Comparison of VASP-phosphorylation assay to light-transmission aggregometry in assessing inhibition of the platelet ADP P2Y12 receptor

Agnieszka Pampuch; Chiara Cerletti; Giovanni de Gaetano

doi:10.1160/TH06-09-0491

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2006; 96(06): 767-773
DOI: 10.1160/TH06-09-0491

Platelets and Blood Cells

Schattauer GmbH

Comparison of VASP-phosphorylation assay to light-transmission aggregometry in assessing inhibition of the platelet ADP P2Y₁₂ receptor

Agnieszka Pampuch

¹Laboratory of Cell Biology and Pharmacology of Thrombosis, Research Laboratories, “John Paul II” Center for High Technology Research and Education in Biomedical Sciences, Catholic University, Campobasso, Italy

,

Chiara Cerletti

¹Laboratory of Cell Biology and Pharmacology of Thrombosis, Research Laboratories, “John Paul II” Center for High Technology Research and Education in Biomedical Sciences, Catholic University, Campobasso, Italy

,

Giovanni de Gaetano

¹Laboratory of Cell Biology and Pharmacology of Thrombosis, Research Laboratories, “John Paul II” Center for High Technology Research and Education in Biomedical Sciences, Catholic University, Campobasso, Italy

› Author Affiliations

Abstract

Summary

There is need to improve platelet function testing to monitor the response to antiplatelet drugs. We compared flow-cytometric analysis of intraplatelet vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) to light-transmission aggregometry for the detection of drug-induced in-vitro inhibition of the platelet P2Y₁₂ ADP receptor on 22 healthy subjects (10 males, 12 females, 28.5 ± 6.6 years). The platelet reactivity index (PRI) of VASP was calculated both from mean fluorescence intensity (MFI) and percent of fluorescence-positive platelets in the presence of PGE₁ with or without ADP (10 µM). Platelet aggregation was induced by ADP (1.25, 2.5 and 5 µM). Cangrelor, a competitive inhibitor of the P2Y₁₂ receptor, preincubated 5 minutes, induced a concentration-dependent inhibition of platelet ADP-receptor function in both tests. Indeed PRI (%) based on either MFI or percent platelets gated were highly correlated with each other (r = 0.97, p<0.0001) and with aggregation in- duced by ADP. The IC₅₀ of cangrelor against each of the three ADP concentrations used in aggregometry increased from 5.8 ± 3.4 nM to 23.1 ± 4.0 nM and to 98 ± 25 nM, respectively. The IC₅₀ of cangrelor based on VASP-P was within the same range (25.5 ± 7.7 nM). No correlation was observed between IC₅₀ values of cangrelor and ADP concentrations giving 50% effect (EC₅₀) in the absence of the drug. However, at 10 nM cangrelor seven subjects could be identified by the VASP-P assay as “low responders” to the drug (PRI> 50%), and six of them also had an aggregation response to 5 µM ADP > 50%. These six subjects showed the lowest ADP EC₅₀ values in the absence of the drug, possibly reflecting high sensitivity of their platelet P2Y₁₂ receptors to ADP. In conclusion, both the VASP-P assay and light-transmission aggregometry detect in a comparable way in-vitro pharmacological inhibition of the platelet P2Y₁₂ ADP receptor and its individual variability.

Keywords

VASP phosphorylation - platelet aggregation - P2Y₁₂ ADP receptor antagonist - cangrelor

Full Text

References

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