Comparison of a new ELISA assay with the flow cytometric assay for platelet vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation in whole blood to assess P2Y₁₂ inhibition

Summary

Thienopyridines and other agents target the platelet P2Y₁₂ receptor and inhibit several platelet activities mediated by adenosine diphosphate (ADP). The measurement of vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation, expressed as platelet reactivity index (PRI), mirrors the degree of P2Y₁₂ receptor inhibition and can detect the well-known variable response to clopidogrel. The commercially available VASP assay uses flow cytometry (FC) and requires that the test be run within 48 hours of blood collection. A new ELISA VASP assay offers the advantages of using more widely available technology and the potential to freeze and store samples before analysis. The objectives of the present study were to compare the performance of the ELISA and FC methods and to describe the relative flexibility of the ELISA-based assay. Human blood samples encompassing a wide range of levels of P2Y₁₂ blockade achieved in vitro by preincubation with P2Y₁₂ antagonists or in vivo from patients treated with clopidogrel were included, reflecting the wide spread of values reported in clinical studies. The correlation between the PRI measured by ELISA and FC was highly significant (r=0.95, p<0.001), (n=80). After the initial activation, samples were stable for at least four weeks when frozen (−20°C) prior to analysis by ELISA. Frozen samples from patients treated with clopidogrel appeared stable for up to nine weeks. Based on these results, the ELISA-based assay appears to provide a reliable and more flexible alternative to the FC method to determine P2Y₁₂ receptor blockade and may enable more extensive utilisation of the VASP assay in clinical studies.

Note: All work done at BioCytex, Marseille, France and Daiichi Sankyo, Tokyo, Japan.

Keywords

VASP - ELISA - flow cytometry - clopidogrel - prasugrel

• References

• 1 Jakubowski JA, Winters KJ, Naganuma H. et al. Prasugrel: a novel thienopyridine antiplatelet agent. A review of preclinical and clinical studies and the mechanistic basis for its distinct antiplatelet profile. Cardiovasc Drug Rev 2007; 25: 357-374.
• 2 Gachet C.. P2 receptors, platelet function and pharmacological implications.. Thromb Haemost 2008; 99: 466-472.
• 3 Cattaneo M.. New P2Y(12) inhibitors.. Circulation 2010; 121: 171-179.
• 4 Wallentin L, Becker RC, Budaj A. et al. Ticagrelor versus clopidogrel in patients with acute coronary syndromes.. N Engl J Med 2009; 361: 1045-1057.
• 5 Gurbel PA, Bliden KP, Antonino MJ. et al. The effect of elinogrel on high platelet reactivity during dual antiplatelet therapy and the relation to CYP2C19*2 genotype: first experience in patients.. J Thromb Haemost 2010; 8: 43-53.
• 6 Angiolillo DJ, Fernandez-Ortiz A, Bernardo E. et al. Variability in individual responsiveness to clopidogrel: clinical implications, management, and future perspectives.. J Am Coll Cardiol 2007; 49: 1505-1516.
• 7 Gurbel PA.. The relationship of platelet reactivity to the occurrence of post-stenting ischemic events: emergence of a new cardiovascular risk factor. Rev Cardiovasc Med 2006; 7 (Suppl. 04) S20-S28.
• 8 Jakubowski JA, Payne CD, Li YG. 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 2008; 99: 215-222.
• 9 Cattaneo M.. Light transmission aggregometry and ATP release for the diagnostic assessment of platelet function.. Semin Thromb Hemost 2009; 35: 158-167.
• 10 Michelson AD.. Methods for the measurement of platelet function. Am J Cardiol 2009; 103: 20A-26A.
• 11 Michelson AD, Frelinger III AL, Furman MI.. Current options in platelet function-testing. Am J Cardiol 2006; 98: 4N-10N.
• 12 Jakubowski JA, Payne CD, Li YG. 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.
• 13 Barragan P, Bouvier JL, Roquebert PO. et al. Resistance to thienopyridines: clinical detection of coronary stent thrombosis by monitoring of vasodilator-stimulated phosphoprotein phosphorylation.. Catheter Cardiovasc Interv 2003; 59: 295-302.
• 14 Blindt R, Stellbrink K, de TA. et al. The significance of vasodilator-stimulated phosphoprotein for risk stratification of stent thrombosis. Thromb Haemost 2007; 98: 1329-1334.
• 15 Bonello L, Paganelli F, Arpin-Bornet M. et al. Vasodilator-stimulated phosphoprotein phosphorylation analysis prior to percutaneous coronary intervention for exclusion of postprocedural major adverse cardiovascular events.. J Thromb Haemost 2007; 5: 1630-1636.
• 16 Barragan P, Paganelli F, Camoin-Jau L. et al. Validation of a novel ELISA-based VASP whole blood assay to measure P2Y12-ADP receptor activity.. Thromb Haemost 2010; 104: 410-411.
• 17 Angiolillo DJ, Gibson CM, Cheng S. et al. Differential effects of omeprazole and pantoprazole on the pharmacodynamics and pharmacokinetics of clopidogrel in healthy subjects: randomized, placebo-controlled, crossover comparison studies.. Clin Pharmacol Ther 2011; 89: 65-74.
• 18 Bonello L, Tantry US, Marcucci R. et al. Consensus and future directions on the definition of high on-treatment platelet reactivity to adenosine diphosphate.. J Am Coll Cardiol 2010; 56: 919-933.