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

Authors


Marco Cattaneo, Unità di Medicina III, Ospedale San Paolo, Dipartimento di Medicina, Chirurgia e Odontoiatria, Università degli Studi di Milano, Via di Rudinì 8, 20142 Milan, Italy.
Tel.: +39 250323095; fax: +39 250323089.
E-mail: marco.cattaneo@unimi.it

Platelets possess two P2Y receptors for ADP: P2Y1, which is coupled to Gq, mediates ADP-induced platelet shape change and initiates ADP-induced aggregation, and P2Y12, which is coupled to Gi and amplifies the platelet aggregation response [1]. Concomitant activation of both the Gq and Gi pathways by ADP is necessary to elicit normal aggregation. P2Y12 is important for both normal hemostasis and pathologic thrombosis, as proven by the observations that drugs targeting P2Y12, such as the thienopyiridines (ticlopidine, clopidogrel and prasugrel) and the direct, reversible inhibitor ticagrelor, reduce the risk of major cardiovascular events and increase the risk of bleeding [2], and that patients with congenital P2Y12 deficiency have a bleeding diathesis [1].

In 1992, the first patient (V.R.) with congenital, severe P2Y12 deficiency was described [3]. He had a lifelong history of excessive bleeding and prolonged bleeding time. His platelet aggregation response to ADP was severely impaired, even at very high ADP concentrations (> 10 μm), whereas ADP-induced platelet shape change was normal. Other typical platelet abnormalities were: (i) no inhibition by ADP of prostaglandin (PG)E1-stimulated increase in platelet cAMP, but normal inhibition by epinephrine; and (ii) the presence of approximately 30% of the normal number of binding sites for [33P]2MeS-ADP on fresh platelets [3] or [3H]ADP on formalin-fixed platelets [4], suggesting that the P2Y1 component (about 30%) was normally represented, whereas the P2Y12 component (about 70%) was severely deficient. Additional patients, with similar characteristics, were later described [5–7]. Another patient (A.C.) with abnormal P2Y12-mediated platelet responses to ADP was also described, whose platelets display normal number of dysfunctional P2Y12 receptors, owing to compound heterozygosity of two missense mutations affecting TM6 and EL3 of the P2Y12 molecule [8]. The response of his platelets to very high concentrations of ADP, albeit impaired, was slightly more pronounced than that observed in platelets that are severely deficient in the receptor, and was further decreased by a P2Y12 antagonist, indicating the presence of some residual receptor function.

Although defects of P2Y12 receptor can be easily suspected, on the basis of the observation that ADP, even at high concentrations, induces very slight and rapidly reversible platelet aggregation, the laboratory tests that are necessary to confirm the diagnosis in suspected cases are not easily available to most laboratories. Of the two diagnostic confirmatory tests, that is, (i) lack of ADP-mediated inhibition of the cAMP increase induced by adenylyl cyclase (AC) stimulation, and, (ii) deficiency of P2Y12-associated binding sites for ADP (usually measured by the use of radiolabeled 2MeS-ADP), the first one is preferable because it is sensitive to both quantitative and qualitative defects of the receptor. However, inhibition of the AC-induced increase in cAMP by ADP is a time-consuming and cumbersome assay, involving: preparation of platelet-rich plasma (PRP) from whole blood; its exposure to PGE1, PGI2 or other stimulators of AC, in the presence or absence of different concentrations of ADP; centrifugation of PRP samples to obtain a platelet pellet; lysis of the platelet pellet; extraction of cAMP from platelel lysates; and measurement of cAMP with an immunoassay. An alternative, less time-consuming, faster and cheaper method for measuring the effect of ADP on AC involves measurement of the degree of phosphorylation of vasodilator-stimulated phosphoprotein (VASP), an intracellular actin regulatory protein, by cAMP-dependent protein kinase A [9]. The P2Y12-mediated inhibition of AC by ADP prevents VASP phosphorylation induced by agents that increase platelet cAMP, such as PGE1. Levels of VASP phosphorylation/dephosphorylation thus reflect P2Y12 function.

The aim of our study was to test whether a commercially available flow cytometric VASP phosphorylation assay [10] is useful for diagnosing congenital mild-to-severe defects of the platelet P2Y12 receptor.

We evaluated four groups of subjects: 20 healthy blood donors (median age 42 years, range 20–66 years, 10 men and 10 women); two patients with severe P2Y12 deficiency (V.R., a 68-year-old man [3], and M.G., a 56-year-old woman [6]); one patient with dysfunctional P2Y12 function (A.C, a 63-year-old man [8]); two patients with heterozygous P2Y12 defects (G.L, a 23-year-old boy, son of M.G. [6], and F.C, a 34-year-old man, son of A.C. [8]), whose platelets had mild-to-moderate abnormalities of P2Y12-dependent platelet function [6,8]. The number of P2Y12-associated binding sites for 2MeS-ADP in G.L.’s platelets was about 60% of the lower limit of the normal range and about 35% of the median normal value [6].

Blood samples were collected from an antecubital vein and mixed 9 : 1 with sodium citrate (10.9 mm, final concentration). The VASP phosphorylation analysis was performed immediately after blood collection, using Platelet VASP (Diagnostica Stago, Asnières, France), according to the manufacturer’s instructions. Briefly, blood samples were incubated with PGE1 and ADP, alone and in combination, for 10 min and fixed with paraformaldehyde. Platelets were then permeabilized with a non-ionic detergent, labeled with a primary monoclonal antibody (16C2), and then with a secondary fluorescein isothiocyanate-conjugated polyclonal goat anti-mouse Ig antibody (BioCytex, Marseille, France). Analyses were performed using a Becton Dickinson (Plymouth, UK) FACS Calibur flow cytometer. The platelet population was identified from its forward and side scatter distribution, and 10 000 platelets were gated. The platelet reactivity index (PRI) was calculated from the median fluorescence intensity (MFI), reflecting VASP phosphorylation, of samples incubated with PGE1 or PGE1 plus ADP, according to the formula: PRI = [(MFI(PGE1) − MFI(PGE1 + ADP))/MFI(PGE1)] × 100. The analysis was completed in about 30 min after blood sampling.

The PRI ranged between 76.7% and 95.3% in 20 blood donors. Patients with severe P2Y12 defects had severely decreased PRI (V.R., 0.7%; M.G., 0%); patient A.C. (with dysfunctional P2Y12) had a low PRI (47.7%); and patients with heterozygous P2Y12 defects had PRIs that were indistiguishable from those of healthy blood donors (G.L., 80.4%; F.C., 85.4%) (Fig. 1).

Figure 1.

 Platelet reactivity index, as measured by analysis of vasodilator-stimulated phosphoprotein phosphorylation, in 20 blood donors, patients with severe P2Y12 deficiency, a patient with dysfunctional P2Y12 and patients with heterozygous P2Y12 defects.

Therefore, the flow cytometric analysis of VASP is a fast and useful technique for diagnosing patients with severe or moderate P2Y12 receptor defects, but is unable to identify patients with heterozygous defects of the receptor. Our results might also be relevant to the use of the VASP phosphorylation assay for monitoring individual response to clopidogrel, which has been recommended for identifying patients who are poor responders to the drug [2]. The VASP phosphorylation assay has been used for this purpose in many studies [10,11], but the cut-off value for the identification of patients who are poor responders or resistant to the drug has not been clearly identified yet. From our observation that the assay is insensitive to mild defects of P2Y12, it can be concluded that clopidogrel-treated patients who display a normal PRI with the VASP phosphorylation assay should also not be considered resistant to clopidogrel, because they may have a high percentage of their platelet receptors inhibited by the drug.

Disclosure of Conflict of Interests

The authors state that they have no conflict of interest.

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