Abstract 12847: A New Assay of Measuring Platelet Reactivity Using Monoclonal Antibody Against Activated Platelet Glycoprotein IIb/IIIa in a Whole Blood
Introduction: Clopidogrel binds to P2Y12 ADP receptors of platelets to inhibit aggregation. However, in some patients with coronary artery diseases, platelet aggregation is not inhibited despite taking clopidogrel. Although VerifyNow P2Y12 assay is commonly used to measure residual platelet reactivity, it is a kind of light transmission aggregometry. Activation and prothrombin binding of platelet glycoprotein (GP) IIb/IIIa is a final common pathway of platelets aggregation. If the reactivity of platelet GP IIb/IIIa is directly measured, that will be a more accurate assay to evaluate residual platelet reactivity.
Hypothesis: PAC1, a monoclonal antibody against activated platelet GP IIb/IIIa, and flow cytometric analysis would be used to measure platelet reactivity.
Methods: Twenty seven patients with coronary artery disease who were taking clopidogrel were enrolled in this study. Whole blood was withdrawn into a sodium citrate coated tube. FITC-conjugated PAC1 15 μL (3 μg) was incubated for 15 minutes with 5 μL of whole blood in a mixture, consisted of 5 μL of ADP, and 25 μL of isotonic HEPES buffer (total volume 50 μL). Final concentrations of ADP were 1, 10, or 20 μM. After fixation with 1% paraformaldehyde, flow cytometric analysis was performed with BD FACSCalibur. Mean fluorescence intensity (MFI) and % positive platelets (PP) were measured and binding index (BI: MFI x %PP/100) was calculated. P2Y12 reaction unit (PRU) and %inhibition of VerifyNow assay were also measured in a usual manner.
Results: PRU of VerifyNow assay correlated significantly with MFI, %PP, and BI at 10 μM (r=0.59, 0.73, and 0.60, all p<0.005) or 20 μM of ADP (r=0.61, 0.75, and 0.63, all p<0.005). % inhibition of VerifyNow assay also correlated significantly with MFI, %PP, and BI at 10 μM (r=-0.60, -0.69, and -0.59, all p<0.005) or 20 μM of ADP (r=-0.63, -0.71, and -0.62, all p<0.005).
Conclusions: Direct measurements of the reactivity of platelet GP IIb/IIIa were feasible using PAC1 and flow cytometry. Cut-off values of this assay to define residual platelet reactivity are needed to be determined in the clinical study.
Author Disclosures: S. Joo: None. J. Choi: None. S. Kim: None. K. Kim: None. Y. Kim: None.
- © 2014 by American Heart Association, Inc.