‘VASPFix’ for measurement of VASP phosphorylation in platelets and for monitoring effects of P2Y₁₂ antagonists

Summary

Vasodilator-stimulated phosphoprotein (VASP) is phosphorylated and dephosphorylated consequent to increases and decreases in cyclic nucleotide levels. Monitoring changes in VASP phosphorylation is an established method for indirect measurement of cyclic nucleotides. Here we describe the use of an innovative cocktail, VASPFix, which allows sensitive and reproducible measurement of phosphorylated VASP (VASP-P) in a simple, single-step procedure using cytometric bead technology. Frozen VASPFix-treated samples are stable for at least six months prior to analysis. We successfully used VASPFix to measure VASP-P in platelets in both platelet-rich plasma and blood in response to compounds that increase (dibutyryl cAMP, adenosine, iloprost, PGE₁) and decrease (ADP, PGE₁) cAMP, and to determine the effects of certain receptor antagonists on the results obtained. The change in VASP-P brought about by adding ADP to PGE₁-stimulated platelets is a combination of the effect of ADP at the P2Y₁₂ receptor and of PGE₁ at both IP and EP3 receptors. For iloprost-stimulated platelets EP3 receptors are not involved. A procedure in which iloprost, ADP and VASPFix were used to determine effectiveness of clopidogrel and prasugrel in patients was compared with an established commercial procedure that uses PGE₁ and ADP; the latter produced higher platelet reactivity values that were the result of PGE₁ interacting with platelet EP3 receptors. We conclude that VASPFix can be used both as a research tool and for clinical investigations and provides better specificity for P2Y₁₂ receptor inhibition. The latter confers a distinct advantage over existing methods used to monitor effects of P2Y₁₂ antagonists on platelet function.

Keywords

VASP phosphorylation - cyclic nucleotides - cytometric bead assay - P2Y₁₂ antagonists - platelets

• References

• 1 Schwarz UR, Walter U, Eigenthaler M. Taming platelets with cyclic nucleotides. Biochem Pharmacol 2001; 62: 1153-1161.
• 2 Nolte C, Eigenthaler M, Schanzenbacher P. et al. Comparison of vasodilatory prostaglandins with respect to cAMP-mediated phosphorylation of a target substrate in intact human platelets. Biochem Pharmacol 1991; 42: 253-262.
• 3 Geiger J, Brich J, Honig-Liedl P. et al. Specific impairment of human platelet P2Y(AC) ADP receptor-mediated signalling by the antiplatelet drug clopido-grel. Arterioscler Thromb Vasc Biol 1999; 19: 2007-2011.
• 4 Sudo T, Ito H, Kimura Y. Phosphorylation of the vasodilator-stimulated phosp-hoprotein (VASP) by the anti-platelet drug, cilostazol, in platelets. Platelets 2003; 14: 381-390.
• 5 Yamamoto H, Takahashi K, Watanabe H. et al. Evaluation of the antiplatelet effects of cilostazol, a phosphodiesterase 3 inhibitor, by VASP phosphorylation and platelet aggregation. Circ J 2008; 72: 1844-1851.
• 6 Yamanouchi J, Hato T, Niiya T. et al. Vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay for platelet response to cilostazol. Platelets 2010; 22: 135-142.
• 7 Aktas B, Utz A, Hoenig-Liedl P. et al. Dipyridamole enhances NO/cGMP-me-diated vasodilator-stimulated phosphoprotein phosphorylation and signaling in human platelets: In vitro and in vivo/ex vivo studies. Stroke 2003; 34: 764-769.
• 8 Chakrabarti S, Blair P, Wu C. et al. Redox state of dipyridamole is a critical determinant for its beneficial antioxidant and antiinflammatory effects. J Cardiovasc Pharmacol 2007; 50: 449-457.
• 9 Schwarz UR, Geiger J, Walter U. et al. 1999 Flow cytometry analysis of intracellular VASP phosphorylation for the assessment of activating and inhibitory signal transduction pathways in human platelets - Definition and detection of ti-clopidine/clopidogrel effects. Thromb Haemost 1999; 82: 1145-1152.
• 10 Barragan P, Bouvier J-L, Roquebert P-O. 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.
• 11 Aleil B, Ravanat C, Cazenave JP. 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.
• 12 Blindt R, Stellbrink K, de Taeye A. et al. The significance of vasodilator-stimulated phosphoprotein for risk stratification of stent thrombosis. Thromb Hae-most 2007; 98: 1329-1334.
• 13 Bonello L, Paganelli F, Arpin-Bornet M. et al. Vasodilator-stimulated phospho-protein phosphorylation analysis prior to percutaneous coronary intervention for exclusion of postprocedural major adverse cardiovascular events. J Thromb Haemost 2007; 5: 1630-1636.
• 14 Schäfer A, Weinberger S, Flierl U. 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.
• 15 Bouman HJ, Parlak E, van Werkum JW. et al. Which platelet function test is suitable to monitor clopidogrel responsiveness? A pharmacokinetic analysis on the active metabolite of clopidogrel. J Thromb Haemost 2010; 8: 482-488.
• 16 Kleiman NS. Will measuring vasodilator-stimulated phosphoprotein phos-phorylation help us optimize the loading dose of clopidogrel?. J Am Coll Cardiol 2008; 51: 1412-1414.
• 17 Bonello L, Laine M, Baumstarck K. et al. A randomized trial of platelet reactivity monitoring-adjusted clopidogrel therapy versus prasugrel therapy to reduce high on-treatment platelet reactivity. Int J Cardiol. 2013 Epub ahead of print.
• 18 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.
• 19 Geiger J, Brandmann T, Hubertus K. et al. A protein phosphorylation-based assay for screening and monitoring of drugs modulating cyclic nucleotide pathways. Anal Biochem 2010; 407: 261-269.
• 20 Jakubowski JA, Bourguet N, Boulay-Moine D. et al. Comparison of a new ELISA assay with the flow cytometric assay for platelet vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation in whole blood to assess P2Y(12) inhibition. Thromb Haemost 2012; 107: 388-395.
• 21 Abtan J, Silvain J, Kerneis M. et al. Identification of poor response to P2Y12 inhibitors in ACS patients with a new ELISA-based vasodilator-associated stimu- lated phosphoprotein (VASP) phosphorylation assay. Thromb Haemost 2013; 110: 1055-1064.
• 22 Hobl EL, Jilma B, Derhaschnig U. et al. Comparison of a new ELISA-based with the flow cytometric assay for vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation to assess P2Y12 -inhibition after ticagrelor intake. Cy-tometry B Clin Cytom. 2013 Epub ahead of print.
• 23 Butt E, Abel K, Krieger M. et al. cAMP-dependent and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in-vitro and in intact human platelets. J Biol Chem 1994; 269: 14509-14517.
• 24 Iyu D, Juttner M, Glenn JR. et al. PGE(1) and PGE(2) modify platelet function through different prostanoid receptors. Prostagland Lipid Med 2011; 94: 9-16.
• 25 Wiviott SD, Braunwald E, McCabe CH. et al. Prasugrel versus clopidogrel in patients with acute coronary syndromes. New Engl J Med 2007; 357: 2001-2015.
• 26 Sugidachi A, Ogawa T, Kurihara A. et al. The greater in vivo antiplatelet effects of prasugrel as compared to clopidogrel reflect more efficient generation of its active metabolite with similar antiplatelet activity to that of clopidogrel’s active metabolite. J Thromb Haemost 2007; 5: 1545-1551.
• 27 Jaremo P, Lindahl TL, Fransson SG. et al. Individual variations of platelet inhibition after loading doses of clopidogrel. J Intern Med 2002; 252: 233-238.
• 28 Judge HM, Buckland RJ, Sugidachi A. et al. Relationship between degree of P2Y12 receptor blockade and inhibition of P2Y12-mediated platelet function. Thromb Haemost 2010; 103: 1210-1217.
• 29 Cattaneo M. The platelet P2Y12 receptor for adenosine diphosphate: congenital and drug-induced defects. Blood 2011; 117: 2102-2112.
• 30 Zighetti ML, Carpani G, Sinigaglia E. et al. Usefulness of a flow cytometric analysis of intraplatelet vasodilator-stimulated phosphoprotein phosphorylation for the detection of patients with genetic defects of the platelet P2Y12 receptor for ADP. J Thromb Haemost 2010; 8: 2332-2334.