Korean Circ J.  2015 Sep;45(5):378-385. 10.4070/kcj.2015.45.5.378.

An Assay of Measuring Platelet Reactivity Using Monoclonal Antibody against Activated Platelet Glycoprotein IIb/IIIa in Patients Taking Clopidogrel

• Joo SJ
• Choi JH
• Kim SY
• Kim KS
• Kim YR
• Kang SH

• KMID: 2223801
• DOI: http://doi.org/10.4070/kcj.2015.45.5.378

Abstract

BACKGROUND AND OBJECTIVES
Residual platelet reactivity in patients who are taking clopidogrel is commonly measured with VerifyNow assay, which is based on the principle of light transmission aggregometry. However, to evaluate the residual platelet reactivity, it would be more accurate if the reactivity of platelet glycoprotein (GP) IIb/IIIa is directly monitored. In this study, PAC1, a monoclonal antibody against activated platelet GP IIb/IIIa, was used to measure the residual platelet reactivity.
SUBJECTS AND METHODS
Twenty seven patients with coronary artery disease taking clopidogrel were enrolled. Platelets in whole blood were stained with fluorescein isothiocyanate (FITC)-conjugated PAC1. Mean fluorescence intensity (MFI) and % positive platelets (PP) were measured with flow cytometry, and the binding index (BI; MFI x %PP/100) was calculated. P2Y12 reaction unit (PRU) and % inhibition of VerifyNow assay were also measured in the usual manner.
RESULTS
PRU of VerifyNow assay correlated significantly with MFI, %PP, and BI at 10 microM (r=0.59, 0.73, and 0.60, respectively, all p<0.005) and 20 microM of adenosine diphosphate (ADP; r=0.61, 0.75, and 0.63, respectively, all p<0.005). The % inhibition also correlated significantly with MFI, %PP, and BI at 10 microM (r=-0.60, -0.69, and -0.59, respectively, all p<0.005) and 20 microM of ADP (r=-0.63, -0.71, and -0.62, respectively, all p<0.005).
CONCLUSION
Direct measurements of the reactivity of platelet GP IIb/IIIa were feasible using PAC1 and flow cytometry in patients taking clopidogrel. Further clinical studies are required to determine the cut-off values which would define high residual platelet reactivity in patients on this treatment protocol.

Keyword

Blood platelets; Glycoprotein IIb/IIIa; Platelet function test; Flow cytometry; Clopidogrel

MeSH Terms

Adenosine Diphosphate
Blood Platelets*
Coronary Artery Disease
Flow Cytometry
Fluorescein
Fluorescence
Glycoproteins*
Humans
Platelet Function Tests
Adenosine Diphosphate
Fluorescein
Glycoproteins

Figure

• 

  Fig. 1 Flow cytometric analysis of whole blood. (A) Platelets were separated from erythrocytes and white blood cells, on the basis of their characteristic forward- and side- scatter profiles, and a narrow gate was placed around the platelets. (B) Staining of resting platelets with PE-conjugated anti-CD41, a monoclonal antibody against glycoprotein IIb. (C) Staining of platelets with control FITC-conjugated mouse IgMκ. (D) Staining of platelets with FITC-conjugated PAC1. Antibody-positive platelets were defined as those with a fluorescence intensity >99.0% to 99.5%, of non-stimulated platelets stained with FITC-conjugated mouse IgM κ. SSC: side-scatter, FSC: forward-scatter, FITC: fluorescein isothiocyanate, PP: positive platelet.

• 

  Fig. 2 Examples of flow cytometric data and analysis in a patient with (A) low residual platelet reactivity (B) high residual platelet reactivity. A patient with high residual platelet reactivity showed much greater mean fluorescence intensity (MFI), % positive platelets (%PP), and binding index (BI) at 10 µM or 20 µM of ADP. ADP: adenosine diphosphate, FITC: fluorescein isothiocyanate.

• 

  Fig. 3 Changes of mean fluorescence intensity (MFI), % positive platelets, and binding index after ADP stimulation. Values are mean±standard error of mean. They reached maximal values at 10 µM concentration. ADP: adenosine diphosphate.

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  Fig. 4 Correlation between mean fluorescence intensity (MFI), % positive platelets, and binding index with VerifyNow PRU at (A) 10 µM or (B) 20 µM of ADP. PRU: P2Y12 reaction unit, ADP: adenosine diphosphate.

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  Fig. 5 Correlation between mean fluorescence intensity (MFI), % positive platelets, and binding index with VerifyNow P2Y12 % inhibition at (A) 10 µM or (B) 20 µM of ADP. ADP: adenosine diphosphate.

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