromes. The Task Force for the diagnosis and treatment of non-ST-segment elevation acute coronary syndromes for the European Society of Cardiology. Eur Heart J 2007; 28: 1598-1660.
2 Van de Werf F. et al. Management of acute myocardial infarction in patients presenting with persistent ST-segment elevation. Eur Heart J 2008; 29: 2909-2945.
3 Anderson JL. et al. ACC/AHA guidelines for the management of patients with unstable angina/non-ST-elevation myocardial infarction: executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Revise the 2002 Guidelines for the Management of Patients with Unstable Angina/Non-ST-Elevation Myocardial Infarction). Circulation 2007; 116: 803-877.
4 Antman EM. et al. 2007 focused update of the ACC/AHA 2004 Guidelines for the Management of Patients With ST-Elevation Myocardial Infarction: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Group to Review New Evidence and Update the 2002 ACC/AHA 2004 guidelines for the Management of Patients with ST-Elevation Myocardial Infarction). Circulation 2008; 117: 296-329.
5 King SB III. et al. 2007 focused update of the ACC/AHA/SCAI 2005 Guideline Update for Percutaneous Coronary Intervention: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines: (2007 Working Group to Review New Evidence and Update the 2005 ACC/AHA/SCAI Guideline Update for Percutaneous Coronary Intervention). J Am Coll Cardiol 2008; 51: 172-209.
6 Hirsch AT. et al. ACC/AHA 2005 practice guidelines for the management of patients with peripheral arterial disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Association for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Peripheral Arterial Disease). Circulation 2006; 113: e463-e654.
7 Sacco RL. et al. Guidelines for prevention of stroke in patients with ischemic stroke or transient ischemic attack: a statement for healthcare professionals from the American Heart Association/American Stroke Association Council on Stroke, cosponsored by the Council on Cardiovascular Radiology and Intervention. Stroke 2006; 37: 577-617.
8 Gurbel PA. et al. Clopidogrel for coronary stenting: response variability, drug resistance, and the effect of pretreatment platelet reactivity. Circulation 2003; 107: 2908-2913.
9 Gurbel P, Tantry U. Drug insight: clopidogrel nonresponsiveness. Nat Clin Pract Cardiovasc Med 2006; 3: 387-395.
10 Siller-Matula J. et al. Thienopyridines in cardiovascular disease: focus on clopidogrel resistance. Thromb Haemost 2007; 97: 385-393.
11 Neubauer H. et al. How to optimise clopidogrel therapy? Reducing the low-response incidence by aggregometry-guided therapy modification. Thromb Haemost 2008; 99: 357-362.
12 De Miguel A. et al. Clinical implications of clopidogrel resistance. Thromb Haemost 2008; 100: 196-203.
13 Serebruany V. et al. Baseline platelet activity and response after clopidogrel in 257 diabetics among 822 patients with coronary artery disease. Thromb Haemost 2008; 100: 76-82.
14 Béres BJ. et al. Analysis of platelet α2-adrenergic receptor activity in stable coronary artery disease patients on dual antiplatelet therapy. Thromb Haemost 2008; 100: 829-838.
15 Gremmel T. et al. Comparison of methods to evaluate clopidogrel-mediated platelet inhibition after percutaneous intervention with stent implantation. Thromb Haemost 2009; 101: 333-339.
16 Sibbing D. et al. Impact of proton pump inhibitors on the antiplatelet effects of clopidogrel. Thromb Haemost 2009; 101: 714-719.
17 Ferreiro JL, Angiolillo DJ. Clopidogrel response variability: Current status and future directions. Thromb Haemost 2009; 102: 7-14.
18 Elsenberg EHAM. et al. The influence of clinical characteristics, laboratory and inflammatory markers on ‘high on-treatment platelet reactivity’ as measured with different platelet function tests. Thromb Haemost 2009; 102: 719-727.
19 Zürn CS. et al. ADP-receptor blockade: A case for personalised pharmacotherapy?. Thromb Haemost 2010; 103: 496-506.
20 Williams CD. et al. Application of platelet function testing to the bedside. Thromb Haemost 2010; 103: 29-33.
21 Sibbing D. et al. Clopidogrel response status assessed with Multiplate point-of-care analysis and the incidence and timing of stent thrombosis over six months following coronary stenting. Thromb Haemost 2010; 103: 151-159.
22 Montalescot G. et al. Prasugrel compared with high-dose clopidogrel in acute coronary syndrome. The randomised, double-blind ACAPULCO study. Thromb Haemost 2010; 103: 213-223.
23 Sofi F. et al. Clopidogrel non-responsiveness and risk of cardiovascular morbidity. An updated meta-analysis. Thromb Haemost 2010; 103: 841-848.
24 Malinin A. et al. Validation of a VerifyNow-P2Y₁₂ cartridge for monitoring platelet inhibition with clopidogrel. Methods Find Exp Clin Pharmacol 2006; 28: 315-322.
25 Craft RM. et al. A novel modification of the Thromboelastograph assay, isolating platelet function, correlates with optical platelet aggregation. J Lab Clin Med 2004; 143: 301-309.
26 Schwarz UR. et al. Flow cytometry analysis of intracellular VASP phosphorylation for the assessment of activating and inhibitory signal transduction pathways in human platelets – definition and detection of ticlopidine/clopidogrel effects. Thromb Haemost 1999; 82: 1145-1152.
27 Gurbel PA. et al. Platelet reactivity in patients and recurrent events post-stenting. Results of the PREPARE POST-STENTING study. J Am Coll Cardiol 2005; 46: 1820-1826.
28 Hochholzer W. et al. Impact of the degree of peri-interventional platelet inhibition after loading with clopidogrel on early clinical outcome of elective coronary stent placement. J Am Coll Cardiol 2006; 48: 1742-1750.
29 Pampuch A. et al. Comparison of VASP-phosphorylation assay to light-transmission aggregometry in assessing inhibition of the platelet ADP P2Y₁₂ receptor. Thromb Haemost 2006; 96: 767-773.
30 Jakubowski JA. et al. A comparison of the antiplatelet effects of prasugrel and high-dose clopidogrel as assessed by VASP-phosphorylation and light transmission aggregometry. Thromb Haemost 2007; 99: 215-222.
31 Paniccia R. et al. Different methodologies for evaluating the effect of clopidogrel on platelet function in high-risk coronary artery disease patients. J Thromb Haemost 2007; 5: 1839-1847.
32 Van Werkum JW. et al. A comparison between the Plateletworks™ assay and light transmittance aggregometry for monitoring the inhibitory effects of clopidogrel. Int J Cardiol 2010; 140: 123-126.
33 Bal dit Sollier C. et al. Functional variability of platelet response to clopidogrel correlates with P2Y₁₂ receptor occupancy. Thromb Haemost 2009; 101: 116-122.
34 Born GV, Cross MJ. The aggregation of blood platelets. J Physiol 1963; 168: 178-195.
35 Savi P. et al. P2Y₁₂, a new platelet ADP receptor, target of clopidogrel. Biochem Biophys Res Commun 2001; 283: 379-383.
36 Savi P, Herbert JM. Use of radiolabeled 2-methylthio-ADP to study P2Y receptors on platelets and cell lines. Methods Mol Biol 2004; 273: 115-124.
37 Aleil B. et al. Flow cytometric analysis of intraplatelet VASP phosphorylation for the detection of clopidogrel resistance in patients with ischemic cardiovascular diseases. J Thromb Haemost 2005; 3: 85-92.
38 Schäfer A. et al. ADP-induced platelet aggregation frequently fails to detect impaired clopidogrel-responsiveness in patients with coronary artery disease compared to a P2Y₁₂-specific assay. Thromb Haemost 2008; 100: 618-625.
39 Blindt R. et al. The significance of vasodilator-stimulated phosphoprotein for risk stratification of stent thrombosis. Thromb Haemost 2007; 98: 1329-1334.
40 Butt E. et al. cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphorylation (VASP) in vitro and in intact human platelets. J Biol Chem 1994; 269: 14509-14517.
41 Geiger J. et al. Specific impairment of human platelet P2Y(AC) ADP receptor-mediated signalling by the antiplatelet drug clopidogrel. Arterioscler Thromb Vasc Biol 1999; 19: 2007-2011.
42 Jakubowski JA. et al. The use of the VerifyNow P2Y₁₂ point-of-care device to monitor platelet function across a range of P2Y₁₂ inhibition levels following prasugrel and clopidogrel administration. Thromb Haemost 2008; 99: 409-415.
43 Varenhorst C. et al. Assessment of P2Y₁₂ inhibition with the point-of-care device VerifyNow P2Y₁₂ in patients treated with prasugrel or clopidogrel coadministered with aspirin. Am Heart J 2009; 157: 562.e1-562.e9.