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. 2018 Feb 16:11:10-18.
doi: 10.1016/j.plabm.2018.02.002. eCollection 2018 Jul.

Assessment of a flow cytometry technique for studying signaling pathways in platelets: Monitoring of VASP phosphorylation in clinical samples

N Mallouk  1   2 , J Varvat  3 , A Berger  4   5 , M Epinat  3   6 , S Accassat  1 , A Garcin  7 , A Montmartin  6 , G Li  8 , P Garnier  3 , P Mismetti  1   6 , C Lambert  4   9
Affiliations

Assessment of a flow cytometry technique for studying signaling pathways in platelets: Monitoring of VASP phosphorylation in clinical samples

N Mallouk et al. Pract Lab Med. .

Abstract

A recently released kit (PerFix EXPOSE) was reported to improve the measurement of the degree of phosphorylation of proteins in leukocytes by flow cytometry. We tested its adaptation for platelets to monitor vasodilator-stimulated phosphoprotein (VASP) phosphorylation, which is the basis of a currently used test for the assessment of the pharmacological response to P2Y12 antagonists (PLT VASP/P2Y12). The PerFix EXPOSE kit was compared to the PLT VASP/P2Y12 kit by using blood samples drawn at 24 h post clopidogrel dose from 19 patients hospitalized for a non-cardio-embolic ischemic stroke and treated with clopidogrel monotherapy for at least five days in an observational study. The platelet PerFix method was based on adaptation of the volume of the sample, the centrifugation speed and the incubation temperature. Poor agreement between prevention by adenosine diphosphate (ADP) of PGE1-induced cAMP-mediated VASP phosphorylation and ADP induced aggregation assessed by Light Transmittance Aggregometry was found. We found a significant correlation between the PLT VASP/P2Y12 kit and the PerFix EXPOSE kit. The PerFix EXPOSE kit may also be helpful to monitor adverse effects of second-generation tyrosine kinase inhibitors on platelets.

Keywords: Clopidogrel; Flow cytometry; Platelet signaling; VASP.

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Figures

Fig. 1
Fig. 1
Gates used for the analysis of platelets identified as CD61 + population (A–C). Whole blood samples were drawn from healthy volunteers. Representative histograms for PGE1 (white) versus PGE1 + ADP conditions (gray, B, D). PLT VASP/P2Y12 kit (A–B) and the PerFix EXPOSE kit (C-D). Ab = antibody.
Fig. 2
Fig. 2
Correlation between PRI values obtained with the PLT VASP/P2Y12 and the PerFix EXPOSE kits (A). R2 = 0.7, slope = 0.98 (n = 19). Bland – Altman plot (B).
Fig. 3
Fig. 3
Illustration of good (A, B, E, F and I) and poor clopidogrel response profiles (C, D, G, H and J). Gates used for the analysis of platelets identified as CD61 + population (A-C-E-G). Representative histograms for PGE1 (white) versus PGE1 + ADP conditions (gray, B, D, F and H). PLT VASP/P2Y12 (A–D) versus PerFix EXPOSE kits (E–H). Platelet aggregation was induced by ADP 10 μM (I–J). Maximum platelet aggregation was measured.
Fig. 4
Fig. 4
The Gate used for the analysis of platelets was identified as CD61 + population (A). Whole blood sample was drawn from a healthy volunteer. Representative histograms for PGE1 with the PerFix EXPOSE kit (B). CD61- = CD61 negative population;.
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