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

Authors

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

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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