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
1   Laboratory 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
1   Laboratory 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
1   Laboratory 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
› Institutsangaben
Financial support: This work was partially supported by the Italian Ministry of Health (MINSAL Ricerca Finalizzata 2002, Convenzione 241) and by MIUR (Programma Triennale Ricerca, decreto 1588).
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Publikationsverlauf

Received 01. September 2006

Accepted after resubmission 28. Oktober 2006

Publikationsdatum:
29. November 2017 (online)

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.

 
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